YES Guide YES Meeting 2010

Book of Abstracts
PRESENTING STUDENTS ABSTRACTS
Oncology
PRESENTING STUDENTS ABSTRACTS
PS 131 PS 233
THE ROLE OF GSTM1 AND GSTT1 POLYMORPHISM IN PATIENTS WITH RENAL SYNTHESIS AND ANTITUMOR ACTIVITY OF DIHYDROPYRANO DERIVATIVES OF
CELL CARCINOMA . . . . . . . . . . . . . . . . . . . . . . . . . . 6 BAICALEIN AND 3,7-DIHYDROXYFLAVONE . . . . . . . . . . . . . . . 19
PS 137 PS 234
GLUCOSE UPTAKE BY BREAST CANCER CELLS: IMPLICATIONS ON CELL EVALUATION OF THE INHIBITION OF MONOCARBOXYLATE TRANSPORTERS IN
METABOLISM AND SURVIVAL . . . . . . . . . . . . . . . . . . . . 7 GLIOMAS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
PS 146 PS 249
IS PHOSPHORYLATION OF THE ANDROGEN RECEPTOR OF CLINICAL EPIGENETICS - A NEW TARGET THERAPEUTIC APPROACH IN CANCER . . . 21
SIGNIFICANCE IN THE DEVELOPMENT AND PROGRESSION OF PROSTATE PS 259
CANCER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 INFLUENCE OF HOXA9 EXPRESSION IN THE RESPONSE OF GLIOBLASTOMA
PS 154 CELLS TO TEMOZOLOMIDE . . . . . . . . . . . . . . . . . . . . . . 22
COMPARATIVE FEATURES OF EXPERIMENTAL FIBROSARCOMA AT THREE PS 261
HAMSTER SPECIES . . . . . . . . . . . . . . . . . . . . . . . . . 9 CORRELATION BETWEEN GLUT EXPRESSION AND IN VITRO 18F-FDG UPTAKE
PS 156 IN BREAST CANCER CELL LINES . . . . . . . . . . . . . . . . . . . 23
LOW DOSES OF IONIZING RADIATION PROMOTE TUMOR GROWTH AND PS 270
METASTASIS BY ENHANCING ANGIOGENESIS . . . . . . . . . . . . . 10 AVIAN PATOGENIC ESCHERICHIA COLI ISOLATED FROM BACTEREMIA IN
PS 190 HUMANS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
EFFECT OF RANK-RANKL SIGNALLING PATHWAYS AND MMP1 EXPRESSION PS 277
IN THE INVASIVE ABILITY OF BREAST AND PROSTATE CANCER CELL LINES IN ASCORBIC ACID AND ITS POTENTIALITY AS AN ANTICANCER AGENT: IN VITRO
VITRO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 AND IN VIVO STUDIES IN HUMAN COLORECTAL CANCER . . . . . . . . . 25
PS 197 PS 280
NICOTINE EFFECTS ON BONE METABOLISM: IN VITRO STUDIES WITH HUMAN EXPRESSION OF DNA METHYLTRANSFERASES IN THE MOUSE HEART
OSTEOCLASTS AND CO-CULTURES OF OSTEOCLASTS AND OSTEOBLASTS . . 12 FOLLOWING MATERNAL PROTEIN UNDERNUTRITION . . . . . . . . . . 26
PS 198 PS 307
MITOCHONDRIA AS A NEW THERAPEUTIC TARGET IN CANCER . . . . . . 13 POTENTIAL THERAPEUTIC OF BATIMASTAT IN MULTIPLE MYELOMA CELLS A
PS 203 PRELIMINARY STUDY . . . . . . . . . . . . . . . . . . . . . . . . 27
EXPRESSION OF P53 PROTEIN IN INFILTRATING DUCTAL CARCINOMA OF THE PS 310
BREAST . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 PREVENTION OF BLADDER CANCER BY GREEN TEA IN MUS MUSCULUS . . 28
PS 211 PS 315
LEAP OF FAITH: UNDERSTANDING THE RELATIONSHIPS BETWEEN BRCA1 AND IN VITRO EFFECT OF MELOXICAM ON T24 AND 5637 INVASIVE BLADDER
ESTROGEN RECEPTOR IN HEREDITARY BREAST CANCER . . . . . . . . . 15 CANCER CELL LINES . . . . . . . . . . . . . . . . . . . . . . . . . 29
PS 215 PS 317
CELLULAR AND MOLECULAR CHARACTERIZATION OF THE PARACRINIC EFFECTS PROSPECTIVE ANALYSIS OF PATIENTS WITH HEPATITIS VIRUSES AND CHRONIC
OF HUMAN BREAST CANCER CELL LINES ON OSTEOCLASTOGENESIS . . . 16 LYMPHOPROLIFERATIVE DISORDERS . . . . . . . . . . . . . . . . . 30
PS 218 PS 326
ENHANCED SIALYLATION OF SURFACE ANTIGENS ON BLADDER CANCER CELL DISTINCT CHARACTERISTICS OF UPTAKE OF DIETARY FOLATES BY TWO BREAST
LINES INDUCES DENDRITIC CELL IMMUNE TOLERANCE IN VITRO. . . . . 17 CANCER CELL LINES: T47D AND MCF7 CELLS . . . . . . . . . . . . . . 31
PS 222 PS 334
CD40 AND CD26 EXPRESSION: IMPLICATION IN ORAL CANCER AND DRUG THE INFLUENCE OF DIETILNITROSAMINE IN MICE PHYSIOLOGICAL
RESPONSE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

2 Abstracts Oncology 3
 YES Meeting 2010
YES Guide

Book of Abstracts
PRESENTING STUDENTS ABSTRACTS
Oncology
PRESENTING STUDENTS ABSTRACTS

PARAMETERS . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
PS 353
POTENTIAL MUTAGENICITY OF TXA1, A SMALL MOLECULE POTENT INHIBITOR
OF HUMAN TUMOR CELL GROWTH . . . . . . . . . . . . . . . . . . 33
PS 357
MELANOCORTIN 5 RECEPTOR TARGETING TO CELL SURFACE: THE ROLE OF THE
N-TERMINUS . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
PS 359
TRAIL- A NEW APPROACH IN MYELOID LEUKEMIAS . . . . . . . . . . . 35

4 Abstracts Oncology 5
 YES Guide YES Meeting 2010
PS 131 PS 137
THE ROLE OF GSTM1 AND GSTT1 POLYMORPHISM IN PATIENTS WITH GLUCOSE UPTAKE BY BREAST CANCER CELLS: IMPLICATIONS ON CELL
RENAL CELL CARCINOMA METABOLISM AND SURVIVAL
VESNA C 1, 2- L. MOREIRA, 1- A. CORREIA-BRANCO, 1- F. MARTEL, 1- E. KEATING
FACULTY OF MEDICINE, UNIVERSITY OF BELGRADE 1 - DEPARTMENT OF BIOCHEMISTRY, FACULTY OF MEDICINE, UNIVERSITY OF PORTO, 2 - FACULTY OF SCIENCES, UNIVERSITY OF
PORTO
AIM
AIM
The aim of this study was to test the association between GSTM1 and GSTT1 polymorphism and susceptibility to renal cell carci-
noma (RCC), independently or in conjunction with known risk factors. The aims of this work were to characterize glucose uptake by the human breast cancer cell line MCF7 and to investigate the effect
of the flavonoids quercetin (QUE) and epigallocatechin galate (EGCG) upon glucose uptake and metabolism and upon MCF7 cell
INTRODUCTION viability and proliferation.
Members of glutathione transferase (GST) superfamily exhibit polymorphic expression. GSTs are investigated as biomarkers of risk INTRODUCTION
for various cancers, including renal cell carcinoma (RCC).
Breast cancer is the most common type of cancer amongst women in Western Europe and North America and is the second leading
METHODS cause of cancer deaths in this population. Tumour cells, when compared with normal differentiated cells, have a higher demand of
glucose. Indeed, higher glucose uptake and metabolism are obligatory to the malignant transformation process and are positively
Genomic DNA was isolated from 182 controls and 76 patients with RCC. GSTM1 and GSTT1 genotypes were determined by multi- correlated with tumour cell invasiveness. Nonetheless, knowledge on glucose uptake and metabolism in breast cancer cells is
plex PCR. Data obtained were analyzed with respect to RCC risk factors including smoking and occupational exposure. still largely unexplored. A recent work from our group demonstrated that the green tea flavonoids QUE and EGCG inhibit glucose
RESULTS uptake by placental cells. So, we hypothesized that these flavonoids could also inhibit glucose uptake in breast cancer cells, impair-
ing glucose metabolism and decreasing cell viability and proliferation.
The frequency of GSTM1-null genotype was higher in patients with RCC (60.5%) compared to controls (47.2%). GSTT1-null
genotype was found in 28.6% controls and 27.6% cases. GSTM1-null individuals exhibit 1.9-fold increased risk of RCC (95% CI: METHODS
1.06-3.33). The presence of GSTT1 active genotype was associated with increased risk of RCC in occupationally exposed subjects In order to test our hypothesis, we studied the transport characteristics of 3H-2-deoxy-D-glucose (3H-DG) in MCF7 human breast
when unexposed GSTT1-null subjects were used as a comparison group (OR: 2.48; 95% CI: 1.05-5.86). No association was found cancer cell line. Then, we investigated the effect of QUE and EGCG upon 3H-DG uptake in this cell line, glucose metabolism (by
between inactive form of GSTM1 and GSTT1 and smoking in RCC patients. measuring lactate production), cell viability (by using LDH assay) and proliferation (by measuring 3H-thymidine incorporation).
CONCLUSION RESULTS
In Serbian cohort of patients, the presence of GSTM1 active genotype is protective against RCC, whereas GSTT1 active genotype Our results show that 3H-DG uptake by MCF7 cells is: a) time-dependent, b) saturable, c) sodium-independent, d) inhibited by the
increases RCC risk in occupationally exposed subjects. GLUT inhibitor cytochalasin B and e) partially stimulated by insulin. Our results also demonstrate that 3H-DG uptake by MCF7 cells
REFERENCES is potently and concentration-dependently inhibited by QUE and EGCG (IC50s of 10.8 and 44.1 M for QUE and EGCG respectively).
QUE was shown to be more potent than EGCG both in the short- (26 min) and in the long-term (4 h) exposure experiments
and both flavonoids acted as competitive inhibitors of 3H-DG uptake. In addition, in the short-term exposure, these flavonoids
synergistically inhibited 3H-DG uptake by MCF7 cells. With respect to glucose metabolism, both compounds completely blocked
lactate production by MCF7 cells, either in 26 min or in 4 h treatments. Moreover, a 4 h-treatment with QUE was shown to decrease
MCF7 cell viability and proliferation. On the other hand, a 4 h-treatment with EGCG decreased MCF7 cell proliferation, although it
increased cell viability.
CONCLUSION
We can conclude that 3H-DG uptake by MCF-7 cells is mediated by members of the GLUT family of glucose transporters (most
probably GLUT1, given its characteristic expression in breast cancer and in MCF7 cells, with a slight contribution by the insulin-
dependent GLUT4), and does not involve the sodium-dependent glucose transporter (SGLT1). We can also conclude that QUE and
EGCG potently impair 3H-DG uptake and metabolism by this cancer cell line. We propose that these effects impact negatively on
MCF7 cell viability and proliferation rates. In the near future, we will investigate the effect of QUE and EGCG upon GLUT1 gene
expression.
ACKNOWLEDGEMENTS

6 Abstracts Oncology 7
 YES Guide
PS 146
IS PHOSPHORYLATION OF THE ANDROGEN RECEPTOR OF CLINICAL
SIGNIFICANCE IN THE DEVELOPMENT AND PROGRESSION OF PROSTATE
CANCER
ADAMS C, MCCALL P, EDWARDS J
SECTION OF SURGERY, DIVISION OF CANCER SCIENCES AND MOLECULAR PATHOLOGY, UNIVERSITY OF GLASGOW, MCGREGOR
BUILDING, WESTERN INFIRMARY, GLASGOW G11 6NT
AIM
In order to study if phosphorylation of serine residues is associated with the development of hormone-nave prostate cancer,
immunohistochemistry was performed for the expression of total androgen receptor and androgen receptor phosphorylated at
serine-81 in hormone-nave tissue samples.
INTRODUCTION
Prostate cancer is one of the most common malignancies found in men, ranking second in the UK behind lung cancer as a cause
of malignancy-related mortality. P. McCall et al. (BJC 2008) presented evidence that increased phosphorylation of the androgen
receptor at serine-210 in the transition from hormone-nave to hormone-refractory disease was associated with decreased
disease-specific survival. Studying expression levels and phosphorylation status of the androgen receptor at the time of diagnosis
of prostate cancer will enable the development of a better understanding of the pathogenesis of this disease and could prove to be
of use in developing better prognostic markers.
METHODS
Immunohistochemistry was employed to measure the expression of total androgen receptor and androgen receptor phosphoryl-
ated at serine-81 in hormone-nave prostate cancer samples from a cohort of 114 patients. The expression of these proteins was
assessed using the weighted histoscore technique. Histoscores were compared with patient Gleason grade, time to recurrence and
overall survival. All of this information was readily available for each patient.
RESULTS
Expression of androgen receptor phosphorylated at serine-81 was largely shown to not be statistically significant with any clinical
outcome measures. Cytoplasmic expression of phosphorylated serine-81 was found to correlate with nuclear phosphorylated
serine-81 expression (p = 0.001). Expression of nuclear total androgen receptor was shown to correlate with the presence of
metastases at initial diagnosis (p = 0.022) and with disease-specific survival (p < 0.001). Expression of total androgen receptor
was not found to correlate with any other clinical variables.
CONCLUSION
In summary, increased total androgen receptor expression in the nucleus is associated with decreased disease-specific survival.
This could therefore serve as a prognostic marker for patients with hormone-nave prostate cancer. However, androgen receptor
phosphorylated at serine-81 could not be used as a suitable prognostic marker.
REFERENCES
8 Abstracts
YES Meeting 2010
PS 154
COMPARATIVE FEATURES OF EXPERIMENTAL FIBROSARCOMA AT THREE
HAMSTER SPECIES
HRUBIK N.
FACULTY OF MEDICINE, UNIVERSITY OF NOVI SAD
AIM
Experimental hamster fibrosarcoma represents in vivo inoculated permanent cell line BHK 21-C13, obtained from kidney fibro-
blasts of newborn Syrian golden hamster. Cell line inoculated subcutaneous at hamster grows without intention and order cor-
responding to tumor. Success of tumor development depends of tumor cell features, immunological system condition of recipient
and way of tumor induction. Dexamethasone is fluored glucocorticoid with strongest antiinflammatory and immunosuppressive
effect within corticosteroid drugs, also has antiproliferative effect.
INTRODUCTION
Investigation of biological conduction and morphological features of tumor at three hamsters species at immunocompetent and
individuals treated with dexamethasone to affirm dexamethasone effect on their immune response and tumor development.
METHODS
Experimentaly was comprised 18 hamsters from three species into two groups: control and treated with dexamethasone in dose
defined with Clarck formule in duration of 28 days. At all hamsters on the 14th day was inoculated tumor. After sacrifice tumor
preparations stained with H&E method and compared tumor features between groups and species.
RESULTS
It was found large difference of morphological tumor features within groups and species. At Syrian golden hamster tumor dont
prompt immune response and its pressent infiltrative growth, siberian clearly recognize tumor and react with prolific lymphocytes
and plasmocytes production, until Roborowski with own immunity absolutely neutralize tumor development. Dexamethasone
have different effect on tumor: at Syrian golden has antiproliferative, at syberian manage infiltrative growth with totally elimina-
tion immune response, and at Roborowski development small tumor without infiltrative growth.
CONCLUSION
Inoculating cells of BHK-fibrosarcoma approved tumor at different species within same family isnt equal tumorigenic. Dexametha-
sone showed racily antiproliferative effect at Syrian golden, but at two other species immunosuppressive effect and favorize tumor
growth.
REFERENCES
Oncology 9
 YES Guide
PS 156
LOW DOSES OF IONIZING RADIATION PROMOTE TUMOR GROWTH AND
METASTASIS BY ENHANCING ANGIOGENESIS
1 - INS SOFIA VALA, 1 - RAQUEL NUNES, 1 - ANDR ROCHA, 1 - JOS RINO, 2 - CURZIO REGG, 3 -
ISABEL MONTEIRO GRILLO, 4 - MARC MAREEL AND 1 - SUSANA CONSTANTINO ROSA SANTOS
1 - INSTITUTO DE MEDICINA MOLECULAR, FACULDADE DE MEDICINA DA UNIVERSIDADE DE LISBOA, PORTUGAL. 2 - DIVISION
OF EXPERIMENTAL ONCOLOGY, CENTRE PLURIDISCIPLINAIRE DONCOLOGIE, FACULTY OF BIOLOGY AND MEDICINE, UNIVERSITY
OF LAUSANNE, SWITZERLAND. 3 - SERVIO DE RADIOTERAPIA DO HOSPITAL DE SANTA MARIA, PORTUGAL. 4 - DEPARTMENT OF
RADIOTHERAPY, GHENT UNIVERSITY HOSPITAL, BELGIUM.
AIM
To investigate the biological effects of low doses of Ionizing radiation (IR) on the vasculature surrounding the tumor area.
INTRODUCTION
Radiotherapy is a widely used local treatment for malignant tumors. However, there are clinical and experimental observations
indicating that IR might promote a metastatic behavior of cancer cells and that the irradiated host microenvironment might exert
tumor-promoting effects [1,2]. Recently, different studies have focused on the mechanisms by which IR activates cellular targets
potentially contributing to invasion and metastasis [3,4,5,6]. Doses of IR causing such stimulating effects are classically delivered
inside the tumor target volume in daily small fractions in order to limit damage of healthy tissues and until a potentially curative
dose has accumulated inside the tumor volume. Furthermore, the delivery in small fractions and the isodose distributions of
external beam radiotherapy result in lower doses of IR outside the tumor target volume. The biological effects of these low doses
of IR on the healthy tissue surrounding the tumor area remain largely to be determined.
METHODS
Lung Human Microvascular Endothelial Cells (HMVEC-L), anesthetized zebrafish or mice received single doses of IR, performed at
room temperature using a linear accelerator X-rays photon beam, operating at 6 MV (Varian Clinac 2100 CD) with a dose rate of
300 MU/min.
RESULTS
We found that doses of IR lower or equal to 0.8 Gy enhance endothelial cell migration without impinging on cell proliferation or
survival. Moreover, we show that low-dose IR induces a rapid phosphorylation of several endothelial cell proteins, including the
Vascular Endothelial Growth Factor (VEGF) Receptor-2 and induces VEGF production in hypoxia mimicking conditions. By activat-
ing the VEGF Receptor-2, low-dose IR enhances endothelial cell migration and prevents endothelial cell death promoted by an
anti-angiogenic drug, bevacizumab. In addition, we observed that low-dose IR accelerates embryonic angiogenic sprouting during
zebrafish development and promotes adult angiogenesis during zebrafish fin regeneration. Using an orthotopic breast cancer
model, we show that low-dose IR promotes metastasis and that these effects were prevented by the administration of a VEGF
receptor tyrosine kinase inhibitor immediately before IR exposure.
CONCLUSION
These findings demonstrate a new mechanism to the understanding of the potential pro-metastatic effect of IR and may provide a
new rationale basis to the improvement of current radiotherapy protocols.
REFERENCES
10 Abstracts
YES Meeting 2010
PS 190
EFFECT OF RANK-RANKL SIGNALLING PATHWAYS AND MMP1 EXPRESSION
IN THE INVASIVE ABILITY OF BREAST AND PROSTATE CANCER CELL LINES
IN VITRO
CARVALHO C; TEIXEIRA R; CASIMIRO S.
FACULTY OF MEDICINE, UNIVERSITY OF LISBON
AIM
Our main purpose was to contribute to unravel the effect of RANK-RANKL on the behaviour of breast and prostate cancer cells in
vitro. More specifically, we aimed to analyse the effect of RANK or MMP1 gene knock-down in the activation of RANK downstream
MAPK pathways in breast or prostate cancer cell lines in vitro.
INTRODUCTION
Bone metastases are highly prevalent and cause severe morbidity and mortality in patients with advanced breast or prostate can-
cer. In physiologic bone remodelling, the amount of bone resorbed is compensated by the amount of bone that is newly formed.
However, in bone metastases there is an increased osteoclastic activity with consequent development of osteolytic lesions. It is
well established that RANK-RANKL signalling pathways are associated with osteoclastogenesis and osteoclastic activity. Recently,
it was found that bone metastatic cells from breast and prostate cancers for example, may express RANK receptor. Moreover,
stimulation of these cells with RANKL induces cell migration and invasion through a collagen matrix. Despite the availability of
biphosphonates as a therapeutic option against skeletal complications of malignancy, a medical need exists for a more convenient,
effective and safe therapy and the inhibition of RANK-RANKL signalling pathway seems to be a promising therapeutic target. An
anti-tumoural effect of anti-resorptive drugs targeting RANK-RANKL and osteoclasts, like the monoclonal antibody denosumab,
may contribute to an improved clinical outcome of patients with advanced bone metastatic disease.
METHODS
Human prostate cancer cell line PC3 and breast cancer cell line MDA-MB-231-BO2f11 were used. RANK or MMP1 knock-down were
performed by shRNA technique, in PC3 or MDA-MB-231-BO2f11 cells, respectively. Cells were stimulated with 1 g/ml hRANKL and
nuclear and citoplasmatic protein extracts were obtained before stimulation and at 10, 20, 40, 60 and 120 min post stimulation.
-Actin, RANK, MMP1, p65, and the phosphorylated and total forms of JNK1/2/3, ERK1/2, p38 and AKT1/2/3 proteins expression
was analysed by Western Blot.
RESULTS
RANKL stimulation of RANK knock-down PC3 cells leads to a non-activation of MAPK pathways downstream to RANK, namely
ERK1/2, p38 and AKT1/2/3, pathways that are activated in parental cells following the same stimuli. JNK1/2/3 pathway shows
RANKL-dependent activation, comparable to parental cells. MMP1 knock-down in MDA-MB-231-BO2f11 cells does not affect
RANKL-dependent activation of JNK1/2/3, ERK1/2, p38 or AKT1/2/3 pathways.
CONCLUSION
RANK knock-down abrogates the downstream MAPK pathways, demonstrating that they are RANKL-activated. On the other hand,
MMP1 knock-down does not have any effect upstream on RANK-RANKL-activated MAPK pathways. RANK and MMP1 knock-down
cell lines are important tools to assess the effect of RANK stimulus on MMP1 expression and cancer metastatic cell invasive ability.
REFERENCES
Oncology 11
 YES Guide
PS 197
NICOTINE EFFECTS ON BONE METABOLISM: IN VITRO STUDIES WITH HUMAN
OSTEOCLASTS AND CO-CULTURES OF OSTEOCLASTS AND OSTEOBLASTS
1 - ROCHA I., 2 - COSTA-RODRIGUES J., 3 - FERNANDES M.H.
1 - LABORATRIO DE FARMACOLOGIA E BIOCOMPATIBILIDADE CELULAR, FACULDADE DE MEDICINA DENTRIA, UNIVERSIDADE
DO PORTO; FACULDADE DE ENGENHARIA, UNIVERSIDADE DO PORTO, 2 - LABORATRIO DE FARMACOLOGIA E BIOCOMPATIBI-
LIDADE CELULAR, FACULDADE DE MEDICINA DENTRIA, UNIVERSIDADE DO PORTO, 3 - LABORATRIO DE FARMACOLOGIA E
BIOCOMPATIBILIDADE CELULAR, FACULDADE DE MEDICINA DENTRIA, UNIVERSIDADE DO PORTO
AIM
The aim of this work is to characterize the effects of nicotine on osteoclastogenesis, in cultures of human osteoclastic cells and
co-cultures of osteoclastic and osteoblastic cells.
INTRODUCTION
Bone is a dynamic tissue, whose metabolism involves the coordinated action of the bone resorbing osteoclasts and the bone
synthesizing osteoblasts. Imbalances in those activities can lead to bone metabolic disorders, namely involving bone loss. Smoking
is associated with deleterious effects in bone metabolism, usually related to periodontal bone loss and delay in bone regeneration
processes. Among the cigarette smoke components, nicotine appears to be a major responsible for these effects. Several studies
have described the effects of nicotine on osteoblastic cells. However, the knowledge regarding the behaviour of osteoclastic cells
in the presence of nicotine is almost negligible. In this work, osteoclastic precursors, isolated or co-cultured with osteoblastic cells,
were exposed to a range of salivary and plasmatic nicotine concentrations.
METHODS
Osteoclastic precursors (PBMC) were isolated from human peripheral blood. Cells were cultured isolated or co-cultured with
osteoblast-like MG63 cells for 21 days, in the presence of 1.28x10^-6 - 0.5mg/mL nicotine. PBMC cultures were performed in the
absence or presence of exogenous osteoclastogenic stimulus. Cultures were tested at days 7, 14 and 21 for tartarate-resistant acid
phosphatase activity and histochemical staining, presence of multinucleated cells with actin rings and expressing vitronectin and
calcitonin receptors, and ability to resorb bone.
RESULTS
In PBMC cultures, nicotine stimulates osteoclastogenesis at low doses. This effect was observed for cultures performed either in the
absence or presence of exogenous osteoclastogenic stimulus. High doses of nicotine were lethal. In co-cultures of osteoclastic and
osteoblastic cells, nicotine decreases osteoclastogenesis, in a concentration-dependent way.
CONCLUSION
Results regarding PBMC cultures suggest that nicotine has the ability to act directly on osteoclastic precursors, inducing (low
concentrations) or inhibiting (high concentrations) osteoclastogenesis. However, in a osteoclast/osteoblast co-culture system, the
inductive effects of low doses of nicotine were not observed, suggesting involvement of osteoblast-mediated mechanisms. Taken
together, this work shows that nicotine has the potential to affect osteoclast development in different ways, and that the interplay
between these effects can ultimately lead to significant changes in bone metabolism.
12 Abstracts
YES Meeting 2010
PS 198
MITOCHONDRIA AS A NEW THERAPEUTIC TARGET IN CANCER
1 - A. BARBOSA RIBEIRO; 1 - A.L. FERREIRA; 1,2 - A. C. GONALVES; 2,3 - S. NEVES; 1 - A. M. ARAJO; 1
- F. CARVALHO; 1 - J. CARVALHO; 1 - R. M. SANTOS, 1 - V. ALVES; 1 - T. SILVA, 2,4,5 - J. M. NASCIMENTO
COSTA; 1,2,3 - A. B. SARMENTO RIBEIRO
1 - FACULTY OF MEDICINE, UNIVERSITY OF COIMBRA (FMUC), PORTUGAL; 2 - CENTER OF INVESTIGATION ON ENVIRONMENT
GENETIC AND ONCOBIOLOGY - CIMAGO, FMUC, PORTUGAL; 3 - CENTER FOR NEUROSCIENCE AND CELL BIOLOGY, COIMBRA,
PORTUGAL; 4 - MEDICINE SERVICE AND HEPATOLOGY UNITY, UNIVERSITY HOSPITAL OF COIMBRA, PORTUGAL; 5 - HEMATOLOGY
CLINICAL UNIVERSITY, FMUC, PORTUGAL
AIM
Our goal is to evaluate the therapeutic potential of dequalinium (DQA) in hematological and solid malignancies, namely in B-cell
Chronic Lymphocytic Leukaemia (B-CLL), Acute Promyelocytic Leukaemia (APL) and Hepatocelular Carcinoma (HCC).
INTRODUCTION
Cancer is one of the most important causes of death worldwide and despite great advances on treatment for these malignancies
there is still a long way to go until treatment is really effective. The hepatocellular carcinoma (HCC) is the most and the third
most common cause of cancer mortality worldwide. On the other hand B-CLL is the most common lymphoid leukemia and these
cells have a low proliferative capability, though being resistant to apoptosis, whereas APL is an acute leukemia whose cells are
characterized by a blockage in the maturation process thus having a high proliferative rate. Mitochondria plays a major role in
sustaining normal and tumor cellular functions mainly due to ATP synthesis. It is also the main site for Reactive Oxygen Species
(ROS) production, which has been reported to have a relevant role in inducing apoptosis. On the other hand, neoplastic cells have
a higher mitochondrial potential than normal cells, which may be explored in developing new approaches on treating cancer.
Lipophilic cations possessing a delocalized positive charge (i.e., delocalized lipophilic cations or DLCs) penetrate the hydrophobic
barriers of the plasma and mitochondrial membranes and accumulate in mitochondria in response to the negative inside trans-
membrane potentials. The higher plasma and/or mitochondrial membrane potentials of neoplastic vs normal cells, in general,
account for greater uptake of these compounds in neoplastic cells and may be a way to selectively target these cells since DLCs
exhibit mitochondrial toxicity at high concentrations.
METHODS
For this purpose we used three cell lines, EHEB (B-CLL), HL-60 (APL) and HUH-7 (HCC), to evaluate the effect of different concentra-
tions of DQA either by single dose administration, by daily dose administration and by association with conventional anticarcino-
genic agents. Cell viability and death was determined by the resazurin assay, optical microscopy and by flow cytometry. The latter
was also used to evaluate mitochondrial membrane potential, ROS levels (H2O2; O2-) and the antioxidant defense, Reduced
Glutathione (GSH), using fluorescent probes.
RESULTS
We found that DQA induced a decrease in cell viability inducing cell death by late apoptosis/necrosis in a time, dose and cell type
dependent manner, with and IC50 of 2.5, 4.7 and 7.5 M at 48h of exposure, respectively to HL-60, HUH-7 and EHEB. These effects
may be mediated by oxidative stress as we have observed and increase in ROS production and a decrease in GSH levels and in
mitochondrial membrane potential. We also observed that if DQA is administered on a daily basis a much lower concentration is
required to induce the same effect. On the other hand, the association of DQA with the conventional drug induces a synergistic
effect, because lower concentration of both drugs is required to obtain the same effect.
CONCLUSION
In summary, our results suggest that DQA may be used as new therapeutic approach in these malignancies both in monotherapy
and in association with the conventional therapy. However, new administration schemes, should be tested in order to improve
therapeutic efficacy.
Oncology 13
 YES Guide YES Meeting 2010
PS 203 PS 211
EXPRESSION OF P53 PROTEIN IN INFILTRATING DUCTAL CARCINOMA OF LEAP OF FAITH: UNDERSTANDING THE RELATIONSHIPS BETWEEN BRCA1
THE BREAST AND ESTROGEN RECEPTOR IN HEREDITARY BREAST CANCER
1- KISELICKI D., 2-VLAJNIC T. 1-MESQUITA D., 2-BONTE D., 2-LE GUILLOU M., 2, 3-FEUNTEUN J.
FACULTY OF MEDICINE, UNIVERSITY OF NOVI SAD 1-FACULTY OF SCIENCES, UNIVERSITY OF PORTO, 2--UMR 8200 STABILIT GNTIQUE ET ONCOGENSE, INSTITUT DE CANC-
ROLOGIE GUSTAVE ROUSSY, FRANCE, 3-UNIVERSIT PARIS SUD-XI, FRANCE
AIM
AIM
The aim of the study was to determine the frequency of p53 protein expression in infiltrating ductal carcinoma of the breast as
well as to determine if there is a correlation between over-expression of p53 protein and standard prognostic factors such as Understand the events triggered by BRCA1 depletion in vitro, and the eventual consequences on ER- pathway, which activity is
tumor size, histological grade and lymph node status. followed by the expression of pS2 estrogen-dependent protein. Two cell lines with different ER- status were used throughout this
work: MCF-7, a breast cancer cell line (ER-+), p53 protein wt, and HeLa, a cervical cancer cell line (ER--) expressing low amounts
INTRODUCTION of p53 protein. After BRCA1 depletion, a possible cell dedifferentiation (ER-+ into ER--) will be investigated, as well as the
consequences of contradictory signals received by the cells (estrogens pushing to S-phase and BRCA1 depletion inducing growth
The p53 gene is a tumor suppressor gene,located on the short arm of chromosome 17. P53 mutations remain the most common arrest by p53 activation)
genetic change identified in human neoplasia. P53 mutation is associated with more aggresive forme of the breast cancer and
worse overall survival. INTRODUCTION
METHODS Hereditary breast cancers were shown to be related to germline mutations in one of the cancer susceptibility genes, BRCA1. Up to
80% of women presenting this germ-line mutation ultimately develop breast cancer. BRCA1 was identified as a tumor suppressor
A total of 120 cases of breast carcinoma,who underwent surgery for breast carcinoma at the Surgical clinic for operative gene, since loss of the wild-type allele occurs in the majority of cancers that develop in mutation carriers. These tumors are
Oncology,Institute of Oncology Vojvodine, Sremska Kamenica, were included in this study. The immunohistochemical staining for characterized by a basal-like triple negative phenotype, which means they are ER-, PR-, and HER2-. However, its unknown why
p53 protein were evaluated, using the DAKO DO-T, M 7001 antibodies. such mutations only predispose to cancer of hormone responsive tissues. This predisposition may result from a tumor-promoting
RESULTS interaction between BRCA1 loss and estrogen pathway. Estrogen exposure tends to increase the probability of mutations due to
enhanced proliferation and direct genotoxic effects of estrogen metabolites, which may be involved in the loss of BRCA1 second
P53 protein overexpression was present in 10 out of 63 T1 lesions (16%) , in 13 out of 44 T2 lesions(30%), in 4 out of 6 T4 lesions allele. So, the combined effects of estrogen-promoted cell proliferation with the loss of BRCA1-DNA repair pathway might increase
(67%), while in T3 lesions not found. Protein overexpression was found in 11 out of 21 grade III tumors (52%) and 16 out of 72 the mutation rate leading to genomic instability and then to carcinogenesis in breast cells
grade II tumors (22%).Overexpression was not detected in grade I tumors.
METHODS
CONCLUSION
BRCA1 siRNA was used to knockdown BRCA1 expression at 100nM. For immunofluorescence and immunoblotting, anti-BRCA1,
Statistically significant correlation was found between p53 protein overexpression with large tumor size and high histological anti-ER and anti-pS2 antibodies were used
grade, ( p<0,01;chi-square test), but there was no positive correlation with axillary nodal status and tumor supresor gene. Our
findings showed that the expression of p53 protein might point out an aggressive tumor phenotype. This marker migh help to RESULTS
stratify patients for an appropriate therapy on an individual basis and, thus, offer the possibility of a more effectively tailored MCF-7 were cultured in estrogen-free medium and pS2 expression was used as a reporter of E2 stimulation. When MCF-7 were
treatment program. cultured in estrogen-free medium for 72h, pS2 protein became undetectable by immunoblotting. The protein was restored after
REFERENCES 24h of E2 stimulation. Interestingly, we observed a decrease in ER- expression upon restoration of pS2. Then, MCF-7 and HeLa
cells were used to investigate the effects of BRCA1 depletion on ER- status. For both cell lines, we obtained a depletion efficiency
of more than 40%. In MCF-7 cells, this was correlated to an absence of ER- in the cells that were specifically negative for BRCA1. A
30% increase of ER- negative cells was estimated. It was shown that BRCA1 siRNA transfected MCF-7 stopped proliferating while
HeLa cells grew normally. The cell viability was also slightly decreased for MCF-7 after transfection.
CONCLUSION
pS2 protein appears as a good read out of E2 stimulation. BRCA1 absence has an effect in ER- pathway signaling and on ER-
expression level. MCF-7 cells depleted for BRCA1 stopped proliferating, probably due to activation of the G1-S checkpoint mainly
controlled by p53, which induces cell arrest. Whereas HeLa cells, grew exponentially. We can conclude that BRCA1 knockdown
leads to a proliferation arrest of ER-a +/- and wt p53 cells, it will be important to determine if this growth arrest is due to a block
of the cells in G1-phase and/or to the decrease of ER-a
REFERENCES

14 Abstracts Oncology 15
 YES Guide
PS 215
CELLULAR AND MOLECULAR CHARACTERIZATION OF THE PARACRINIC
EFFECTS OF HUMAN BREAST CANCER CELL LINES ON OSTEOCLASTOGENESIS
1,2 - MONIZ K. 1- COSTA-RODRIGUES J. , 1- FERNANDES M.H.
1- FACULDADE DE MEDICINA DENTRIA, UNIVERSIDADE DO PORTO , 2- FACULDADE DE CINCIAS, UNIVERSIDADE DO PORTO
AIM
The aim of this work is to evaluate the osteoclastogenic paracrinic potential of four human breast cancer cell lines on osteoclast
precursors and to characterize the underlying intracellular mechanisms.
INTRODUCTION
Although with a rigid structure, bone tissue is frequently affected by tumor metastasis. Bone metastasis appears as distinct
types: osteolytic, osteoblastic or mixed. Among the tumors that origin bone metastasis, breast cancer is one of the most frequent,
originating usually osteolytic metastasis. Osteoclastogenesis is a complex process, involving osteoblastic-mediated mechanisms.
However, in order to have conditions suitable for tumor cell proliferation in the bone, this tissue has to be previously remodeled,
by activation of osteoclastic cells. In this context, paracrinic communications between osteoclasts (or their precursors) and tumor
cells appear as a potentially key player in the development of osteolytic metastasis. In this work, the paracrinic effects of different
human breast cell lines on osteoclastogenesis were characterized.
METHODS
Osteoclastic precursors (PBMC) were isolated from human peripheral blood. PBMC cultures were performed in the presence of
10% or 20% of conditioned media from four different human breast cell lines (SK-BR-3, HTB-22, HTB-126 and HTB-133), collected
after 48 hours, 7 days and 14 days of culture. When indicated, cell cultures were also treated with different signaling osteoclasto-
genic pathways inhibitors, namely, MEK and NFkB pathways inhibitors and a PGE2 synthesis inhibitor. Cells were analyzed at days
7, 14 and 21 for tartarate-resistant acid phosphatase activity and histochemical staining, presence of multinucleated cells with
actin rings and expressing vitronectin and calcitonin receptors and ability to resorb bone.
RESULTS
All the breast cancer cell lines displayed a high osteoclastogenic potential. Among them, HTB-133 promoted osteoclast differentia-
tion at a higher extent, followed by SK-BR-3, HTB-126 and HTB-22. However, the effects of the different conditioned media varied
with the concentration and the culture period. Regarding the involved osteoclastogenesis signaling pathways, the NFkB signaling
was essential for the osteoclastogenic response induced by every tested cell line. PGE2 production was important in all the tested
conditions, particularly in the case of the cell line HTB-133. Although important, the MEK pathway seems to be less activated in
the case of conditioned medium from HTB-126.
CONCLUSION
The tested breast cancer cell lines induced a high degree of osteoclastogenic differentiation and activation, by paracrinic associated
mechanisms. Interestingly, although there are some common osteoclastogenic signaling pathways activated by those cells, the
mechanisms underlying the effects of each cell type are not exactly the same, which suggests that different breast cancers can
induce osteoclastogenesis, at least partially, by different mechanisms. These results might contribute to a better clarification of the
osteolytic metastasis process associated to breast cancer.
16 Abstracts
YES Meeting 2010
PS 218
ENHANCED SIALYLATION OF SURFACE ANTIGENS ON BLADDER CANCER
CELL LINES INDUCES DENDRITIC CELL IMMUNE TOLERANCE IN VITRO.
HELENA GOUVEIA, MYLENE CARRASCAL, HELIO CRESPO, GUADALUPE CABRAL, PAULA VIDEIRA
FACULTY OF MEDICAL SCIENCES, NEW UNIVERSITY OF LISBON. CEDOC CHRONIC DISEASES RESEARCH CENTRE.
AIM
Our main aim is to demonstrate that sialylation of tumor cells hinders anticancer immune response by dendritic cells.
INTRODUCTION
Aberrant glycosylation of surface structures or secreted molecules is a hallmark of cancer. This way, tumor cells establish prefer-
ential interactions with their neighboring matrix and cells thus enabling them to proliferate, invade and eventually metastasize.
In this setting, dendritic cells (DCs) professional antigen(Ag)-presenting cells can either patrol the resident tissue for
aberrantly expressed Ags and induce an immune response against tumor cells or accumulate within the tissue, as immature and
non-migratory, and mediate immunologic tolerance to tumor Ags. In the clinical setting, superficial bladder carcinoma standard
treatments such as resection and adjuvant immunotherapy fail in 30% of patients. No tumor markers predicting positive response
to therapy have yet been identified. Expression of the pancarcinoma carbohydrate epitope Tn (Thomson-Friedenreich precursor
Ag) in its sialylated form (sTn) by bladder cancer cells correlates with more aggressive tumor biology and poorer prognosis. Such
sialylation is obtained by a Golgi-anchored sialyltransferase, ST6GalNAc I, which transfers sialic acid to O-linked GalNAc residues on
the carbohydrate chain. We hypothesize that such changes in sialylation of Tn alters tumor recognition by immune cells, therefore,
potentiating uncontrollable tumor progression.
METHODS
In this project, sTn-absent MCR cell lines were transduced either with ST6GalNAc I plasmids (ST6) or empty vectors negative
control (NC). DCs were obtained from monocytes isolated from buffy coats of healthy volunteers provided by the Portuguese Blood
Institute. DCs were incubated with MCR-ST6 and -NC for 2 hours at 37C. After removing non-adherent DCs, the co-culture was
trypsinized and MCR cells and adhering DCs collected. Cells were labeled with anti-HLA-DR, CD80 and CD86 fluorescent antibodies
and analyzed by flow cytometry. Gene expression of cytokines and co-stimulatory molecules was analyzed using real-time PCR
technology. Statistical analysis was performed by GraphPad software.
RESULTS
Our results show that expression TGF-b and IL-10 increased and TNF-a and IL-6 decreased in DC:MCR ST6 co-cultures. In concerns
to adhesion assays, there were no significant differences of DC adherence to MCR ST6 or NC cells (P=0,25). However, HLA-DR mean
fluorescence intensity (MFI) signal in initial DCs, non-adhering DCs and adhering DCs progressively increased and that differences
between HLA-DR MFI signal in DC:MCR ST6 and MCR:NC co-cultures were statistically significant (P=0,0043).
CONCLUSION
These findings show that enhanced sialylation of surface molecules on MCR cells do not alter DC adherence but affects DC matura-
tion phenotype and cytokine expression. Thus, tumor Ag-presenting capability to T cells in secondary lymphoid organs is likely to
be affected. Such data strongly supports our hypothesis of a correlation between tumor aberrant sialylation and immune tolerance
and translational research is warranted to confirm its applicability in clinical setting.
Oncology 17
 YES Guide
PS 222
CD40 AND CD26 EXPRESSION: IMPLICATION IN ORAL CANCER AND DRUG
RESPONSE
BATISTA J1, NEVES S2,3, GONALVES AC2,3, ALVES V4, SARMENTO RIBEIRO AB2,3,5, DOURADO M3,6
1. MASTERS STUDENT OF BIOCHEMISTRY, FCTUC, PORTUGAL;
AIM
The aim of this study was to evaluate the cellular expression of CD26 and CD40 in two OC cell lines, in situ and metastatic, before
and after cell treatment with cisplatin.
INTRODUCTION
Oral squamous cell carcinoma (OC) is an aggressive disease and is difficult to treat with conventional therapies as they are often ac-
companied by a decrease in patients quality of life. Although improvements have been achieved in surgical techniques, radiation
and chemotherapeutic protocols, the overall 5-year rate for this disease remains at 50% without any significant improvement in
the last decades. Understanding the underlying molecular pathogenesis of OC may afford new opportunities for future treatments.
Some regulators of the immune system, such as CD40/CD40L and CD26/DPPIV, are involved in mechanisms of tumour growth,
angiogenesis and also in immunologic mechanisms of anti-tumour defence and metastization.
METHODS
For this purpose, two human OC cell lines, BICR10 (OC in situ) and HSC3 (OC metastatic) cells, were incubated with cisplatin in
different concentrations. Cell morphology was evaluated by light microscopy examination of May-Grenwald Giemsa stained cells
and cell viability was estimated by alamar blue test. Cell death was evaluated by annexin V/propidium iodide (AV/PI) incorporation
and detected by flow cytometry. CD26, CD40 and CD40L expression were detected by flow cytometry using monoclonal antibodies
labelled with fluorescent probes.
RESULTS
Preliminary results show that CD26 expression was higher in BICR10 than in HSC3 (32,4 vs 21,6, MIF). On the opposite, CD40 was
higher in HSC3 cells than in BICR10 (74,6 vs 33,92, MIF). On the other hand, after treatment with cisplatin we didnt observe any
significant variation in CD26 expression. However, the expression of CD40 and CD40L increases in both cell lines, that could be
related with apoptotic cell death detected by morphology and cytometry studies, in agreement with others studies (Vonderheide
et al, 2007; Vonderheide, 2007; Khalil et al, 2007; Eliopoulos et al, 2004).
CONCLUSION
Our results suggest that CD40, CD40L and CD26/DPPIV can be involved in oral progression/metastization and drug response and
could constitute a new molecular target for oral cancer diagnosis/prognosis and treatment.
REFERENCES
18 Abstracts
YES Meeting 2010
PS 233
SYNTHESIS AND ANTITUMOR ACTIVITY OF DIHYDROPYRANO DERIVATIVES
OF BAICALEIN AND 3,7-DIHYDROXYFLAVONE
1,2 - J. FLOR; 1,2 - M. PERRO NEVES; 1,2 - S. CRAVO; 1,2 - H. CIDADE; 3 - K. CHOOSANG; 4 -P. PAKKONG;
5,6 - M.H. VASCONCELOS ; 1,2 - M. PINTO
1 - LABORATORY OF ORGANIC AND PHARMACEUTICAL CHEMISTRY, DEPARTMENT OF CHEMISTRY, FACULTY OF PHARMACY, UNI-
VERSITY OF PORTO, PORTUGAL; 2 - RESEARCH CENTRE OF MEDICINAL CHEMISTRY OF THE UNIVERSITY OF PORTO (CEQUIMED-
UP), FACULTY OF PHARMACY, UNIVERSITY OF PORTO, PORTUGAL; 3 - INTER DEPARTMENTAL MULTIDISCIPLINARY GRADUATE
PROGRAM IN BIOSCIENCE, FACULTY OF SCIENCE, KASETSART UNIVERSITY, BANGKOK 10900, THAILAND; 4 - APPLIED RADIATION
AND ISOTOPES DEPARTMENT, FACULTY OF SCIENCE, KASETSART UNIVERSITY, B
AIM
Molecular modification of two flavonoids (baicalein and 3,7-dihydroxyflavone) in order to improve their tumor cell growth inhibi-
tory activity.
INTRODUCTION
Prenylflavonoids, including pyrano derivatives, have been reported to mediate several interesting biological activities, namely
antitumor. Although the oxygenation pattern of these derivatives can play an important role in their biological activity, when
compared with their non-prenylated analogs the presence of the prenyl side chains also seem to enhance the interaction with
biological membranes and/or to increase affinity for target proteins. In fact, it was demonstrated that isoprenylation of flavones
significantly increased the growth inhibitory effect against several human tumor cell lines [1,2].
METHODS
The synthetic strategy involved the use of Montmorillonite K10 clay, to catalyze direct condensation of baicalein and 3,7-dihy-
droxyflavone with prenyl bromide and the use of microwave irradiation as a source of heating [3]. Structures were established by
IR and NMR (1H, 13C, COSY, HSQC and HMBC). The effect of the synthesized compounds on the growth of three human tumor cell
lines (MCF-7, breast; NCI-H460, lung; and A375-C5, melanoma) was evaluated according to the procedure adopted in the NCIs in
vitro anticancer drug screening that uses the SRB assay to assess growth inhibition [4].
RESULTS
Two angular dihydropyrano derivatives were obtained: 5,6-dihydroxy-7,8-(2,2-dimethylchromano)flavone and 3-hydroxy-7,8-
(2,2-dimethylchromano)flavone. Both derivatives were shown to be strong inhibitors of the growth of the three human tumor cell
lines referred above.
CONCLUSION
Two angular dihydropyrano flavones were synthesized for the first time. Both derivatives were shown to be strong inhibitors of the
growth of MCF-7, NCI-H460, and A375-C5 cell lines.
REFERENCES
Oncology 19
 YES Guide
PS 234
EVALUATION OF THE INHIBITION OF MONOCARBOXYLATE TRANSPORTERS
IN GLIOMAS
CLIA MARIA PEIXOTO DE SOUSA, VERA GONALVES, RUI MANUEL REIS, FTIMA BALTAZAR
SCHOOL OF HEALTH SCIENCES, UNIVERSITY OF MINHO
AIM
To assess the in vitro effect of monocarboxylate transporters (MCTs) inhibitors, acids -cyano-4-hydroxycinnamic (CHC) and 4,
4-diisothiocyanostilbene-2, 2-disulfonic (DIDS), in glioblastomas cell proliferation, program cell death and migration.
INTRODUCTION
Glial tumor cells produce high levels of lactic acid that must be exported from cells to avoid excessive reduction of the intracellular
pH. MCTs are transmembrane proteins identified as carriers of lactate in tumor cells. Thus, these cells present a high degree of
dependence of MCTs for survival.
METHODS
We used the human glioma cell line U251 maintained in DMEM (Dulbeccos modified Eagles medium) supplemented with fetal
bovine serum (FBS) and antibiotics (penicillin and streptomycin. A Western blot assay was performed to evaluate the expression
of MCTs, with CHC and DIDS; the effects of these inhibitors at the level of cellular metabolism were evaluated using cellular me-
tabolism assays; wound healing assays were performed to assess the effect of these inhibitors on cell migration; a flow cytometry
assay was done for CHC in order to characterize the type and percentage of death and a cell cycle assay was also made to assess the
effects of these inhibitors on tumor cell cycle.
RESULTS
In Western blot, for CHC there was an increase in the expression of MCT 1, with dose administered (5mM-IC50- and 10mM-2xIC50),
whereas for MCT 4 changes were not as pronounced. It is believed that the cells increase the expression of MCT 1 as a way to
counteract the accumulation of intracellular lactate, with CHC. For DIDS, occurred also an increased expression of MCT 1, according
to the dose of administered drug. For MCT 4, the results are not coherent. In cellular metabolism, the total glucose consumed up
to 48 hours was higher in control in relation to the populations treated with CHC and DIDS. For lactate, There was a significant
inhibition of export of lactate. However, with DIDS, the inhibition is not significant. From the results, DIDS is possibly a less specific
inhibitor of MCTs than CHC. In wound healing assay, in cell populations treated with CHC and DIDS was obtained, up to 48 hours, a
decrease of only about 20% of the wound created, whereas in controls the decrease has been about 50%. In cell cycle assay, none
of the drugs had a very noticeable effect on cell cycle and there was only an increase in cell death in relation to the control, with
IC50 and 2xIC50 of drugs. In flow cytometry assay, the percentage of cell death was double in relation to controls and the death
occurred by necrosis or late apoptosis.
CONCLUSION
Inhibitors exert their action especially on the induction of death of tumor cells. Results of wound healing assay can also demon-
strate that these inhibitors may have a significant inhibitory action on tumor migration, and who knows in tumor metastasis.
However, the results are only preliminary. Therefore, CHC and DIDS may be strong allies in treatment of glioblastomas and possibly
in other type of tumors, acting as adjuvant treatments of radio or chemotherapy.
ACKNOWLEDGEMENTS
20 Abstracts
YES Meeting 2010
PS 249
EPIGENETICS - A NEW TARGET THERAPEUTIC APPROACH IN CANCER
1 - J.A. CARVALHO , 1,2,3 - E. CORTESO , 1 - J. D. BRANCO , 1,3 - A.C. GONALVES , 1,3,4 - S. NEVES ,
1 - F. CARVALHO , 1 - A. RIBEIRO , 1 - V. ALVES , 1 - T. SILVA , 1,3 - M. DOURADO, 1,2,3 - J.M. NASCI-
MENTO COSTA , 1,3,4 - A.B. SARMENTO RIBEIRO
1 - FACULTY OF MEDICINE, UNIVERSITY OF COIMBRA (FMUC), PORTUGAL; 2 - UNIVERSITY HOSPITAL OF COIMBRA (HUC),
PORTUGAL; 3 - CENTER OF INVESTIGATION ON ENVIRONMENT GENETICS AND ONCOBIOLOGY (CIMAGO), FMUC, PORTUGAL;
4 - CENTER FOR NEUROSCIENCE AND CELL BIOLOGY (CNC), COIMBRA, PORTUGAL - THESE AUTHORS CONTRIBUTE EQUALLY TO
THE STUDY
AIM
With this work we intend to evaluate the potential therapeutic effect of the histone deacetylase inhibitor, Trichostatin A (TSA) and/
or the hypometilant agent, decitabine (DEC), in hematological neoplasias, namely MDS and Chronic Lymphocytic Leukemia (CLL)
and in solid tumors, in particular, hepatocellular carcinoma (HCC).
INTRODUCTION
Epigenetic has been known to be involved in several diseases, namely in cancer. It is related with heritable changes in gene
expression that do not involve changes in DNA sequence. Two important epigenetic changes known to contribute to disease are
abnormal DNA methylation patterns (ex. hypermethylation of the CpG islands) and chromatin histones modifications (ex. histone
deacetylation). Unlike genetic changes, which are irreversible, epigenetic changes are reversible, allowing the expression or
repression of a gene that might reverse malignant phenotype. Despite hypomethylating agents are already used in the treatment
of some subtypes of Myelodysplastic Syndromes (MDS), the role of histone deacetylase inhibitors and the combination of both
drugs in hematological and solid tumors is still unclear.
METHODS
For this purpose, the cell lines EHEB (B-CLL), F36P (MDS) and HUH-7 (HCC) were maintained in culture in absence and presence of
TSA and/or DEC, a demethylating drug. The density and cell viability was assessed using Trypan Blue and/or Alamar test. Cell death
was evaluated by flow cytometry using Annexin V and optic microscopy.
RESULTS
Our results show that epigenetic modulators, TSA and DEC, induce a decrease in cell proliferation and viability in a dose, time,
administration scheme and cell type dependent manner, inducing cell death by apoptosis. Besides TSA was also more effective in
monotherapy, when administered to cells 3/4 hours before than DEC, or in a daily dose it was observed an increase in the cytotoxic
effect. On the other hand, MDS seems to be the less sensitive cells and the higher cytotoxic effect is achieved earlier and at lower
doses in HUH-7 cells than in EHEB cells, for the same drug concentration.
CONCLUSION
This study suggests that epigenetic modulators may constitute a new therapeutic approach in cancer. However, the schedule of
drugs administration and cancer cell type may interfere with their therapeutic efficacy. So, the choice of the optimal schedule of
drugs administration may be crucial to the success of the therapy.
ACKNOWLEDGEMENTS
Oncology 21
 YES Guide
PS 259
INFLUENCE OF HOXA9 EXPRESSION IN THE RESPONSE OF GLIOBLASTOMA
CELLS TO TEMOZOLOMIDE
GONCALVES T., COSTA B.M., REIS R.M.
LIFE AND HEALTH SCIENCES RESEARCH INSTITUTE, SCHOOL OF HEALTH SCIENCES, UNIVERSITY OF MINHO, BRAGA
AIM
Evaluate the functional relevance of HOXA9 expression in the response of glioblastoma cells to the chemotherapeutic agent
temozolomide.
INTRODUCTION
Glioblastomas are the most common and most aggressive type of primary brain tumors in adults, accounting for about 70%
of malignant gliomas (1). Although different treatment strategies have been tried in the past years, the median survival of
these patients still remains approximately 14 months. Much of the ongoing research is based on understanding the biology of
the disease and identifying characteristic molecular alterations that can define prognostic subgroups. While several molecular
markers with putative prognostic value have been suggested (e.g., MGMT promoter methylation status, EGFR expression, PI3K/
PTEN/Akt activation), none is still being used in the clinical management of glioblastoma patients (2). HOXA genes encode crucial
transcriptional regulators of embryonic development that have also been shown to be altered in a wide variety of human tumors.
A recent study of our group demonstrated that reactivation of HOXA9 expression is a novel, independent, and negative prognostic
factor in glioblastoma patients (3). As the standard chemotherapeutic agent used in these patients is temozolomide (TMZ), we
hypothesized that HOXA9 may modulate the response of glioblastoma cells to TMZ.
METHODS
To assess the influence of HOXA9 in the response to TMZ, we used an established human glioblastoma cell line (U87-MG), and
immortalized human astrocytes (hTERT/E6/E7) as putative glioblastoma precursor cells. Since both cell lines do not express en-
dogenous HOXA9, they have been previously genetically engineered with retroviral vectors to obtain stable HOXA9-overexpressing
cells. The effect of HOXA9 expression in the cellular response to TMZ was measured at the levels of cellular proliferation and
apoptosis. Proliferation measurements were performed by the trypan blue assay and apoptosis was analyzed with the annexin V
and propidium iodide assay
RESULTS
Cells overexpressing HOXA9 showed higher proliferation indexes after TMZ exposure than cells without HOXA9 expression. In addi-
tion, cells overexpressing HOXA9 also displayed lower levels of TMZ-induced cell death.
CONCLUSION
Our preliminary results indicate that glioblastoma cells overexpressing HOXA9 are more resistant to TMZ treatment, which may
partly explain the correlation between HOXA9 overexpression and worse clinical outcome previously reported in glioblastoma
patients. These findings may have clinical relevance in the future assisting the clinician in prognostication of patients and in
therapy decisions.
REFERENCES
22 Abstracts
YES Meeting 2010
PS 261
CORRELATION BETWEEN GLUT EXPRESSION AND IN VITRO 18F-FDG
UPTAKE IN BREAST CANCER CELL LINES
M. C. MARTINS1,2,3*, A. M. ABRANTES1; 4, A. C. GONALVES5,4, M. C. RODRIGUES6, A. C. MAMEDE1,
S. D. TAVARES1, G. COSTA2; J. M. LIMA2; A. RODRIGUES6, A. B. SARMENTO-RIBEIRO5,4; BOTELHO MF1;
4; 5
1.
NUCLEAR MEDICINE DEPARTMENT, COIMBRA UNIVERSITY HOSPITAL, COIMBRA, PORTUGAL; 3.
Ceramics and Glass Engineering Department, University of Aveiro, Aveiro, Portugal; 4.
CENTRE OF INVESTIGATION ON ENVIRONMENT GENETICS AND ONCOBIOLOGY (CIMAGO), FACULTY OF MEDICINE,
UNIVERSITY OF COIMBRA, COIMBRA, PORTUGAL; 5.
Biochemistry department, Faculty of Medicine, University of Coimbra, Coimbra,
Aim
In this context, the main purpose of this study is to determine the pattern of 18F-FDG uptake in various breast
cancer cell lines, which present different hormonal and HER2 receptors expressions and to set a correlation with the
expression of GLUTs 1 and 3 in each of these cell lines.
Introduction
Positron emission tomography (PET) using the radiolabeled glucose analogue 18F-FDG allows uncovering glycolysis
processes within cancer cells and reveals high value in diagnosis, staging, detection of recurrence and evaluation
of response to therapy in several malignancies. Breast cancer is the most common form of cancer among women
with an increasing prevalence. Among the different laboratory tests available to assess response to therapy, the
search of the expression of estrogens and progesterone receptors by tumour cells is essential for the implementa-
tion of appropriate chemotherapy associated or not to hormonal therapy. The HER2/neu receptor has recently been
introduced as a new predictive marker of prognosis. Breast cancer presents considerable variability when it comes to
18F-FDG uptake, which results in different sensitivity and specificity values related to this technique. Its diagnostic
usefulness is, therefore, called into question. 18F-FDG enters the cells by the same mechanisms of glucose mem-
brane transport, glucose transporters (GLUTs).
Methods
Three different human breast cancer cell lines, MCF-7, HCC1806 and HCC1954, purchased from ATCC, were used.
MCF-7 express estrogen and progesterone receptors, HCC1806 cells are triple negative and HCC1954 are negative for
hormonal receptors, but overexpress HER2/neu. 18F-FDG uptake studies were performed with these three cell lines.
GLUT-1 and GLUT-3 expressions were assessed by flow cytometry.
Oncology 23
 YES Guide
PS 270
AVIAN PATOGENIC ESCHERICHIA COLI ISOLATED FROM BACTEREMIA IN
HUMANS
1 NEDER NETO C., SANTOS ACM., CAGGEGI D., SILVA RM., 2 PIGNATARI ACC., 3 VIDOTTO MC.,
KOBAYASHI R.
1 - DEPARTMENT OF MICROBIOLOGY, IMMUNOLOGY AND PARASITOLOGY FEDERAL UNOVERSITY OF SAO PAULO, 2 - DEPART-
MENT OF INFECTIOUS AND PARASITIC DISEASES - FEDERAL UNIVERSITY OF SAO PAULO, 3 - UNIVERSIDADE ESTADUAL DE
LONDRINA
AIM
The aim of the present study was to verify the avian origin of E. coli strains isolated from the blood stream of humans with
bacteremia.
INTRODUCTION
Extra-intestinal pathogenic Escherichia coli (ExPEC) cause infections both in humans and animals. The avian pathogenic E. coli
(APEC) presents a variety of virulence determinants, some of which are chromosomal and others are plasmid born. The plasmidial
pathogenic determinants are: traT and iss, for serum complement resistance; iucD, iroN and sitB, for iron uptake; the cvaC gene,
for colicin production; and the tsh gene for adhesion to eukaryotic cells. In a previous work a screening of 75 E. coli strains isolated
from human bacteremia showed that 28% of the strains had some of these virulence markers. This fact raised the question if avian
pathogenic E. coli could cause extra-intestinal infections in humans.
METHODS
Human E. coli strains isolated from bacteremia and presenting APEC virulence markers are from a laboratory collection. APEC strain
09, and neonatal meningitis E. coli strain RS218 were used as positive controls. Non-pathogenic strains HB101 and C600 were used
as negative controls. Antimicrobial resistance was assessed by the disk diffusion method. Plasmid profile was determined by the
alkaline lyses method followed by electrophoresis in agarose gel. Serotyping was carried out by PCR and primers specific for some
of the most frequent APEC somatic O antigens. In vivo virulence tests were done in one day old chicken by intradermal inoculation
of broth cultures containing 108 bacterial cells per mL.
RESULTS
Nineteen strains presented plasmids more than 100 Kbp in size. So far, only three of those strains were completely studied and
showed to harbor the APEC virulence genes iss, traT, iucD, cvaC, iroN, and sitB. These strains presented multiple drug resistance to:
sulfonamide, amoxicillin, ampicillin, streptomycin, trimetoprim, and cotrimoxazole, as well as a plasmid band of 145 Kbp. These
three clinical isolates did not belong to the APEC serogroups, even so they were positive in the virulence animal model used. The
transference of the 145 Kbp plasmid to E. coli C600 rendered it positive for all the virulence and resistance markers present in the
donor strains, but was not sufficient to turn the E. coli C600 virulent in the animal model.
CONCLUSION
To date, it is possible to conclude that ExPEC isolated from human bacteremia can carry virulence traits characteristic of APEC. As
in APEC, these traits are present in conjugative plasmids which, in this case also carry resistance to antimicrobial drugs of routinely
clinical use facilitating the co-selection of virulence and multiple drug resistance. Finally, although the clinical strains do not
belong to common APEC serogroups they were fully virulent in the animal model used, suggesting that although this plasmid is
not sufficient to render a strain virulent, as shown by the virulence assay with the transconjugants, it can be complemented by
chromosomal genes present in human strains.
ACKNOWLEDGEMENTS
24 Abstracts
YES Meeting 2010
PS 277
ASCORBIC ACID AND ITS POTENTIALITY AS AN ANTICANCER AGENT: IN
VITRO AND IN VIVO STUDIES IN HUMAN COLORECTAL CANCER
1 - MAMEDE AC, 2 - TAVARES SD, 3 - ABRANTES AM, 4 - PIRES AS, 5 - GONALVES AC, 6 - SERRA E, 7 -
SARMENTO-RIBEIRO AB, 8 - MAIA J, 9 - BOTELHO MF
1 - BIOPHYSICS/BIOMATHEMATICS INSTITUTE, IBILI, FACULTY OF MEDICINE, UNIVERSITY OF COIMBRA, FACULTY OF HEALTH
SCIENCES, UNIVERSITY OF BEIRA INTERIOR, 2 - BIOPHYSICS/BIOMATHEMATICS INSTITUTE, IBILI, FACULTY OF MEDICINE, FAC-
ULTY OF SCIENCES AND TECHNOLOGY, UNIVERSITY OF COIMBRA, 3 - BIOPHYSICS/BIOMATHEMATICS INSTITUTE, IBILI, FACULTY
OF MEDICINE, UNIVERSITY OF COIMBRA, 4 - BIOPHYSICS/BIOMATHEMATICS INSTITUTE, IBILI, FACULTY OF MEDICINE, FACULTY
OF SCIENCES AND TECHNOLOGY, UNIVERSITY OF COIMBRA, 5
AIM
To evaluate vitamin C metabolism and mechanisms of action and show their cytotoxic effects in colorectal adenocarcinoma cells by
nuclear medicine imaging and molecular and cell biology techniques.
INTRODUCTION
Vitamin exists in two forms: the reduced form (ascorbic acid - AA) and oxidized form (dehydroascorbic acid - DHA). As an antioxi-
dant, the main role of vitamin C is neutralize free radicals by donating their electrons to them, reducing oxidative stress. However,
this nutrient may have a pro-oxidant activity, promoting the formation of reactive oxygen species (ROS) that can induce cell death
in cancer cells. This factor, coupled with the decrease of antioxidant enzymes and increased decompartmentalized transition met-
als in tumor cells may result in the selective cytotoxicity of vitamin C and the subsequent revelation of its therapeutic potential.
METHODS
AA was labeled with technetium-99m (99mTc) using iron (III) chloride (FeCl3) as reducing agent in order to obtain a radioactive
complex (99mTc-AA) that can be used in nuclear imaging. The labeling efficiency was controlled by HPLC in order to differenti-
ate 99mTc-AA, free pertechnetate, AA and FeCl3. The radiopharmaceutical developed was applied in uptake studies in colon
adenocarcinoma cells (WiDr, ATCC). In vitro studies also allowed us to evaluate the cytotoxicity of vitamin C through the evaluation
of cell proliferation by spectrophotometry, clonogenic assays and flow cytometry. In vivo studies with Balb/c and Balb/c nu/nu
mices were performed in order to verify the results obtained in the quality control of 99mTc-AA and obtain information about their
biodistribution and pathways of metabolism and excretion. To this end, the animals were injected with 99mTc-AA and dynamic
and static acquisitions until 360 minutes after administration were achieved. The animals were sacrificed and several organs
including tumor were excised and counted in a well count. The AA was also used as therapy that consisted of daily intraperitoneal
injection of a solution of AA in Balb/c nu/nu mices with xenografts whose aim was to verify if there was stabilization of tumor
growth after twelve days.
RESULTS
The labelling efficiency, higher than 95%, was obtained using 200mg of AA, 0,2mL of FeCl3 0.1 N (in HCl 0,1N) with a pertechne-
tate activity of 222MBq and pH of 6,5. The pharmaceutical formulation, reproducible and stable over time, was not captured by
WiDr, confirming that AA is not transported directly by tumor cells. By MTT studies we verified that vitamin C induces an inhibitory
effect on cells under study, being this effect accompanied by a cytotoxic effect verified by flow cytometry. This technique revealed
a decrease in cell viability and the consequent increase in cell death by apoptosis and necrosis, as well as an increased production
of ROS. The biodistribution studies show that the 99mTc-AA elimination are done by renal and hepatobiliary excretion and has a
high labeling efficiency, supported by biodistribution and imagiologic studies. Uptake in tumor tissue is not high, a factor which
may be explained by the same reasons why tumor cells do not capture the pharmaceutical formulation in the studies of uptake.
The therapy developed in Balb/c nu/nu xenografts also proved that AA stabilizes tumor growth.
CONCLUSION
The reduced form of vitamin C, which is only captured by tumor cells if extracellularly was oxidized to DHA, induces an anti-
proliferative and/or cytotoxic effect in tumor cells and may eventually represent a new therapeutic approach in the treatment of
colorectal cancer.
Oncology 25
 YES Guide
PS 280
EXPRESSION OF DNA METHYLTRANSFERASES IN THE MOUSE HEART
FOLLOWING MATERNAL PROTEIN UNDERNUTRITION
DYKE B.
FACULTY OF MEDICINE, UNIVERSITY OF SOUTHAMPTON
AIM
To elucidate the role of epigenetic mechanisms in the developmental programming of adult disease using a mouse model of
maternal protein undernutrition.
INTRODUCTION
Coronary heart disease and diabetes mellitus constitute a significant burden of disease in the UK and across the world. Epidemio-
logical and experimental evidence supports the assertion that these chronic diseases of adulthood have their foundation in early
environment experience. Sub-optimal maternal nutrition and other maternal or fetal stressors during gestation alter the develop-
mental programme and predict a range of adverse health outcomes including obesity, glucose intolerance and hypertension which
significantly contribute to cardiovascular and diabetes risk. Epigenetic mechanisms, such as DNA methylation, are hypothesised to
underlie such environmental influences because they can induce stable changes to gene expression profile without affecting DNA
sequence. Maternal low protein diet (LPD) in the rat has been shown to affect liver expression of DNA methyltransferase 1, one of
the agents of methylation, and to alter its modulation of gene expression. This effect was apparent even when dietary restriction
was limited to the preimplantation period of blastocyst development. Here we have explored the effect of LPD on programming of
DNA methyltransferase expression profile in the mouse heart.
METHODS
Pregnant mice were fed Low Protein throughout gestation (LPD), Low Protein limited to the preimplantation period (Emb-LPD) or
Control (NPD) diets. At 28 weeks, offspring heart tissue was harvested. Heart expression of DNA methyltransferases (Dnmts) 1,3a
and 3b was confirmed by endpoint PCR. Relative expression of Dnmt1 and Dnmt3a mRNA was measured using real-time reverse
transcriptase PCR (RT qPCR). DNMT1 protein expression was quantified by western blotting.
RESULTS
Dietary treatment limited to the preimplantation period (Emb-LPD) did not affect methyltransferase expression but Dnmt1 mRNA
was upregulated in LPD subjects (females P<0.036, males P<0.027). No significant between-treatment differences were detected
for Dnmt3a mRNA or DNMT1 protein expression. Consistent with previous studies, Emb-LPD tended to elevate offspring systolic
blood pressure (P<0.04). A trend towards decreased heart size relative to body weight was also noted in female LPD subjects but
did not achieve statistical significance.
CONCLUSION
Our findings reflect results from a similar model in the mouse kidney and further reports in the literaure in support of a role for
DNMT1 in the developmental programming of adult disease. The absence of an effect in Dnmt3a mRNA probably reflects the
subtle differences in function of this enzyme. Additionally this study provides the first report of Dnmt changes in the rodent heart.
Global and gene-specific methylation assays are required to confirm whether altered mRNA expression is linked to DNMT enzymic
activity and whether this confers increased disease risk.
ACKNOWLEDGEMENTS
26 Abstracts
YES Meeting 2010
PS 307
POTENTIAL THERAPEUTIC OF BATIMASTAT IN MULTIPLE MYELOMA CELLS
A PRELIMINARY STUDY
1- ANA SOFIA FERNANDES PAIS; 1,2- ANA CRISTINA GONALVES; 1,3- CATARINA GERALDES; 1,2- ANA
BELA SARMENTO RIBEIRO.
1- FACULTY OF MEDICINE, UNIVERSITY OF COIMBRA, PORTUGAL; 2- CENTER OF INVESTIGATION ON ENVIRONMENT GENETICS
AND ONCOBIOLOGY (CIMAGO), FACULTY OF MEDICINE, UNIVERSITY OF COIMBRA PORTUGAL; 3- UNIVERSITY HOSPITAL OF
COIMBRA, PORTUGAL.
AIM
Our main goal is to evaluate the therapeutic potential of a metalloproteinase inhibitor, batimastat (BB-94), in a multiple myeloma
cell line in culture.
INTRODUCTION
Multiple myeloma (MM) is a plasma-cell neoplasm that is characterized by skeletal destruction, renal failure, anaemia, and hyper-
calcemia. It accounts for 10% of all blood system malignancies. The first pathogenic step is a premalignant monoclonal gamopathy
of undetermined significance (MGUS). With progression of MGUS to myeloma, complex genetic/epigenetic events occurs in the
neoplastic plasma cell, and in the bone marrow microenvironment. However, many of the biologic processes in MM remain to be
defined. Complex interactions between the MM cells and the microenvironment clearly play important roles in the pathogenesis
of the disease including the induction of angiogenesis, the suppression of cell-mediated immunity, and the development of
paracrine signalling loops involving cytokines such as interleukin-6 and vascular endothelial growth factor. The resultant interac-
tions of myeloma cells, bone marrow stromal cells, and microvessels contribute to persistence of the tumour and its resistance to
drugs. Matrix metalloproteinases (MMPs) play a critical rule in bone remodeling (osteolitic lesions) and tumor invasion, and could
be a new therapeutic target in MM. The knowledge of the MM biology has lead to the development of new drugs as bortezomib,
a proteasome inhibitor, and the immunomodulatory and anti-angiogenic drugs, thalidomide and lenalidomide. However, it is
important to investigate other therapeutic strategies designed specifically against a relevant molecular target, which can permit a
therapeutic approach directed to the molecular profile and consequently to the patients needs.
METHODS
For this purpose a MM cell line, the NCI-H929 [H929] cells were cultured in absence and presence of different concentrations of the
MMP inhibitor, BB-94 during different periods of time. Cell viability and death was determined by the alamar blue assay and by
flow cytometry using the annexin V/propidium iodide incorporation.
RESULTS
Our preliminary results show that BB-94 has an antiproliferative effect in MM cells, that seems to be independent of concentration
in a broad range of drug concentration. On the other hand this effect is accompanied of a cytotoxic one (IC50= 1 a 5M), as we
observed an increase in apoptosis.
CONCLUSION
Our preliminary study suggests that batimastat could be a new therapeutic approach in multiple myeloma.
ACKNOWLEDGEMENTS
Oncology 27
 YES Guide
PS 310
PREVENTION OF BLADDER CANCER BY GREEN TEA IN MUS MUSCULUS
1-VIDEIRA-HENRIQUES A.,1- FERREIRA S., 1-ALMEIDA I., 1,2-PIRES MJ., 3-NUNES F., 3-FERREIRA AM,
1,2-COLAO A., 1,2-OLIVEIRA PA.
1- DEPARTMENT OF VETERINARY SCIENCES, UNIVERSITY OF TRS-OS-MONTES AND ALTO DOURO, VILA REAL, PORTUGAL, 2-
CECAV, DEPARTMENT OF VETERINARY SCIENCES, UNIVERSITY OF TRS-OS-MONTES AND ALTO DOURO, VILA REAL, PORTUGAL, 3
-CQVR, DEPARTMENT OF CHEMISTRY, UNIVERSITY OF TRS-OS-MONTES AND ALTO DOURO, VILA REAL, PORTUGAL
AIM
The aim of this experimental work was to evaluate the potential effect of the consumption of green tea in the induction of bladder
cancer in Mus musculus ICR, male and female, through the experimental model of chemical carcinogenesis with N-butyl-N-(4-
hydroxybutyl) nitrosamine (BBN).
INTRODUCTION
One of the mortality and morbidity leaders in the industrialized western society is bladder cancer, is very usual in the urinary
tract and the pathology incidence is higher in men then in women. Food chemoprevention has acquired special attention under
the new preventive approaches. Green tea is one of the beverages more consumed in the entire World. Due to biochemical and
pharmacological proprieties and possible association with cancer decrease incidence, this product has been studied in vivo and in
vitro models.
METHODS
Animals were divided in three groups in both sexes. The groups I and IV were exposed to BBN and green tea, the groups II and V
were exposed to BBN and tap water and the groups III and VI were exposed only to green tea. The BBN was administered by gavage
during 10 weeks. Green tea was provided daily at a concentration of 0.5%. After 20 weeks animals were sacrificed and samples of
bladder, liver, spleen, lungs, kidneys and heart are collected for histopathological study. Blood samples were collected to evaluate
possible alterations in some biochemical parameters. The chemical evaluation of green tea consisted on total phenol, flavonoids
and antioxidant activity assays and the characterization by HPLC.
RESULTS
Males obtained a lower weight gain comparatively with females. We identified four types of preneoplastic lesions, simple
hyperplasia, dysplasia, papilloma and squamous metaplasia. In males, group I (BBN and Green Tea) had presented an higher
incidence of lesions, with 75% of simple hyperplasia, 87,5% of dysplasia and 25% of papilloma. However, in females the opposite
was observed, group V (BBN) was the group that presented higher incidence, with 66,7% of simple hyperplasia and dysplasia and
16,7% of both squamous metaplasia. We observed in all groups of both sexes incidence of inflammatory infiltrate. This incidence
was higher in group exposed to BBN, followed by group treated with BBN and green tea and at last green tea group with a less
incidence. These observations were concordant for both sexes. In biochemical parameters of hepatic function we also observed
differences between sexes. Chemically, green tea has a concentration of 70,492,03 mg gallic acid/g tea leaves of total phenol
content, 6,1230,809 mg kercetin/g tea leaves of flavonoids and a antioxidant activity of 385,8818,2 mg trolox/g tea leaves.
Individual phenolic compounds were analyzed by reversed-phase HPLC (C18) with photodiode-array detection. The flavonoids
were identified by the retention time and UV-spectrum.
CONCLUSION
Polyphenol composition of green tea is associated with relevant antioxidant activity and its bioavailability is influenced by geo-
graphic localization, climate and preparation. Differences observed between sexes in incidence of urothelial lesions may be related
with gender differences. Green tea may have a protector effect on urothelial lesions of females, which wasnt observed on lesions
of males. Its extremely important the study of gender differences and how they influence in the genetic factors and in the action
of compounds, so that the therapies can be most appropriate to both sexes.
28 Abstracts
YES Meeting 2010
PS 315
IN VITRO EFFECT OF MELOXICAM ON T24 AND 5637 INVASIVE BLADDER
CANCER CELL LINES
R ARANTES-RODRIGUES1, R PINTO LEITE2, ML CARDOSO3, A COLAO1, PA OLIVEIRA1
1DEPARTMENT OF VETERINARY SCIENCES, CECAV, UNIVERSITY OF TRS-OS-MONTES AND ALTO DOURO, VILA REAL, PORTUGAL
2GENETIC SERVICE, CYTOGENETIC LABORATORY, HOSPITAL CENTER OF TRS-OS-MONTES AND ALTO DOURO, VILA REAL, PORTU-
GAL 3DEPARTMENT OF BIOCHEMISTRY, FACULTY OF PHARMACY, UNIVERSITY OF PORTO, PORTO, PORTUGAL
AIM
In vitro study of meloxicam on proliferation of invasive bladder cancer cell lines.
INTRODUCTION
Non-steroidal anti-inflammatory drugs (NSAIDs), are among the most usually used medications worldwide. These drugs are
considered effective to treat rheumatic diseases and inflammation symptoms. Actually, is considered that inflammatory conditions
predispose to cancer. Correlations between NSAIDs use and lower incidence of cancer have been suggested recently by several
researchers. Meloxicam, is a drug that inhibit the cyclooxygenase-2, has an inhibitory effect on nonsmall-cell lung cancer cells,
colorectal cells and osteosarcoma cells. New studies have been suggesting a similar effect of NSAIDs in many other types of malig-
nant diseases. However, there are no studies published until the moment correlating the effect of meloxicam on invasive bladder
cancer cell lines. To study the effect of meloxicam on cell proliferation, we use two invasive bladder cancer cell lines, T24 and 5637.
The T24 cell line has been established from a highly malignant grade III human urinary bladder carcinoma and the 5637 cell line
are a transitional cell carcinoma established in 1974.
METHODS
T24 and 5637 cell lines were grown as a monolayer in RPMI 1640 culture medium, supplemented with 10% heat-inactivated
fetal calf serum (FCS), 100U/mL penicillin and 100g/mL streptomycin, in 5% CO2 at 37C. Cells were routinely subcultured by
trypsination. Using a Neubauer counting chamber, and after verifying cell viability by trypan blue dye exclusion, cells were seeded
in 96-well microtiter plates at a density of 2x104 cells/well in 100 L of medium. The plate was incubated for 24 hours to allow
adherent cell growth. The medium was removed and cells were treated with different concentrations of meloxicam for 72 hours.
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to assess cell viability after exposure
to meloxicam. Mitochondrial dehydrogenases of viable cells cleave the tetrazolium ring, yielding purple formazan crystals which
are insoluble in aqueous solution. The crystals were dissolved in 100 L DMSO/well and the resulting purple solution was spec-
trophotometrically measured at 492 nm using an ELISA plate reader. The proliferation inhibitory rate percentage was calculated
as follows: % growth inhibition = 100-[(absorbance of experimental well - absorbance of positive control) x100]/(absorbance of
negative control - absorbance of positive control). Each experimental data points represent the average values obtained from 3
replicates.
RESULTS
Meloxicam inhibits the growth of T24 and 5637 invasive bladder cancer cell lines. Significant correlation was found between cell
proliferation and meloxicam concentration in both cell lines used (T24: r=0.957; p<0.01 and 5637: r=0.961; p<0.01).
CONCLUSION
The results obtained demonstrate that the cell proliferation was indirectly proportional to meloxicam concentration. Meloxicam
has an inhibitory effect on invasive bladder cancer cell lines proliferation.
Oncology 29
 YES Guide
PS 317
PROSPECTIVE ANALYSIS OF PATIENTS WITH HEPATITIS VIRUSES AND
CHRONIC LYMPHOPROLIFERATIVE DISORDERS
1 - ION I.M., DELCEA C., NISTOR S.I., ILIESCU M.C., 1,2 - CIUFU C. MD, VLADAREANU A.M. MD PHD, 1,3
- ARAMA V. MD PHD
1 - CAROL DAVILA UNIVERSITY OF MEDICINE AND PHARMACY BUCHAREST, 2 - DEPARTMENT OF HEMATOLOGY, UNIVERSITY
EMERGENCY HOSPITAL, BUCHAREST, 3 - DEPARTMENT OF INFECTIOUS DISEASES, MATEI BALS HOSPITAL, BUCHAREST
AIM
The objective of our study is to evaluate the prevalence of hepatitis viruses in patients with CLD, as well as their impact on the
clinical and biological evolution.
INTRODUCTION
Hepatitis viruses are both hepatotropic and lymphotropic and a striking association between hepatitis B, C, D viruses infection and
chronic lympfoproliferative disorders (CLD) has been shown.
METHODS
In this prospective analysis, we studied 639 patients with CLD admitted between January 2008 - May 2010 at the Hematology
Department of the University Emergency Hospital Bucharest. The cases consisted of: B-cell non-Hodgkins lymphomas (B-NHL),
T-cell non-Hodgkins lymphomas (T-NHL), Hodgkins disease (HD), chronic lymphocytic leukemia (CLL), multiple myeloma (MM),
Waldenstrom macroglobulinemia (WM), adult T-cell leukemia/lymphoma (ATLL). The diagnosis of CLD was established according
to WHO classification: histopathology and immunohistochemical tests on lymph node/bone marrow biopsy completed by im-
munophenotyping analysis when necessary. All patients had serology test for hepatitis detection.
RESULTS
In the studied population, 11.58% (74 out of 639) patients tested positive for hepatitis viruses. The mean age for the group
infected with hepatitis B virus (HBV) was 54.31 years 11.13, significantly lower than the mean age for hepatitis C virus (HCV)
group: 64.79 years 9.3, p = 0.0004. Male/female ratio was 43/31. 43 patients were infected by HCV (55.8%), 29 by HBV (41.6%)
and only 2 (2.6%) patients presented double/triple infection. The prevalence of hepatitis viruses was higher in patients with
B-NHL (21%, 50/239 cases), WM (20%, 2/10), ATLL (10%, 1/10) than in patients with MM (5%, 7/139), CLL (5.38%, 9/167), HD
(6.66%, 5/75). Considering the subgroup of NHL, the prevalence was higher in follicular lymphoma (26.31%, 5/19), diffuse large
B-cell lymphoma (24.35%, 19/78) than in small B-cell lymphoma (18.58%, 21/113), T-NHL (17.85%, 5/28). HBV infection was
significantly associated with more aggressive histological lymphoproliferations: (OR=3.1, p=0.02), particularly with diffuse large
B-cell lymphoma and also with higher fibrinogen levels, increased ALT levels, higher ESR (p < 0.05). On the other hand, HCV was
more frequently associated with adenopathies revealed on clinical examination (61.53%, 24/39 patients with adenopathies, p =
0.03) and feminine gender (65.11%, 28/43). Thrombocytopenia was revealed in 32 out of 74 patients (32.43%), mainly associated
with HBV infection (p = 0.01). The majority of the patients had lymphocytosis (59.45%, 44/74), particularly the ones with HCV
infection and indolent types of CLD (p = 0.003). Hepatic cytolysis was associated predominantly with indolent types of CLD, and
with HCV infection. During the evolution, 73% of patients with hepatitis viruses had worsening or stationary anemia and the
prevalence of leucopenia increased in the HCV group. 21 patients (28.37%) died before the study was finished. Chemotherapy was
given in 48/74 patients with CLD (64.86%), as monotherapy or combined chemotherapy/chemoimmunotherapy.
CONCLUSION
The analysis showed a higher incidence of CLD with hepatitis viral infection in women over 50 years old, with a predominance of
HCV infection, which was predominantly linked to: indolent CLD subtypes, adenopathies, lymphocytosis, and liver dysfunction.
The particularity of our study is that HBV was found in a large number of patients and it was associated with more aggressive CLD,
especially diffuse large B-cell lymphoma.
30 Abstracts
YES Meeting 2010
PS 326
DISTINCT CHARACTERISTICS OF UPTAKE OF DIETARY FOLATES BY TWO
BREAST CANCER CELL LINES: T47D AND MCF7 CELLS
1- CORREIA-BRANCO A.,1,2- MOREIRA L., 1- KEATING E. AND 1- MARTEL F.
1- DEPARTMENT OF BIOCHEMISTRY (U38/FCT), FACULTY OF MEDICINE, UNIVERSITY OF PORTO, PORTUGAL, 2- FACULTY OF
SCIENCES, UNIVERSITY OF PORTO, PORTUGAL
AIM
Little is known concerning the characteristics of uptake of folates by breast cancer cells, so the aim of this study was to characterize
the uptake of two folate forms (FA and 5-MTHF) by two human breast cancer cells, the T47D and the MCF7 cell lines.
INTRODUCTION
Folate compounds, such as 5-methyltetrahydrofolate (5-MTHF) and folic acid (FA), belong to the B vitamin family. These
compounds function as one-carbon units donors in the biosynthesis of pirimidines and purines required for DNA synthesis[1].
Anti-folate chemotherapy agents such as methotrexate and fluorouracil reduce proliferation of neoplastic cells by inhibiting DNA
synthesis[2]. Paradoxically, epidemiological data suggests an inverse relationship between dietary folate intake and incidence of
certain types of neoplastic disease such as breast cancer[3-4].
METHODS
Uptake of 3H-FA (20 nM) and 14C-5-MTHF (5 uM) in T47D and MCF7 breast cancer cell lines was characterized at acidic (5.0) and
physiological (7.5) pHs. Also, the expression of folate receptor- (FR-), reduced folate carrier-1 (RFC1), and proton-coupled folate
transporter (PCFT) in T47D and MCF7 cells was determined by RT-PCR.
RESULTS
Uptake of 3H-FA and 14C-5-MTHF by T47D cells was shown to be time-dependent at both pH values, and pH-dependent (with
uptake increasing at lower extracellular pH). This pH-dependence was much slighter for 14C-5-MTHF uptake. 3H-FA uptake by
T47D cells was: 1) inhibited by folate analogs (methotrexate (MTX), FA and 5-MTHF) at both pH values; 2) more strongly inhibited
by the anion transport inhibitor 4-acetamido-4-isothiocyano-2,2-disulfonic acid stilbene (SITS) at pH 5.0 than at pH 7.5; and
3) less potently inhibited by the RFC1 inhibitor thiamine pyrophosphate (TPP) at pH 5.0 than at pH 7.5. On the other hand,
14C-5-MTHF uptake by T47D cells was: 1) inhibited by MTHF and SITS at both pH values; and 2) inhibited by MTX and TPP only
at pH 7.5. Regarding MCF7 cells, uptake of 3H-FA was: 1) pH-dependent (but, inversely to T47D cells, uptake increased at higher
extracellular pH), 2) time-dependent at pH 7,5 (no time-dependence for uptake at pH 5.5 was observed). Finally FR-, PCFT and
RFC1 mRNAs were detected in both cell lines.
CONCLUSION
In conclusion, both 3H-FA and 14C-5-MTHF uptake by T47D cells seems to be mediated mainly by PCFT at pH 5.0 and by RFC1 at pH
7.5. Interestingly enough, PCFT doesnt seem to be functionally present in MCF7 cell line. This is very intriguing, as both cell lines
express the three folate transporters and are known to have similar phenotypes (e.g. epithelial morphology, estrogen-receptor
positivity). Further research is needed to clarify the mechanism of folate uptake in breast cancer cell lines, and its pharmacological
modulation, in order to understand the influence of these processes in breast carcinogenesis.
REFERENCES
Oncology 31
 YES Guide
PS 334
THE INFLUENCE OF DIETILNITROSAMINE IN MICE PHYSIOLOGICAL
PARAMETERS
1*-PAULA-SANTOS NM, 1*-CONCEIO-PEREIRA I, 1-PEREIRA FO,1-PIRES MJ, 2-PALOMINO LF2,
3-LOPES C, 1-COLAO A, 1-OLIVEIRA PA
1-CECAV, DEPARTMENT OF VETERINARY SCIENCES, UNIVERSITY OF TRS-OS-MONTES E ALTO DOURO, 5001-911 VILA REAL,
PORTUGAL, 2-DEPARTMENT OF PHYSIOLOGY, VETERINARY FACULTY, UNIVERSITY OF SANTIAGO DE COMPOSTELA, LUGO, SPAIN,
3-ICBAS, UNIVERSITY OF OPORTO, PORTUGAL
AIM
Evaluate the effect of N-diethylnitrosamine (DEN) on 5 weeks old male ICR mice physiologic parameters.
INTRODUCTION
Cancer is a severe public health problem. Hepatocellular carcinoma (HCC) accounts for 85 to 90% of primary liver cancers being
responsible for more then 600 000 deaths/year worldwide (1). Laboratory mice (Mus musculus) are often used for cancer research
due to physiology and genetic similarities between rodents and humans (2). Animal models that mimic human diseases are quite
important to understand biopathology mechanisms underlying HCC and the mouse model of DEN induced HCC has a histology and
genetic signatures similar to human HCCs (3; 4). Hepatocarcinogenesis can be induced by intraperitoneal administration of the
genotoxic carcinogen N-diethylnitrosamine (DEN). This chemical carcinogenic is converted by a P-450 dependent mono-oxygenase
into highly reactive molecules which react with DNA molecules affecting primarily the liver.
METHODS
During this experimental protocol 60 mice received food and water ad libitum. Intraperitoneal injections were administrated
during 8 weeks to 30 male ICR mice (5 weeks age old). Pentobarbital was used to euthanize mice groups at 7, 14 and 21 weeks
after last DEN injection. A completely necropsy was performed. Macroscopic evaluation of organs was carried out. Organ weight
was registered and blood was collected by cardiac puncture for hematocrit and serum biochemistry analysis. Mean and standard
deviation were calculated for each of the parameters. To assess the statistical significance of intergroup differences for quantitative
data, Bonferronis multiple comparison test was used after one-way ANOVA. Calculations were performed with SPSS (v 17.0). P
value < 0.05 was considered significant.
RESULTS
Mice exposed to DEN developed visible alterations in the liver and lungs (2nd 3rd euthanasias). The hematocrit at 3rd euthanasia
was statistically significant (p=0.005). Significant values in biochemistry parameters between control/DEN groups were
determined to alanine transaminase (p=0.044) and total bilirrubin (p=0.026). Mean weight values were also significant between
groups in the first euthanized mice (p=0.048). Food consumption on control group was higher than DEN group; nonetheless the
last group consumed more water than the control group.
CONCLUSION
Despite no external signs of toxicity, alterations were found on mice after 29 weeks since the beginning of the experimental
protocol. Some physiologic parameters presented significant variations. The macroscopic alterations found in liver and lungs are
compatible with tumors.
REFERENCES
32 Abstracts
YES Meeting 2010
PS 353
POTENTIAL MUTAGENICITY OF TXA1, A SMALL MOLECULE POTENT
INHIBITOR OF HUMAN TUMOR CELL GROWTH
D. VIEIRA 2* AND A. S. GOMES 1,2*, E. SOUSA1,2, C. AFONSO 1,2, M. PINTO1,2, F. CARVALHO3AND A.
SANTOS-SILVA4,5
1 DEPARTMENT OF CHEMISTRY, LABORATORY OF ORGANIC AND PHARMACEUTICAL CHEMISTRY, FACULTY OF PHARMACY,
UNIVERSITY OF PORTO, RUA ANBAL CUNHA, 164, 4050-047 PORTO, PORTUGAL 2 CEQUIMED-UP, CENTER OF MEDICINAL CHEM-
ISTRY UNIVERSITY OF PORTO, PORTUGAL 3 REQUIMTE, TOXICOLOGY DEPARTMENT, FACULTY OF PHARMACY, UNIVERSITY OF
PORTO, RUA ANBAL CUNHA, 164, 4050-047 PORTO, PORTUGAL 4 DEPARTMENT OF BIOLOGY, LABORATORY OF BIOCHEMISTRY,
FACULTY OF PHARMACY, UNIVERSITY OF PORTO, RUA AN
AIM
The objective of this work was to investigate the genotoxicity of TXA1, a small molecule potent inhibitor of human tumor cell
growth by the micronucleus in vivo assay.
INTRODUCTION
The need for a continued search for new anticancer drugs is of great importance, since many of the current drugs are insufficiently
effective, often highly toxic and resistances may be developed. (Thio)xanthones are of documented relevance as antitumor agents
[1,2]. TXA1 is a new promising antitumor agent with a thioxanthonic scaffold that has been already synthesized by the research
group CEQUIMED-UP. This compound showed a potent inhibitory action with a GI50 <10 M against the growth of several tumor
cell lines, namely MCF-7, NCI-H460, A375-C5, and K562. There is no information about the antitumor mechanism of action of this
newly synthetised compound. Thus, there is a strong need for the characterization of the molecular and cellular mechanisms of
this molecule so that this molecule (or/and its derivatives) may become potential antitumor drugs.
METHODS
The mutagenicity activity was evaluated by the rodent erythrocyte micronucleus assay. Four groups of four male CD1 mice were
treated with three different concentrations of the TXA1 hydrochloride (TXA1.HCl) (5.75 mM, 11.5 mM and 23 mM) and negative
control was injected with saline NaCl (0.9%). The solutions were administrated by intraperitoneal injection and mice were sacri-
ficed with 24 h of delay. Smears were prepared with peripheral blood and stained by Wrights stain. At least 1000 total cells were
scored per slide under optical microscope [3]. Statistical analysis was performed using GraphPad Prism 5 software and statistically
significant data were considered for P<0.05.
RESULTS
Treatment with TXA1.HCl increased significantly (P<0.05) the frequency of micronucleated cells at 0.340.16% and 0.290.12%
for the investigated concentrations 11.5 mM and 23 mM, respectively. In contrast, the lowest concentration (5.75 mM) was not
significantly different (P>0.05) relatively to the negative control. Treatment with TXA1.HCl didnt show any significantly increase
(P>0.05) of the frequency of reticulocytes in peripheral blood for all the investigated concentrations.
CONCLUSION
These findings may suggest a direct DNA reactivity of TXA1.HCl to concentrations higher than 5.75 mM resulting in a clue to further
investigate the antitumor mechanism of action of these derivatives.
REFERENCES
Oncology 33
 YES Guide
PS 357
MELANOCORTIN 5 RECEPTOR TARGETING TO CELL SURFACE: THE ROLE OF
THE N-TERMINUS
1,2- SOUSA D, 1- RODRIGUES AR, 1-ALMEIDA H, 1,3- GOUVEIA AM
1- FACULDADE DE MEDICINA DO PORTO AND INSTITUTO DE BIOLOGIA MOLECULAR E CELULAR, UNIVERSIDADE DO PORTO
2- UNIVERSIDADE DO MINHO, DEPARTAMENTO DE BIOLOGIA, 3- FACULDADE DE CINCIAS DA NUTRIO E ALIMENTAO, OF COIMBRA (FMUC), PORTUGAL; 2BIOLOGY DEPARTMENT, FACULTY OF TECHNOLOGY AND SCIENCE,
UNIVERSIDADE DO PORTO
AIM
The study aims to identify the amino acid residues of Melanocortin 5 Receptor (MC5R) crucial for its targeting to cell surface.
INTRODUCTION
The melanocortin system is a complex network of molecules that comprises five receptors (MC1R-MC5R) and their diverse agonists
and antagonists1. The melanocortin receptors belong to the family of G protein-coupled receptors (GPCRs). MC5R was the last
receptor to be cloned and its function and intracellular trafficking mechanisms are poorly understood. It was shown that the
N-terminal 20 amino acids of the human MC5R could be removed without interfering with the receptor binding to the agonist2.
However, previous results from our group highlighted the crucial role of the MC5R N-terminus on its cell surface expression3 and
informatics approaches revealed putative important residues at the N-terminus that can undergo post-translational modifications
like glycosylation.
METHODS
Since the N-terminus of MC5R seems to be important to the receptor cell-surface expression, our approach was to specifically
analyze the function of N-terminus 2 to 21 residues of rat MC5R. Receptor mutants were generated by site-directed mutagenesis
using MC5R tagged with GFP as template. We obtained 8 different constructs that were used to transfect HeLa cells. The total
expression levels of these mutant forms of MC5R were determined by cell western quantification of GFP levels. The targeting
efficiency to the plasma membrane was evaluated by a two step procedure. First, all proteins of cell surface were labeled with a
biotinylated cross-linker. Then, after neutravidin binding of biotinylated proteins, GFP levels were quantified.
RESULTS
By site-directed mutagenesis we obtained 8 mutant forms of MC5R-GFP, with diverse alterations of the first 21 amino acids of the
N-terminus. At the plasma membrane the levels of the mutated proteins relatively to the non-mutated receptor are significantly
decreased (around 60%) when the residues His6 and Leu7 were mutated to alanines. Moreover, the mutation of the amin oacids
12 to 18 slightly decreases the MC5R targeting to cell surface. The remaining amino acids did not influence the MC5R expression at
the plasma membrane. Cell western analysis using anti-GFP antibodies revealed that the total expression levels of all constructs is
similar to the full-length MC5R-GFP. This data supports the fact that the reduction in the cell-surface expression of the mutants is
not due to differences in total receptor expression.
CONCLUSION
In this work we concluded that a specific domain comprising amino acids His6 and Leu7 is essential for the correct targeting of
MC5R to cell surface. Understanding the molecular and cellular mechanisms controlling GPCR intracellular routing is essential for
preventing or correcting the conformational abnormalities associated with disease-causing misfolded receptors.
REFERENCES
34 Abstracts
YES Meeting 2010
PS 359
TRAIL- A NEW APPROACH IN MYELOID LEUKEMIAS
FILIPA CARVALHO1, ANA SOFIA COELHO2, JOO A. CARVALHO1, ANDR RIBEIRO1, RUI SANTOS1,
CRISTINA GONALVES3,4, VERA ALVES5, T. SILVA1, MARLIA DOURADO1,4, J. M. NASCIMENTO
COSTA1,4,5,6, ANA BELA SARMENTO RIBEIRO1,3,4,6,7 STUDENTS, FACULTY OF MEDICINE, UNIVERSITY
UNIVERSITY OF COIMBRA, PORTUGAL; 3APPLIED MOLECULAR BIOLOGY/BIOCHEMISTRY INSTITUTE,
FMUC, PORTUGAL; 4CENTER OF INVESTIGATION
1MEDICAL STUDENTS, FACULTY OF MEDICINE, UNIVERSITY OF COIMBRA (FMUC), PORTUGAL; 2BIOLOGY DEPARTMENT, FACULTY
OF TECHNOLOGY AND SCIENCE, UNIVERSITY OF COIMBRA, PORTUGAL; 3APPLIED MOLECULAR BIOLOGY/BIOCHEMISTRY INSTI-
TUTE, FMUC, PORTUGAL; 4CENTER OF INVESTIGATION ON ENVIRONMENT GENETICS AND ONCOBIOLOGY (CIMAGO), UNIVERSITY
OF COIMBRA, PORTUGAL; 5IMMUNOLOGY INSTITUTE, FMUC; 5MEDICINE SERVICE, UNIVERSITY HOSPITAL OF COIMBRA,
PORTUGAL; 6HEMATOLOGY, FMUC, PORTUGAL; 7CENTER OF NEUROSCIENCE AND CELL BIOL
AIM
The aim of this work is to analyse the therapeutic efficacy of TRAIL in haematological cancers, namely in myeloid leukemias in
monotherapy and/or in combination with conventional anticarcinogenic drugs.
INTRODUCTION
Recent progress has broadened us to think that deficient apoptosis is a major cause in the development and progression of cancer,
namely in leukemias, and it plays a significant role in the resistance to conventional therapeutic regimes. On this way, apoptosis
may be a useful target for therapeutic intervention. The tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is a
member of the TNF superfamily that can induce apoptosis by binding to their death receptors (TRAIL-R1 and TRAIL-R2) in cancer
cells, with low cytotoxity in normal cells.
METHODS
For this purpose we use two different myeloid leukemia cell lines, such as: HL-60 cells (Acute Promyelocitic Leukemia) and K562
(Chronic Myeloid Leukemia in blast crisis). These cell lines were treated with increasing concentrations of TRAIL during different
periods of time. The viability was acessed using the trypan blue method. Cell death analysis was performed by flow cytometry
(Annexin V and Propidium Iodide) and by optical microscopy. The expression of TRAIL and its receptors was determined by flow
cytometry using monoclonal antibodies stained with fluorescent probes.
RESULTS
Our results show that rhAPO2L/TRAIL induces a decrease in cell viability and apoptotic cell death in a dose, time and cell type
dependent manner. In fact, rhTRAIL as single agent is more efficient in HL-60 cells than in K-562 cells, were we didnt find a
significant cytotoxic or antiproliferative effect. However when rhTRAIL is added to ATRA or IMATINIB, we observe a potentiation of
the cytotoxic and antiproliferative effect, but more pronounced in HL-60 cells. These results may be correlated with the differential
TRAIL receptors expression found in these cell lines. Besides K562 cells show higher TRAIL-R2 expression than HL-60 cells, they
also have higher levels of the anti- apoptotic TRAIL receptors, TRAIL-R3 and TRAIL-R4, and a lower ratio between pro- and anti-
apoptotic TRAIL receptors, which may contribute for the decrease in therapeutic efficacy in this cell line.
CONCLUSION
With this study we can conclude that rhAPO2L/TRAIL may constitute a useful therapeutic approach in myeloid leukemia, as single
agent and/or in combination with ATRA an IMATINIB. However, cell sensitivity to apoptosis and the posologic drug squeme, is
fundamental for the therapeutic efficacy.
ACKNOWLEDGEMENTS
Oncology 35
 YES Guide YES Meeting 2010

Book of Abstracts Physiology &

Immunology
PRESENTING STUDENTS ABSTRACTS
PS 27
THE EFFECT OF GAG REFLEX ON CARDIAC SYMPATOVAGAL TONE . . . . . 38
PS 32
INFLUENCE OF MAST-CELL STABILIZATION WITH SODIUM CROMOGLYCATE PRESENTING STUDENTS ABSTRACTS
ON THE PULMONARY VESSEL WALL REMODELLING IN HYPOXIC PULMONARY
HYPERTENSION . . . . . . . . . . . . . . . . . . . . . . . . . . 39
PS 90 PS 251
ACUTE HAEMODYNAMIC EFFECTS OF TEZOSENTAN IN RATS WITH AGE-ASSOCIATED SHIFTS IN THE RESPONSIVENESS OF THE CENTRAL
MONOCROTALINEINDUCED PULMONARY HYPERTENSION. . . . . . . . 40 CATABOLIC MELANOCORTIN SYSTEM IN RATS . . . . . . . . . . . . . 51
PS 151 PS 257
ASSESSMENT OF FACTORS AFFECTING LYMPHOCYTE ACTIVATION IN THE DRAWING-UP THE CARDIAC STEM CELL(S) NICHE UNDER NORMALCY VS.
NEONATE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 MYOCARDIAL INFARCTION . . . . . . . . . . . . . . . . . . . . . 52
PS 175 PS 269
EFFECT OF CELLULAR FOLATE AVAILABILITY ON ADIPOCYTE LIFE CYCLE AND STABILITY OF VITAMIN C IN ORANGE JUICE UNDER DIFFERENT TREATMENTS
METABOLISM . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 IN A SHORT PERIOD OF TIME . . . . . . . . . . . . . . . . . . . . . 53
PS 182 PS 281
EFFECTS OF CENTRAL ALPHA-MSH INFUSION IN RATS OF VARIOUS COMPARATIVE STUDY OF THE PREVALENCE, CLINICAL FEATURES,
NUTRITIONAL STATES . . . . . . . . . . . . . . . . . . . . . . . . 43 SENSITISATION PROFILES AND RISK FACTORS FOR ALLERGIC RHINITIS
BETWEEN ELDERLY AND YOUNG ADULTS IN COVA DA BEIRA . . . . . . . 54
PS 184
CHARACTERIZATION OF PLACENTAL UPTAKE OF THE AMINO ACID PS 295
L-METHIONINE . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 ANGIOTENSIN 1-7 EFFECTS ON MIOCARDIAL CONTRACTILITY AFTER HYPOXIA-
REOXYGENATION. . . . . . . . . . . . . . . . . . . . . . . . . . 55
PS 195
ALLOSTERIC MODULATION OF ANGIOTENSIN II RECEPTORS BY ROSIGLITAZONE PS 331
IN THE SYMPATHETIC NEURONS OF THE RAT HEART . . . . . . . . . . 45 THE EFFECTS OF CELERY EXTRACTS ON SPONTANIOUS AND ACETYLCHOLINE
INDUCED CONTRACTIONS OF RATS DUODENUM . . . . . . . . . . . . 56
PS 209
ENDOCRINE FUNCTIONS OF THE HUMAN STOMACH SEROTONIN AND PS 340
GHRELIN IMMUNOREACTIVE CELLS OF THE GASTRIC MUCOSA . . . . . . 46 MORPHOFUNCTIONAL EVALUATION OF THE EFFECTS OF CHRONIC
CONSUMPTION OF BEER IN RAT LIVERS . . . . . . . . . . . . . . . . 57
PS 229
A STUDY OF 147 EXTENDED HAPLOTYPES CARRYING THE C282Y HFE MUTATION: PS 348
A NOVEL APPROACH TO EXPLAIN THE INVOLVEMENT OF THE MHC-CLASS I CHARACTERIZATION OF THE DIASTOLIC RESPONSE TO ACUTE HEMODYNAMIC
REGION IN THE SETTING OF CD8+ T LYMPHOCYTE NUMBERS IN HUMANS. 47 OVERLOAD AND ITS MODULATION BY ISCHEMIA MECHANISMS UNDERLYING
ISCHEMIC DIASTOLIC DYSFUNCTION . . . . . . . . . . . . . . . . . 58
PS 239
AUTONOMIC CHANGES DUE TO MIDDLE CEREBRAL ARTERY PERFUSION PS 349
DEFICIT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 UROCORTIN 2 RELAXES THE IRIS SPHINCTER MUSCLE THROUGH CRF2 AND
PKC PATHWAY . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
PS 241
PULL DOWN AND EXPANSION OF NAVE CMV-SPECIFIC T-CELLS . . . . . 49 PS 352
NEBIVOLOL REDUCES PORTAL HYPERTENSION IN RATS WITH BILE DUCT
PS 243 LIGATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
COMPARATIVE STUDY OF THE PREVALENCE, CLINICAL FEATURES,
SENSITISATION PROFILES AND RISK FACTORS FOR BRONCHIAL ASTHMA
BETWEEN ELDERLY AND YOUNG ADULTS IN COVA DA BEIRA PORTUGAL . 50

36 Abstracts Physiology & Immunology 37

 YES Guide
PS 27
THE EFFECT OF GAG REFLEX ON CARDIAC SYMPATOVAGAL TONE
S. MEHRAN HOSEINI, ALI REZA MALEKI(PRESENTER)
DEPARTMENT OF PHYSIOLOGY, FACULTY OF MEDICINE, GOLESTAN UNIVERSITY OF MEDICAL SCIENCES, GORGAN, IRAN
AIM
Using the heart rate variability (HRV) analysis, the effect of gagging on cardiac sympatovagal balance was studied.
INTRODUCTION
Heart velocity may be influenced by gagging. The medulla oblongata receives the afferents of gag reflex. Neuronal
pools of vomiting, salivation and cardiac parasympathetic fibers are very close in this area. So, their activities may be
changed by spillover from each other.
METHODS
ECG was recorded from 18 healthy non-smoker volunteer university students for ten minutes in the sitting position
between 10-11 AM. Gagging was elicited by tactile stimulation of posterior pharyngeal wall. At 1 kHz sampling
rate, HRV was calculated and normalized on the basis of very low frequency component (0-0.04); the mean of heart
rate, low and high frequencies (LF: 0.04-0.15; HF: 0.15-0.4 Hz) were compared before and after the stimulus.
RESULTS
The mean of average heart rate, LF and HF in normalized units (nu) and the ratio of them (LF/HF) before and after
the gagging were 89.9 3 and 95.2 3 bpm (p=0.021, df: 8); 44.2 5.8 and 21.2 4 (p=0.010, df: 8); 31.1
5.3 and 39.4 3.8 (p=0.179, df: 8); 1.7 0.3 and 0.6 0.2 (p=0.015, df: 8) respectively.
CONCLUSION
Gagging increased heart velocity and had differential effect on two branches of cardiac autonomic nerves. Despite
of some increase in parasympathetic activity there was about fifty percent reduction in normalized LF and a similar
decrease in the LF/HF ratio. The paradoxical relation between average heart rate and HRV indexes of sympatovagal
tone may be due to unequal rate of change in autonomic fibers activities which is masked by five minute interval
averaging.
38 Abstracts
YES Meeting 2010
PS 32
INFLUENCE OF MAST-CELL STABILIZATION WITH SODIUM CROMOGLYCATE
ON THE PULMONARY VESSEL WALL REMODELLING IN HYPOXIC PULMO-
NARY HYPERTENSION
1 - NOVOTNY T., 1 - KREJCI J., 1 - SVEHLIK V., 1 - WASSERBAUER R., 1,2 - MALIKOVA J., 1 - UHLIK J.,
1 - VAJNER L.
1 - DEPARTMENT OF HISTOLOGY AND EMBRYOLOGY, CHARLES UNIVERSITY IN PRAGUE, SECOND FACULTY OF MEDICINE, 2 -
DEPARTMENT OF PAEDIATRICS, CHARLES UNIVERSITY IN PRAGUE, SECOND FACULTY OF MEDICINE
AIM
We suppose the effective stabilization of MC could result in different remodelling of pulmonary vessels as well as in
different re-remodelling during the recovery period.
INTRODUCTION
The environment of lowered content of oxygen leads to development of hypoxic pulmonary hypertension as the
result of pulmonary vessel remodelling. Having returned back to normoxic environment, pulmonary vascular bed
reveals re-remodelling to original shape. Both processes are performed by action of the mast-cell (MC) products
depending on MC distribution and interstitial collagenase production. We assessed morphological changes of
pulmonary vessels as well as of MC distribution depending on the hypoxia duration, recovery duration and various
timing of the sodium cromoglycate (SC) administration.
METHODS
Total 112 Han Wistar rat males were divided in 14 groups by 8 individuals and exposed to an acute (4 days) and
chronic (21 days) normobaric hypoxia (10 % of oxygen), respectively. If need was, they were allowed to recover
for a short term (4 days) or for a long term (21 days) of the chronic hypoxia under concurrent variously timed
intraperitoneal administration of SC. Histological changes of lung sections were assessed using a light microscope.
MC distribution was assessed using an image analyser software.
RESULTS
During the acute hypoxia, CS administration causes very restricted remodelling of pulmonary vessels (neomuscu-
larization of prealveolar arteries and the oblique-segment formation in smooth muscles of peripheral arteries).
During the chronic hypoxia, early CS administration causes only partial neomuscularization of prealveolar arteries
and symmetrical hypertrophy of the tunica media (TM) in peripheral arteries. During the chronic hypoxia, late
CS administration causes neomuscularization of prealveolar arteries and non-symmetrical hypertrophy of TM
together with the oblique-segment formation in peripheral arteries. Having passed 4-day recovery with the CS
administration at the beginning of hypoxia exposure, practically complete re-remodelling of all pulmonary vessels
has revealed. Having passed 21-day recovery with the CS administration at the beginning of hypoxia exposure,
complete re-remodelling of pulmonary vessels has revealed. Having passed 4-day recovery with the concurrent CS
administration, no re-remodelling of pulmonary vessels has revealed. Having passed 21-day recovery with the CS
administration at the beginning of recovery, belated and still continuing symmetrical re-remodelling of pulmonary
vessels has revealed.
CONCLUSION
CS administration at the beginning of hypoxia restricts conspicuously the morphological remodelling of pulmonary
vessels and enables re-remodelling. CS administration at the beginning of recovery restricts re-remodelling of
pulmonary vessels into their original normoxic shapes.
REFERENCES
Physiology & Immunology 39
 YES Guide
PS 90
ACUTE HAEMODYNAMIC EFFECTS OF TEZOSENTAN IN RATS WITH MONO-
CROTALINEINDUCED PULMONARY HYPERTENSION.
FIGUEIREDO PINTO D., FONTOURA D., SILVA D., PINTO J., VASQUES-NVOA F., LOURENO A. P. AND
LEITE-MOREIRA A. F.
1 - FACULTY OF MEDICINE, UNIVERSITY OF PORTO
AIM
To evaluate the acute haemodynamic effects of tezosentan in chronic experimental pulmonary hypertension.
INTRODUCTION
Tezosentan, a short-acting intravenous (iv) dual endothelin-1 (ET-1) receptor blocker [1], may be useful for acute
haemodynamic control in patients with chronic pulmonary hypertension (PH). Our goal was to evaluate the haemo-
dynamic effects of tezosentan in monocrotaline (MCT)-induced chronic PH.
METHODS
Wistar rats (180-200g) were randomly injected with either 60mg.Kg-1 MCT or vehicle, subcutaneously. Thereafter a
subgroup of MCT-injected rats was gavaged daily with 300mg.Kg-1 bosentan (M+B, n=13) while another subgroup
(M, n=20) and control rats (C, n=7) received vehicle. At the 28th day after injection, and 24 hours after interrupt-
ing bosentan, right (RV) and left ventricular (LV) pressures were continuously recorded after thoracotomy under
anaesthesia with fentanil and sevoflurane, mechanical ventilation, and 0.6 mL.Kg-1.h-1 saline as fluid replacement.
Tezosentan was administered in cumulative iv doses of 0.5, 1, 2, 5, 10, and 20mg.Kg-1, after stable effect of the
previous dose. Statistical analysis was performed by two-way repeated measures ANOVA. Quantitative variables are
presented as mean SEM.
RESULTS
During follow-up, 63% and 46% of M and M+B rats died, respectively, and 1 rat from group M died during haemo-
dynamic evaluation. Compared with C, RV systolic pressure (SP) and relaxation constant were increased in M and
M+B, while LVSP was decreased in M. RVSP was dose-dependently reduced by tezosentan in M (from 58.81.0, at
baseline, to 48.61.0 mmHg, with 20mg.Kg-1) and M+B (51.11.1 to 40.01.3 mmHg), with no change in LVSP
or heart rate, while no changes were observed in C (32.71.0 to 31.21.0 mmHg.
CONCLUSION
Tezosentan acutely reduces RVSP in chronic experimental PH. This effect is preserved despite previous chronic ET-1
receptor blocker therapy. Tezosentan may prove to be useful for haemodynamic handling of PH patients during
cardiac surgery or in critical care.
REFERENCES
40 Abstracts
YES Meeting 2010
PS 151
ASSESSMENT OF FACTORS AFFECTING LYMPHOCYTE ACTIVATION IN THE
NEONATE
KOLLR S
FIRST DEPT. OF PEDIATRICS, SEMMELWEIS UNIVERSITY
AIM
The aim of this study was to assess the relation between the elements of adaptive immunity and chief factors lead-
ing to preterm birth (preeclampsia (PE), premature rupture of membranes (PROM)), as well as gender and prenatal
steroid treatment (PS) during the first postnatal week.
INTRODUCTION
Alterations of the adaptive immune system play a central role in the development of perinatal complications. The
aim of this study was to assess the relation between the elements of adaptive immunity and chief factors leading to
preterm birth (preeclampsia (PE), premature rupture of membranes (PROM)), as well as gender and prenatal steroid
treatment (PS) during the first postnatal week.
METHODS
We enrolled 22 female and 21 male preterm infants born before the 33rd week of gestation and with less than 2000
g weight at birth into our study. Peripheral blood samples were drawn at birth (from cord blood) as well as on the
1st, 3rd, and 7th postnatal days of life. We characterized the prevalence of major lymphocyte subsets (CD4, CD8, Th1
(CXCR3+), Th2 (CCR4+)) and that of activated lymphocytes (CD69+, CD25+, CD62L+) using flow cytometry. The
independent effects of postnatal age, PE (n = 8), PROM (n = 13), PS (n = 25) and gender were analyzed using the
mixed effect model method. Where an effect was noticed, Mann-Whitney test was applied to determine the extent
of the alteration.
RESULTS
The prevalence of CD62L+ lymphocytes was higher in male than in female infants. The prevalence of CD25+ cells
was increased in cases of PROM. The prevalence of CD4 and CD8 cells and CD4/CD8 ratio were decreased in PE.
Postnatal age and PS did not affect the prevalence of investigated markers. Prevalence of other lymphocyte subsets
investigated was not influenced by the above factors.
CONCLUSION
The gender of patients and the ground for preterm birth do influence the elements of adaptive immunity. Based
on clinical experience, severe perinatal complications occur more frequently in cases when PROM or PE is present.
Furthermore, it is known that perinatal morbidity of male infants is elevated compared to female infants. Our obser-
vations indicate that alterations affecting the elements of adaptive immunity investigated in this study contribute
to these phenomena.
REFERENCES
Physiology & Immunology 41
 YES Guide
PS 175
EFFECT OF CELLULAR FOLATE AVAILABILITY ON ADIPOCYTE LIFE CYCLE
AND METABOLISM
1,2 - MARQUES C., 1 - TEIXEIRA D., 1 - CUNHA A., 1 - PESTANA D., 1 - MEIRELES M., 1 - KEATING E., 1 -
CALHAU C., 1 - MONTEIRO R., 1,3 - FARIA A.
1 - DEPARTMENT OF BIOCHEMISTRY (U38-FCT), FACULTY OF MEDICINE, UNIVERSITY OF PORTO, 2 - FACULTY OF NUTRITION AND
FOOD SCIENCES, UNIVERSITY OF PORTO, 3 - CHEMISTRY INVESTIGATION CENTRE (CIQ), FACULTY OF SCIENCES, UNIVERSITY OF
PORTO;
AIM
To investigate the effect of cellular folate availability on preadipocyte proliferation, and on adipocyte differentiation
and glucose uptake.
INTRODUCTION
The role of folate in obesity and metabolic syndrome is beginning to be investigated but is far from being fully
understood. Many studies have drawn attention to the association of folate status and plasma homocysteine levels
[1], an established independent risk factor for cardiovascular disease development. Recently, epidemiologic data
shows an inverse association between serum folate and body mass index [2], which has been associated with a
lower intake of vegetables. However, there is no straight evidence about the effects of folate on adipose tissue.
METHODS
3T3-L1 preadipocytes were cultured as described [3] to evaluate proliferation by sulforhodamine B staining and
methyl-3H-thymidine incorporation, after 24h or 48h of treatment with methotrexate (MTX, 0.1 and 10 M), an
inducer of a low cellular folate status. Preadipocytes were induced to differentiate with an appropriate adipogenic
cocktail in the presence or absence of MTX, and adipogenesis was determined by measuring lipid accumulation after
staining with oil red O [2].3H-2-Deoxyglucose uptake was determined by liquid scintillation counting [4].
RESULTS
MTX treatment for 24 and 48h reduced culture protein content and methyl-3H-thymidine incorporation in a time-
and concentration-dependent manner (P< 0.05). At the end of treatment, there was even a lower protein content
than in the beginning of treatment for the highest concentration of MTX (10 M) used (P< 0.05). In differentiated
adipocytes, MTX treatment increased lipid accumulation and the effect was much more pronounced for the highest
concentration of MTX. Also, MTX (10 M) increased basal glucose uptake (P< 0.05 vs control and MTX 0.1 M).
However, in MTX (10 M)-treated adipocytes, insulin-stimulation did not result in an increase of glucose uptake,
contrary to what was observed in untreated adipocytes.
CONCLUSION
According to the new understanding of obesity therapeutic strategies with the aim of reducing associated metabolic
complications, adipocyte hypertrophy and dysfunction could be prevented if adipose tissue preserves the ability
to recruit preadipocytes to differentiate, so that fat content can be distributed among the new adipocytes [5]. Our
results showed that, when folate availability is compromised by MTX treatment, there was a stimulation of preadi-
pocyte differentiation, but this stimulation was accompanied by a decrease in preadipocyte number. Furthermore,
the results obtained in glucose uptake studies suggested that these cells were resistant to insulin stimulation. In
conclusion, our results suggest that folate deprivation can interfere with adipocyte proliferation, differentiation
and metabolism and promote the hypertrophic growth of adipocytes, which may contribute to the development of
obesity and metabolic syndrome.
REFERENCES
42 Abstracts
YES Meeting 2010
PS 182
EFFECTS OF CENTRAL ALPHA-MSH INFUSION IN RATS OF VARIOUS NUTRI-
TIONAL STATES
TORE SCHJTTELVIK, ILDIKO ROSTAS, VERONIKA SIPOS
DEPARTMENT OF PATHOPHYSIOLOGY AND GERONTOLOGY, MEDICAL SCHOOL, UNIVERSITY OF PCS, HUNGARY
AIM
The study was conducted to observe the effects of different nutritional states on the responsiveness of the hypo-
thalamic melanocortin system within the same age-group of 6-month old rats using a central alpha-melanocyte
stimulating hormone (MSH) infusion. Then results were compared to those obtained with similar infusions of
leptin.
INTRODUCTION
Long-term regulation of energy balance shows two characteristic trends: obesity in the middle-aged and weight
loss leading to cachexia and sarcopenia (20-40% loss of skeletal muscles) in aged individuals (1). Complex periph-
eral and central regulatory mechanisms may contribute to these age-related changes in body weight and body
composition. Leptin is one of the major peripheral catabolic hormones, acting primarily in the arcuate nucleus, the
central effects of which may change significantly with age. We have observed previously that the catabolic effects
of a central leptin infusion vary in different nutritional states within the same age-group of young adult rats (2).
Among the central neuropeptides influenced by leptin, melanocortins (MC) may be the key catabolic mediators.
MCs show a co-ordinated catabolic effect: alpha-melanocyte stimulating hormone (MSH), an intrinsic agonist of
the system, decreases food intake (FI), increases metabolic rate (MR) and reduces body weight (BW).
METHODS
Parameters of energy balance [FI, BW, core temperature (Tc), heart rate (HR)] of three groups of 6-month old male
Wistar rats [calorie-restricted (CR6), normally fed (NF6) and a high-fat diet-induced obese (HF6) group] were
recorded in a biotelemetric system (VitalView) during a 7-day (1 g/l/h) intracerebroventricular infusion of MSH
using Alzet osmotic minipumps. Each group contained at least 10-12 rats. Data were sampled every five minutes
and averaged for 12-h periods. For statistical analysis of the data, repeated-measures ANOVA was used.
RESULTS
The MSH-induced transient drop in FI was most pronounced in HF6, less in NF6 rats and weakest in the CR6 group
similarly to the effects of a leptin infusion. A consequent BW loss occurred in NF6, CR6, but not in HF6 rats due to
MC administration. Unlike leptin, MCs induced a significant drop of BW in CR6 animals and failed to do so in the HF6
rats. A simultaneous increase in the mean daytime Tc and HR induced by MCs was observed in all groups (indicating
a rise in daytime MR). These changes were most pronounced in the CR6, but also remarkable in the NF6 and HF6
groups. Similar results were obtained using a leptin infusion. MCs however elicited a stronger Tc and HR response in
HF6 rats.
CONCLUSION
In contrast to the leptin effects, some of the MSH-induced catabolic effects were maintained in obese HF6 animals.
In CR6 rats, where leptin failed to decrease BW, MSH caused a significant weight loss. Our data suggest that the
effects of leptin and those of MSH are not changing parallel to one another. Despite obesity-induced leptin resist-
ance, MC-responsiveness is maintained for core temperature and heart rate.
REFERENCES
Physiology & Immunology 43
 YES Guide
PS 184
CHARACTERIZATION OF PLACENTAL UPTAKE OF THE AMINO ACID L-ME-
THIONINE
1-ARAJO J.R., 1-CORREIA-BRANCO A., 2-RAMALHO C., 1-KEATING E. AND 1-MARTEL F.
1-DEPARTMENT OF BIOCHEMISTRY (U38-FCT), FACULTY OF MEDICINE, UNIVERSITY OF PORTO, PORTO, PORTUGAL; 2-DEPART-
MENT OF GYNAECOLOGY AND OBSTETRICS, HOSPITAL S. JOO, PORTO, PORTUGAL.
AIM
Despite the extreme importance of metionine (L-Met) for fetal development, placental transport of this amino acid
is still largely unexplored. So, the aim of this work was to characterize the placental transport of L-Met.
INTRODUCTION
The placenta is a fetal organ essential for fetal development and pregnancy health (1). Maternal-to-fetal transfer
of amino acids across the placenta is essential for intrauterine growth, since these compounds are important for
carbon and nitrogen accretion, energy metabolism, and biosynthesis of nucleotides, neurotransmitters and heme
(2). Methionine (L-Met) is a nutritionally essential large neutral amino acid critical for brain (3) and liver develop-
ment (4).
METHODS
For this, we studied the characteristics of 14C-L-Met apical uptake in two human syncytiotrophoblast cell models:
the Bewo human choriocarcinoma cell line and primary cultures of human cytotrophoblasts (NTB cells).
RESULTS
Uptake of 14C-L-Met (250 nM) in both BeWo and NTB cells was: 1) time-dependent and linear for the first 6 min of
incubation; 2) saturable; 3) partially Na+-dependent; 4) strongly (about 50%) inhibited by the non-metabolizable
amino acid analogue BCH (2-aminobicyclo[2,2,1]heptane-2-carboxylic acid) (1 mM) and 5) only slightly (11-17%)
inhibited by the small neutral amino acid L-alanine (L-Ala) (100 M). Interestingly enough, Na+-dependence of
14C-L-Met uptake was higher in NTB cells when compared to Bewo cells (20-25% vs. 7% reduction in the absence of
NaCl, respectively).
CONCLUSION
In conclusion, our results show that L-Met uptake occurs through a similar process in both BeWo and NTB cells,
which seems to involve: 1) the BCH-sensitive system L, mainly represented by its L-Ala-insensitive subtype, LAT1;
2) the BCH-insensitive component system bo+ and 3) the BCH-insensitive and Na+-dependent component y+ L.
Finally, we conclude that system y+ L is more active in NTB than in BeWo cells.
REFERENCES
44 Abstracts
YES Meeting 2010
PS 195
ALLOSTERIC MODULATION OF ANGIOTENSIN II RECEPTORS BY ROSIGLITA-
ZONE IN THE SYMPATHETIC NEURONS OF THE RAT HEART
FERREIRA I, PEREIRA L, PINHEIRO H, MOURA D
INSTITUTE OF PHARMACOLOGY AND THERAPEUTICS, FACULTY OF MEDICINE, UNIVERSITY OF PORTO
AIM
This study examines the effect of the peroxisome proliferator-activated receptor- (PPAR) agonist rosiglitazone on
the facilitatory action of angiotensin II on noradrenaline release in the heart.
INTRODUCTION
Rosiglitazone is a PPAR agonist used in the treatment of type 2 diabetes mellitus. It modulates the transcription
of target genes, inducing partial reversal of insulin resistance. However, treatment with rosiglitazone increases by
unknown mechanisms the risk of myocardial infarction and adverse cardiovascular events. Since telmisartan, an
angiotensin II receptor antagonist, acts as a partial agonist of PPAR, we looked whether the PPAR agonist rosigli-
tazone might act on angiotensin II receptors in sympathetic neurons increase noradrenaline release.
METHODS
Slices of the left ventricle of male normotensive Wistar rats were loaded with 0.2 M 3H-noradrenaline for 1 hour,
mounted in small chambers and perifused with Krebs-Henseleit solution. Cocaine (12 M) was added to the solu-
tion at t=60 min of perifusion. From t=80 min the perifusion fluid was collected in samples of 5 min until the end of
the experiment for scintillation counting. In the experiments using angiotensin II, transmural electrical stimulation
(300 pulses, 1 Hz, 2ms, 50 mA) was applied at min 100 and 150. The first period was taken as control (Scontrol).
The second stimulation was applied in the presence of angiotensin II (Sdrug). In the experiments with rauwolscine
and phentolamine, two further periods of electrical stimulation were applied (t=200 and t=250 min). Drugs were
added at increasing concentrations before the second, third and fourth stimulation periods. Tritium overflow in-
duced by electrical stimulation was calculated in the absence (Scontrol) and presence of drugs under study (Sdrug).
The effect of drugs was measured as the ratio Sdrug /Scontrol.
RESULTS
Angiotensin II caused a concentration-dependent increase of tritium overflow induced by electrical stimulation
[pEC30%=8.60.2 (meanSEM, n=18); maximum increase=1108%]. Rosiglitazone (0.3-3 M) had no direct
effect. The concentration-response to angiotensin II in the presence of fixed concentrations of rosiglitazone was
shifted to the left with increase of the maximum increase (pEC30%=8.80.2, 9.20.2 and 9.30.3; maximum
increase=11814%, 14613% and 14816%, in the presence of 0.3, 1 and 3 M of rosiglitazone, respectively,
n=4-6, each). The release-enhancing effects of neither rauwolscine nor phentolamine (1-300 nM) were changed by
rosiglitazone. Noradrenaline release induced by electrical stimulation in the presence of 10 nM angiotensin II and
1 M rosiglitazone (Sdrug/Scontrol = 2.770.21) was reduced by 1 M of the PPAR antagonist GW9662 (Sdrug/
Scontrol = 2.060.29, p<0.05).
CONCLUSION
Rosiglitazone increased the facilitatory action of angiotensin II on noradrenaline release in a concentration-
dependent manner, without changing the release-enhancing effect of 2-receptor antagonists. Potentiation of
angiotensin II by rosiglitazone was antagonized by GW9662. The results suggest that a positive allosteric modula-
tion of angiotensin II receptors by PPAR agonists occurs in sympathetic terminals. This action may contribute to the
cardiovascular adverse effects of rosiglitazone.
Physiology & Immunology 45
 YES Guide
PS 209
ENDOCRINE FUNCTIONS OF THE HUMAN STOMACH SEROTONIN AND
GHRELIN IMMUNOREACTIVE CELLS OF THE GASTRIC MUCOSA
POPOVA L., PENKOVA N., KOEVA Y., ATANASSOVA P., PORYAZOVA E., ANDONOV V.
FACULTY OF MEDICINE, UNIVERSITY OF PLOVDIV
AIM
The aim of the current study was an immunohistochemical proof of the presence and localization of serotonin- and
ghrelin-producing cells in the mucosa of the stomach.
INTRODUCTION
One of the regulative systems in the human body is the gastro-entero-pancreatic endocrine system. With its variety
of cell types and hormones that they produce this system is a subject to many studies. Serotonin as a neurotransmit-
ter and gut hormone is an important element of the not yet completely examined brain gut axis. Being one of the
regulators of the gut motility, secretion and visceral sensitivity serotonin is the trigger of pathophysiological mecha-
nisms of some of the symptoms of the gastro-intestinal disorders. Ghrelin, a novel growth hormone (GH)-releasing
acylated peptide, was recently isolated from rat stomach. It stimulated the release of GH from the anterior pituitary
through the GH secretagogue receptor. However, its action in regulating the fed and fasted motor activity of the
digestive tract is not fully understood. The type of the endocrine cells that secrete ghrelin has not yet been identified
and the morphological characteristics of the ghrelin-producing cells is yet to be discovered.
METHODS
Biopsy specimens from the different regions of the stomach mucosa: body and antrum were taken by fibrogas-
troscopy. The paraffin sections for immunochistochemical study were investigated by ABC method with primary
antibody for MAB352 serotonin (rabbit polyclonal antibody Chemicon USA) and with primary antibody for ghrelin
(rabbit polyclonal antibody Ghrelin - Santa Cruz Biotechnology USA).
RESULTS
The immunohistochemical study of the biopsy specimens established a significant number of serotonin containing
cells from the antral mucosa. Some of the serotonin positive cells were singly observed in the covering epithelium.
Others were localized in the column and body of the pyloric glands. The immunohistochemical investigation
revealed positive ghrelin expression in the endocrine cells of main glands of the stomach body, mainly in its corpus
and rarely in the bottom.
CONCLUSION
The immunihistochemistry establishes expression of serotonin and ghrelin in endocrine cells in the gastric body and
antrum. The localization of the serotonin- and ghrelin-producing cells along the gastrointestinal tract is important
for a better understanding of its functions.The results of our research could help the understanding of the pathologi-
cal processes of the gastrointestinal diseases.
REFERENCES
46 Abstracts
YES Meeting 2010
PS 229
A STUDY OF 147 EXTENDED HAPLOTYPES CARRYING THE C282Y HFE MUTA-
TION: A NOVEL APPROACH TO EXPLAIN THE INVOLVEMENT OF THE MHC-
CLASS I REGION IN THE SETTING OF CD8+ T LYMPHOCYTE NUMBERS IN
HUMANS.
1 - MORAIS S., 2 - COSTA M., 3 - BETTENCOURT A., 2,4 - CRUZ E., 2 - ALMEIDA S., 3 - SILVA B. M., 2,4,5
- PORTO G.
1 - ABEL SALAZAR INSTITUTE FOR BIOMEDICAL SCIENCES (ICBAS), PORTO, PORTUGAL. 2 - IRON GENES AND THE IMMUNE
SYSTEM (IRIS) GROUP, INSTITUTE FOR MOLECULAR AND CELL BIOLOGY (IBMC), PORTO, PORTUGAL. 3 - IMMUNOGENETICS
LABORATORY, ABEL SALAZAR INSTITUTE FOR BIOMEDICAL SCIENCES (ICBAS), MULTIDISCIPLINARY BIOMEDICAL RESEARCH
UNIT (UMIB), PORTO, PORTUGAL. 4 - CLINICAL HEMATOLOGY, SANTO ANTNIO HOSPITAL, PORTO, PORTUGAL. 5 - MOLECULAR
IMMUNOLOGY AND PATHOLOGY, ABEL SALAZAR INSTITUTE FOR BIOMEDICAL
AIM
To clarify the relative impact of the HLA specificities or the whole haplotype on the genetic transmission of CD8+T
lymphocytes in humans.
INTRODUCTION
The numbers of peripheral blood CD8+ T lymphocytes are known to be genetically determined in the context of
genes at the MHC-class I region. The present study analyses, for the first time, the inheritance of CD8+ T lympho-
cytes in the context of extended haplotypes defined between HFE and HLA-B by segregation analysis in families of
Hereditary Hemochromatosis (HH) patients.
METHODS
A total of 71 unrelated C282Y homozygous HH patients and 61 of their family members were studied for extended
haplotype analysis, performed by segregation analysis for the SNP markers on PGBD1, ZNF193 and ZNF165 (defining
the restricted SNP haplotypes AAT and GGG) and for the HLA-A and -B loci, all correlated with the numbers of CD8+
T lymphocytes. In addition, 123 DNA samples from an independent population of C282Y homozygous subjects were
tested to confirm the frequencies of the SNP markers in the Portuguese HH population.
RESULTS
The relative frequencies of the AAT and GGG haplotypes in the population of C282Y homozygous HH patients was
94.3% and 4.9% respectively. We confirmed the strong association of the most common HLA-A*03, -B*07 and the
A3B7-AAT ancestral haplotype with the inheritance of low CD8+T lymphocytes. The HLA-A*03 was the only HLA al-
lele increased in frequency in patients with high lymphocytes. The definition of extended haplotypes allowed us to
construct a model with all patients chromosomes classified as carrying a low or high CD8+ trait and to compare
the distribution of HLA alleles and haplotypes between the two groups. The results clearly showed a greater allelic
and haplotypic variability in the high vs the low CD8+ trait group supporting the hypothesis of a different
recombination history.
CONCLUSION
The study of extended haplotypes carrying the C282Y HFE mutation offers a new model to explain the contribution
of the MHC region to the setting of CD8+T lymphocyte numbers. The results support the hypothesis of a putative
still unidentified trait localized centromeric to HLA-A.
ACKNOWLEDGEMENTS
Physiology & Immunology 47
 YES Guide
PS 239
AUTONOMIC CHANGES DUE TO MIDDLE CEREBRAL ARTERY PERFUSION
DEFICIT
1 - SOUSA D., 2 - ESCALDA A., 3 - RODRIGUES F., 4 - ROCHA I.
1,3 - FACULTY OF MEDICINE, MD STUDENT; 2,4 - INSTITUTE OF PHYSIOLOGY, AUTONOMIC NERVOUS SYSTEM UNIT, PHD
AIM
In an animal model of ischemic stroke, we intended to evaluate the acute changes (up to 1 hour) of cardiovascular
autonomic function related to Middle Cerebral Artery infarction.
INTRODUCTION
Stroke is a major cause of mortality and morbidity in the world and refers to a group of acute diseases causing neu-
rological and motor deficit. Most strokes (85%) are ischemic in nature and, among all vascular territories involved,
the middle cerebral artery (MCA) is the most commonly affected. Previous studies have shown that in acute stroke,
changes in baseline autonomic activity may underline some end organ dysfunction that is observed in these pa-
tients (eg, arrhythmias, myocardial infarction or heart failure) assuming a major importance the characterization of
the consequent autonomic changes mainly those related to the cardiovascular system. The evaluation of autonomic
cardiovascular function involves the application of Fourier transform (FFT) that provides a spectrum with 2 main
bands - LF and HF - related to sympathetic and parasympathetic activity, respectively, as well as the evaluation of
changes on blood pressure and heart rate due to peripheral baroreflex stimulation.
METHODS
In Wistar male rats (n = 7), anaesthetized (pentobarbital sodium, 60mg/kg), under neuromuscular blockade
(vecuronium, 4mg/kg/h) and artificial ventilation, a craneotomy was performed to allow MCA ligation. Blood
pressure (BP), ECG and heart rate (HR) derived from ECG were continuously monitored throughout the experiment.
Baroreflex stimulation was performed peripherally, with phenylephrine (25g/mL), observing the evoked changes
in BP and HR, in 3 periods: before ischaemia (CRT) and after ischaemia, at 10th and 40th minute and the baroreflex
index (BRI) was calculated for each stimulation. To evaluate sympatho-vagal balance throughout the experimental
procedure, fast Fourier transform (FFT) was applied to RR signal at the following periods: before and after MCA
laqueation (in this case at 5, 35 and 55min after stroke) and LF, HF and the LF / HF were calculated for each period
(Marques-Neves et al, 2004). For statistical analysis, Student-t test was used and differences considered significant
where p<0.05. At the end of the experiment, the animal was killed with an overdose of anaesthetic.
RESULTS
Results did not show significant differences for LF, HF and LF/HF, BP and HR between CRT and the periods after
MCA ligation. However, there is a trend towards an increase of parasympathetic activity, which is accompanied by
a tendency of HR, BP and LF/HF ratio decrease. Also, a comparative analysis of sympathetic and parasympathetic
activity over time, showed in 5/7 animals the same evolution of the autonomic (sympathetic and parasympathetic)
pattern of response till the 40min after stroke that, in 3/5 animals, was maintained until the end of the experiment.
BRI profile accompanies the change in LF / HF ratio.
CONCLUSION
In conclusion, the variation in results, offset by the trends, imposes the need to increase the sample to conclude
about the acute autonomic changes evoked by stroke.
REFERENCES
48 Abstracts
YES Meeting 2010
PS 241
PULL DOWN AND EXPANSION OF NAVE CMV-SPECIFIC T-CELLS
RAZ Y., HOMBRINK P., VON DEM BORNE P.A., FALKENBURG J.H.F., HEEMSKERK M.H.M.
DEPARTMENT OF EXPERIMENTAL HEMATOLOGY, LEIDEN UNIVERSITY MEDICAL CENTER
AIM
-
INTRODUCTION
Allogeneic bone marrow transplantation is a valuable treatment for several hematological malignancies, such as
leukemia. Post-transplantation cytomegalovirus (CMV) reactivation constitutes a serious risk of this treatment,
especially when CMV-negative donors are used. CMV reactivation can be successfully treated with adoptive T-cell
therapy. The extremely low frequency of antigen-specific nave T-cells has challenged us to develop a sensitive and
effective method to study the nave T-cell repertoire. In this study, we present a novel approach for the generation of
CMV-specific T-cell lines, covering multiple HLA class I alleles, derived from the nave T-cell repertoire.
METHODS
CMV-specific T-cell lines were generated from CMV-negative individuals. One hundred million peripheral blood
mononuclear cells were stained with a panel of CMV-specific class I major histocompatibility complex tetramers.
Tetramer positive T-cells were pulled down by magnetic-activated cell sorting (MACS) and cultured in the presence
of autologous feeders and anti-CD3/CD28 stimulation beads. To increase the frequency of CMV-specific T-cells in
culture, pull down was repeated after ten days.
RESULTS
Using this approach we generated CMV-specific T-cell lines from four out of four CMV-negative individuals. The
cell lines were generated within four weeks and covered multiple CMV epitopes. The frequency of CMV-specific
T-cells varied between 20 to 80% of total cultured T-cells. To analyze the peptide specificity and functionality of
the different T-cell populations, we generated CMV-specific T-cell clones. These clones were tested in addition to
the complete cell-lines in a cytokine secretion and cytotoxicity assay. CMV-specific T-cell populations demonstrated
variable cytotoxicity upon endogenous peptide presentation. As might be expected in CMV-negative individuals,
both high and low affinity T-cell populations were isolated. In addition, different cytokine secretion profiles were
observed after stimulation.
CONCLUSION
The fast and effective approach described here can be utilized for the generation of CMV-specific T-cell lines from
CMV-negative individuals. This method may be of potential clinical use in adoptive cellular immunotherapy for
treatment of CMV reactivation.
Physiology & Immunology 49
 YES Guide
PS 243
COMPARATIVE STUDY OF THE PREVALENCE, CLINICAL FEATURES, SENSITI-
SATION PROFILES AND RISK FACTORS FOR BRONCHIAL ASTHMA BETWEEN
ELDERLY AND YOUNG ADULTS IN COVA DA BEIRA PORTUGAL
MOREIRA SM, RIBEIRO AC, FONSECA M, LOURENO O, TABORDA-BARATA L
FACULTY OF HEALTH SCIENCE - UNIVERSITY OF BEIRA INTERIOR; CICS - HEATH SCIENCES RESEARCH CENTRE
AIM
The aim of the present study was to compare the prevalence, pattern of aeroallergen sensitization and clinical
features of bronchial asthma between elderly and young adults. In addition, we also wanted to analyse possible risk
factors for the development of asthma.
INTRODUCTION
Bronchial asthma is one of the most common allergic diseases worldwide. Very few studies have analysed whether
the prevalence of asthma decreases with age.
METHODS
This study followed a cross-sectional design. A standardised allergy and asthma questionnaire and skin prick tests
(SPT) were carried out in all volunteers. The study population included two groups of individuals: elderly (aged 65
years and older) and young adults (aged between 18-35 years) from Beira Interior. The sample was selected by
simple randomization, after calculating a confidence interval of 95% and an estimated error below 5%. Statistical
analysis was carried out using Chi-square tests, Mann-Whitney U test, univariate regression analysis, binary logistic
regression analysis and Odds Ratio. A p value less than 0.05 was considered statistically significant. All patients
signed a written informed consent and the study was approved by the Regional Health Authority Ethics Committee.
RESULTS
A total sample of 1460 volunteers was included. Thus far, we have analyzed a total of 27.6% of elderly volunteers
in the Health Care Centre (median age = 73 years; 41.5% males) and 14.1% of the young group (median age =
28 years; 44.2% males). A short questionnaire was applied by telephone to 11% of the total sample. We found
significant differences in the prevalence of bronchial asthma between elderly and young adults (19.7% vs 29.4%,
respectively; p=0.02; Chi-square test). In addition, the prevalence of atopic bronchial asthma was also significantly
lower in elderly than in young adult patients (32.1% vs 57.1%, respectively; p=0.02, Chi-square test). Both groups
were mostly sensitised to Dermatophagoides pteronyssinus and Dermatophagoides farinae. Significant differ-
ences were found in terms of sensitisation to grass pollens amongst elderly and young patients (11.8% vs 70.8%,
respectively; p=0.0006, Chi-square test). The majority of the asthmatic patients were sensitised to more than one
allergen. Finally, differences were observed in terms of risk factors for bronchial asthma between both groups, with
gender as a significant risk factor in elderly patients and concomitant rhinitis in young patients. The presence of
rhinitis augmented the risk of having asthma 3.2 times, in the group of young adults.
CONCLUSION
Our preliminary results suggest that the prevalence of asthma is lower in elderly people. The sensitisation profile
and the risk factors for this disease are different between elderly and young adult patients.
50 Abstracts
YES Meeting 2010
PS 251
AGE-ASSOCIATED SHIFTS IN THE RESPONSIVENESS OF THE CENTRAL CATA-
BOLIC MELANOCORTIN SYSTEM IN RATS
SCHMIDT A.T., SZABAD A.O.
FACULTY OF MEDICINE, UNIVERSITY OF PECS, HUNGARY
AIM
We studied age-related alterations in the effects of an intracerebroventricular alpha-MSH infusion on parameters of
energy balance in rats.
INTRODUCTION
Hypothalamic melanocortins like alpha-melanocyte stimulating hormone (alpha-MSH) play an important role in
the regulation of energy balance: they suppress food intake (FI), enhance metabolic rate (MR) and decrease body
weight (BW). Aging is characterized by two major trends: obesity in the middle-aged, later anorexia of aging. The
melanocortin-system may participate in these alterations.
METHODS
Male Wister rats aged 2-, 4-, 12- and 24-months represented the human juvenile, young, middle-aged and old
age-groups, respectively. Body temperature (Tc), spontaneous activity (ACT), heart rate (HR, indicator of MR) were
recorded in a biotelemetric system. FI and BW were measured daily. An Alzet osmotic minipump was implanted into
a lateral cerebral ventricle providing a 7-day-long infusion of 1 ug/ul/h alpha-MSH.
RESULTS
Decline of FI and BW during the infusion was weakest in the 12- and most pronounced in the 24-month-old rats.
Elevations of the mean 12-h day- and nighttime Tc values showed a different pattern. No effects were seen in
juvenile rats, modest daytime changes occurred in the young and old groups, both values increased significantly
in the middle-aged animals. Tachycardia developed only in the 2 older groups. In the old rats, however, the rise
in nighttime HR lasted only for 4 days, whereas the middle-aged animals were able to maintain high night- and
daytime HR throughout the infusion. ACT did not change in any group.
CONCLUSION
Middle-aged rats appear to be insensitive to the anorexigenic but not to the metabolic effects of alpha-MSH, their
slight weight loss may be attributed to MR-rise, indicated by rises in Tc and HR. On the other hand, FI and BW are
particularly sensitive to melanocortins in aged rats. The effects of alpha-MSH on different parameters of energy
balance do not change parallel to one another during aging.
ACKNOWLEDGEMENTS
Physiology & Immunology 51
 YES Guide
PS 257
DRAWING-UP THE CARDIAC STEM CELL(S) NICHE UNDER NORMALCY VS.
MYOCARDIAL INFARCTION
*1- VALENTE M., *1-NASCIMENTO D., 1-FREIRE A., 2-CARVALHO I., 1-ZILHO N., 3-CAIADO F., ABREU
C., 1-OLIVEIRA M.J., 3-DIAS S. AND 1-PINTO-DO- P.
1-INSTITUTO DE ENGENHARIA BIOMDICA, PORTO, PORTUGAL; 2-INSTITUTO DE BIOLOGIA MOLECULAR E CELULAR, PORTO,
PORTUGAL; 3-ANGIOGENESIS LABORATORY, INSTITUTO PORTUGUS DE ONCOLOGIA FRANCISCO GENTIL, LISBOA, PORTUGAL.
AIM
By using a combination of approaches, we aim at providing a clear illustration of how the cardiac stem/progeni-
tor cells (CSC/CPC) distribute throughout the heart while following the cardiac niche dynamics. This will highlight
whether the putative CSC engages into regeneration/repair and how such potential is affected by myocardial
infarction (MI).
INTRODUCTION
The heart was recently acknowledged as an organ that contains cells displaying stemness properties, potentially
responsible for cardiomyocyte renewal. Although the existence of CSC/CPC has been steadily reported, knowledge
on how these cells are nested in the cardiac tissue and their relation with neighboring cells is still scarce. Moreover,
the current paradigm in which stem cells are arranged in discrete histofunctional units (niches) which contribute for
cell-fate regulation, demand for a detailed characterization of how the architecture of the niche changes overtime
and/or under stress conditions.
METHODS
An experimental MI mouse model was established by permanent ligation of the left anterior descending (LAD)
coronary artery. Functional analysis was performed by transthoracic echocardiography and heart histology evalu-
ated by Massons trichrome (MT) staining. Quantification of MI extension was performed by area and midline length
measurement. Immunofluorescence (IF) stainings were done by indirect detection method and after streptavidin/
biotin amplification. For the zymography analysis, protein extracts from cardiac tissue lysates were loaded on a
10% SDS-PAGE containing gelatin or beta-casein as substrates. Zymograms were run under nondenaturing condi-
tions and proteolytic activity was visualized after Coomassie Blue stain.
RESULTS
The established MI model was validated by histological and functional analysis. Echocardiography indicated an
accentuated decrease of heart shortening and ejection fractions as a result of left ventricle (LV) remodelling. This
was further confirmed by MT stain that revealed the thinning of the LV wall and collagen deposition. Morphometric
analysis of tissue sections allowed quantification of MI extension by measuring area and midline length of the LV
wall affected by MI. By IF staining, an increase on MDR1 expression, particularly in the cardiomyocytes affected by
ischemia was observed 2 and 7 days after MI. A clear increase in c-Kit expression, evidenced 2 days after LAD liga-
tion, was still observed 7 days post-surgery. Lastly, sca-1 expression appeared to be augmented in the heart a week
after MI induction. Expression of basement membrane proteins (fibronectin, laminin and collagen IV), showed an
increase not only in the MI area but also throughout the myocardium. Lastly, MMP9, 2, 7 and 1/3 activities were
increased already at 48h, although differences were more pronounced at 7 days post-MI.
CONCLUSION
This work constitutes the basis for an integrative study of the cardiac stem/progenitor cells niche composition ad-
dressed in a clinically relevant MI model.
ACKNOWLEDGEMENTS
52 Abstracts
YES Meeting 2010
PS 269
STABILITY OF VITAMIN C IN ORANGE JUICE UNDER DIFFERENT TREAT-
MENTS IN A SHORT PERIOD OF TIME
1- SILVA-BRANDO R. R., 2- SOARES R. A. M., 3- ARAS J. A. G.
1, 2 E 3- FACULTY OF PUBLIC HEALTH, UNIVERSITY OF SAO PAULO
AIM
This work is important for better targeting of consumption of vitamin C from natural orange juice, helping doctors
and nutritionists in dietary counseling and disease prevention. Thus, it becomes important to analyze the stability
of Vitamin C in orange juice in a short period of time (60 minutes) for different conditions usually found in orange
juice consumption, to support better dietary.
INTRODUCTION
Ascorbic acid is an essential nutrient in the human diet that is present in several food matrices. It performs impor-
tant antioxidant functions, acting also as an enzymatic cofactor. Processing of orange such as peeling, cutting, mix-
ing, may interfere in the concentration of ascorbic acid and influence the stability of the juice during its subsequent
handling due to enzymatic oxidation.
METHODS
Ascorbic acid was determined in orange juice by the method of potassium iodate titration. The conditions studied
were: ambient temperature; refrigeration (6C); stirring for 2 minutes in blender and rest at the ambient; vacuum
processing for 10 seconds and sealing during the experiment. The conditions were followed by 60 minutes with
sampling in 15, 45 and 60 minutes immediately after the first titration. For statistic analysis of the results, the
software SPSS was employed. The one way analysis of variance was used to compare differences between means
during the time period studied with a significance level of p 0.05.
RESULTS
There was no difference between the mean values of ascorbic acid between the titrations in the period followed and
neither between all treatments. This study demonstrated that natural loss of vitamin C in orange juice in different
conditions after its production is not statistically significantly for at least 60 minutes.
CONCLUSION
The common recommendation of ingesting orange juice as quickly as possible for not losing vitamin C, that is a
common-sense among the Nutrition professionals and laymen, has no support by the present results.
REFERENCES
Physiology & Immunology 53
 YES Guide
PS 281
COMPARATIVE STUDY OF THE PREVALENCE, CLINICAL FEATURES, SENSITI-
SATION PROFILES AND RISK FACTORS FOR ALLERGIC RHINITIS BETWEEN
ELDERLY AND YOUNG ADULTS IN COVA DA BEIRA
RIBEIRO A.C.G., MOREIRA S.R., LOURENO O., FONSECA A.M., LOPES N.T., TABORDA-BARATA L.
CICS, CENTRO DE INVESTIGAO EM CINCIAS DA SADE, UNIVERSITY OF BEIRA INTERIOR, COVILH, PORTUGAL; DEPARTMENT
OF ALLERGY & CLINICAL IMMUNOLOGY, CENTRO HOSPITALAR COVA DA BEIRA, COVILH, PORTUGAL.
AIM
The aim of the present study was to compare the prevalence, clinical characteristics, pattern of aeroallergen sensiti-
sation and risk factors for AR between elderly and young adults.
INTRODUCTION
Allergic Rhinitis (AR) is the most common allergic disease worldwide. Very few studies have analysed whether the
prevalence of AR decreases with age. There is also a lack of publications assessing the risk factors for AR in elderly
people.
METHODS
This was a cross-sectional study using a simple random sample. The population consisted of 2 groups of individuals
from Beira Interior: one of young adults (aged between 18 and 35 years) and another of elderly individuals (aged
65 years or older). A standardised allergy and rhinitis questionnaire as well as skin prick tests (SPT) were carried
out in all volunteers, except for those who answered the questionnaire by telephone. All patients signed a written
informed consent and the study was approved by the Regional Health Authority Ethics Committee. Chi-square test,
Mann Whitney U test and logistic regression test were used for statistical analysis. A p value less than 0.05 was
considered statistically significant.
RESULTS
Our study sample included 1460 volunteers. To date, 473 volunteers have been analysed (312 elderly (median age =
72 years; 168 females) and 161 young adults (median age = 29 years; 85 females). Both groups were paired regard-
ing gender. The prevalence of AR was significantly lower in elderly (26.7%) than in young adult volunteers (40.6%)
(p=0.0194; Chi-square test). For both groups, association between overall positivity of SPT and self-reported
symptoms of AR was highly significant (p<0.0001 for elderly; p=0.0069 for young adults). Significant differences
were observed in the sensitisation patterns between the two groups with the elderly being mostly sensitised to Der-
matophagoides pteronyssinus (11.0%), Parietaria judaica (10.5%) and Olive tree (7.3%) and young adults mostly to
Dermatophagoides pteronyssinus (25.7%), Dermatophagoides farinae (15.8%) and cereal pollen (16.8%). A signifi-
cant association was found between AR and urban residence in the elderly group (p=0.047; Logistic regression).
CONCLUSION
Our data suggest that the prevalence of AR decreases with age and also that there may be differences in the profile
of sensitisation to aeroallergens between young and elderly individuals as well as in risk factors for both groups.
54 Abstracts
YES Meeting 2010
PS 295
ANGIOTENSIN 1-7 EFFECTS ON MIOCARDIAL CONTRACTILITY AFTER HY-
POXIA-REOXYGENATION.
MENDONCA L., PINTALHAO M., CERQUEIRA R., CASTRO CHAVES P., LEITE MOREIRA A.
FACULTY OF MEDICINE, UNIVERSITY OF PORTO
AIM
The aim of the present study is to assess Ang1-7 ability to modulate myocardial function in a hypoxia-reoxygenation
model.
INTRODUCTION
Angiotensin 1-7 (Ang1-7) is a bioactive heptapeptide of the renin-angiotensin system (RAS) that has received an
increasing attention for its ability to balance angiotensin II actions in the cardiovascular system, mainly through
the binding to Mas receptor [1]. Both angiotensin converting enzyme 2, the main enzyme responsible for Ang1-7
synthesis and Mas receptor are highly expressed in the heart, suggesting that this new renin-angiotensin system
pathway may have a key role on cardiac pathophysiology [2].
METHODS
Ang1-7 effects were evaluated using right ventricular rabbit papillary muscle preparations, immersed in a modified
Krebs-Ringer solution and electrically stimulated (1.8mM Ca^2+; 35C; 0.6Hz). After an initial stabilization proce-
dure, we added Ang1-7 (ang1-7 group, 10^-5M, n=6) or the same volume of vehicle (control group, H2O, n=6) to
the superfusing solution, followed by 30 min of hypoxia (replacement of the initial 95%O2 + 5%CO2 gas mixture
with a 95%N2 + 5%CO2 one), after which there was a 40 min reoxygenation period (95%O2 + 5%CO2). The
contractile response to increasing concentrations of isoproterenol (10^-8 10^-6M) was then evaluated. The same
protocol was also performed in the presence of the Mas receptor antagonist A-779 (10^-5M) followed by the addi-
tion of Ang1-7 (10^-5M), (A-779+ang1-7 n=6). The results are presented as mean+/-standard error (p<0.05).
RESULTS
We observed no statistically significant differences in contractility after hypoxia-reoxygenation between the several
groups. In the first protocol, pre-treatment with Ang1-7 improved the inotropic and lusitropic responses to beta-
adrenergic stimulation. For the higher isoproterenol concentration, we observed a greater increase of the contractile
parameter active tension in Ang1-7 group when compared to the control group (434,4%+/-69,0% vs 238,6%+/-
26,9%). Additionally, and also for the higher isoproterenol concentration, the Ang1-7 group showed an accelerated
relaxation as observed by an increase in the dP/dtmin when compared to the control group (552,9%+/-74,9% vs
333,2%+/-40,8%). The observed increase in contractility after isoproterenol addition was significantly attenuated
in the presence of the Mas receptor antagonist A-779, corresponding, at the higher tested concentration of isopro-
terenol, to an increase in AT of 227,9+/-49,6%. In the presence of A-779, it was also observed a significant decrease
in the dP/dtmin comparing to the Ang1-7 group, 293,9+/-68,4% vs 552,9+/-74,9%, respectively.
CONCLUSION
In conclusion, in this animal species, Ang1-7 improved the contractile response to beta-adrenergic stimulation after
hypoxia-reoxygenation which seems to be mediated through Mas receptor stimulation. These results reinforce the
role of this new mediator in modulating cardiac function, manly through its ability to improve myocardial inotropic
reserve. Therefore, these observations support Ang1-7 potential beneficial effects in the contractile dysfunction
associated with ischemia.
REFERENCES
Physiology & Immunology 55
 YES Guide
PS 331
THE EFFECTS OF CELERY EXTRACTS ON SPONTANIOUS AND ACETYLCHO-
LINE INDUCED CONTRACTIONS OF RATS DUODENUM
1-DZAMBAS J.,2-SPASIC M.,3-ANTIC M.
FACULTY OF MEDICINE, UNIVERSITY OF NIS
AIM
The aim of our research is to find out the possible acute effects of different extracts of celery leaf, water and
ethanol, on duodenal motility of the rat.
INTRODUCTION
Celery (Apium graveolens) is crunchy biennial plant whose seeds, leaves, roots and trees are increasigly used in
medicine because of its curative effects. Medical effects of celery are based on flavonoids luteolin and apigenin,
the most common compounds of its chemical structure. The effects of those flavonoids in human are hypotensive,
antiinflamative, antispasmolitic, antidiarrheal and anticanerogenic. It is also experimentaly shown on guinea pig,
mouse, rat and rabbit that celery has antispasmolytic effects on ileum and colon after the stimulation by acetylcho-
line, barium chloride or histamin, but mechanisms of the effects are not known yet. Besides these known chronic
effects, there are no data in the literature about acute effects of celery.
METHODS
Wistar rats, sacrificed by cervical dislocation, weight between 170 and 200g, both genders, were used in this experi-
ment. The part of rats duodenum, 2-3cm long, was put in a water bath for isolated organs according to Magnus
method. The adaptation lasted for 30 minutes. After the adaptation, substasnces were added on serosal part of the
duodenum. In control series of the research we found out the effects of different concentration of acetylcholine.
In experimental series we found out the effects of different concentration of celery with and without adding
acetyhcholine after it. The period between two doses was 20 to 30 minutes. The contraction of longitudal muscles
was registrated on physiograph. All the data were statisticaly analysed using standard deviation and T-test.
RESULTS
Both water and ethanol extracts of celery cause relaxation of duodenum, but only ethanol extracts cause concen-
tration-depended relaxation. There is a statistically significant difference between the effects of those two extracts.
Extracts also reduce contractions of duodenum intensified by acetylholine. 0,3g/l of water extract doesnt give any
significant results (ED50=4x10-8 M to 4.9x10-8 M) but 1g/l of extract gives stetistically significant concentration-
depended decreasing of acetylholines effects (ED50=1.1x10-7). Contractility inhibition caused by acetilholine is
concentration-depended and statistically more significant in relation with ethanol extract (ED50=1.6x10-7 M with
series of 0.3 g/l and ED50=3x10-6 M with series of concetration 1g/).
CONCLUSION
The extracts of celery have relaxant effect on duodenal motility of rats with three times stronger effects of ethanol
extract. It is assumed that the effects are realized throw muscarin receptors.
REFERENCES
56 Abstracts
YES Meeting 2010
PS 340
MORPHOFUNCTIONAL EVALUATION OF THE EFFECTS OF CHRONIC CON-
SUMPTION OF BEER IN RAT LIVERS
1,2 - SILVA JL, 1 - TEIXEIRA D, 1,3 - FARIA A, 1 - MEIRELES M, 1 - CUNHA A, 1 - CALHAU C, 1 - MON-
TEIRO R, 1 - PESTANA D
1 DEPARTMENT OF BIOCHEMISTRY (U38-FCT), FACULTY OF MEDICINE, UNIVERSITY OF PORTO, PORTO, PORTUGAL, 2 HEALTH
SCIENCES SCHOOL, UNIVERSITY OF MINHO, BRAGA, PORTUGAL, 3 CHEMISTRY INVESTIGATION CENTRE (CIQ), FACULTY OF
SCIENCES, UNIVERSITY OF PORTO, PORTO, PORTUGAL.
AIM
The aim of the present study was to evaluate the effect of chronic consumption of beer on hepatic tissue, from a
morphological, functional and molecular standpoint, focusing on redox, inflammatory and metabolic status mark-
ers.
INTRODUCTION
The liver is the most important organ involved in the detoxification of ethanol which probably relates to the fact
that it is also a main target of ethanol deleterious effects. It also plays a critical role on redox, metabolic and
inflammatory balance regulation and, therefore, its dysfunction is likely to have systemic repercussions. Beer is
currently one of the most widely consumed beverages in Portugal. Apart from its content in ethanol, beer is also a
rich source of polyphenols, compounds with recognized health protective effects which has been attributed to their
antioxidant, anti-inflammatory, anti-proliferative and endocrine properties. Taking this into account, there is some
controversy on whether the chronic consumption of beer results in deleterious effects in the liver.
METHODS
In order to do so, three groups of 6 male Wistar rats were treated for 8 weeks with: water (control, C); ethanol solu-
tion (5.4% ethanol, E); lager beer (with 5.4% ethanol, B). Beverages were supplied to the animals as the sole fluid
source. Food and fluid intake, as well as body weight gain were monitored through the study. The amount of etha-
nol and beer ingested corresponded to moderate consumption in humans. Livers were collected at the end of the
study and stored at -80C for evaluation of i) DNA oxidation through determination of 8-hydroxydeoxyguanosine
(8-OHdG), ii) transcription levels of the cell death-related receptor Fas or iii) processed for histological assessment (
i) paraffin-embedded, sectioned and stained with hematoxylin and eosin for overall morphological evaluation, ii)
fixed with OCT and stained with oil red O for quantification of lipid inclusions).
RESULTS
No effects of prolonged treatment with beer or ethanol were detected on DNA oxidation, Fas transcription levels or
general morphological features of hepatic tissue. Regarding hepatic lipid accumulation, ethanol and beer intake
resulted in an increase of lipid inclusion diameter. However, after normalization of total lipid area by the number
of cells on each section, ethanol-treated rats tended to have a higher lipid inclusion area, and beer-drinking rats
lower lipid inclusion area than that of control rats. In this sense, prolonged beer consumption seems to result in
the accumulation of less lipid inclusions, but with macrovesicular characteristics. Interestingly, macrovesicular
steatosis, in contrast with microvesicular steatosis, is related to a lower degree of beta-oxidation impairment and
pro-inflammatory stimulation.
CONCLUSION
In conclusion, data obtained in this study do not allow establishing a relationship between chronic consumption of
beer and deleterious hepatic effects. Furthermore, regarding lipid accumulation, the effects of beer were distinct
from those of ethanol, suggesting the involvement of non-alcoholic components of the beverage.
ACKNOWLEDGEMENTS
Physiology & Immunology 57
 YES Guide
PS 348
CHARACTERIZATION OF THE DIASTOLIC RESPONSE TO ACUTE HEMODY-
NAMIC OVERLOAD AND ITS MODULATION BY ISCHEMIA MECHANISMS
UNDERLYING ISCHEMIC DIASTOLIC DYSFUNCTION
NEVES J.S., FERREIRA R., LADEIRAS-LOPES R., PINTALHO M., CARVALHO R., LEITE-MOREIRA A.
DEPARTMENT OF PHYSIOLOGY, FACULTY OF MEDICINE, UNIVERSITY OF PORTO, PORTUGAL
AIM
To evaluate and characterize the diastolic response to an acute hemodynamic overload and its modulation during an
ischemic insult.
INTRODUCTION
Acute myocardial stretch induces an adaptive response both at systolic (immediate and time-dependent increase
in contractility) and diastolic levels (time-dependent decrease in myocardial stiffness). Myocardial infarction leads
to stretch of ventricular wall being the adaptive response to stretch impaired during an ischemic situation. The
mechanisms responsible for the failure of diastolic adaptation remain largely unknown.
METHODS
Rabbit papillary muscles (modified Krebs solution, 0.2Hz, 1.8mM Ca2+, 30C) were mechanically overloaded from
92% Lmax to 100% Lmax (length at which maximal force is developed), under non-ischemic conditions (Basal
group; n=9); under non-ischemic conditions in presence of Rp-8-Br-PET-cGMPS, an inhibitor of Protein Kinase G
(PKG) (iPKG group; 10-5M; n=7); during an ischemic insult (Isch group; stretch during ischemia, followed by reper-
fusion; n=8); during an ischemic insult in the presence of 8-Bromo-cGMP, a selective PKG agonist (Isch-cGMP group;
10-5M; n=8) and during an ischemic insult in the presence of 2,3-butanedione monoxime (BDM), an inhibitor of
actin-myosin interaction. (Isch-BDM group; 3%; n=8). Immediate and delayed responses to muscle stretch were
evaluated. Results as mean standard error (p 0.05).
RESULTS
In the Basal group, despite the immediate increase in myocardial passive tension induced by acute stretch (from
1.70.4 to 18.22.2 mN mm-2), afterwards this parameter showed a significant and time-dependent decrease
down to 8.21.1 mN mm-2 (-55%) at 60 minutes. This time-dependent decrease in myocardial stiffness is
significantly attenuated by PKG inhibition (iPKG group). In the Isch group this response was completely abolished
throughout the ischemic period. On the contrary, when the 8-Bromo-cGMP (Isch-cGMP group) was present, there
was a significant decrease in myocardial stiffness during the ischemic period. In the Isch-BDM group there was also
a significant decrease in passive tension compared to Isch group.
CONCLUSION
Normal myocardium demonstrates a significant and time-dependent decrease in myocardial stiffness in response
to acute stretch. Its attenuation by PKG inhibition and its absence under ischemic conditions (partially reverted
by cGMP) highlights the possibility of an active and energy dependent process. During ischemia, the addiction
of BDM is responsibly for partial recovery of the adaptive response to stretch, which suggests that the formation
of actin-myosin rigor bonds may also contribute to the impaired diastolic adaptation. Our results also highlight
the cGMP-PKG signaling pathway and potentially the actin-myosin interactions, as important therapeutic targets
in myocardial ischemia given their role in the ischemic diastolic dysfunctional response to acute hemodynamic
overload.
58 Abstracts
YES Meeting 2010
PS 349
UROCORTIN 2 RELAXES THE IRIS SPHINCTER MUSCLE THROUGH CRF2 AND
PKC PATHWAY
PINTO S., TAVARES-SILVA M., CARDOSO S., FONSECA S., ROCHA-SOUSA A.A., LEITE-MOREIRA A.
FACULTY OF MEDICINE, UNIVERSITY OF PORTO
AIM
The aim of our study is to observe if CRF2 is the receptor involved in the action of UCN2 in the iris sphincter muscle
and, if so, to describe the subcellular pathways responsible for this effect.
INTRODUCTION
Urocortin 2 (UCN2) is an endogenous peptide of the corticotropin releasing factor family which presents many
physiological effects at different levels in the organism. At the ocular level, UCN2 is expressed in the retinal
pigmented epithelium and has a protective effect in retinal degeneration pos-ischemia. Also in the eye, UCN2 has a
vasodilator effect in the retinal circulation, specifically, in retinal resistance arterioles. As we have described before,
in the anterior segment of the eye UCN2 promotes relaxation of the iris sphincter muscle, mediated by the produc-
tion of prostaglandins and nitric oxide. The only receptor described to be activated by UCN2 is CRF2, which regulates
several different subcellular pathways (PKC, adenilcyclase, PI3K, MAPK).
METHODS
We tested the effect of increasing concentrations of UCN2 (10e-10-10e-6M) on carbacol-precontracted (10e-6M)
rabbit iris sphincter muscles (n=9) in the presence of the CRF2 selective antagonist antisauvagine-30 (10e-6M;
n=8) and in the presence of the PKC inhibitor cleritrine (10e-5M; n=7).
RESULTS
UCN2 promoted a decrease on active tension of the precontracted iris sphincter muscles, with maximal effect at 10-
6M (12,94,1%). This effect was blunted with antisauvagine-30 (2,53,4%) and with cleritrine (4,43,4%).
CONCLUSION
The action of UCN2 on the iris sphincter muscle is mediated through the CRF2 receptor. At the subcellular level, we
verified that PKC pathway is involved in the muscular relaxation mediated by UCN2.
REFERENCES
Physiology & Immunology 59
 YES Guide YES Meeting 2010
PS 352
NEBIVOLOL REDUCES PORTAL HYPERTENSION IN RATS WITH BILE DUCT
LIGATION
ELISABETE FRANCO1, BRUNO COLAO1,3, ANA PINTO2, AURA COLAO1,2, MARIA JOO PIRES1,2
1 CECAV, CENTRO DE ESTUDOS DE CINCIA ANIMAL E VETERINRIA, UNIVERSIDADE DE TRS-OS-MONTES E ALTO DOURO,
VILA REAL, PORTUGAL; 2 DEPARTAMENTO DE CINCIAS VETERINRIAS, UNIVERSIDADE DE TRS-OS-MONTES E ALTO DOURO,
VILA REAL, PORTUGAL; 3 DEPARTAMENTO DE ZOOTECNIA, UNIVERSIDADE DE TRS-OS-MONTES E ALTO DOURO, VILA REAL,
PORTUGAL.

AIM
Evaluate the effects of Nebivolol on portal hypertension in a rat model of secondary biliary cirrhosis.
INTRODUCTION
Portal hypertension is an almost unavoidable complication of cirrhosis, and provides the driving force for most of its
complications, Because of this, there is a great interest in strategies to revert portal hypertension, since these would
prevent portal hypertension-related complications, clinical decompensation, and death (Graham e Smith, 1981). An
endothelial dysfunction, associated with decreased production of nitric oxide (NO) in the intrahepatic microcircula-
tion, has been extensively documented in cirrhotic liver, and these defects could directly contribute to the increased
intrahepatic resistance typical of portal hypertension (Clemens, 1999). Therefore, the modification of the systemic
and/or intrahepatic NO bioavailability may be a logical approach for the treatment of portal hypertension. Nebivolol
is a new [beta]1-selective adrenergic receptor antagonist that has vasodilating properties attributable to its ability
to increase NO bioavailability (Ignarro, 2004). Thus we explored the potential of nebivolol as a therapy for portal
hypertension by testing the effects of the drug on portal hypertension in a rat model of secondary biliary cirrhosis.
METHODS
Twenty-four adult male Wistar rats were subjected to either BDL or sham operation (SO) using aseptic techniques.
Three days after surgery, rats were divided into three experimental groups: SO: Sham-operated (n=8); BDL: Bile
duct ligated without treatment (n=8); BDL+N: BDL receiving nebivolol in drinking water (8 mg/Kg of body weight)
(n=8). After 4 weeks of treatment, animals were anaesthetized with sodium pentobarbital and intrasplenic pres-
sure, an indirect measure of portal pressure, was determined.
RESULTS
As expected, portal pressure was increased in the groups with BDL, when compared with SO group. The portal pres-
sure of the nebivolol treated group was significantly lower than that of the untreated group.
CONCLUSION
The results of this preliminary study suggest that nebivolol may have beneficial effects on portal hypertension.
REFERENCES

60 Abstracts Physiology & Immunology 61

 YES Guide YES Meeting 2010

Book of Abstracts
PRESENTING STUDENTS ABSTRACTS
Internal Medicine
PRESENTING STUDENTS ABSTRACTS
PS 81 LAVAGE FLUID SAMPLE PREPARATION . . . . . . . . . . . . . 77
PHARMACOECONOMICS AND GASTROENTERITIS INFECTION TREATMENT 64 PS 341
PS 82 LONGTERM CLINICAL OUTCOME OF OVERLAPPING DRUG-ELUTING STENTS -
LONG AXIS M-MODE AMPLITUDE AS A PREDICTOR OF MORTALITY IN SIROLIMUS VERSUS PACLITAXEL . . . . . . . . . . . . . . . 78
MEDICALLY TREATED PATIENTS WITH CONGESTIVE HEART FAILURE DUE TO PS 304
REDUCED SYSTOLIC FUNCTION . . . . . . . . . . . . . . . . 65 THE NACIONALISM AND THE SCIENTIFIC SELECTION OF THE IMMIGRANT IN
PS 87 BRAZIL (1930-1945) . . . . . . . . . . . . . . . . . . . 79
PARAMETARS OF RENAL FUNCTION IN ACUTE MYOCARDIAL INFARCTION 66 PS 317
PS 168 ASSESSMENT OF RELATIONSHIP BETWEEN ANTIPHOSPHOLIPID ANTIBODIES
SATISFACTION AND PERCEIVED PERFORMANCE QUALITY OF AN ELECTRONIC AND MIGRAINE A RETROSPECTIVE ANALYSIS OF PATIENTS WITH
MEDICAL RECORD (EMR) SYSTEM IN A TERTIARY HOSPITAL IN OMAN . . 67 INFLAMMATORY CONNECTIVE TISSUE DISEASES. . . . . . . . . . 80
PS 172
GEOGRAPHICAL DISTRIBUTION OF HOSPITAL ADMISSION AND FATALITY RATE
OF ISCHEMIC HEART DISEASE . . . . . . . . . . . . . . . . 68
PS 178
FAMILY HISTORY OF DIABETES: THE ROLE OF GRANDPARENTS DATA TO
IDENTIFY ADOLESCENTS AT DIABETES RISK . . . . . . . . . . . 69
PS 200
APPLICATION OF PREVENTION QUALITY INDICATORS TO PORTUGUESE DATA: A
PRELIMINARY ANALYSIS OF FINANCIAL AND MORTALITY BURDEN . . . 70
PS 254
LEVEL OF ORAL HYGIENE AMONG THE SMOKERS . . . . . . . . . 71
PS 274
M. TUBERCULOSIS INFECTION AMONG MEDICAL STUDENTS FROM THE FEDERAL
UNIVERSITY OF SO PAULO AS ASSESSED BY ELISPOT-TB AND TUBERCULIN
SKIN TEST . . . . . . . . . . . . . . . . . . . . . . . 72
PS 282
DUFFY GENOTYPE AND CLINICAL MANIFESTATIONS OF SICKLE CELL
DISEASE . . . . . . . . . . . . . . . . . . . . . . . . 73
PS 285
CLINICAL PROFILE OF HYPERTENSIVE COMPLICATIONS IN PATIENTS ADMITTED
IN GOVERNMENT STANLEY HOSPITAL, INDIA. . . . . . . . . . . 74
PS 287
SMOKING: A CROSS-SECTION STUDY . . . . . . . . . . . . . . 75
PS 309
MECONIUM ASPIRATIONS TREATMENT, A TRUE PARTICULARITY OF NEONATAL
REANIMATION AND A CHALLENGE FOR THE EMERGENCY DOCTOR . . . 76
PS 327
PROTEOMIC ANALYSIS IN PULMONARY DISEASES: BRONCHOALVEOLAR

62 Abstracts Internal Medicine 63

 YES Guide YES Meeting 2010
PS 81 PS 82
PHARMACOECONOMICS AND GASTROENTERITIS INFECTION TREATMENT LONG AXIS M-MODE AMPLITUDE AS A PREDICTOR OF MORTALITY IN MEDI-
CALLY TREATED PATIENTS WITH CONGESTIVE HEART FAILURE DUE TO RE-
1. VLAJANKOV A. , 2. HULALI M. , 3. POPOVIC D. DUCED SYSTOLIC FUNCTION
FACULTY OF MEDICINE, UNIVERSITY OF NOVI SAD
1- IBRAHIMI P., 1- JASHARI F., 1- BYTYQI D., 1- SHABANI B., 1- JASHARI H., 1- PROF.BAJRAKTARI G.
AIM
FACULTY OF MEDICINE, UNIVERSITY OF PRISHTINA
The therapeutic efficiency of the antibacterial drugs used in treating gastroenteritis has been assessed as well as the pharmaco-
economic justification of the application. Efficiency and the price of the physicians therapy of choice and the therapy according to AIM
the pharmacoeconomic guidelines in developed countries were compared. We investigated the value of echocardiogrpahic parameters on mortality in patients with chronic heart failure due to LV systolic
INTRODUCTION dysfunction.

There are no strict guidelines for treating bacterial infections in our country. Antibacterial drugs are a personal choice of the
physician, which is not always in accordance with the recommended therapy in the countries with a developed pharmacoeco-
nomic practice. This study reviews the difference in implementation of everyday gastroenteritis infection therapy and the therapy
conducted according to pharmacoeconomic guidelines.
METHODS
A study was conducted at the Infectious Disease Clinic of the Vojvodina Clinical Center which included 100 patients diagnosed for
gastroenteritis, divided into two groups. The efficiency and the costs of the therapy were established for the sample group as well
as the research group.
RESULTS
Based on the compared efficiency and the cost of gastroenteritis therapy before and after the introduction of the pharmacoeco-
nomic guidelines, it was established that there were no significant differences in the efficiency of the used therapies, while there
were significant savings in the cost.
CONCLUSION
Efficiency of the physicians antibacterial therapy of choice for gastroenteritis infections and the therapy according to the devel-
oped countries pharmacologic guidelines are equal. The treatment proved to be financially cheaper in the group of patients healed
according to the developed countries pharmacoeconomic guidelines.
REFERENCES
INTRODUCTION
The mortality of patients with heart failure (HF) remains high despite new achievements in its pharmacological treatment. The HF
due to left ventricular (LV) systolic dysfunction has higher mortality compared to HF with preserved LV systolic function.
METHODS
This study included 75 consecutive patients (age: 59.311.3 years, %female) with congestive heart failure due to reduced LV
systolic function without rheumatic valve disease. Mean follow-up was 3213 months. Routine 2-dimensional, M-mode, Doppler
and tissue Doppler parameters were performed.
RESULTS
The LV-end systolic dimension (ESD) and end diastolic dimension (EDD) were higher (6.10.9 vs. 5.21.0 cm, P<0.001, and
7.10.9 vs. 6.50.7, p=0.006, respectively), and LV shortening fraction (SF) and ejection fraction (EF) were lower (145 vs.
194 % and 2810 vs. 358, p<0.001, for both), in non-survivors compared to survivors. The lateral and right long axis
amplitudes were also lower in non-survivors (0.60.2 vs. 0.90.2 cm, P<0.001, and 1.80.4 vs. 2.40.7, p=0.008, respectively).
Multivariate analysis identified the septal M-mode long axis amplitude (OR=0.001, 95% CI 0.000-0.814; P=0.043), as the only
independent correlate of mortality.
CONCLUSION
In medically treated patients with nonrheumatic chronic heart failure due to left ventricular systolic dysfunction, the M-mode long
axis amplitude is an independent predictor of mortality.

64 Abstracts Internal Medicine 65

 YES Guide
PS 87
PARAMETARS OF RENAL FUNCTION IN ACUTE MYOCARDIAL INFARCTION
1-MILJKOVI M., 2-RAKI M.,3-PEI M.
1-FACULTY OF MEDICINE, UNIVERSITY OF NI, 2- FACULTY OF MEDICINE, UNIVERSITY OF NI, 3 - FACULTY OF MEDICINE,
UNIVERSITY OF NI
AIM
To check is the worse prognosis in patients with azotemia, and compare urea and creatinine like prediction parametars for death.
INTRODUCTION
The renal function in AIM is based on score of creatinine and urea, but clirens of creatinine is better.
METHODS
Retrospective study included 304 patients, hospitalized in Department for Cardiovascular Disease Clinical Centre Ni in 2004. There
were 60.86% men and 39.14% women. The average age was 70.5 10.2.We used the protocol of 47 parameters.
RESULTS
In the group of patients with normal urea mortality in- hospital was 9.76% , its more then 2.5 times less then in the group of
patients with higher urea 25.71%. Patients with normal creatinine had mortality 9.26%, and mortality was near 4 times higher in
the group with higher creatinine, it was 37.21% (p< 0.0001). In the group with normal urea, plasma glucose was 8.764.29, and
its less then 11.096.86 mmol/L (p =0.00047) in the group with higher urea. The group with normal creatinine had lower score of
white blood cell (WBC) (10.61 8.13) then in the group with higher creatinine (13.96 14.47), p=0.0423.
CONCLUSION
In-hospital mortality was near 2.5 times higher in patients with higher urea, and near 4 times higher in patients with higher
creatinine, what is the proof that those parametars are good predictors of death.
REFERENCES
YES Meeting 2010
PS 168
SATISFACTION AND PERCEIVED PERFORMANCE QUALITY OF AN ELECTRON-
IC MEDICAL RECORD (EMR) SYSTEM IN A TERTIARY HOSPITAL IN OMAN
1-OTHMAN M., 2-AL MUJAINI A., 3- AL FARSI Y., 4- AL MUNIRI A., 5- GANESH A.
FACULTY OF MEDICINE, SULTAN QABOOS UNIVERSITY
AIM
To evaluate the knowledge, attitude and practice of physicians at Sultan Qaboos University Hospital (SQUH) in Oman towards the
new EMR system, that was implemented in the year 2006.
INTRODUCTION
Electric Medical Record (EMR) systems are employed to improve quality of care by supporting medical decision making, promoting
use of standard guidelines, and increasing coordination between different health care providers. There is a wide variation in sat-
isfaction and improvement of care among care givers after incorporation of EMR systems. Our hospital adopted a fully integrated
EMR system for patient care and administrative purposes in June 2006. To date a formal evaluation of the system adopted by
Sultan Qaboos University Hospital (SQUH) has not been performed.
METHODS
A cross-sectional survey was conducted. We developed a questionnaire to assess the knowledge, attitude, and practice of physi-
cians towards the EMR system. The validity and reliability was assessed by conducting a pilot study on a focused group. The final
version was considered to have adequate validity and reliability by independent assessors. The questionnaire was distributed
among physicians of various specialties at SQUH. Information was analyzed using SPSS software.
RESULTS
Out of 200 distributed questionnaires, 141 (70.5%) questionnaires were received from multiple departments. Overall, only 22
physicians (15.6%) rated the current EMR system as an effective tool. The majority rated it as either fair (78; 55.3%) or poor (41;
29.1%). A substantial proportion (26.3%) of respondents considered EMR not worth the time and effort required to use it, and
31.2% reported poor satisfaction with EMR. The majority (67.4%) reported no improvement or even increasing difficulty with the
performance of work after applying EMR. The overall quality of work has either never changed (41.2%) or declined (27.4%). The
low satisfaction and underperformance was found to be associated with younger age (p= 0.032), junior designation (p= 0.041),
and low familiarity with computers (p= 0.047)
CONCLUSION
We report low satisfaction and low perceived quality of work among physicians at SQUH with the current EMR system. The main
barrier appears to be related to software that is not user-friendly leading to inappropriate and inadequate usage of the system. We
have undertaken further studies to explore practical obstacles.
ACKNOWLEDGEMENTS

66 Abstracts Internal Medicine 67

 YES Guide
PS 172
GEOGRAPHICAL DISTRIBUTION OF HOSPITAL ADMISSION AND FATALITY
RATE OF ISCHEMIC HEART DISEASE
FERREIRA-PINTO LM., TEIXEIRA-PINTO A.
FACULTY OF MEDICINE, UNIVERSITY OF PORTO
AIM
In this study we analyse the geographical distribution of hospital admissions and in-hospital fatality of patients with Ischemic
Heart Disease and examine its association with demographic and economical factors and health care resources at the regional
level.
INTRODUCTION
Regional variations in mortality from Ischemic Heart Disease (IHD) have been observed between [1,2] and within European coun-
tries [3,4]. Several factors may explain these differences such as prevalence of cardiovascular risk factors, demographic factors,
socioeconomic factors, environmental factors and unequal distribution of health care resources.
METHODS
We used data from a national database containing all hospital admissions to Portuguese State Hospitals between 2000 and 2007.
Data was analyzed at county (Concelho) level. Standardized mortality rate and standardized admissions rate were computed for
each county adjusting for age and sex. Linear regression was used for further adjustment regarding economic development (ipc)
and health resources distribution (doctors per 100.000 inhabitants and medical appointments per inhabitant per year).
RESULTS
Non adjusted mortality and hospital admission showed an inner/coastal pattern but no North/South gradient was clear. 7.5%
was the mean of hospital fatality, being Aguiar da Beira the county presenting the highest fatality rate (18.6%) and Vizela the
one which presenting the lowest mortality rate (0%). When adjusted for age, sex, economic development and health resources
distribution, there was still unexplained geographical variation of fatality and hospital admission rate. No significant differences
were found between the health care resources distribution. Counties with higher economical development had significantly higher
fatality and admissions rates.
CONCLUSION
There was marked regional variation of IHD fatality and hospital admission rates. This variation was still observed after adjusting
for age, sex, economic development and availability of health resources distribution. An interesting finding was the inexistence of
association between the health care resources and both IHD fatality and admissions rate. This type of analysis may be very helpful
for the definition of national health policies in particular to define priority regions for disease prevention and guidelines for health
care resources distribution.
REFERENCES
68 Abstracts
YES Meeting 2010
PS 178
FAMILY HISTORY OF DIABETES: THE ROLE OF GRANDPARENTS DATA TO
IDENTIFY ADOLESCENTS AT DIABETES RISK
BRANDO MR, LOPES C, RAMOS E
PORTO MEDICAL SCHOOL, UNIVERSITY OF PORTO
AIM
To evaluate the role of grandparents history of Diabetes Mellitus (DM) on defining family history (FH) of DM and to identify adoles-
cents at high risk of diabetes. We also aimed to determine the prevalence of adolescents with impaired fasting glucose (IFG) and to
evaluate its associated characteristics.
INTRODUCTION
Both Type 1 and Type 2 DM prevalences are rising all over the world among children and adolescents. As the age of onset of Type
2 DM is becoming younger and the patients life span is longer, there is a bigger propensity to the development of the multiple
chronic complications of DM. Thus, an early identification would help to deliver earlier and proper care and to minimize the conse-
quences of the disease. Family history of DM is a well known risk factor for diabetes. Thus, using FH information as a screening tool
is appealing because it is easy and inexpensive to collect in both the clinical and community setting. Previous data suggest that a
family DM history in grandparents is associated with increased risk of disease in grandchildren. However in literature, there is not
yet a consensus about the role of grandparental DM history data to the identification of adolescents in risk of diabetes.
METHODS
We evaluated 1276 population based adolescents, aged 13-year-old, from the EPITeen cohort in Porto, Portugal. Data was col-
lected by self-reported questionnaires, a clinical evaluation was performed including anthropometric measurements and a fasting
blood sample was taken. We measured fasting plasma glucose (FPG) and fasting plasma insulin. We have divided the subjects in
two groups according to FPG, using three criteria: American Diabetes Association (ADA), World Health Organization (WHO) and
our own criteria (75th percentile of FPG). Insulin resistance was measured by the Homeostasis Model (HOMA-IR) method and
its sensitivity by the Quantitative Insulin Sensitivity Check Index (QUICKI) method. We have also classified the subjects based on
Parental FH, which included only information from parents and Total FH that combined FH information from both parents and
grandparents. FH was considered positive when, at least, one of the adolescents relatives reported a diagnosis of DM.
RESULTS
The prevalence of IFG/DM was 3.7% using ADA criteria and 0.55% using WHO criteria. There was no relationship between
adolescents categories of body mass index and FPG. However, we found that overweight and obese adolescents had higher fasting
plasma insulin levels and were more insulin resistant. The prevalence of positive Parental FH was 8%, but it increased to 45% when
we used both parental and grandparental information (Total FH). After adjustment for sex and parents education, the Odds Ratio
for having FPG75th percentile considering only Parental FH was 0.91 (95%CI: 0.57-1.47) and combining data from parents and
grandparents the Odds Ratio was 1.17 (95%CI: 0.83-1.65).
CONCLUSION
Combining parental with grandparental history it lead to a 5.5 fold increase in the identification of adolescents with a positive
family history. However, there is no significant association between a FPG75th percentile and a positive family history, both
considering only data from parents and when data from grandparents was also taking in account. Follow-up studies should be
made to define if identifying adolescents based on their grandparental DM family history, would target who is more at-risk of
developing IFG or DM.
Internal Medicine 69
 YES Guide
PS 200
APPLICATION OF PREVENTION QUALITY INDICATORS TO PORTUGUESE
DATA: A PRELIMINARY ANALYSIS OF FINANCIAL AND MORTALITY BURDEN
SOARES-DOS-REIS R., FREITAS J.A.S., COSTA-PEREIRA A.
DEPARTMENT OF BIOSTATISTICS AND MEDICAL INFORMATICS, FACULTY OF MEDICINE, UNIVERSITY OF PORTO, PORTUGAL &
CINTESIS, CENTER FOR RESEARCH IN HEALTH TECHNOLOGIES AND INFORMATION SYSTEMS, UNIVERSITY OF PORTO, PORTUGAL
AIM
Apply the Agency for Healthcare Research Quality (AHRQ) Prevention Quality Indicators (PQIs) to a nationwide Portuguese
inpatient administrative database and evaluate the impact of avoidable hospitalizations (AH) on the Portuguese national health
system.
INTRODUCTION
More than 30 years ago, at Alma-Ata, Primary Health Care (PHC) was put forward as part of a comprehensive national health
system and in coordination with other sectors. Preventive care and health promotion were the main focus of this international
conference (1). However, in 2008, the World Health Organization (WHO) issued a World Health Report entitled Primary Health
Care Now More than Ever, in an effort to revive the original spirit of the Alma-Ata declaration and proposing four core principles:
universal coverage, people-centeredness, health public policies and leadership (2). This brings us to the field of healthcare evalu-
ation and indicators. In order to monitor performance of PHC systems, identify areas which need further improvement and follow
trends, the AHRQ developed PQIs. These are based on Ambulatory Care Sensitive Conditions (ACSCs), conditions for which timely
and adequate outpatient care can prevent/reduce the need for hospitalization. Thus, hospitalization rates for these conditions
reflect the quality and access to care in a particular region (3).
METHODS
PQIs, according to AHRQ criteria, were calculated using a Portuguese mainland public hospitals inpatient database comprising
circa 8 million records (2000-2007). The analysis was stratified by Health Administrative Region (ARS) influence area. Hospitaliza-
tion rates, expenses/quality ratios and mortality burden were computed and compared over time. All measures were age and sex
standardized using direct standardization.
RESULTS
: Comparison with USA data reveals lower, but comparable values of AH in Portugal. AH region rank is as follows: Alentejo < North
= Algarve < Lisbon < Centre. Proportional mortality was lowest in the North and highest in Alentejo. The cost-quality ratio was
best in the Northern ARS and worst in the Centre. The overall trend for AH rates is to increase over the 8year period. The mean
yearly cost for all AH episodes was 200 million (assessed by diagnosis related group (DRG) reimbursement values).
CONCLUSION
PQIs are applicable to Portuguese data. There are differences in AH rates, hospitalization outcomes (mortality) and resource man-
agement among Portuguese regions. Further study is needed to give insight into the true meaning of this heterogeneity, which
can be a result of true differences in PHC quality (quality of care and access to care), or reflect coding issues, socio-economical and
urbanization differences, varying prevalence of underlying risk factors and of propensity to seek care.
REFERENCES
70 Abstracts
YES Meeting 2010
PS 254
LEVEL OF ORAL HYGIENE AMONG THE SMOKERS
1-VOSTINIC V., 2-GARIC N., 3-SPASIC M.
1-FACULTY OF MEDICINE, UNIVERSITY OF NIS, 2-FACULTY OF MEDICINE, UNIVERSITY OF NIS, 3-FACULTY OF MEDICINE,
UNIVERSITY OF NIS
AIM
The aim of the present study was to determine clinical condition of gingival and periodontal tissues in smokers and non-smokers,
as well as to investigate the knowledge about the importance of maintaining oral hygiene.
INTRODUCTION
The main cause of gingivitis and periodontal disease is dental plaque, but tobacco smoke is basic risk factor for development of
chronic periodontitis. Periodontal disease is three times more common in smokers than nonsmokers, regardless of the level of oral
hygiene. However, smokers have more progressive periodontal disease than non-smokers.
METHODS
Ninety subjects were included in the study, which had been examined at Dental Clinic, Medical faculty in Nis. After the anamnesis
was taken and the diagnosis was established, two groups were formed: I group of smokers or former smokers, II group of non-
smokers. Clinical examination was included: Index of oral hygiene, Gingival and Periodontal indexes (Pl, IZK, Ikon, Gi, Ikrv, PDI i
CPITN).All of the patients filled the questionarie.
RESULTS
The examined sample consisted of 90 subjects. There was 50 (55.56%) smokers of the total number. In the group of smokers, 46.0
% were female, and 54.0 % were male. The average age of smokers was 39. 62..12. 69 lower than non-smokers aged 42. 68
13. 85. The values of all the studied parameters were higher in the group of smokers, but the plaque index, Index of supragingival
and Index of subgingival calculus were statistically higher (p<0.01). All of the examinees wash their teeth by toothbrush and
dentifrice two times per day. Out of 50 smokers, 36 ( 72.0 %) used some of the additional tools of the oral hygiene. There is higher
percent of people who use chewing gum in the smokers group ( 46.0 %).
CONCLUSION
After the results were obtained the following conclusions were found: the values of all the studied parameters were higher in
the group of smokers, but the plaque index, Index of supragingival and Index of subgingival calculus were statistically higher (p
<0.01). All examinees used the tooth paste and dentifrice. There is higher percent of people who use chewing gum in the smokers
group ( 46.0 %). The knowledge level about oral hygiene of smokers is good, but permanent education and motivation are neces-
sary.
REFERENCES
Internal Medicine 71
 YES Guide
PS 274
M. TUBERCULOSIS INFECTION AMONG MEDICAL STUDENTS FROM THE
FEDERAL UNIVERSITY OF SO PAULO AS ASSESSED BY ELISPOT-TB AND
TUBERCULIN SKIN TEST
1-MONTEIRO, L.M.C.; 2-PIMENTEL, L.G.M.; 3-YAMADA, R.K.F.; 4-LOURENATTO, A.B.; 5-VIANA, P.O.;
6-PINTO, M.I.M.
PAULISTA SCHOOL OF MEDICINE, FEDERAL UNIVERSITY OF SO PAULO
AIM
Study M. tuberculosis infection rate among medical students from the Federal University of So Paulo and to compare ELISPOT-TB
test with the tuberculin skin test (TST).
INTRODUCTION
Tuberculosis is a severe infectious disease transmitted by aerosols, caused by Mycobacterium tuberculosis. It is the second leading
cause of death worldwide. In Brazil, the incidence rate was 37.1 per 100,000 inhabitants in 2008. It is common knowledge that
healthcare professionals are more exposed to infections, mainly due to contact with patients. Currently, the main method for diag-
nosis of latent M. tuberculosis infection is the TST (tuberculin skin test). Some years ago, ELISPOT-TB was launched. It is supposed
to be more specific and sensitive than TST. In order to investigate M. tuberculosis infection rate among healthcare professionals,
our study was conducted with medical students at the Federal University of So Paulo, in So Paulo, Brazil, in different periods of
the medical course, using both TST and ELISPOT.
METHODS
Recruitment of students, who answered a questionnaire, were submitted to the TST and ELISPOT test. TST was considered positive
if equal to or greater than 5mm as assessed 72 hours after PPD administration.
RESULTS
Two hundred and fifteen medical students were included. The average age was 22.9 years. There were 49.7% female students.
Seventy-eight students were at the 1st and 2nd grade of the medical course, 96, at the 3rd and 4th grade and 41 students at the
5th and 6th grade. The majority of students belonged to socio- economic levels A and B. Twenty-seven students had no BCG scar,
none of them reported history of tuberculosis and 9 students reported previous household contact with a baciliferous tuberculosis
patient. In the hospital environment, 46 students reported contact with a patient with baciliferous tuberculosis: 28 students from
the 3rd and 4th grades and 16 students from the 5th and 6th grades. Prior history of TST testing was only reported by 35.3% of the
students. Thirteen out of the 215 students participating in the study did not return for TST reading. Among students who attended
the TST reading,90.1% had negative results. As for ELISPOT, there were 5.1% indeterminate results. Among the remaining 204,
89,7% were negative.
CONCLUSION
A progressive increase in rates of positive results in both TST and ELISPOT throughout the medical course was observed, which
was concomitant with the increase in the frequency of contact with baciliferous patients in the hospital. However, statistical
significance was only seen using the ELISPOT as a diagnostic method. There is no laboratory test for diagnosis of latent tuberculosis
infection that can be considered as a gold standard. As a result, the known contact with an infectious patient with tuberculosis
has often been used as a parameter to evaluate new tests. The rationale for that would be that the risk of tuberculosis infection
is greater the larger and more intense the contact with the bacillus. The ELISPOT proved to be a test with higher sensitivity and
specificity compared to the TST and it may be an alternative for identification of M. tuberculosis infection in populations at risk
such as healthcare professionals.
REFERENCES
72 Abstracts
YES Meeting 2010
PS 282
DUFFY GENOTYPE AND CLINICAL MANIFESTATIONS OF SICKLE CELL
DISEASE
HAYASHIDA, D.Y.; MECAB G.; NETO, F.M.; FIGUEIREDO, M.S.
ESCOLA PAULISTA DE MEDICINA, UNIVERSIDADE FEDERAL DE SO PAULO
AIM
To find out if there is association between Duffy genotype and clinical manifestations in a group of Brazilian Sickle Cell Anemia
patients.
INTRODUCTION
It is well known that Duffy antigen can modulate the function of inflammatory responses. The glycoprotein binds inflammatory
mediators that act as chemoattractant and facilitators of leukocyte adhesion, both in leukocyte and endothelium. Studies suggest
that the Duffy antigen on erythrocytes may act to prevent activation of leukocytes in the systemic circulation and to reduce the
spread of chemokines from blood to organs. Sickle cell anemia (SCA) patients are predominantly African American and the Duffy-
negative phenotype is more common in this population as this phenotype is protective against Plasmodium vivax infection. The
lack of expression of Duffy antigens has been hypothesized as associated with more severe clinical course in these patients.
METHODS
We evaluated 87 SCA patients followed by Hereditary Anemias Outpatient Clinic of the Universidade Federal de So Paulo
(UNIFESP/EPM). The median age was 30 years old (SD:10), 41% male and 59% female. Forty-eight percent of the patients
were in use of hydroxyurea, and they were similarly distributed among the Duffy phenotype groups. DNA was extracted with
standard techniques, and Duffy genotyping was done as described before with minor modifications. The patients were considered
Duffy-negative when genotyping showed two mutated GATA site (FY*B-33) or when they had one FY*B-33 associated with one
of the other mutations observed on the Duffy gene. Total Blood Count, Lactate Dehydrogenase (LDH) and microalbuminuria de-
terminations were obtained during steady state. Patient clinical data was obtained in a standard questionnaire, and the followed
manifestations were considered: Pulmonary hypertension (PH), stroke and renal function.
RESULTS
Seventy-seven of 87 (88%) patients realized Doppler echocardiography, and they were considered as having PH when tricuspid re-
gurgitant jet velocity was equal or above 2.5 m per second. Hematological, biochemical and clinical data were compared between
Duffy-negative and Duffy-positive SCA patient groups. The frequency of Duffy-negative in this population of SCA patients (25.3%)
probably reflects the characteristic racial admixture observed in Brazil. The hemoglobin level was 8.5 1.22 in Duffy-negative and
9.1 1.36 in Duffy-positive patients (p=0.04). There was difference in LDH levels between the two groups of patients: a signifi-
cantly higher LDH in Duffy-negative patients was observed (646.4 370.3 vs. 468.8 350.3; p=0.009). PH was more frequent in
Duffy-negative group (66.7% vs. 32.1%; p=0.009).
CONCLUSION
As suggested by Kato et al. (2007), SCA patients could be divided in two different (SCA) phenotypes: one associated with hemolytic
manifestations and other related to hyperviscosity and vasoocclusion. PH, priapism, leg ulcers and stroke can be linked to the
intensity of hemolysis and seem to be more related with the hemolytic phenotype. We observed a higher frequency of PH in the
group of patients with Duffy-negative phenotype, which is in agreement with the higher LDH levels observed in this group. We
believe, however, that studies with larger number of patients are necessary to clarify this question.
REFERENCES
Internal Medicine 73
 YES Guide YES Meeting 2010
PS 285 PS 287
CLINICAL PROFILE OF HYPERTENSIVE COMPLICATIONS IN PATIENTS AD- SMOKING: A CROSS-SECTION STUDY
MITTED IN GOVERNMENT STANLEY HOSPITAL, INDIA. 1- KARAKURT S.,2- NKFAM S.,3- KOCAKAYA D.,4- OLGUN TANDODU S.,5-CEYHAN B.,6-ELIKEL T.
1-MALAVIKA PRASAD, 2- DR.S.SHIVAKUMAR. 1 FACULTY OF MEDICINE, UNIVERSITY O MARMARA PULMONARY MEDICINE DEPARTMENT
1-STUDENT OF MEDICINE, GOVERNMENT STANLEY HOSPITAL,2- FACULTY OF MEDICINE,GOVERNMENT STANLEY HOSPITAL
AIM
AIM to assess smoking and smoking dependency in population
To study the target organ damages (cardiovascular, neurological and renal complications) in hypertensives and identify the
prevalent end organ damage and the enhancement effect of co-morbid risk factors on hypertension. There is scarce information INTRODUCTION
about the clinical profile of hypertensives with complications in conditions of clinical practice. With this aim, a retrospective As almost 60% of people in Turkey are cigarette smokers, we as pulmonary medicine department, take the responsibility of giving
epidemiological survey was designed. advise,encouraging and supporting people to cease smoking.in this study we wondered the rate of dependency among smokers
and to learn the number of people who need medical support to cease smoking.
INTRODUCTION
Hypertensive emergencies are referred to as target organ damage due to hypertension(HT). It is responsible for 57% of stroke METHODS
deaths and 24% of coronary heart disease(CAD) deaths. HT is the most important modifiable risk factor for end organ damage We want to investigate smoking habitues and degree of dependency in general population in different part of Istanbul. We use
and is also influenced by co-morbid risk factors like diabetes, hypercholesterolemia, smoking and alcohol. We will analyze the fagestrom scale and measurement of CO level in exhaled air and record cigarette use in pack-year.
incidence of end-organ damage and study the co-morbid risk factors.
RESULTS
METHODS
2116 people whose mean age 3712 year (man 296, woman 497) was investigated. The 67% was man, 1711 pack-year,
Patients admitted with hypertension >160/100mm/Hg(JNCVII) aged >18years were analyzed (n=100) for:Congestive Heart (man 1710, woman 1615 ). Fagestrom scale was more than 6 point in 61% of people, and more than 10 point in 12% of
Failure (CHF) and Acute Coronary Syndrome; Stroke due to infarct or hemorrhage; and renal failure. The data was obtained from people. CO level in exhaled air was higher than 10 ppm in 82% of man (3213 ppm) and 23% of woman (2212 ppm).
the case records and during the assessment period, secondary cases of HT were excluded; target organ damage from BP was evalu-
ated and any co-morbid risk factors that add to the burden were identified.We analyzed by Clinical examination;Blood Glucose and CONCLUSION
Urea;Serum Cholesterol and Creatinine;Sodium and Potassium levels;EKG;CT-Scan and Chest X-ray.
The smoking in pack-year and dependency rate was higher in the population. So, the people may need medical assistance during
RESULTS smoking cessation period.
100 cases analyzed, majority of which were men (70%) and 30% women with mean age 64.2 and 60.3 respectively. The prevalent REFERENCES
SBP was 160-169mm/Hg in 33% and DBP was 100-109mm/Hg in 38%cases evaluated. 20.3% had hypercholesterolemia, 22.8%
diabetes, 30.1% and 26.8% were current smokers and alcoholics respectively. The most common was Cardiovascular complications
detected in 57% of which CAD was 81% which includes: Myocardial Infarction in 52%, Unstable Angina in 48%, Stable Angina
in 7%; and CHF in 12% of the patients. Stroke was detected in 28%- stroke due to infarct in 57% and due to hemorrhage in 43%.
Renal failure was the least prevalent with 15% evaluated. 56% were known hypertensives of which 69.6% of patients had HT for
1-3years and 53.5% were on irregular anti-hypertensive medication.The risk thus rises progressively with increasing pressures. The
data of known hypertensives highlight that after a long asymptomatic period, persistent HT developed into complicated HT.The
roles of co-morbid risk factors in predisposition, precipitation and perpetuation of HT have been outlined. Hypercholesterolemia
in cardiac,smoking for stroke and alcohol for renal complications emerged as the major risk factors. The data also highlights that
diabetic hypertensive patients have a high risk for developing end-stage renal disease.
CONCLUSION
We not only identified the prevalent end-organ complication recorded as CAD(most common being Myocardial
Infarction)>Stroke>Renal Failure, but also outlined the importance to promote healthy lifestyle modifications, as co-morbid risk
factors are powerful predictors of future hypertension, to decrease the prevalence of HT in the general population. We observed
that a comprehensive strategy must be undertaken which must include prevention strategies, earlier detection and adequate
treatment as a relatively small reduction in BP will affect the incidence of end-organ complications on a population basis.
REFERENCES

74 Abstracts Internal Medicine 75

 YES Guide
PS 309
MECONIUM ASPIRATIONS TREATMENT, A TRUE PARTICULARITY OF NEO-
NATAL REANIMATION AND A CHALLENGE FOR THE EMERGENCY DOCTOR
SIMONA SCORUS, SIMONA BRATU
FACULTY OF GENERAL MEDICINE, UNIVERSITY OF MEDICINE AND PHARMACY, TIRGU MURES
INTRODUCTION
Meconium is the first substance eliminated in the gastro-intestinal tract in the neo-natal period. Meconium aspiration is a
frequent emergency of the neonate and a challenge for the emergency doctor, due to its different reanimation protocol, the great
incidence of late complications and mortality. This study has been made to reveal the frequency of this pathology and to underline
the importance of the emergency doctor in evaluating a neonate with meconium aspiration, making a quick diagnosis and the
necessary steps for a correct emergency treatment and an efficient reanimation, without late complications.
METHODS
The retrospective study has been done for the period between January 2006 and December 2008 on a total number of 321 neo-
nates. The datas have been collected from the air transport and special neonatal reanimation car registries, from the Emergency
Department SMURD, Tirgu-Mures, Romania. 229 patients (71,3 %) were transported with the neonatal reanimation car and the
rest, of 92 (28,7 %) with the helicopter.
RESULTS
Out of the 321 cases that have been studied, 20 (6,23%) were caused by airway obstruction, and out of these, 14 (70%) were
caused by meconium aspiration. In the same period there have been noted 177 (55,15 %) cases of breathing distress, 86 (26,8%)
of premature babies, 24 (7,5%) newborns with stroke and 14 (4,36%) suffering from hypothermia. Among the 321 cases, there
were 10 deaths (3%) out of which one was caused by meconium aspiration.
CONCLUSION
Meconium aspiration is the most frequent cause of airway obstruction in neonates and the mortality rate is approximately 10%.
Meconium aspiration is much more frequent in overdue pregnancies and the number of boys and girls affected was approximately
equal. Although the diagnosis of this pathology is relatively easy and its based on a clinical observations and a thorough patient
history analysis, the neonates resuscitation with such a pathology represents a reanimation particularity and a challenge for the
emergency doctor.
76 Abstracts
YES Meeting 2010
PS 327
PROTEOMIC ANALYSIS IN PULMONARY DISEASES: BRONCHOALVEOLAR
LAVAGE FLUID SAMPLE PREPARATION
NOGAL A. 1/5, MOLINA-PINELO S. 1 JAIME JC. 1/2, MARTIN-JUN J. 3, CARNERO A. 4, PAZ-ARES L. 1/2,
PASTOR MD. 1
1- MOLECULAR ONCOLOGY AND NEW THERAPIES GROUP, BIOMEDICINE INSTITUTE OF SEVILLE (INSTITUTO DE BIOMEDICINA
DE SEVILLA IBIS), SPAIN; 2- MEDICAL ONCOLOGY SERVICE, HOSPITAL UNIVERSITARIO VIRGEN DEL ROCO, SEVILLE, SPAIN;
3- RESPIRATORY DISEASE MEDICAL AND CHIRURGICAL UNIT, HOSPITAL UNIVERSITARIO VIRGEN DEL ROCO, SEVILLE, SPAIN
4- MOLECULAR BIOLOGY OF CANCER GROUP, BIOMEDICINE INSTITUTE OF SEVILLE (IBIS), SPAIN; 5-BIOMEDICAL SCIENCES
INSTITUTE OF ABEL SALAZAR, OPORTO, PORTUGAL
AIM
To generate a workflow that would allow for a small sample of BALF to be used in different proteomic analysis methods such as
iTRAQ and 2-D electrophoresis.
INTRODUCTION
Proteomic profiling is starting to prove helpful in several illnesses, such as pulmonary diseases. Serum, plasma and bronchoal-
veolar lavage fluid (BALF) are useful in the detection of various diseases and search for biomarkers that can be used in early
diagnosis. BALF is usually used for differential cell counting or the assessment of specific inflammatory mediators. The analysis of
BALF protein content may reveal changes in lung protein expression and secretion during the course of pulmonary diseases, such
as lung cancer. Sample preparation is a critical step in proteomic analysis of BALF. Procedures such as iTRAQ, 2-D Electrophoresis
and Mass Spectrometry require a sample with specific characteristics and BALF samples, which present low protein content with
a wide range of protein concentrations, high amounts of salts (due to the buffer instilled during flexible bronchoscopy) and lipids,
need to be carefully prepared in order to obtain consistent and reproducible results.
METHODS
Bronchoalveolar lavage fluid samples from patients with pulmonary diseases were obtained by instillation and aspiration of
40-60 ml aliquots of 0.9% sterile saline in the bronchopulmonary segment. Recovered fluid was immediately passed through a
100 m sterile nylon filter to remove mucus and centrifuged at 4C and 1800g for 10 minutes. The supernatant was collected and
frozen in 2 ml aliquots at 80C until further processing. Four to eight ml of supernatant of each sample were thawed on ice with
a protease inhibitor. In order to obtain a more concentrated BALF sample, acetone precipitation and a vacuum concentrator were
tested. Protein quantitation was assessed by the RCDC method. High abundance plasma proteins are present in BALF samples and
can mask the detection of low abundance ones, therefore the depletion of plasma proteins is necessary. Several affinity resin/
bead columns were examined: ProteoMiner (Bio-Rad), Proteoprep Immunoaffinity Albumin and IgG Depletion Kit (SIGMA) and
Albumin and IgG Depletion SpinTrap (GE Healthcare). The removal of salts and other contaminants is essential to prepare a sample
for 2-D electrophoresis and iTRAQ experiments followed by mass spectrometry. To clean the samples, a 2-D Clean-Up kit (GE
Healthcare) was used.
RESULTS
Concentrating samples by speed vac resulted in a relatively smaller protein loss than acetone precipitation, thus speed vac
concentration became the first step in our protocol. Although effective in reducing the amount of the major plasmatic proteins,
ProteoMiner columns (Bio-Rad) equalize the concentration of all the proteins present in a sample, making them disadvanta-
geous for our studies. In the present work we present the final workflow obtained from our experiments and the best choices in
each case depending on the experiment to perform. Protein quantitations (RCDC) of two samples along all steps of the protocol
were assessed to monitor protein loss. The samples, among others treated by the same procedure, were successfully submitted to
iTRAQ labelling and 2-D electrophoresis followed by MS, showing no interfering substances or contaminations.
CONCLUSION
The workflow we propose allows for limited BALF samples to be used in different proteomic analysis methods such as iTRAQ and
2-D electrophoresis.
ACKNOWLEDGEMENTS
Internal Medicine 77
 YES Guide
PS 341
LONGTERM CLINICAL OUTCOME OF OVERLAPPING DRUG-ELUTING STENTS
- SIROLIMUS VERSUS PACLITAXEL
1 - M. BIGOTTE VIEIRA, 1 - R. BAETA BAPTISTA, 2 - E. INFANTE DE OLIVEIRA, 2 - P. ALMEIDA, 2 - HM.
GABRIEL, 2 - J. DUARTE, 2 - J. MARQUES DA COSTA, 2 - P. CANAS DA SILVA, 2 - MG. LOPES
1 - UNIVERSITY OF LISBON, FACULTY OF MEDICINE, LISBON, PORTUGAL ,2 - HOSPITAL LISBON NORTH, HOSPITAL SANTA MARIA,
LISBON, PORTUGAL
AIM
Objective: Study the influence of antiproliferative drug type in longterm clinical outcome of overlapping drug eluting stents (DES) -
sirolimus eluting stent (SES) versus paclitaxel eluting stent (PES).
INTRODUCTION
Percutaneous treatment of long lesions or diffuse coronary disease frequently requires the use of overlapping drug-eluting stents
(DES). Concerns have been raised about persistent inflammation and delayed re-endothelialization at overlapping compared to
non-overlapping sites due to drug toxicity or polymer instability. Animal pathologic studies found evidence of increased inflam-
mation and delayed healing in overlapping DES sites. Clinically, overlapping DES are associated with greater late lumen loss, more
frequent angiographic restenosis and stent thrombosis. Small series reports or subset analysis of randomized clinical trials and
prospective observational studies comparing DES to bare metal stents found that the magnitude of the restenosis benefit of DES
was similar in overlapping and single stents. However, concerns were raised regarding stent thrombosis.
METHODS
Retrospective pool analysis of consecutive patients submitted to coronary stenting in one center. Define two groups of patients
treated with two overlapping DES according with type of antiproliferative drug sirolimus or paclitaxel. Minimum follow-up of
four years. Primary end-point defined as the combination of all major adverse cardiovascular events (MACE) death, myocardial
infarction, stent thrombosis, target lesion revascularization.
RESULTS
68 patients received two overlapping drug-eluting stents (SES 24; PES 44). There were no significant differences between groups
regarding major cardiovascular risk factors, left ventricular function, maximum stent length and maximum stent diameter. At 24
months, 7 patients achieved the primary end-point [SES 2 (8.3%) vs. PES 5 (11.4%), NS]. At 48 months, 13 patients achieved the
primary end-point [SES 4 (16.7%) vs. PES 9 (20.5%), NS]. Kaplan-Meyer analysis for primary end-point was not significant. There
were no significant differences between groups regarding separate MACE analysis at 24 or 48 months.
CONCLUSION
In this long-term retrospective non-randomized study, clinical outcomes of overlapping DES were not influenced by the type of
antiproliferative drug (sirolimus versus paclitaxel).
REFERENCES
78 Abstracts
YES Meeting 2010
PS 304
THE NACIONALISM AND THE SCIENTIFIC SELECTION OF THE IMMIGRANT IN
BRAZIL (1930-1945)
NICHTERWITZ F.
FACULDADE DE CINCIAS HUMANAS E HUMANIDADES - CAMPUS GUARULHOS, UNIVERSIDADE FEDERAL DE SO PAULO -
UNIFESP
AIM
This scientific research is concerned with history and eugenics in Brazil, especially its use to deal the improvement of immigrants
hereditary as different races. In Brazil this practice was headed by the federal government to avoid certain groups considered as
subversives,especially at the time of Second World War (1938-1945). The brazilian government, based on the medical reserches
of that time, was worried about the considered danger personality that this immigrants were suposed to have. This way, they
were arrested because the eugenics in Brazil was concerned with biometrics: the immigrants who were arrested were submitted
to medical examination and body measuring to prove their illness or good health: it could mean their freedom or their extradition.
After that, this historical research intent to alert the medical community to the present uses of molecular biology and the worry of
eugenics.
INTRODUCTION
Studying the brazilian history, especially between 1930 and 1945, was possible to observe that eugenics was reponsable to the
body measuring (biometrics) and the medical examinations of the imigrants who were considered dangerous in Brazil at that
time. Medical researches based the arguments of the brazilian government to avoid and persue these people until their extradition
or death. In this case, the japanese (asiatics in general) and germans were considered the most dangerous groups of immigrants in
Brazil because of their body characteristics.
METHODS
The methods in this research was concerned with read, analysis and invetigation of the documents produced in 1930 to 1945
(newspaper, federal constiutions - 1934 and 1937, and police promptuary) and its confrontation with the bibliografy choosed to
base this research.
RESULTS
Is safe to say that in Brazil, especially in 1930-1945, the imigrants were persued by the federal government based on the medical
researches in eugenics and biometrics. The groups that were considered most dangerous were not able to be free in the country:
they had to have themselves hide or communicate any displacement of their own.
CONCLUSION
In Brazil, as in other countries at 1930-1945, the medical researches and their authors were able to control and manipulate some
of the government decisions. In this case, the immigrants who were considered by the society as subversives or dangerous had
to be tested and measured by medical doctors concerned with eugenics and biometrics.These tests could prove the illness or the
good health of a person in race and body characteristics terms. After that, the considered dangerous people were not able to live in
the country or had to report themselves to the police.
REFERENCES
Internal Medicine 79
 YES Guide YES Meeting 2010
PS 317
ASSESSMENT OF RELATIONSHIP BETWEEN ANTIPHOSPHOLIPID ANTIBOD-
IES AND MIGRAINE A RETROSPECTIVE ANALYSIS OF PATIENTS WITH IN-
FLAMMATORY CONNECTIVE TISSUE DISEASES.
ION IM, NISTOR SI, DELCEA C, ILIESCU MC, TANASESCU R MD PHD.
CAROL DAVILA UNIVERSITY, FACULTY OF GENERAL MEDICINE, NEUROLOGY DEPARTMENT

INTRODUCTION
The antiphospholipid syndrome (APS) is defined by the constant presence of antiphospholipid antibodies (aPL), together with
various systemic manifestations like thrombosis and recurrent spontaneous abortions. The aim of this study was to obtain a new
insight into possible relation between aPL and migraine.
METHODS
In this retrospective analysis, we studied 428 patients with inflammatory connective tissue diseases admitted between 1998-2001
at Colentina Hospital Bucharest. APS was confirmed by the presence of laboratory markers for aPL, demonstrated by ELISAs for
antibodies against phospholipids and associated phospholipids-binding cofactor proteins and/or circulating lupus anticoagulant
(LA).
RESULTS
In the studied population, headache is not significantly associated with aPL. On the other hand, the association between migraine
and aPL is statistically significant: P=0.0186, OR=1.941. The presence of cerebral ischemia was also studied in patients with
headache and aPL. In systemic lupus erythematosus (SLE) patients, headache is significantly associated with positive titers of
aPL, and cerebral ischemic lesions are significantly encountered; P<0.0001, OR=5.860. For the SLE patients with migraine, there
is a significant association between aPL and cerebral ischemia: P=0.0055, OR=9.048, 95% CI: 1.912-42.824. The trend of the CI
suggests that the association power will increase for a larger studied group.
CONCLUSION
Although it is true that migraine and a high aPL value can both occur in patients with immune systemic disease we conclude that
cerebral ischaemic lesions associated with these symptoms clearly define a sub-group at risk. Changes in migraine characteristics
and MRI appearance of new cerebral ischemic lesions plead as strong arguments in favour of anticoagulant therapy.
REFERENCES

80 Abstracts Internal Medicine 81

 YES Guide YES Meeting 2010

Book of Abstracts
PRESENTING STUDENTS ABSTRACTS
Surgery
PRESENTING STUDENTS ABSTRACTS
PS 88 PS 339
DRUNKEN STATE IN FATALLY INJURED DRIVERS OF MOTOR VEHICLES . . 84 IN VITRO EXAMINATION OF DENTAL EROSION . . . . . . . . . . . . . 97
PS 152 PS 454
HEPCIDIN LEVELS AND CHARACTERISTICS OF IRON HOMEOSTASIS IN DIGITAL PHOTOPLETISMOGRAPHY IN THE DIAGNOSTICS OF DEEP VENAL
PREECLAMPSIA . . . . . . . . . . . . . . . . . . . . . . . . . . 85 THROMBOSIS . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
PS 186
NEW ANTIBIOTIC FILLED CAPSULES FOR THE TREATMENT OF BONE AND
INTRAMEDULLARY INFECTIONS: AN EXPERIMENTAL STUDY IN RABBITS. . 86
PS 214
LUNG FUNCTION IN INFANTS AFTER THE REPAIR OF CONGENITAL
DIAPHRAGMATIC HERNIA . . . . . . . . . . . . . . . . . . . . . . 87
PS 223
EVALUATING ANTI-CARBOHYDRATE AUTOANTIBODIES FOR COLORECTAL
CANCER SCREENING . . . . . . . . . . . . . . . . . . . . . . . . 88
PS 248
THREE-DIMENSIONAL (3D) DENTAL CASTS EVALUATION OF UNILATERAL
CLEFT LIP AND PALATE (UCLP) PATIENTS TREATED WITH ACTIVE VS. PASSIVE
PLATES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
PS 260
PRESERVATION SOLUTIONS FOR LIVER TRANSPLANTATION IN ADULTS:
CELSIOR (CS) VS CUSTODIOL (HTK). A SYSTEMATIC REVIEW AND META-
ANALYSIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
PS 289
SIGNIFICANCE OF THE FINE NEEDLE ASPIRATION CITOLOGY IN THE DIAGNOSIS
OF THIROID GLAND NODULAR LESIONS . . . . . . . . . . . . . . . . 91
PS 299
PATIENTSMATE: THE IMPLEMENTATION AND . . . . . . . . . . . . . 92
EVALUATION OF AN ONLINE PROSPECTIVE AUDIT . . . . . . . . . . . 92
SYSTEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
PS 306
ANASTOMOTIC ANEURYSM AFTER VASKULAR RECONSTRUCTION IN FEMORAL
REGION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
PS 329
THE SIGNIFICANCE OF TRANSITION OF BACTERIAL FLORA OF MAXILLARY
SINUS IN PATIENTS WITH ORO-ANTRAL COMMUNICATION . . . . . . . 94
PS 333
THE RESULTS OF THE MULTIMODAL TREATMENT IN ORAL CAVITY CANCERS 95
PS 336
PERINATAL OUTCOME IN PREGNANCIES COMPLICATED WITH
THROMBOCYTOPENIA . . . . . . . . . . . . . . . . . . . . . . . 96

82 Abstracts Surgery 83
 YES Guide YES Meeting 2010
PS 88 PS 152
DRUNKEN STATE IN FATALLY INJURED DRIVERS OF MOTOR VEHICLES HEPCIDIN LEVELS AND CHARACTERISTICS OF IRON HOMEOSTASIS IN
PREECLAMPSIA
1-DJUROVIC G., 2-RANCIC N.
1,2 - TOLDI G., 2 - STENCZER B.
FACULTY OF MEDICINE, UNIVERSITY OF BELGRADE
1 - FIRST DEPT. OF PEDIATRICS, 2 - FIRST DEPT. OF OBSTETRICS AND GYNECOLOGY, SEMMELWEIS UNIVERSITY
AIM
Analysis of frequency and other important medicolegal characteristics of drunken state in drivers of motor vehicles. AIM
We aimed to characterize hepcidin levels and their association with iron homeostasis in preeclampsia.
INTRODUCTION
Consumption of alcohol may be an important causative factor in traffic accidents, particularly in categories of drivers and INTRODUCTION
pedestrians. Preeclampsia is an inflammatory complication of pregnancy characterized by hypertension and proteinuria. This syndrome affects
5-8% of all pregnancies worldwide, and is a major risk factor for both maternal and fetal morbidity and mortality. Plasma iron
METHODS levels were found to be elevated in preeclampsia, contradicting the ongoing inflammation. The link between iron homeostasis and
We analysed autopsies performed in the Institute of Forensic Medicine Belgrade during 2008 and 2009. Data were obtained from inflammation is a recently described acute phase protein, hepcidin. The physiological role of hepcidin is to decrease plasma iron
autopsy protocols, results of toxicological examinations, and police reports about circumstances of traffic accidents. levels through the internalization and degradation of the iron transporter molecule, ferroportin. We aimed to characterize hepcidin
levels and their association with iron homeostasis in preeclampsia.
RESULTS
METHODS
Out of all 84 fatally injured drivers, drunken state was proved in 31 (36,9 %). The majority of them were males (30 or 97 %), while
only one female driver was under influence of alcohol. The most often drunken drivers were in the third life decade (10 or 32,2 We took peripheral blood samples from 30 preeclamptic (gestational age: 36,5 [24-40] weeks [median, range]) and 37 healthy
%). In the most cases the injured alcoholised drivers died immediately after the accident (83,8 %). In most of them blood alcohol pregnant women (gestational age: 36 [28-39] weeks [median, range]). Plasma hepcidin levels were measured with a modified
concentration (BAC) was higher than 2 (58,1 %), most frequently (12) BAC varied between 2,1 and 2,5 , and the highest method of mass spectrophotometry developed at our laboratory. We further determined IL-6 levels, complete blood cell count and
determined BAC was 3,85 . The majority of accidents occurred between midnight and 6 a.m. (54,8 %), mostly during working parameters describing iron homeostasis. Mann-Whitney test was used for statistical analysis.
days (77,4 %). The most frequent ways of occurring of traffic accidents were driving side away from road and strike from behind to
other vehicle on the road. RESULTS
Plasma hepcidin, IL-6, iron and ferritin levels were elevated, whereas plasma transferrin levels, total iron binding capacity and
CONCLUSION mean corpuscular hemoglobin concentrations were lower in preeclampsia compared to healthy pregnancy. No difference was
The obtained results poin out to great importance of drunken state in drivers of motor vehicles as a caustaive factor in traffic acci- revealed in other parameters investigated.
dents in our population. The potentially effective preventive measure could have been absolute prohibition of alcohol consumption
for all drivers. CONCLUSION
Plasma iron levels are elevated in spite of high hepcidin levels in preeclampsia, thus our finding might indicate a resistance to the
REFERENCES iron-decreasing action of hepcidin. This mechanism may be an important contributing factor to the pathogenesis of preeclampsia
due to the elevated generation of reactive oxygen species and the exacerbation of the ongoing inflammation. Our results raise the
notion that the need for iron supplementation is to be reconsidered in preeclamptic pregnancies, and the appropriate level of iron
intake should be set individually for preeclamptic pregnant women based on their actual iron homeostasis.
REFERENCES

84 Abstracts Surgery 85
 YES Guide
PS 186
NEW ANTIBIOTIC FILLED CAPSULES FOR THE TREATMENT OF BONE AND
INTRAMEDULLARY INFECTIONS: AN EXPERIMENTAL STUDY IN RABBITS.
1-JANCSIK V, 2-BRZSEI L, 1-KERESKAI L, 1-KOCSIS B, 1-FLP A, 1-BORSICZKY B.
1-UNIVERSITY OF PCS, FACULTY OF MEDICINE; 2-KAPOSI MR HOSPITAL, KAPOSVR
AIM
The research aimed at developing a new medical tool.
INTRODUCTION
The Department of Surgical Research and Techniques at the University Medical School of Pecs, launched a new research pro-
gramme, for testing a new capsule form of the widely-known polymethyl-metacrylate (PMMA), as a drug delivery system. The
research aimed at developing a new medical tool.
METHODS
To evaluate our system, S. Aureus (2109 CFU) was used to induce tibial osteomyelitis in 110 mixed sex New Zealand rabbits.
The follow up of the sepsis, included the monitoring of the animalsphysical state, blood test, checking body temperature and the
state of the wounds made. Infected areas were detected using radiological, microbiological, and histological methods.Six weeks
after the preparation time the implantation of the capsules was carried out. The procedure included treating all the animals with
surgical debridement, and then the randomly grouped animals underwent the following procedures: tygacillin, gentamycin,
tobramycin, amikin and clindamycin was administered on one and only surgical debridement was applied on their other legs. 7
weeks after inoculum the tibias were explanted sterilely, and sent for radiological, microbiological, hystological, and hematologi-
cal analyses.
RESULTS
The radiological score showed that the lowest and best score was observed in animals treated with amikin, and debridement. Our
histology results showed that tygacillin, and amikin reached the best results in comparison with the other groups. The microbio-
logical plating after the treatment has proven the lack of the presence of S. Aureus. The analysis of the systemic blood serum could
verify only in one case the systemic antibiotic concentration.
CONCLUSION
The current findings showed that the tested device might effectively lead to S. Aureus infection healing after surgical debridement
and immediate implantation.
YES Meeting 2010
PS 214
LUNG FUNCTION IN INFANTS AFTER THE REPAIR OF CONGENITAL DIA-
PHRAGMATIC HERNIA
1-KUKLOV P., 2-ULC J., 1-PCHA K., 3-STRAK Z., 2-SVOBODOV T., 1-NAJDAUF J., 1-RYGL M.
1 - CHARLES UNIVERSITY IN PRAGUE - 2ND FACULTY OF MEDICINE, DEPARTMENT OF PAEDIATRIC SURGERY, 2 - CHARLES UNI-
VERSITY IN PRAGUE - 2ND FACULTY OF MEDICINE, DEPARTMENT OF PAEDIATRICS, 3 - CHARLES UNIVERSITY IN PRAGUE - 3RD
FACULTY OF MEDICINE, DEPARTMENT OF NEONATOLOGY
AIM
Aim of present study is to evaluate the lung function in children after the repair of congenital diaphragmatic hernia during the first
three years of their life.
INTRODUCTION
Congenital diaphragmatic hernia (CDH) is in most cases associated with lung hypoplasia. There is a paucity of data regarding lung
growth and function in survivors during early childhood.
METHODS
Infant lung function tests (ILFT) were assessed using babybodyplethysmography (BBP), rapid thoracic compression (RTC) and tidal
breathing analysis (TBA), which allows measurements of lung volume and airway resistance in non-cooperating infants and tod-
dlers up to patients 13 kg of BW. ILFT were performed on special computer-driven system (MS PAED, Jaeger, VIASYS, Germany).
The following parameters were used for ILFT assessments: Functional residual capacity (FRCp), specific effective airway resistance
(sReff), maximum expiratory flow rate at FRC (VmaxFRC) and time to peak expiratory flow/total expiratory time (tPTEF%tE).
Frequencies of particular ILFT indices were analyzed in 18 children after the repair of the CDH of average age 18.2 months (range
11- 33 months) and average weight 10.1 kg (range 7.7 kg 13 kg). Oral chloralhydrate (80ml/kg) was used for sedation. ILFT find-
ings were related to the childs medical history and the clinical status. Statistical analysis was performed by Fisher exact test.
RESULTS
ILFT assessment by BBP was completed successfully in all (18/18), TBA in 94 % (17/18) and RTC in 89 % (16/18). ILFT revealed
lower airway obstruction in 72.2 % (13/18), upper intrathoracic airway obstruction in 50 % (9/18), and lung hyperinflation in 66.7
% (12/18). Restrictive pattern was found only in one patient (5.6 %). All measured indices of lung function were normal in only
2 infants (11 %). Lung function (mean +/- SD of z scores) were: FRCp [% pred.]: 133.8 +/- 27.4, sReff [% pred.]: 81.2 +/- 46.1,
tPTEF%tE [%]: 20.3 +/- 3.9 and VmaxFRC[ml*s-1]: 128.7 +/- 51.9. Significantly higher lung morbidity was found in children
with postnatal pulmonary hypertension (p= 0.0275). No complications occurred during or after the ILFT.
CONCLUSION
The presented lung function tests are a feasible and safe diagnostic method of establishing lung function in early childhood. The
results of this pilot study show an increased incidence of airway obstruction, rather than restrictive pattern in infants and toddlers
after the repair of the CDH. Successful detection of lung dysfunction in early childhood enables early initiation of targeted and
effective therapy.
ACKNOWLEDGEMENTS

86 Abstracts Surgery 87
 YES Guide
PS 223
EVALUATING ANTI-CARBOHYDRATE AUTOANTIBODIES FOR COLORECTAL
CANCER SCREENING
1- BLOK E., 1- LONARDI E., 1- LAMBERTZ U., 2- MESKER W., 2- TOLLENAAR R, 1- DEELDER A., 1-
WUHRER M.
1 - DEPARTMENT OF PARASITOLOGY, LUMC, THE NETHERLANDS. 2 - DEPARTMENT OF CLINICAL ONCOLOGY, LUMC, THE NETHER-
LANDS
AIM
To screen sera from a cohort of colorectal cancer patients and controls using a new designed glycan-microarray, to evaluate the
auto-antibody response against tumor associated glycans.
INTRODUCTION
Colorectal carcinomas are one of the most prevalent carcinomas with an annual mortality of 600,000 people worldwide. Early
stage diagnosis has proven to have a beneficial effect on 5-year survival. Because of their ability to induce a humoral immune
response, tumor-associated glycans can possibly be used as biomarkers in screening and diagnosis. Previously, our group has
developed an immobilization technique based on fluorescent labels that covalently bind an epoxy-activated solid surface, by
means of their amino-group. With this immobilization technique, we designed a microarray platform which we employed in the
screening of sera from a cohort of patients and controls, to evaluate the auto-antibody response.
METHODS
Protein-linked N-glycans and glycan moieties from glycolipids were enzymatically released from colorectal tumor tissues, labeled
with 2-aminobenzoic acid (AA), and fractionated by HPLC. These fractions were immobilized on epoxide-coated glass slides in
nanoliter aliquots to create microarrays.A cohort consisting of 24 controls, 17 stage 0/1 patients, 24 stage 2 patients and 22 stage
3 patients (age and sex matched) was kindly provided by the Dept of Clinical Oncology (LUMC, The Netherlands). The microarrays
were incubated with serum samples (diluted 1:50) and overlaid with secondary antibodies against human IgG and IgM.Obtained
results are currently being validated on a new cohort of 29 controls, 33 stage 0/1 patients, 39 stage 2 patients and 17 stage 3
patients.
RESULTS
The auto-antibody profile of patients and healthy controls differed significantly for numerous glycan fractions. These antibody
responses were used to create models to differentiate between colorectal cancer patients (in separate tumor stages) and healthy
controls. These models are currently tested in a validation study, which is expected to be finished by July 2010. Preliminary results
suggest a confirmation of at least 15 different glycan fractions.
CONCLUSION
Profiling of anti-glycan autoantibodies in serum of colorectal cancer patients has indicated that these profiles are suitable in
distinguishing between cancer patients and controls. Moreover, a differentiation between disease stages was observed, and these
results are currently being corroborated for use in early diagnosis. Notably, the glycan fractions on the array contained a mixture of
several glycans. In the future, the fractions with significant differences in antibody response will be further deconvoluted to create
a new generation of glycan microarrays with less heterogeneous fractions. With these arrays it will be possible to identify the most
relevant glycans involved in inducing the auto-antibody response and create a simple diagnostic test. Structural characterization of
these glycans will furthermore shed light on their suitability in glycan-based anti-cancer vaccine.
REFERENCES
88 Abstracts
YES Meeting 2010
PS 248
THREE-DIMENSIONAL (3D) DENTAL CASTS EVALUATION OF UNILATERAL
CLEFT LIP AND PALATE (UCLP) PATIENTS TREATED WITH ACTIVE VS. PAS-
SIVE PLATES
1- DE MENEZES M.* , 1-2- BRESCIANI E., 3- ZAPATA A.M.C, 3- PALACIO A.M.L., 1- SFORZA C.
1 - DIPARTIMENTO DI MORFOLOGIA UMANA E SCIENZE BIOMEDICHE CITT STUDI, UNIVERSIT DEGLI STUDI DI MILANO,
MILANO, ITALY, 2 DIPARTIMENTO DI BIOINGEGNERIA, POLITECNICO DI MILANO, MILANO, ITALY, 3 DEPARTMENT OF PEDIAT-
RIC DENTISTRY AND PREVENTIVE ORTHODONTICS, UNIVERSIDAD CES, MEDELLN, COLOMBIA
AIM
The aim of this study was to compare the three-dimensional (3D) changes of alveolar segments in UCLP children who received
active or passive pre-surgical plate treatment previous to rhinocheiloplasty.
INTRODUCTION
Cleft lip and/or palate represent the most frequent congenital malformation of the head and neck. Although the treatment of
children with cleft lip/palate has improved over the years, deficient growth of the maxilla is still common. Passive and active plates
have been used as pre-surgical orthopedic treatment in patients with Unilateral Cleft Lip and Palate (UCLP) to reduce the separa-
tion of the alveolar segments.
METHODS
Within the collaboration between the University of Milan (Italy) and the University CES of Medellin (Colombia), 32 children (15
girls and 17 boys) from Fundacin Clnica Noel - Medelln (Colombia) with UCLP were evaluated. The patients were divided in
two groups, 16 of them were treated with passive plates (PP) and 16 with active plates (AP). Impressions of the maxillary arches
were taken at three different times: A, prior to pre-surgical orthopedic treatment (mean age 9.6 days); B, before rhinocheiloplasty
(mean age 5.8 months); and C, after rhinocheiloplasty (mean age 8.5 months). Their maxillary dental casts were obtained and
digitized using a 3D stereophotogrammetric imaging system (Vectra). Three-dimensional measurements (cleft width, depth and
length) were made separately for the larger and shorter cleft segments on the digital dental cast surface between landmarks,
previously marked. To compare the palatal measurements between the two types of plates, and to compare each type of plate at
the three different time points, a two-factor ANOVA with repeated measures on one factor was performed.
RESULTS
Children treated with a PP had a significantly deeper cleft at the larger segment than the group treated with AP. No inter-group
significant differences were found for the other distances. For all distances, there was a significant effect of time within treatment
group. The anterior width of the cleft, the width of the cleft at the canine area, and the depth of the cleft at the shorter segment
had additional significant time x treatment interactions.
CONCLUSION
In conclusion, the maxillary segments of UCLP patients during the early postnatal period can respond to pre-surgical orthopedic
treatment. Treatment with AP was most effective in reducing the segments of the cleft than PP therapy.
Surgery 89
 YES Guide YES Meeting 2010
PS 260 PS 289
PRESERVATION SOLUTIONS FOR LIVER TRANSPLANTATION IN ADULTS: SIGNIFICANCE OF THE FINE NEEDLE ASPIRATION CITOLOGY IN THE DIAG-
CELSIOR (CS) VS CUSTODIOL (HTK). A SYSTEMATIC REVIEW AND META- NOSIS OF THIROID GLAND NODULAR LESIONS
ANALYSIS JASMINA JELI
GLORIA LUCA LEMA Z, MD1; EDUARDO SERNA A, MD, MSC1; JOHN J. ZULETA T, MD, MSC1,2. MEDICAL FACULTY NOVI SAD, UNIVERSITY OF NOVI SAD,
1 HOSPITAL PABLO TOBN URIBE, MEDELLN-COLOMBIA, 2 UNIVERSIDAD DE ANTIOQUIA, MEDELLN-COLOMBIA
AIM
INTRODUCTION To determine diagnostic relevance of FNAC and its role in thyroid gland diseases
: University of Wisconsin (UW) solution has been recognized as the gold standard in liver preservation, but its limitations are
becoming obvious, such as risk of biliary complications and its high cost. Alternatively, there are others solutions as Celsior (CS) and INTRODUCTION
Custodiol (HTK), which have modified some of their components to improved organ preservations. Fine Needle Aspiration Cytology (FNAC) is important diagnostic test for the evaluation of goiter, carcinoma of the thyroid and
preoperative diagnosis of Solitary Thyroid Nodule (STN). Its used as a screening method for the selection for surgery and its use
METHODS considerably lowered the number of surgeries.
We searched electronic databases, abstracts from scientific conferences, bibliographies of relevant articles and contact with
experts. We included randomized and quasi-randomized controlled trials, which compared the efficacy and safety of Celsior vs UW METHODS
and Custodiol vs UW solutions for liver transplantation in adults with their impact on primary dysfunction (PDF) including non pri- Research includes 69 randomly chosen cases of thyroid nodules that underwent thyroid surgery at the Oncology Institute of
mary function (PNF) and initial poor function (IPF), isquemic-type biliary lesions (ITBL) and patient and graft survival rate for 30, Vojvodina in May 2008./May 2009. and which had records of preoperative thyroid FNA. Biopsy was performed under ultrasound
90 and 120 days after transplant. Two authors independently screened all of abstracts and titles. Full text articles were reviewed control. Aspirated material was air-fixed, stained by May-Grnwald-Giemsa and Papanicolau method, and examined with light
if they met inclusion criteria. Any disagreement among authors, were settled by discussion with a third author until a consensus microscope. Thyroid tissue samples, attained by surgical resection, were fixed in formalin, molded in paraffin, stained with Haema-
was reached. Reasons were reported for those articles that did not meet inclusion criteria. Duplicate trials were excluded. Two toxylin and Eosin and examined with light microscope.
independent authors abstracted data and double tool entrance data from Review Manager 5 were used. We contacted some
investigators for missing data or to clarify information. Comprehensive meta-analysis version 2.0 program was used to obtain RESULTS
indirect comparison due that direct comparison about this issue was not found.
In a examined population females were significantly numerous, average age was 49. In 62, 32% patients were diagnosed by
RESULTS FNA as having benign lesions of the thyroid, 13,4% as having suspicious of benign neoplasm, in 14,49% carcinoma (11,59%) or
suspicious of malignant neoplasm. Two cases were false negative : hystopatological analysis has shown follicular and papillary
We identified randomized controlled trials (n=3) and quasi-randomized controlled trials (n=1) that had acceptable methodologi- carcinomas. Correlation test had shown statisticaly significant connection between FNAC and histopatologic findings (p < 0,001).
cal quality to pool in a meta-analysis for Celsior vs UW. Donor and recipient variables were similar in the two groups. Episodes of
PDF were numerically lower in the Celsior group (7.4%) than UW group (9.8%) but the difference was not statistically significant CONCLUSION
(RR= 0,66 IC95%:0,22-2,00). We found a Cochrans Q test p=0, 18 and I2 43 % suggesting heterogeneity. According to this, we
used random model effects method. Two randomized controlled trials (n=2) that compared Custodiol vs UW had low methodo- Results confirmig that FNAC is rapid, secure and cost-effective procedure in the diagnosis and evaluation of patients with thyroid
logical quality, for them, donor and recipient variables were similar in the two groups. Episodes of PDF were numerically lower in nodules. The main goal is to distinguish nodules that require surgery from those that dont.
the Custodiol group (3%) than UW group (8.4%) but the difference was not statistically significant (RR= 0,36 IC 95%:0,08-1,70).
The Cochrans Q test and I2 suggested homogeneity. According to this, we used fixed model effects method. Patient, grafts
survivals and presence of ITLB were similar for both comparisons, however, UW when was compared to Custodiol solution showed
an increase tender for one and three months survival period with important limitations for this outcome. Indirect comparison for
main outcome did not show differences between solutions (RR= 1.88 IC 95%: 0,57-6.16). Limitations: Although bias publication
was not possible to evidence, due to the small studies number mainly for Custodiol vs UW comparison, we cannot eliminate this
possibility. Further limitation, there was not enough powered trials to detect difference in addition to a low incidence events.
CONCLUSION
Results from this meta-analysis suggests that Celsior and Custodiol solutions are similar to UW as preservation solutions in the
clinical setting of liver transplantation. Adequately powered RCTs are required to evaluate the real effect of these solutions.Key-
words: liver transplantation, Celsior, Custodiol, University of Wisconsin solution, Primary dysfunction (PDF), Primary non function
(PNF), Initial poor function (IPF), Isquemic- Type Biliary Lesions( ITBL).

90 Abstracts Surgery 91
 YES Guide YES Meeting 2010
PS 299 PS 306
PATIENTSMATE: THE IMPLEMENTATION AND ANASTOMOTIC ANEURYSM AFTER VASKULAR RECONSTRUCTION IN FEMO-
EVALUATION OF AN ONLINE PROSPECTIVE AUDIT RAL REGION
SYSTEM DUAN POPOVI
LOH KP1, MCHUGH SM1,2, CORRIGAN MA1,2, SHEIKH A2, HENNESSY I2, LEHANE E3, HILL ADK1,2 FACULTY OF MEDICINE, UNIVERSITY OF NOVI SAD
1. DEPARTMENT OF SURGERY, ROYAL COLLEGE OF SURGEONS IN IRELAND, DUBLIN 2, IRELAND
2. DEPARTMENT OF SURGERY, BEAUMONT HOSPITAL, DUBLIN 9, IRELAND AIM
3. DEPARTMENT OF NURSING, UNIVERSITY COLLEGE CORK, CORK, IRELAND The aim of study: to determine the frequency of occurrence of anastomotic aneurysms on femoral region considering the total
number of reconstructive vascular procedures done and to determine a period of time between their initial operative treatment
INTRODUCTION ant the development of the aneurism.
The Hospital Inpatient Enquiry (HIPE) is a national programme devoted to the coding INTRODUCTION
and collection of data relating to patients admitted to hospital. Inaccuracies in patient
data coding have the potential to produce far-reaching consequences. The use of an Anastomotic aneurysms represent a type of false ( pseudo) aneurysms. Most often they appear in femoral region and are one
online clinical database updated by healthcare professionals has been shown to of the most serious complications that occur during arterial reconstruction. Most often they occur at the place where artery and
increase coding accuracy. We aimed to develop an online prospective audit system vaskular graft are joined together.
and evaluate its accuracy against the Hospital Inpatient Enquiry (HIPE) system
currently in use. METHODS
METHODS During the ten year period from 1998 to 2007, at the Vascular and Transplantation Surgery Clinic of the Faculty of Medicine in Novi
Sad 1578 patients were operated with a vascular reconstructive procedure done on the femoral region. Out of these 1578 patients,
A web-based outcome audit system Patientsmate for surgical patients was developed using an integrated database sys- 45 developed anastomotic aneurysm in the femoral region.
tem written in PHP. PHP is a dynamic web-based language embedded within HTML and relays information to and from a remote
MySQL database hosted on a secure hospital server. Coding data was collected and its accuracy compared between three different RESULTS
sources over a study period of one month Patientsmate, the HIPE system and the external standard (ES), where data was
recorded directly by a clinician from theatre logbooks, outpatient registers and medical chart review. Finally a Likert-scale based Given the total number of the prosthetic surgical procedures done on femoral region, the anastomotic aneurysm appeared in
survey was constructed to analyse the user-interface of Patientsmate. 2.85% of cases. From the 45 patients included in the research 39 are male, 6 are female (6.5:1 ratio). An average period of time
from the initial operative procedure to the development of aneurism is 4 years and 8 months with variations from 2 months to 10
RESULTS years and 5 months. At 35 (77.78%) patients there were implantated Dacron protesis, in 9 (20%) poli-tetra-fluoro-etilen (PTFE)
and in 1 (2.22%) at arteriovenous anastomosis. Trombendarterectomi were doing in 14 (31.11%) causes. When it comes to the
There were 108 inpatients identified by the ES. Of these 87 (68.5%) had been registered on the HIPE system, and 90 (83%) on general risk factors, 23 patients were smokers (51.11%), 21 (46.67%) were diagnosed with hypertension, 17 (37.78%) with
Patientsmate. The number of procedures indentified by the ES was 88 in total. Patientsmate identified 75 total procedures diabetes mellitus, 9 (20%) with hyperlipidemia.
while the HIPE system identified 60 procedures. Of these 108 patients, 56 (61%) were day cases. The HIPE system identified 41 day
cases, giving a hospital day case rate of 47.1%. The Patientsmate system identified 55 day cases giving a hospital day case rate of CONCLUSION
52%. The average length of stay (AVOL) was 1.77 days as identified by the ES. The HIPE system reported an AVOL of 1.82 days while
Patientsmate reported an AVOL of 1.4 days. With regard to usability of Patientsmate, inputting data for a single patient Anastomotic aneurysms are rare but very serious complication in prosthetic surgery. It has to be dealt with electively since urgent
took 6-7 minutes. In addition, 75% of those surveyed reported feeling comfortable with using the system once only and 100% state can lead to not only a loss of the extremity a fatal outcome. The factors which can influence the development of aneurism are
were satisfied with the layout of the online interface. many but most often a blame falls on the type of prosthetic material, the type of suturing material and endarterectomy during the
course of primary procedure.
CONCLUSION
REFERENCES
Patientsmate is user friendly and allows for increased accuracy in collection and coding of hospital patient data. Its secure,
online structure allows for safe, instant use by multiple centres. It also facilitates collection of procedure-based audit data, which is
essential for good clinical governance.

92 Abstracts Surgery 93
 YES Guide YES Meeting 2010
PS 329 PS 333
THE SIGNIFICANCE OF TRANSITION OF BACTERIAL FLORA OF MAXILLARY THE RESULTS OF THE MULTIMODAL TREATMENT IN ORAL CAVITY CANCERS
SINUS IN PATIENTS WITH ORO-ANTRAL COMMUNICATION MILULESCU AMELIA , ENESCU ELA , PROF. DR. RODICA ANGHEL
1-SPASIC M., 2-VOSTINIC V., 3-DZAMBAS J. CAROL DAVILA UNIVERSITY OF MEDICINE
FACULTY OF MEDICINE, UNIVERSITY OF NIS
AIM
AIM By analyzing different therapeutic protocols, this study aims to determine to what extent simultaneous chiemoradiotherapy can
The aim of this work is to identify aerobic and anaerobic bacterial flora and to examine whether bacterial flora of maxillary sinus assure a better control of the neoplastic disease.
change itself with oro-antral communication(OAC) appearance.
INTRODUCTION
INTRODUCTION We studied the cases of 202 patients with advanced oral cavity cancer treated at Bucharest Oncologic Institute between 2005-
It is still less known fact whether the changing of maxillary sinus bacterial flora in patients with oro-antral communication(OAC) 2008. We analyzed the distribution of the patients by sex, age, urban/rural, risk factors, the time between the onset of the disease
depends on duration of OAC. and the arrival at the oncologic department, treatments performed and their outcome.

METHODS
Bacterial flora of maxillary sinus with oro-antral communication(OAC) has been examined in period up to 72 hours, in patients
with OAC divided in two groups each group has comprised of 20 patients. In first group OAC has lasted up to 48h and in the
second group OAC has lasted 48 to72h. Identification has been done by standard microbiologic technique and by using Vitek Auto
Microbic system (Vitek AMS). Shedler-agar with the use of Gas Pak system (bioMrieux) has been used for anaerobic identification.
Material has been put into incubator for 48-72 hours and for microorganism identification there have been used Api 20A system
for anaerobic microorganism and Vitek AMS.
RESULTS
By microbiologic inspection there has been identified existence of microorganisms of I group: the most frequent aerobic bacteria
that have been isolated pertain to genus: Streptococcus Pneumonia (Streptococcus alfa hemolyticus 17.6%) and Staphylococ-
cus epidermidis (23.9%). The most frequent anaerobic bacteria that have been isolated are: Streptococcus intermedius (34.3%).
Results of II group:the most frequent aerobic bacteria that have been isolated pertain to genus: Staphylococcus epidrmidis (35.5
%) and Staphylococcus aureus(25.0%) and then Streptococcus sanguis (27.5%). The most frequent anaerobic bacteria that have
been isolated are: Streptococcus intermedius (33.0%) and bacteria that pertain to genus Peptostreptoccocus and Clostridium.
There has been identified reduction of percentage of aerobic bacteria in I group 12 (54.1%) in comparison with II group 8 (46.4%).
Percentage of anaerobs in I group was 9 (40.9%) and with time of OAC existence it grew up to 10 (57. 6%).
CONCLUSION
Existence of aerobic and anaerobic bacterial flora in patients with OAC and accession of percentage of anaerobic bacteria with time
of OAC existence show that in this group of patients there have been appeared bacteria which were dominate in chronic sinusitis
before.
REFERENCES
METHODS
The patients were divided into 5 therapeutic protocols :1. Chemotherapy followed by radiotherapy, 2.Radiotherapy followed by
chemotherapy 3. Radiotherapy as sole treatment, 4.Radiotherapy with Cisplatin sensitizer, 5.Chemoradiotherapy simultaneous
followed by chemotherapy. The criteria followed at the end of the protocol were: complete tumor response (CTR), partial tumor
response (PTR) and tumor stagnation ( TS). Also, we have observed the side effects of each protocol.
RESULTS
1. Global radiotherapy 70 Gy CTR 6,9%, PTR 36,2%, TS 56,8%; 2. Radiotherapy >70 Gy CTR 22,2%, RTP 66,6%, TS 11,2%; 3.
Radiotherapy with Cisplatin sensitizer CTR 30,3%, PTR 39,2%, TS 30,4%; 4. Simultaneous Chemotherapy-radiotherapy Cisplatin
with 5FU CTR 56%, RTP 26,8%, TS 17,2% 5. Simultaneous chemotherapy-radiotherapy Taxanes with Cisplatin CTR 71,1%, PTR
26,7%, TS 2,2%.
CONCLUSION
From the analysis of the results of the study we conclude that the therapeutic protocol based on chemoradiotherpay followed
by chemotherapy represents an efficient solution in cases of advanced oral cavity cancers. It assures a better local control of the
neoplasm after one year (56-71%), than radiotherapy (30%) as unique treatment, with a better efficacy when using taxies.The
good results of this therapeutic protocol depend also on how we prevent and remove the side effects of the treatment.

94 Abstracts Surgery 95
 YES Guide
PS 336
PERINATAL OUTCOME IN PREGNANCIES COMPLICATED WITH THROMBOCY-
TOPENIA
1 - DELCEA C., ION I.M., NISTOR S.I., ILIESCU M.C., 1,2 - ONISAI M. MD, VLADAREANU A.M. MD PHD,
BUMBEA H. MD PHD, 2 - NICOLESCU A. MD, 3 HORHOIANU V. MD PHD, 4 - CIORASCU M. MD, 1,4 -
VLADAREANU R. MD. PHD
1 - FACULTY OF MEDICINE, CAROL DAVILA UNIVERSITY OF MEDICINE AND PHARMACY BUCHAREST, 2 - UNIVERSITY EMERGENCY
HOSPITAL BUCHAREST, HEMATOLOGY DEPARTMENT, 3 - UNIVERSITY EMERGENCY HOSPITAL BUCHAREST, OBSTETRICS-GYNE-
COLOGY DEPARTMENT, 4 - UNIVERSITY EMERGENCY HOSPITAL ELIAS BUCHAREST, OBSTETRICS-GYNECOLOGY DEPARTMENT
AIM
The objective of our study is to assess the perinatal outcomes and complications of pregnancy in women presenting with thrombo-
cytopenia, stratified by the etiology of the hematologic condition.
INTRODUCTION
Thrombocytopenia is encountered in 7-8% of all pregnancies. Out of its many possible etiologies, idiopathic thrombocytopenic
purpura and preeclampsia are known to be associated with preterm delivery, low birth weight or perinatal mortality.
METHODS
We retrospectively analyzed 936 pregnant women admitted in the Obstetrics-Gynecology Department, University Emergency
Hospital Bucharest, Romania, from January 1st 2009 to June 1st 2009. 104 patients were diagnosed with thrombocytopenia. The
data was analyzed using Epi Info 3.5.1.
RESULTS
Patients with thrombocytopenia represented 11.11% of the studied population; of these, 63/104 (6.73% of total, respectively
60.57% of thrombocytopenic pregnancies) had gestational etiology, 28/104 (2.99%, respectively 26.92%) - preeclampsia, 6/104
(0.06%, respectively 5.76%) - thrombophilia and 1/104 (0.01% respectively 1.20%) - sepsis. Also 6/104 (0.06%, respectively
5.76%) patients were diagnosed with immune thrombocytopenia, but in 5 cases, therapeutic abortion was performed before 12
weeks, due to severely decreased platelet count; 1 case was lost from the study after therapeutic splenectomy during pregnancy.
Thus, ITP patients are not taken into consideration in further discussion. Platelet count in control lot was 245541.98 60593.08
versus 118975.00 33015.25 in thrombocytopenic group (p=0.00). Mean age in patients was 30.82 5.20 years versus 28.42
5.28 years (p=0.00) control group. Thrombocytopenia was classified in mild (platelets>100000/L), moderate (50000-100000/
L) and severe (<50000/L). Therapy was necessary in 33.65 % of thrombocytopenic patients (corticoids plus specific treatment
to each condition). Cesarian section was performed in 83 patients. Regarding the perinatal outcome, thrombocytopenia was a
risk factor for premature delivery (RR=3.42, p=0.00), with higher risk for severe thrombocytopenia (RR=8.69, p<0.01). Specifi-
cally, patients with thrombocytopenia due to preeclampsia had the highest rate of prematurity (RR=7.97, p=0.00). Babies of
mothers with severe thrombocytopenia were born at 33.60 3.43 weeks vs. 38.74 1.65 weeks (p=0.00) for non-complicated
pregnancies. Out of all thrombocytopenic patients, preeclampsia was more often associated with premature delivery: 35.47 3.31
weeks vs. 38.74 1.65 (p=0.00). Babies of mothers with moderate thrombocytopenia weighted 2720.00 843.66 g (p=0.03),
and those from mothers who had severe thrombocytopenia - 2047.50 938.98 g (p=0.02), vs. 3225.38 496.85 g in controls.
Preeclampsia was significantly associated with small-for-age newborns (RR=2.55, p=0.00). Babies from preeclamptic mothers
weighted 2462.05 794.54 g compared to 3225.38 496.85 g (p=0.00) in normal pregnancies, respectively 2932.37 708.91 in
thrombocytopenic pregnancies.
CONCLUSION
Thrombocytopenia in pregnancy is generally associated with premature delivery and low birth weight. The strongest association
was found between preeclampsia and both prematurity and low birth weight. Also, regardless of the etiology, the lower the
platelet count, the higher the risks for the baby, therefore we strongly recommend close surveillance of thrombocytopenic mothers
and their babies, in order to establish to best moment of intervention.
96 Abstracts
YES Meeting 2010
PS 339
IN VITRO EXAMINATION OF DENTAL EROSION
ILDIK BERZE
FACULTY OF DENTISTRY, SEMMELWEIS UNIVERSITY
AIM
Nowadays, dental erosion proves to be a more central problem than before. As the indexes used to describe erosion in clinical
practice give subjective results, they can only partially be used for scientific research. The aim of this research project was to create
a scientific protocol according to which the appearance and dynamics of dental erosion can in vitro be examined in standard
circumstances.
INTRODUCTION
The following methods are used to measure dental erosion quantitatively: surface profilometry, microcardiography and the
measurement of dissolved calcium and phosphorus. The generally used method for this latter measurement is atom absorption
spectroscopy.
METHODS
During the experiments the quantity of dissolved Ca2+ was measured by an ion selective electrode. To eliminate the uncontrol-
lable effect of acid prior the examination, impacted third molars removed by a surgical operation (n=32) were used. A standard
examination surface (2,01 mm2) was prepared on the largest crown convexity. The remaining surface was covered with acid-proof
varnish. The prepared samples were incubated in a 10 mMol (pH 2) hydrochloric acid solution. The samples were replaced into a
fresh solution every 6 hours in the first 24 hours, and then after 12 hours. The quantity of dissolved Ca2+ in the incubatory solu-
tion was measured by a Radelkis OP-274 pH-ionometer, Ca2+ selective electrode after 6, 12, 18, 24 and 36 hours.
RESULTS
Following a relatively high dissolution rate in the first two testing periods (2,6 mMol/l, SD1,4 and 5,2 mMol/l SD2,2 respec-
tivly), dissolution rate radically decreased, by one order of magnitude (0,45 mMol/l SD0,2 and 0,31 mMol/l, SD0,15) and it
slightly decreased further in the last incubating period, which lasted 2 times as long as the previous incubating periods (0,32
mMol/l SD0,13)
CONCLUSION
Dissolution rate was by one order of magnitude faster in the first 12 hours of incubation than later. The dynamics of further hard
tissue loss became more balanced and the slope of the curve decreased. This difference is probably due to the structural differences
of the enamel but further investigation of the surface morphology of hard tissue are needed. Our results partly contradict the
literature according to which the emerging tooth is covered with a highly acid-proof aprismatic enamel layer of a few ten microns
in thickness. This contradiction must be investigated thoroughly in the future.Our in vitro erosion model could reveal the dynamics
of dental erosion working from the outer enamel layer towards the dentin. In order to clarify the above described anomalies
further tests are needed.
REFERENCES
Surgery 97
 YES Guide YES Meeting 2010
PS 454
DIGITAL PHOTOPLETISMOGRAPHY IN THE DIAGNOSTICS OF DEEP VENAL
THROMBOSIS
LAZIC MILAN, DRAGAN NIKOLI
MEDICINSKI FAKULTET NOVI SAD
AIM
To establish the role of digital photopletismography (D-PPG) in the diagnosis of deep venal thrombosis (DVT) compared with the
golden standard colour-flow duplex imaging (CFDI).
INTRODUCTION
Deep venal thrombosis (DVT) of lower extremities is a usual state in general population, and if not treated, it may lead to fatal
lung embolism; thus DVT diagnosis is essential. Ascendant contrast venography (ACV), as well as Duplex echophlebography (DEF)
are golden standard methods in diagnostics of DVT of lower extremities. Photopletismography is a non-invasive optic technique
used as a simple clinical method for determination of the venal function.
METHODS
Prospective study of 100 out-patients, (103 legs) referred to the Clinic for Vascular and Transplantation Surgery in Novi Sad with
clinically suspected DVT of lower extremities. Each patient was evaluated with CFDI and D-PPG.
RESULTS
n 37 extremities DVT was established by CFDI. All patients with venousrefilling time (RT) larger than 20 s and venous pump (VP)
larger than 35 had regular CFDI. Taking RT values less than 21 s as optimum cut-off point, D-PPG reached the sensitivity of 100%,
negative-predictive value of 100%, specificity of 47% and positive-predictive value of 51%. Using VP values less than 36, as
optimum cut-off point, the sensitivity of 100%, the negative predictive value of 100%, and the specificity of 35% and positive-
predictive value of 46%.
CONCLUSION
The cited results confirm that the use of D-PPG is a useful screening tool in the diagnostic of clinically suspected DVT of lower
extremities. A positive test requires further confirmation of one of the golden standard methods in the diagnostics of DVT, while
negative test effectively excludes DVT.
REFERENCES

98 Abstracts Surgery 99
 YES Guide YES Meeting 2010

Book of Abstracts
PRESENTING STUDENTS ABSTRACTS
Neuroscience
PRESENTING STUDENTS ABSTRACTS
PS 35 PS 253
EFFECTS OF EXPANDED TERRITORY AND ENRICHED ENVIRONMENT ON MORPHOMETRIC EVALUATION OF GREEN TEA AND GREEN TEA EXTRACT
THE NEUROBEHAVIORAL DEVELOPMENT OF NEWBORN RATS EXPOSED TO EFFECTS ON RAT HIPPOCAMPAL CA3 PYRAMIDAL NEURONS DURING AGING
EXCITOTOXIC LESION . . . . . . . . . . . . . . . . . . . . . . . 102 115
PS 40 PS 255
FIRING PATTERN AND COUPLING TO HIPPOCAMPAL ACTIVITY OF VGLUT3- EFFECTS OF CHRONIC ETHANOL TREATMENT AND WITHDRAWAL ON THE
IMMUNOPOSITIVE NEURONS IN THE MEDIAN RAPHE NUCLEUS . . . . 103 EXPRESSION OF NEUROPEPTIDE Y IN THE RAT NUCLEUS ACCUMBENS: AN
PS 53 UNBIASED STEREOLOGICAL STUDY . . . . . . . . . . . . . . . . . 116
AMELIORATIVE POTENTIAL OF PITUITARY ADENYLATE CYCLASE ACTIVATING PS 283
POLYPEPTIDE IN STREPTOZOTOCIN-INDUCED TYPE I DIABETIC RETINOPATHY EFFECTS OF DIURNAL RHYTHM DISTURBANCE AND TROLOX IN ANIMAL MODEL
IN RATS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 OF CHRONIC ALCOHOL INTAKE . . . . . . . . . . . . . . . . . . . 117
PS 111 PS 284
CHARACTERISATION OF MICROGLIAL ACTIVATION IN AN ORGANOTYPIC EFFECT OF DIURNAL RHYTHM DISTURBANCE AND TROLOX ON THE OXIDATIVE
HIPPOCAMPAL MODEL OF A PRIMARY BRAIN TUMOUR . . . . . . . . 105 STRESS LEVEL IN RAT HIPPOCAMPUS . . . . . . . . . . . . . . . . 118
PS 122 PS 290
INFLUENCE OF SEX STEROIDS ON THE MORPHOLOGY OF THE PARVICELLULAR AGE DOES NOT AFFECT THE TOTAL NUMBER OF CHOLINERGIC NEURONS OF
OF THE HIPOTHALAMIC PARAVENTRICULAR NUCLEUS. . . . . . . . . 106 THE PEDUNCULOPONTINE TEGMENTAL NUCLEUS OF THE RAT: AN UNBIASED
PS 133 STEREOLOGICAL STUDY . . . . . . . . . . . . . . . . . . . . . . 119
FOETOPATHOLOGICAL AND EMBRYOLOGICAL ASPECTS OF PS 293
HOLOPROSENCEPHALY (HPE . . . . . . . . . . . . . . . . . . . . 107 INDUCED EXPRESSION OF THE NEURONAL INJURY MARKER ATF3 IN PRIMARY
PS 147 AFFERENTS DURING MONOARTHRITIS: CHARACTERIZATION OF THE NEURONAL
DETAILING THE AGE-RELATED EFFECTS OF MEMANTINE ON LONG-TERM POPULATIONS INVOLVED . . . . . . . . . . . . . . . . . . . . . 120
POTENTIATION . . . . . . . . . . . . . . . . . . . . . . . . . . 108 PS 294
PS 153 AVALIAO DA ATROFIA ENCEFLICA MEDIANTE TAC EM DOENTES COM
ENHANCED AMYGDALA AND HIPPOCAMPUS RESPONSES TO EMOTIONAL PROVVEL DEMNCIA NEURODEGENERATIVA. ESTUDO DE CASOS E
PICTURES IN UNAFFECTED SIBLINGS OF SCHIZOPHRENIA PATIENTS . . 109 CONTROLOS. . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
PS 183 PS 330
THE ROLE OF NAD(P)H OXIDASE IN THE PATHOPHYSIOLOGY OF NEUROPATHIC DESCRIPTIVE STUDY OF THE USE OF VALPROATE AND TOPIRAMATE IN
PAIN UNDER HYPERTENSIVE CONDITIONS . . . . . . . . . . . . . . 110 MIGRAINE PROPHYLAXIS IN THE MEXICAN CHILDREN HOSPITAL. . . . 122
PS 191 PS 343
EXPERIMENTAL STUDY OF THE PHARMACOLOGICAL MODULATION OF UNMASKING THE ROLE OF ALTERNATIVE SPLICING ON MACHADO-JOSEPH
NOCICEPTION IN AN ANIMAL MODEL OF OSTEOARTHRITIS . . . . . . . 111 DISEASE GENE (ATXN3): THE EXPERIENCE OF A MEDICAL STUDENT . . . 123
PS 220 PS 380
THE EVALUATION OF COMATOSE BRAIN WITH FMRI, DTI AND SPECTROSCOPY STUDY OF C-FOS EXPRESSION IN PREFRONTAL REAS AFTER A REVERSAL
112 LEARNING TASK ON RATS WITH PERIPHERAL NEUROPATHY . . . . . . 124
PS 230
BEHAVIOUR DISORDERS IN CD1 MICE CHRONICALLY INFECTED WITH
MYCOBACTERIUM AVIUM . . . . . . . . . . . . . . . . . . . . . 113
PS 235
4-D IN VIVO FETAL MRI BRAIN ATLAS . . . . . . . . . . . . . . . . 114

100 Abstracts Neuroscience 101

 YES Guide
PS 35
EFFECTS OF EXPANDED TERRITORY AND ENRICHED ENVIRONMENT ON THE
NEUROBEHAVIORAL DEVELOPMENT OF NEWBORN RATS EXPOSED TO EXCI-
TOTOXIC LESION
GABOR H., JOZSEF F., ATTILA M., ZSOFIA H., JULIAN Z., DORA R., ANDREA T., ANDREA L., PETER K.
MEDICAL SCHOOL, UNIVERSITY OF PECS, PCS, HUNGARY
AIM
We do research about enriched environment in the Department of Anatomy of the University of Pecs since 2007.
Enriched environment could be an additional therapy beside medications, so we think that our animal models and
experiments could be useful in the future for human experiments and that it will be the treatment of the future,
because enriched environment has no side effects! We checked the effects of enriched environment on different
fields (for instance retina) and we found it can counteract the negative effects of toxic agents. In this experiment we
checked the effects of enriched environment on the general neurological development of the rat pups.
INTRODUCTION
Postnatal neurobehavioral development is well reflected by the appearance of reflexes and performance in motor
tasks. This development is affected by various factors, like nutritional state, neurotrophic factors and environmental
conditions. Enriched environment increases the level of trophic factors, improves cognitive performance and has
profound effects on CNS neurochemistry. Monosodium-glutamate (MSG) is a widespread flavoring substance lead-
ing to severe morphological and functional deteriorations in the developing nervous system.
METHODS
We examined the protective effects of the expanded territory and complex enriched environment in MSG-induced
lesions in newborn rats. Postnatal development was followed by evaluating various parameters: appearance of
eye opening, incisor eruption, negative geotaxis, placing reflexes, grasp reflexes, crossed extensor reflex, sensory
reflexes. Motor coordination and behavior were assessed by rotarod, foot-fault and inclined board tests and by
open-field test.
RESULTS
We found that 2mg/g MSG administered 3 times on days 1, 5 and 9, induced impairments that could be ameliorated
by expanded territory and complex enriched environment. The body weight showed significant differences between
the control and the two enriched groups. These groups performed better also in the foot-fault and rotarod tests.
CONCLUSION
In summary, our results suggest that enriched environment can be protective against nervous system injuries in
newborn rats.
102 Abstracts
YES Meeting 2010
PS 40
FIRING PATTERN AND COUPLING TO HIPPOCAMPAL ACTIVITY OF VGLUT3-
IMMUNOPOSITIVE NEURONS IN THE MEDIAN RAPHE NUCLEUS
DOMONKOS A., HANGYA B., LENGYEL K., BORHEGYI ZS., MTYS F., FREUND T.F., VARGA V.
INSTITUTE OF EXPERIMENTAL MEDICINE, HUNGARIAN ACADEMY OF SCIENCE
AIM
Electrophysiological characterization of VGLUT3-containing neurons in the median raphe nucleus
INTRODUCTION
The median raphe nucleus (MR) is the major source of serotonergic input to the hippocampus. Nevertheless, a dis-
tinct group of MR cells projecting to various brain structures, among others to the hippocampus, has been reported
to lack serotonin (5-HT). The vesicular glutamate transporter type 3 (VGLUT3) was detected in both the 5-HT+
and 5-HT- neuron populations. In a recent study, a novel type of connection exerting fast and powerful 5-HT and
glutamate-mediated effect on hippocampal interneurons has been demonstrated. However, the physiological prop-
erties of neurons giving rise to the non-serotonergic component of the raphe-hippocampal pathway are unknown.
METHODS
Male Wistar rats were anesthetized by urethane. Hippocampal LFP was recorded monopolarly. Neurons in the MR
were recorded employing the juxtacellular technique: glass electrodes pulled from borosilicate capillaries filled with
a solution of 0.5 M NaCl and 2% Neurobiotin were connected to a DC amplifier followed by a signal conditioner.
LFP and unit signals were digitized. The fimbria was stimulated at 1 Hz bipolarly. If a neuron responded with short
latency and high success rate a collision test was carried out unless spontaneous collisions were detected. After the
recordings, neurons were attempted to be filled by the iontophoresis of Neurobiotin. After a short survival period
(10 mins to 2 hours) the rat was transcardially perfused with saline followed by the solution of 4% PFA, 15% picric
acid and 0.05% glutaraldehyde in 0.1 M phosphate buffer. Labeled neurons were identified by multiple immuno-
fluorescence. After successfull identification, the recovered neuron was processed for light microscopic examination
utilizing the peroxidase method. During data analysis the non-theta and theta segments of the recording were
separated and the neurons firing pattern during these hippocampal activity states were determined. Firing rate,
entropy of interspike interval distribution for measuring firing pattern complexity, spike triggered average to detect
coupling to hippocampal sharp waves and phase relationship of spikes to hippocampal theta were calculated.
RESULTS
We registered 9 VGLUT3+, 1 5-HT+, 2 double immunopositive and 4 double immunonegative neurons. VGLUT3+
cells exhibited complex firing pattern expressed as the entropy of interspike intervals during both hippocampal
theta (mean: 2.55) and non-theta (2.45), differing significantly from VGLUT3- cells (1.53 and 1.77). In addition, 7 of
11 VGLUT3-containing neurons changed their discharge during hippocampal slow waves. Diverging from immunon-
egative cells, 6 VGLUT3+ neurons upturned their activity in response to tail pinch, including 1 cell that was activated
during spontaneous hippocampal theta, as well.
CONCLUSION
VGLUT3+ cells are capable of modulating their targets at high temporal resolution by transmitting complex activity
patterns and may contribute to brain state transitions partly in response to intense sensory stimuli.
ACKNOWLEDGEMENTS
Neuroscience 103
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PS 53
AMELIORATIVE POTENTIAL OF PITUITARY ADENYLATE CYCLASE ACTIVAT-
ING POLYPEPTIDE IN STREPTOZOTOCIN-INDUCED TYPE I DIABETIC RETIN-
OPATHY IN RATS
ESZTER BANKI1, KATALIN CSANAKY1, KRISZTINA SZABADFI2, TAMAS ATLASZ3, PETER KISS1, DORA
REGLODI1, MONIKA GRIECS2, ALIZ SZABO4, KRISZTINA KOVACS4, GYORGY JR. SETALO5, BELA JU-
HASZ6, BALAZS VARGA6, ROBERT GABRIEL2
1-DEPARTMENT OF ANATOMY, 2-DEPARTMENT OF EXPERIMENTAL ZOOLOGY AND NEUROBIOLOGY, 3-DEPARTMENT OF SPORT-
BIOLOGY, 4-DEPARTMENT OF BIOCHEMISTRY AND MEDICAL CHEMISTRY, 5-DEPARTMENT OF MEDICAL BIOLOGY, UNIVERSITY OF
PECS; 6-DEPARTMENT OF PHARMACOLOGY, UNIVERSITY OF DEBRECEN, HUNGARY
AIM
The aim of the present study was to investigate the neuroprotective effect of pituitary adenylate cyclase activating
polypeptide (PACAP) in a model of type I diabetic retinopathy induced by streptozotocin in Wistar rats in vivo.
INTRODUCTION
Pituitary adenylate cyclase activating polypeptide (PACAP) has been shown to exert protective effects in models of
neurodegenerative diseases, cerebral ischemia and retinal degeneration. We have provided evidence that PACAP
is neuroprotective in several models of retinal degeneration in vivo. Our previous studies showed that intravitreal
injection of PACAP ameliorated the damaging effects of glutamate-treatment and retinal ischemia. Diabetic
retinopathy, which is classically defined as a microvasculopathy, is being viewed as a neurodegenerative disease of
the retina. Diabetic retinopathy is a leading cause of adult blindness. Alterations in the dopaminergic system are
thought to be among the first significant events in the development of diabetic retinopathy. In the early stages
of diabetes, the dopaminergic amacrine cells characteristically undergo degeneration. Although involvement of
neurotrophins, such as PACAP in diabetic retinopathy has not been characterized yet.
METHODS
Type I diabetes was induced by streptozotocin-injection (70 mg/kg) in Wistar rats. Blood glucose concentration
was measured before the streptozotocin-injection and weekly thereafter. Control animals were injected with equal
volume of saline. Diabetes was confirmed by assaying the glucose concentration in blood obtained from the tail vein
using GlucoTrend Blood Glucose Monitor (Roche). Rats with glucose levels >250 mg/dl were classified as diabetic.
We administered 3 times intravitreal PACAP-injection (100pmol/5l) into the right eye, and the same volume of
saline into the left eye by Hamilton-syringe. Animals were overanesthetized 4 weeks after streptozotocin-injection.
Retinas were evaluated with histologial, different kind of immunohistochemical (vertical sections and whole mount
preparations) and molecular biological methods (Western blot, RT-PCR) and functional analysis (ERG).
RESULTS
Diabetic retinopathy resulted in significant reduction and severe degeneration of dopaminergic amacrine cells in
the inner nuclear layer, as shown by the shape of their soma and their connection by thyrosine hydroxylase (TH)
immunopositivity. Morphometrical analysis showed significant reduction in the number of cells. Neuroprotective
effects of PACAP were observed in streptozotocin-induced retinal degeneration. Intraocular PACAP treatment led
to a nearly intact appearance of the soma, connections and also cell number. According to molecular biological
analyses utilizing pro-, antiapoptotic and TH primary antibody, intensity of immunostaining was altered by PACAP
treatment compared to diabetic retinas. We also made a systematic evaluation of diabetes-, and diabetes + three
times PACAP-treatment-related ERG changes in rats 4 weeks after the streptozotocin-injection.
CONCLUSION
In summary, intravitreal administration of PACAP protected retinal cells, for example, dopaminergic amacrine cells,
demonstrating its therapeutic potential in streptozotocin-induced early diabetic retinopathy.
ACKNOWLEDGEMENTS
104 Abstracts
YES Meeting 2010
PS 111
CHARACTERISATION OF MICROGLIAL ACTIVATION IN AN ORGANOTYPIC
HIPPOCAMPAL MODEL OF A PRIMARY BRAIN TUMOUR
POWELL MR, GATHERER M, JOHNSTON D, NICOLL J, BOCHE D.
CLINICAL NEUROSCIENCES DIVISION, SCHOOL OF MEDICINE, UNIVERSITY OF SOUTHAMPTON
AIM
To characterise the C6 glioma infiltration in an organotypic model. To characterise the microglial activation in this
model.
INTRODUCTION
Gliomas are group of common primary brain tumours associated with poor prognosis. There has been recent interest
in the role that microglia, the cerebral resident macrophages, have in the pathogenesis of this condition. It is known
that microglia infiltrate gliomas in significant numbers, however there is controversy as to whether they represent
a true anti tumour response mounted by the central nervous system, or whether they are recruited by the tumour
to aid its growth. There is increasing evidence for an attenuated immune response by microglia against the glioma.
Greater understanding of this interaction may eventually lead to improved therapy for this condition. Recent hip-
pocampal developments in organotypic modelling have allowed closer representation of the in vivo environment
than traditional in vitro models. Organotypic modelling provides a model which can be manipulated relatively
easily, but retains cellular architecture and function, and thus seem ideally placed to study microglia and their
interaction with gliomas.
METHODS
Rat hippocampal slices were prepared and 7 days later, fluorescent C6 glioma cells were added. At different
time-points (day 1, day 3, day 5 and day 7), the cultures were stopped and the C6 cells infiltration and the level of
cell death within the organotypic slice were analysed using a confocal microscope. Sham and control groups were
prepared in parallel. CD68 (a phagocytic marker) and MHCII (an antigen-presentation marker) immunohistochemis-
try were undertaken on fixed slices at identical time points after C6 glioma application. CD68 levels were quantified
and histological relationships were characterised for both CD68 and MHC II positive microglia.
RESULTS
The confocal analysis shows significant C6 glioma cell infiltration within the hippocampal slices over time
(p<0.001). The number of dead cells per unit volume in the organotypic culture was markedly higher in the
glioma cultures relative to the Sham cultures, and was also correlated to the depth of glioma infiltration. The CD68
quantification shows a significant increase between the different time points in the C6 group (p <0.001) but also
when compared with the sham and control groups. The morphological observation of the CD68 staining also shows
2 different microglial populations: macrophage-like morphology and stellate microglia. Histological examination of
organotypic slice after glioma addition shows significant CD68 positive microglia at the tumour margin. Microglia
migration from the hippocampal slice to within the tumour bulk was also observed. Our preliminary observation of
the MHCII expression shows a weaker staining in the C6 group when compared with the Sham group at day 5 and
day 7.
CONCLUSION
In this study, we have reproduced an in vitro model of brain tumour. Using this model, we have evidence for a sub-
acute toxic effect exerted by the glioma cells. Concurrently increased microglial phagocytic activity and downregu-
lation of the microglial antigen-presentation was observed, which may contribute to glioma growth and invasion.
However further work is needed to characterise the exact microglial contribution to tumour biology.
REFERENCES
Neuroscience 105
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PS 122
INFLUENCE OF SEX STEROIDS ON THE MORPHOLOGY OF THE PARVICELLU-
LAR OF THE HIPOTHALAMIC PARAVENTRICULAR NUCLEUS.
COELHO, S.; SILVA, S.M.
INSTITUTE OF ANATOMY, FACULTY OF MEDICINE, UNIVERSITY OF PORTO
AIM
This study aims to investigate the potential contribution of the dendritic trees for the sex-related differences found
in the volume of the medial parvocellular subdivision of the paraventricular nucleus of the hypothalamus (PVNmp),
as well as the influence that estrogens might have in determining these neuroanotomical sexual dimorphisms.
INTRODUCTION
Gonadal hormones, namely estrogens, affect a variety of reproductive and non-reproductive physiological functions
neurally regulated by numerous structures such as the PVN. The PVNmp is one of the subdivisions involved, among
other functions, in the regulation of the hypothalamic-pituitary-adrenal axis (HPA axis). Previous studies have
found that the volume of the PVNmp is larger in males than in females, although no differences were found in the
total number of its neurons. These differences in the volume may be due to variations in the length of the dendritic
trees or in the number of afferents that reach this nucleus. This last point could be explained by differences in the
concentration of estrogen receptors in PVN neurons.
METHODS
Adult male and female Wistar rats were maintained throughout the experiment under standard laboratory condi-
tions: 12-h light/dark cycle and temperature of 22 C. Solid diet and water were available ad libitum until the day
of sacrifice. Fifteen days before the end of the experiment, two groups of female rats were gonadectomized under
deep anesthesia and the remaining animals were assigned to another two groups: intact males and intact females.
Two days before the sacrifice one group of castrated female rats were subcutaneously injected with estrogen-
benzoate (EB) and the other group with sesame-oil (vehicle). After perfusion, the hipothamali were processed for
Golgi-impregnation. Approximately nine PVNmp neurons were sampled per animal and their dendritic arborizations
were traced by hand using a camera lucida attachment. The drawings were used to estimate the number and to
measure the length of the dendritic segments, as well as to evaluate the length of their terminal segments and the
spine density.
RESULTS
The total length of the dendritic arborizations and the mean length of the terminal segments were significantly
higher in intact females than in intact males. No significant differences were found between gonadectomized
groups. We also found that the spine density was consistently higher in intact females than in males, as well as in
the EB group relative to the vehicle group.
CONCLUSION
These results prove that there is a marked sexual dimorphism in the parvicellular neurons of the PVNmp, with
striking differences in the length of their dendritic arborizations and in spine density. They also reveal that the sex
differences in dendritic length seem to depend only on the organizational effects of sex steroids, whereas those in
spine density also result from the activational effects of estrogens.
ACKNOWLEDGEMENTS
106 Abstracts
YES Meeting 2010
PS 133
FOETOPATHOLOGICAL AND EMBRYOLOGICAL ASPECTS OF HOLOPROSEN-
CEPHALY (HPE
1-KITOVA.T, 2- KITOVA-JOHN.M,3- GAIGI.S,4-KAZAKOV.D, 5- SERTOV.D, 6- MENCHEV
DEPARTMENT OF ANATOMY,HISTOLOGY,EMBRYOLOGY ,UNIVERISTY OF MEDICINE PLOVDIV,2 WESSEX DEANERY SCHOOL
OF GENERAL PRACTICE U.K, 3 DEPARTMENT OF FOETOPATHOLOGY, CENTRE OF MATERNATY AND NEONATOLOGY TUNISIA,
4-5-6-UNIVERSITY OF MEDICINE PLOVDIV
AIM
1.It is foetopathological study of 15 cases of holoprosencephaly and its phenotypes. 2.Describes associated
abnormalies and especially facial ones-the impact on prenatal diagnosis and future family planning 3)Analyzes
the correlation between encephalic and facial phenotype of HPE, based on the anatomic and embryopathogenetic
origin of these structures.
INTRODUCTION
Holoprosencephaly is a congenital abnormality of the central nervous system which affects the development of
the telencephalon in the phase of prosencephalic bubble when it divides into two telencephalic ones. HPE is in the
group of abnormalities of the middle line and it is described by Jakovlev 1959 (1) as a case of cyclopia. Its name is
given by DeMayer (2) when first described. Holoprosencephaly is diverse malformation it can be isolated as well
as associated. Its frequency is 1/250 among fetuses and varies between 1/10 000 20 000 among newborns. Mat-
sunaga & Shiota 1977 (3). This study is exiting because it raises the awareness of diagnosing HPE in prenataly, as it
is lethal in severe cases but the milder cases are frequently missed and difficult to manage post nataly. Holoprosen-
cephalys etiology is heterogeneous. HPE can be monogeneous autosomal dominant according to Benke (4), as well
as autosomal recessive according to Cohen and Gorlin 1969 (5). Often it can also be X-related according to Morse et
al., 1987 (6) Hockey et al. (7). Approximately 50% of all holoprosencephaly cases are cause by cytogenetic abnor-
malities Croen (8), especially chromosome aberrations, the most common of which are trisomy 13, 18 and triploidy
according to Rasmussen et al. 1996 (9). Holoprosencephaly can be associated with malformation syndromes such as
Smith-Lemli_Opitz, Pallister, Rubinsteim-Taybi. Holoprosencephaly is associated with external environmental and
genetic factors and is still not completely elucidated.
METHODS
The method used was autopsy of fetuses with consequent macroscopic and microscopic analyses. The data of
each case was scrutinised according to multiple factors such as details of mother,delievery method, description of
phenotypical characters,autopsy findings,microscopic and histologic findings
RESULTS
All 15 cases of HPE have features of facial dysmorphy , with facial phenotypes of cyclopia, cebo- and ethmocephaly,
which is a confirmation of DeMeyers view 64 (11), that the facial abnormalities predict the brain ones, but not vice
versa (12). Cases of HPEA without facial abnormalities are very rare and are quoted as autosomal recessive Cohen
(13). According to the results of these 15 cases the serious facial dysmorphy can be connected serious brain ones.Re-
gardless of what the type the HPE is, it is always accompanied by middle facial dysmorphy (14, 15).This fetopatho-
logical analysis indicates that all facial phenotypes contain abnormalities of the external nose and the nasal cavity,
related to their form, size and level of development, reaching hypo and agenesis. Some of the nasal abnormalities
are combined with optic (16), palatal or oral (17, 18) defects
CONCLUSION
Thorough and accurate knowledge of the diversity of the correlations of brain and facial phenotype of HPE, and
knowing the place and way of action of genetic and external environmental factors in embryogenesis, could be an
important tool in the hands of the clinician by prenatal diagnosis, comprising mainly a sonographic, nuclear-mag-
netic and genetic analysis for taking proper and preventive actions for the normal development of the fetal brain.
Neuroscience 107
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PS 147
DETAILING THE AGE-RELATED EFFECTS OF MEMANTINE ON LONG-TERM
POTENTIATION
RBEN DUQUE DO VALE 1,2; LUISA VAQUEIRO LOPES 1,2; JOAQUIM ALEXANDRE RIBEIRO 1,2; ANA
MARIA SEBASTIO 1,2; ALEXANDRE VALRIO DE MENDONA 1,2 AND MARIA JOS DIGENES 1,2
1- INSTITUTE OF PHARMACOLOGY AND NEUROSCIENCES, FACULTY OF MEDICINE OF UNIVERSITY OF LISBOA; 2- NEUROCIENCES
UNIT, INSTITUTE OF MOLECULAR MEDICINE, UNIVERSITY OF LISBOA
AIM
In order to understand if memantine could restore the physiological LTP in older rats, we now studied the effect of
memantine on LTP upon ageing.
INTRODUCTION
Memantine is a drug widely used in the treatment of Alzheimers disease (AD). It has cognitive enhancing proper-
ties, in spite of being a partial antagonist of the receptors involved in memory and learning, the NMDA receptors.
However, its exact mechanism of action remains unclear. Experimental studies have been consistently performed
in young or middle aged animals, whereas cognitive deficits are usually observed in aged animals. Previously, we
observed, that long-term potentiation (LTP) - the neurophysiological basis for learning and memory, is increased
in hippocampal slices taken from aged rats (Costenla et al., 1999). This apparent paradox could be related to a
dysfunctional activation of NMDA receptors leading to a consequent dysfunctional increased LTP magnitude.
METHODS
The experiments were performed in hippocampal slices taken from three male adult Wistar rat groups: young adult
(10-15 weeks), old adult (36-40 weeks), and aged rats (80-100 weeks old). Field excitatory postsynaptic potentials
were recorded through extracellular microelectrode (4 M NaCl, 26 M-Ohm resistance) placed in the stratum
radiatum of the CA1 area. Stimulation (rectangular 0.1 msec pulses, once every 10 sec) was delivered through to
the Shaffer collateral/commissural fibers in the stratum radiatum. LTP was induced by -burst protocol consisting
of 4 trains of 100 HZ, 4 stimuli, separated by 200 ms. The effect of memantine was evaluated by comparing the LTP
magnitude of slices pre-incubated with memantine 1M for four hours at room temeprature, with the magnitude of
LTP obtained in slices in the absence of memantine.
RESULTS
When the -burst was applied to hippocampal slices taken from 10-15 week-old rats, a small LTP was observed
(196% increasing in fEPSP slope, n=5). In contrast, when the -burst was applied to hippocampal slices taken
from 36-40 or 80-100 week-old rats, a robust LTP was obtained (511% increasing in fEPSP slope, n=2 or 86 8%
increasing in fEPSP slope, n=2, respectively). The marked increase in LTP magnitude observed in hippocampal slices
from 36-40 (n=2) or 80-100 (n=2) week-old rats was reduced by memantine (1M) whereas, in slices taken from
the youngest group of rats, memantine did not significantly changed LTP magnitude (n=5, p>0.05, student t-test).
CONCLUSION
The results suggest that memantine decreases the dysfunctional increased LTP magnitude in the elder rat hip-
pocampal slices, without compromising the physiological LTP magnitude in slices taken from younger rats. This
work may open a new perspective underlying the mechanisms of action of memantine. On other hand, these results
could be a crucial step to understand the age-related changes of NMDA functioning.
REFERENCES
108 Abstracts
YES Meeting 2010
PS 153
ENHANCED AMYGDALA AND HIPPOCAMPUS RESPONSES TO EMOTIONAL
PICTURES IN UNAFFECTED SIBLINGS OF SCHIZOPHRENIA PATIENTS
1, 2-ANCA E. RAPCENCU, 2-MARIT VAN BUUREN, 2- REN S. KAHN, 2-MATTHIJS VINK
1 - MASTER NEUROSCIENCE & COGNITION, GRADUATE SCHOOL OF LIFE SCIENCES, UTRECHT UNIVERSITY, THE NETHERLANDS,
2 - RUDOLF MAGNUS INSTITUTE OF NEUROSCIENCE, DEPARTMENT OF PSYCHIATRY, UNIVERSITY MEDICAL CENTER UTRECHT,
UTRECHT, THE NETHERLANDS
AIM
The purpose of the present study was to investigate whether the neural systems underlying emotion processing are
disrupted in the unaffected siblings of schizophrenia patients.
INTRODUCTION
Schizophrenia patients display activation abnormalities in the amygdala and the hippocampus as well as behavioral
deficits during emotion processing. Similar to the patients but at an attenuated level, their unaffected siblings show
impaired performance when recognizing emotional cues (Phillips and Seidman 2008). However, to date neuroimag-
ing data in siblings is limited (Habel et al 2004, Rasetti et al 2009).
METHODS
Using functional MRI, brain activity was measured in 25 unaffected siblings of schizophrenia patients and 25
matched controls. Subjects had to view and rate the valence of pictures from the International Affective Pictures
System (IAPS). In addition to whole brain analyses, region of interest (ROI) analyses in the bilateral amygdala and
hippocampus were conducted by averaging the signal across anatomical ROIs. ROI selection was based on Anatomic
Automatic Labeling atlas (Tzourio-Mazoyer et al 2002) for the amygdala and LONI Probabilistic Brain Atlas (Shattuck
et al 2009) for the hippocampus.
RESULTS
Whole brain analyses revealed that, when viewing negative as compared to neutral pictures, siblings showed
increased activation in the left amygdala and left hippocampus, relative to the controls. Subsequent ROI analyses
proved that this aberrant activation pattern in siblings was present bilaterally in the amygdala and hippocampus,
and tended to be higher during negative than during positive pictures viewing. Neutral picture viewing did not yield
any significant group difference.
CONCLUSION
This is the first study to report hyperactivity of the amygdala and the hippocampus in unaffected siblings of schizo-
phrenia patients in response to emotional scenes. Consistent with previous reports in patients, abnormal activity
was particularly prominent during the processing of items with negative valence. Our findings suggest that familial
risk for schizophrenia may be associated with an exaggerated attribution of salience which is specific to emotional
stimuli.
REFERENCES
Neuroscience 109
 YES Guide
PS 183
THE ROLE OF NAD(P)H OXIDASE IN THE PATHOPHYSIOLOGY OF NEURO-
PATHIC PAIN UNDER HYPERTENSIVE CONDITIONS
DORA PINHO1,3, MARINA MORAIS1, DANIELA PATINHA1,3, MARTA COUTO1,3, JOS MARQUES-
LOPES1,3, ISAURA TAVARES2,3, ANTNIO ALBINO-TEIXEIRA1,3
1INSTITUTO DE FARMACOLOGIA E TERAPUTICA, 2INSTITUTO DE HISTOLOGIA E EMBRIOLOGIA, FACULDADE DE MEDICINA;
3INSTITUTO DE BIOLOGIA MOLECULAR E CELULAR, UNIVERSIDADE DO PORTO
AIM
We aimed to evaluate the role of NAD(P)H oxidase in neuropathic pain and clarify hypertension and angiotensin
mechanisms in pain modulation.
INTRODUCTION
The inverse relationship between blood pressure (BP) values and pain sensitivity is widely accepted in acute pain,
but this relationship is ill-defined in chronic pain conditions. Several mechanisms are involved, such as common
anatomical supraspinal areas of cardiovascular and pain control systems, baroreceptor mechanisms, opioids, and
others. Angiotensin II is known to play important roles both in cardiovascular regulation and endogenous pain
control. Many of its actions are elicited through activation of NAD(P)H oxidase and consequent redox dysfunction.
Furthermore, neuropathic pain is frequently accompanied by redox dysfunction, at least at the spinal level, which
might contribute to its induction and maintenance. Therefore, in this study we investigated the effects of apocynin
(a NAD(P)H-oxidase inhibitor) and angiotensin-induced hypertension on the development of spared nerve injury
(SNI) model of peripheral neuropathy in rat.
METHODS
We used 32 male Wistar Han rats weighing 280-300 g. We implanted subcutaneous minipumps for continuous
saline (Sal) or Angiotensin II (Ang, 12 g kg1 h1) infusion. Fourteen days later all animals were submitted to SNI
surgery, which consists of ligation and axotomy of tibial and common peroneal branches of sciatic nerve, while leav-
ing sural branch intact. During the next 2 weeks, apocynin (Apo, 50 mg kg1 h1 in 0.15% DMSO aqueous solution)
or vehicle (Veh, 0.15% DMSO aqueous solution) were orally administered to the rats. Four experimental groups were
formed (each experimental unit consisted of a cage with 2 rats): Sal-Veh (n=4), Sal-Apo (n=4), Ang-Veh (n=4),
Ang-Apo (n=4). Imediatelly before and 7, 14, 21 and 28 days after SNI induction, we monitored the animals blood
pressure (BP), through the non-invasive tail-cuff method, and behavioral responses, through von Frey, acetone and
pinprick tests. The results were analyzed by ANOVA followed by Student-Newman-Keuls.
RESULTS
All neuropathic animals exhibited considerable mechanical and cold allodynia (von Frey and acetone tests) and
mechanical hyperalgesia (pinprick test). Administration of apocynin decreased the developed mechanical allodynia,
both in normotensive and hypertensive rats, although it didnt change the BP levels in either group. Decreased
mechanical hyperalgesia was also observed in apocynin-treated rats, while no differences in cold allodynia could be
detected. Unexpectedly, apocynin-treated hypertensive animals showed more marked mechanical allodynia and
hyperalgesia than the apocynin-treated normotensives.
CONCLUSION
These results suggest that NAD(P)H oxidase activation is involved in the pathophysiology of SNI peripheral
neuropathy. The results concerning the effects of Ang II-induced hypertension suggest a possible inversion of the
physiological relationship between hypertension and pain (hypertension-associated hypoalgesia) in the apocynin-
treated rats. Further studies are currently ongoing to address this issue. Moreover, we plan to investigate the effects
of apocynin intrathecal administration, in order to clarify its mechanisms and site of action.
ACKNOWLEDGEMENTS
110 Abstracts
YES Meeting 2010
PS 191
EXPERIMENTAL STUDY OF THE PHARMACOLOGICAL MODULATION OF NOCI-
CEPTION IN AN ANIMAL MODEL OF OSTEOARTHRITIS
MENDONA M.
INSTITUTE OF HISTOLOGY AND EMBRYOLOGY, FACULTY OF MEDICINE, UNIVERSITY OF PORTO
AIM
By addressing the question of how analgesic drugs administration affect noxious-stimuli evoked behavioral
response we try to better evaluate the usefulness of the CatWalk and Knee-Bend test as clinically relevant tools for
pain evaluation in an animal model of OA.
INTRODUCTION
Pain in osteoarthritis (OA) is characterized by being present at rest but typically worsening with weight bearing and
movement of the affected joint. Despite the high prevalence of pain associated with OA, it remains the least studied
feature of this pathology, with the pharmacological control of OA-associated chronic pain being far from optimal.
Previous results from our group have shown that the Knee-Bend and CatWalk tests are clinically relevant and useful
to assess movement-induced nociception in the mono-iodoacetate (MIA) model of OA.
METHODS
OA was induced by intra-articular injection of 2 mg MIA in the left knee joint of adult male Wistar rats. Control
animals were treated with saline. We investigated the effect of the administration of the opioid morphine (6 mg/
Kg, subcutaneous), the non-steroidal anti-inflammatory drug (NSAID) diclofenac (30 mg/Kg per os) and the local
anaesthetic lidocaine (5 mg, 10% solution, intra-articular) in the nociceptive behavior assessed by those two tests in
control and OA animals. The effect of drug administration on nociceptive behavior was evaluated at days 3, 20 and
31 post MIA administration.
RESULTS
The three drugs significantly reduced the Knee Bend score at all time points, although the diclofenac effect at day
20 was much less pronounced, with no effect at day 31. On the CatWalk test, all drugs showed an increase on the
ipsilateral paw intensity at day 3, but at day 20 only morphine showed to be significantly effective.
CONCLUSION
These results further validate the use of the Knee-Bend and CatWalk tests in the evaluation of movement-induced
nociception, showing their potential usefulness in future studies of the analgesic efficacy of drugs in OA treatment.
REFERENCES
Neuroscience 111
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PS 220
THE EVALUATION OF COMATOSE BRAIN WITH FMRI, DTI AND SPECTROS-
COPY
ANDREA HORVTH
FACULTY OF MEDICINE, UNIVERSITY OF PCS
AIM
Objective evaluation of residual brain function and structure in severely brain-damaged patients
INTRODUCTION
The accurate and reliable assessment of cognitive functions and their structural background in comatose patients
can strongly support an appropriate management and promotes the prediction of future outcomes. However, the
investigation of the level and content of cognitive processing with standard clinical methods is limited. In this recent
study, we introduce a multimodal approach for an objective evaluation of residual brain function and structure in
severely brain-damaged patients.
METHODS
8 comatose patients (6 traumatic, 2 hypoxic injury, all GCS 8) were examined on a 3 T Siemens Trio scanner with
a standard 12 channel head coil. Functional MRI (fMRI), diffusion tensor imaging (DTI), and in several cases, proton
spectroscopy was performed besides the standard routine anatomical scans. Routine anatomical scans comprised
of T1, T2-weighted, FLAIR (Fluid Attenuated Inversion Recovery) and diffusion weighted (DWI) images. To examine
the residual sensory functions, we used standard passive fMRI paradigms: somatosensory, auditory and visual
stimulation in block design setting (alternating active and passive phases). In order to evaluate the integrity of the
neuronal pathways, diffusion tensor imaging (DTI) was also performed. In several cases multivoxel proton spectros-
copy was used to visualize the metabolic profile of the brain. To visualize all measurable metabolites short echo time
was used. The data processing was performed on Siemens Leonardo workstation, and on a separate post processing
computer using MATLAB, Nordic ICE, LC Model and FSL software.
RESULTS
In patients with hypoxic brain injury diffuse morphological changes were visible on both anatomical and DTI scans.
Spectroscopy showed diffuse severe neuronal loss. Brain activity for passive stimuli was detectable by only one of
these cases. In patients with traumatic brain injury different and heterogeneous parenchymal lesions were detected
by routine imaging. In contrast to the hypoxic brain injured patients fMRI revealed some degree of brain activity in
all of the patients. These activities correlated well with the structural damages revealed by DTI. In one case even a
high level of executive cognitive function was detected.
CONCLUSION
While fMRI makes it possible to detect the brain activity of a severely brain damaged patient, DTI provides specific
information about the integrity of the neuronal pathway structures (besides routine imaging), and spectroscopy
reveals its metabolic profile. The combination of these three techniques provides a more complete and objective
method to assess and map the state of the unconscious brain.
112 Abstracts
YES Meeting 2010
PS 230
BEHAVIOUR DISORDERS IN CD1 MICE CHRONICALLY INFECTED WITH MY-
COBACTERIUM AVIUM
BRANCO C., QUINTAS F., MONTEIRO S., CORREIA-NEVES M., ROQUE S.
LIFE AND HEALTH SCIENCES RESEARCH INSTITUTE, HEALTH AND SCIENCES SCHOOL, UNIVERSITY OF MINHO, CAMPUS GUALTAR,
BRAGA, PORTUGAL
AIM
We aimed, in this study, to investigate if M. avium chronic infection causes mood disorders using a mouse model.
INTRODUCTION
A reciprocal interaction between immune and central nervous systems mediated mainly by cytokines (both pro
and anti-inflammatory) has been associated with mood disorders, in particular, depression. It has been shown that
immune-based therapy against cancer or chronic C hepatitis therapy is associated to depression. Furthermore, acute
injection of LPS or chronic administration of pro-inflammatory cytokines in animal models induces the depressive-
like behavior. Depression is then a chronic disease and infections are often chronic but little is known about the
interaction between them. Knowing that mycobacterial infections, such as with Mycobacterium avium infection,
result in prolonged immune system activation. We aimed, in this study, to investigate if M avium chronic infection
causes mood disorders using a mouse model.
METHODS
To reach this objective we submitted M. avium infected CD1 female mice to a set of behavior tests, at the 4th and
13th week after intravenous infection, to assess anxiety and depressive-like behavior as well as spacial reference
memory.
RESULTS
In the forced-swimming test, infected females displayed a significant (p<0,05) decrease of the immobility time
at week 4 but not at week 13 indicating that infected mice have a reduced depressive-like behavior at 4 weeks
post-infection returning to normal behavior in this test latter-4. In contrast, mice spent more time in the center of
open-field arena at week 13, but not at 4th week of infection, suggesting that at latter time points upon infection
mice display a decrease in the anxious-like behavior; however this result was not confirmed by their performance in
the elevated-plus maze test. Assessment of the locomotor and exploratory behaviors in open-field test showed no
differences between the two groups. In Barnes-maze test that evaluates spacial reference memory, infected females
showed a significant (p<0,001)better performance in the test at week 13 post-infection.
CONCLUSION
These data demonstrated that chronic M. avium infection is associated with alterations in behavior resulting in de-
creased anxious and depressive-like phenotypes in CD1 infected female mice as well as to an improve in its cognitive
performance. We will further analyze neurogenesis in these animals to understand if the improvement in cognition
and in depressive-like behavior is a result of neurogenesis changes.
Neuroscience 113
 YES Guide
PS 235
4-D IN VIVO FETAL MRI BRAIN ATLAS
1,2-BOUYSSI-KOBAR M., 2,3-CLOUCHOUX C., 3-GUIZARD N., 2,3-LIMPEROPOULOS C.
1- ENGINEERING SCHOOL OF LUMINY, UNIVERSITY OF THE MEDITERRANEAN AIX-MARSEILLE 2, FRANCE 2- MONTREAL CHIL-
DREN HOSPITAL, MCGILL UNIVERSITY, MONTREAL, CANADA 3- MONTREAL NEUROLOGICAL INSTITUTE, MCGILL UNIVERSITY,
MONTREAL, CANADA
AIM
The aim of this study was to develop an in-vivo 4-D atlas of the fetal brain from a cohort of healthy fetuses that
underwent magnetic resonance imaging (MRI) studies between 25-35 weeks gestational age (GA).
INTRODUCTION
The recent successful application of advanced MRI techniques to the high-risk fetus is providing an unprecedented
opportunity to study in utero brain growth [1].Brain atlases have been shown to be essential neuroimaging tools
[2], used both to capture the average normal brain architecture and for diagnostic purposes. Moreover, age specific
atlases are now emerging in children, which delineate critical developmental changes [3]. In the fetus, static tem-
plates have only recently been created [4,5] however no study to date has developed a dynamic 4-D fetal brain atlas
that captures the rapid and dynamic maturational processes that occur over the second and third trimester.
METHODS
We used MR images of 44 healthy non-sedated fetuses from 25 to 35 weeks GA with 1x1x2 mm resolution. Data
were acquired using a 1.5T scanner and single-shot fast spin echo T2-weighted sequences in sagittal, coronal, and
axial planes. Preprocessing stages included denoising, brain masking, uniformity and motion correction [6]. The
first challenge was to obtain a perfect alignment of all images using iterative rigid transformations (3 translations,
3 rotations), in order to preserve subject size and anatomical variability. We then performed iteratively non-linear
registration averaging with a Kernel-based smoothing method in order to obtain average shape and intensity as a
function of time [7]. Kernel regression enabled us to compute the similarities between fetal GA (weeks) and age-
dependent templates resulting in age specific averaging processes.
RESULTS
The linear registration converged after 4 iterations, and all images were then aligned in the same space. The number
of non-linear registrations varied depending on the GA of the cohort. Specifically, the process converged after
4 iterations on average for templates derived from younger and smoother fetal brains. However, older subjects
demonstrated greater anatomical complexity and required at least 6 iterations before reaching convergence. The
resulting 4-D high resolution atlas accurately delineated the evolving average shape and intensity of fetal cerebral
structures (e.g., cortical grey matter, white matter) over a critical period of brain development.
CONCLUSION
We report for the first time the development of a 4-D fetal MRI brain atlas from in vivo second and third trimester
MRI data. Such an atlas will provide an essential reference tool and offer a comprehensive and quantitative
approach for the study of healthy and high-risk fetal brain development, and ultimately improve the sensitivity,
specificity and prognostic utility of fetal MRI.
REFERENCES
114 Abstracts
YES Meeting 2010
PS 253
MORPHOMETRIC EVALUATION OF GREEN TEA AND GREEN TEA EXTRACT EF-
FECTS ON RAT HIPPOCAMPAL CA3 PYRAMIDAL NEURONS DURING AGING
RODRIGUES J., ASSUNO M.
DEPARTMENT OF ANATOMY, FACULTY OF MEDICINE, UNIVERSITY OF PORTO, ALAMEDA PROF. HERNNI MONTEIRO, 4200-319
PORTO, PORTUGAL.
AIM
In this study we intended to analyze the effects of prolonged consumption of catechins present in large amounts
in green tea and green tea extract on morphologic alterations induced by aging in rat hippocampal CA3 pyramidal
cells.
INTRODUCTION
Aging is associated with biochemical alterations related to oxidative stress that cause progressive cellular damage.
The central nervous system, and particularly the hippocampal formation are very vulnerable to these degenerative
events resulting in deleterious morphological modifications in several neuronal organelles such as mitochondria and
lysosomes and increased lipofuscin accumulation in neuronal cytoplasm. Oxidative injury markedly compromises
hippocampal formation functions affecting learning and memory. As catechins display, among several others,
strong anti-oxidative and anti-inflammatory properties, we intended to investigate if prolonged consumption of
catechin-rich beverages such as green tea or green tea extract could exert neuronal protective effects during aging
in the rat hippocampal formation.
METHODS
Ten male Wistar rats aged 12 months were treated with green tea (GT-19M; n = 5) or green tea extract solution rich
in catechins (GTE-19M; n = 5) as the only liquid source until 19 months of age. Both groups of rats were compared
with control groups of 19 month-old (C-19M; n = 5) or 12 month-old (C-12M; n = 5) to provide baseline data. Using
unbiased stereological methodology in electron microscopy photographs of hippocampal CA3 pyramidal cells, the
volume of lipofuscin granules, mitochondria, lysosomes and multivesicular bodies per neuron was quantified. The
same neurons were also analyzed to estimate the number of pores in the nuclear membrane.
RESULTS
The volume of lipofuscin granules was increased significantly from C-12M to C-19M rats. Interestingly, cytoplasmic
lipofuscin content was reduced by treatment with either green tea or green tea extract when compared with
age-matched controls, to levels similar to those found in young controls. The same pattern of variation was evident
in lysosomal volume. Furthermore, aging was associated with a significant increase in mitochondrial volume, but
not in the number of mitochondria. However, neither of the treatments was able to impede the age-associated
volumetric change in mitochondria. Finally, no differences were detected in the volume of multivesicular bodies or
in the number of pores in the nuclear membrane when all groups were compared.
CONCLUSION
Our results show a remarkable protection of age-associated morphological changes on hippocampal CA3 pyramidal
neurons. Previously demonstrated anti-oxidant and cell signaling-regulating properties of green tea polyphenols
are likely to be involved. In our opinion, further studies are warranted to unravel the modes of action of these com-
pounds and to ascertain the impact of the effects herein demonstrated on functional physiological and pathological
processes involving the hippocampal formation.
ACKNOWLEDGEMENTS
Neuroscience 115
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PS 255
EFFECTS OF CHRONIC ETHANOL TREATMENT AND WITHDRAWAL ON THE
EXPRESSION OF NEUROPEPTIDE Y IN THE RAT NUCLEUS ACCUMBENS: AN
UNBIASED STEREOLOGICAL STUDY
J. R. NEVES, P. A. PEREIRA
INSTITUTE OF ANATOMY, CENTER OF EXPERIMENTAL MORPHOLOGY, FACULTY OF MEDICINE, UNIVERSITY OF PORTO, PORTUGAL
AIM
We examined the effects of chronic ethanol treatment (CET) and withdrawal (W) in the total number of neuropep-
tide Y-immunoreactive (NPY-ir) neurons in the nucleus accumbens (NAc) of rats. It was further investigated if the
administration of nerve growth factor (NGF) would interfere with the content of NPY in the NAc.
INTRODUCTION
The NAc is known for its role in mediating the reinforcing effects of drugs of abuse. Neurons producing NPY are
abundant in the CNS, most notably in the limbic structures. Interestingly, the NAc is one of the brain regions with
highest concentrations of NPY. This neuropeptide displays a pivotal role in anxiety, stress, rewarding processes, and
drug abuse. It may influence ethanol intake by regulating basal levels of anxiety, and by modulating the sedative
effects of ethanol and/or its rewarding properties. In rodents, CET and W alter NPY levels in several brain areas. It
has also been demonstrated in our Institute that the number of NPY-ir neurons of the NAc is reduced in aged rats
and that the subsequent treatment with NGF restores these age-induced changes. NGF belongs to the neurotrophin
family and is essential for neuronal survival under normal conditions. In addition, this neurotrophin, whose levels
are decreased during CET and W, plays a key role in NPY expression.
METHODS
A total 20 Wistar male rats were used. Food and water were available ad libitum until rats were 2-month old. Then,
rats were assigned to control (n=4), young ethanol-treated (n=12) and old ethanol-treated (n=4) groups. Young
ethanol-treated rats received an aqueous ethanol solution as their only available liquid source for 6 months starting
at 2 months of age. At 8 months of age, 8 rats were smoothly shifted from ethanol treatment to water intake during
further 2 months. Four of these rats (W group) received no further treatment whereas the remaining (n=4; W+NGF
group) were submitted to surgery 12 days before the end of the W period for implantation of osmotic minipumps
that allowed intraventricular delivery of NGF. The old group of CET rats was treated as young one, but from 18 to
24 months of age. At the end of the experiments, rats were perfused transcardially. The brains were removed,
coded and serially sectioned in the coronal plane. From each brain, four adjacent series of sections were separately
collected in phosphate-buffered saline. One of these was immunostained for NPY and other was Nissl-stained. The
total number of NPY-ir neurons was estimated by using unbiased stereological methods.
RESULTS
We found no significant effect of CET and W on the total number of NAc NPY-ir neurons of young rats. On contrary, in
alcohol-fed aged rats the expression of NPY was reduced to about 33% of the levels in controls. Our data also show
that NGF treatment significantly increases the expression of NPY in the NAc of W rats treated with NGF relative to
control and W rats.
CONCLUSION
These results demonstrate that age interfere with the effects of CET in the expression of NPY in the rat NAc. They also
corroborate earlier findings showing that NGF regulates the phenotype of NPY-ir neurons. This study might help to
explain the still cryptic role of NPY in alcohol intake, dependence and withdrawal. In addition, it provides hints to
develop new therapies for neuropsychiatric disorders in which the NPY-ergic neurotransmitter system and/or NGF
levels might be disturbed.
ACKNOWLEDGEMENTS
116 Abstracts
YES Meeting 2010
PS 283
EFFECTS OF DIURNAL RHYTHM DISTURBANCE AND TROLOX IN ANIMAL
MODEL OF CHRONIC ALCOHOL INTAKE
STAMATIOS-THEODOROS CHATZOPOULOS
DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY, MEDICAL UNIVERSITY OF SOFIA
AIM
To investigate the effects of Diurnal Rhythm Disturbance (DRD) and Trolox (T) on the spontaneous alcohol consump-
tion in the chronic alcohol intake animal model.
INTRODUCTION
Animal models of chronic alcohol intake are very important from human ethic aspects. In the present investigation,
a model of spontaneous chronic alcohol consumption (A) was developed, with adaptation period of week. The
effects of Diurnal Rhythm Disturbance (DRD) and Trolox (T) on the spontaneous alcohol consumption in this model,
were estimated.
METHODS
Thirty rats (11010 g) were exposed to different living regimes for a period of 3 weeks and were divided in groups
of 5 rats each. The groups were: control group (C), a control group which where administered Trolox (CT), a group
exposed in DRD (D), a group exposed in DRD and administered Trolox(DT), a group provided with alcohol and
exposed to DRD (AD) and a group exposed to DRD, provided with alcohol and administered with Trolox (ADT). C and
CT groups lived at 12/12 hrs light/dark cycle. Groups D, DT, AD and ADT were exposed to 24 hrs light until the end of
the experiment. The adaptation to ethanol was achieved for 7 days, by providing the animals with water solution of
sugar and ethanol, the concentrations varying from 5% to 0 for the sugar and from 0 to 10% for ethanol. At the first
5 days, the animals received 5 g/kg ethanol per os. From the last two days to the end of the experiment, the animals
did not receive alcohol per os anymore. Groups AD and ADT, and A were provided with 10% ethanol only during
the night shift period, while having choice between 10% ethanol and tab water during the day shift period of
the experiment. All other groups received water only, ad libitum. Groups CT, DT and ADT received 200mg/kg Trolox
p.o. every day. All animals were fed with standard rodent chow ad libitum. The spontaneously used alcohol (SUA)
was calculated as grams/kg BW. The statistical significance of relative differences among alcohol intake of different
groups was estimated using INSTAT program package.
RESULTS
During adaptation, the group ADT used spontaneously more ethanol in comparison to groups AD, A, and AT. The
only statistical difference observed was ADT>AD. After the adaptation period, the following statistically significant
differenced among spontaneously consumed alcohol were: AD>>A, AT<A, and ADT>AD.
CONCLUSION
During adaptation, DRD and T did not effect significantly the spontaneous consumption of ethanol, while Trolox lead
to increase the alcohol intake of rats with disturbed diurnal rhythm. After one week of adaptation, the combination
of A with DRD lead to an urge of the rats to drink less alcohol, as this was the only stress factor they can diminish
by themselves. Administration with Trolox decreased the spontaneous alcohol consumption of rats living at normal
light/dark cycle, while increased this parameter for animals living at DRD. Application of antioxidant might be not
always beneficial, if applied to patients with chronic alcohol intake, experiencing DRD.
ACKNOWLEDGEMENTS
Neuroscience 117
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PS 284
EFFECT OF DIURNAL RHYTHM DISTURBANCE AND TROLOX ON THE OXIDA-
TIVE STRESS LEVEL IN RAT HIPPOCAMPUS
LIOUNTMILA ASTASIDI
DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY, MEDICAL UNIVERSITY OF SOFIA
AIM
To investigate the effect of diurnal rhythm disturbance and Trolox on the Oxidative Stress level in Rat hippocampus.
INTRODUCTION
Long lasting OS due to Diurnal rhythm disturbance (DRD) may help to initiate a large variety of pathological condi-
tions and OS- related diseases. Trolox is a soluble form of Vitamin E, which shows very good antioxidant effect.
The effect of Trolox on the OS due to DRD is not yet investigated. The OS evaluation in the hippocampus is useful,
because, hippocampus is an important structure and any damage on it may lead to serious pathological conditions.
The aim of this research is to investigate the effect of Trolox on the OS level in hippocampus of rats exposed to DRD
in vivo. The MDA formation was used as a marker of the OS damage, while the MTT transformation to formazan
indicated the free-radicals formation (FRF) in the hippocampus.
METHODS
20 male Wistar Albino rats (11010 g each) were separated in four groups. The control groups (C, CT) lived at
normal light/dark cycle (12/12 hrs), while two groups (DRD, DRDT) were exposed to a constant light, for a period
of three weeks. The groups CT and DRDT received everyday 200mg/kg Trolox (p.o.). All animals were provided with
fresh food and water ad libitum, and kept at room temperature (221C). At the end of the third week they were
decapitated under anesthesia (ether).The hippocampus was dissected and stored at -81C. The OS was monitored by
measuring the levels of two markers Malondialdehyde (MDA), and conversion of MTT to MTT-formazan due to FRF
in presence of hippocampus homogenate. The amounts of proteins in the hippocampus were determined. Then, the
amounts of MDA and MTT-formazan formed in presence of hippocampus homogenate, for 5 min., were determined
from the Absorbances at 245 and 576 nm for MDA and MTT-formazan, respectively. Data were calculated in moles
per mg. protein, and presented as percentage of these in the Control group.
RESULTS
DRD dramatically increased the formation of free radicals (FR) and MDA in rat hippocampus. The effect of DRD
on both OS markers was: C<DRDT<DRD, this on MDA being much weaker. The use of Trolox resulted in a strong
decrease of FR and substantial decrease of the MDA formation. The relative decrease of OS in the hippocampus due
to Trolox was 30% to 40%.
CONCLUSION
Our results suggested that diurnal rhythm disturbance increased both FRF and oxidative stress damage in the
hippocampus of rat. Application of Trolox (200 mg/kg, p.o.) exhibited a significant in vivo antioxidant effect in the
hippocampus of rat exposed to DRD.
ACKNOWLEDGEMENTS
118 Abstracts
YES Meeting 2010
PS 290
AGE DOES NOT AFFECT THE TOTAL NUMBER OF CHOLINERGIC NEURONS OF
THE PEDUNCULOPONTINE TEGMENTAL NUCLEUS OF THE RAT: AN UNBI-
ASED STEREOLOGICAL STUDY
GOMES, ANA CATARINA; PEREIRA, PEDRO
FACULTY OF MEDICINE, UNIVERSITY OF PORTO
AIM
Since there is not much data about the putative effects of normal aging in cholinergic neurons in the mesopontine
tegmentum, we investigated whether there were any changes in the total number of choline acetyltransferase-
immunoreactive (ChAT-ir) neurons in the Pedunculopontine Tegmental nucleus (PPTg) of aged male Wistar rats.
INTRODUCTION
The mammalian brain cholinergic system is involved in several paramount functions such as memory, attention,
emotion, locomotion and sleep. Cholinergic neurons form two major anatomically distinct nuclear groups, one
located in the basal forebrain (BF) and the other in the brainstem. The latter is mainly confined to the mesopontine
tegmentum, in particular to the pedunculopontine (PPTg) and laterodorsal tegmental (LDT) nuclei. The PPTg is a
morphologically and neurochemically heterogeneous nucleus and its cholinergic projections have been typically
associated with the ascending reticular activating system (ARAS). It must be emphasized that, in addition to
the cholinergic neurons, the nucleus comprises other neuronal populations, namely glutamatergic and gamma
aminobutiric neurons. However, despite the abundant information regarding the BF nuclei, the study of the
pontomesencephalic nuclei has been neglected thus far. Indeed, there is considerable evidence showing that the BF
cholinergic neurons undergo atrophic or degenerative changes with age but it is not well established how brainstem
cholinergic neurons react to this very same condition.
METHODS
The study was carried out in a total of 5 adult male Wistar rats, 6 months old, and 5 aged male Wistar rats, 24
months old, all of which were maintained throughout the experiment under standard laboratory conditions. At the
end of the experimental period, animals were deeply anesthetized with pentobarbital and perfused transcardi-
ally. The brainstems were removed, coded and serially sectioned in the coronal plane. From each brainstem, three
adjacent series of sections were separately collected in phosphate-buffered saline. One of these series of sections
was immunostained for ChAT. The total number of ChAT-ir neurons was estimated on blind-coded slides by using the
optical fractionator.
RESULTS
The PPTg of adult male rats contains a mean of 2013 ChAT-ir neurons, whereas in the old rats the mean number is
1885. Statistical analysis revealed no age-related changes in the total number of PPTg cholinergic neurons [F(1,8) =
0.596392, P = 0.46216].
CONCLUSION
We found no significant differences in the number of ChAT-ir neurons in the PPTg of aged rats, which differs from
what has been reported to happen in the BF cholinergic nuclei. Therefore, the brainstem neurons seem to be more
resistant to age-associated degenerative changes than those in the BF. According to literature, the lower vulner-
ability may be partially explained by the higher expression of mechanisms protecting against oxidative stress,
probably the major aggression inflicted on biological systems during aging. Our study also shows that in spite of
the deluge of research conducted on the brainstem cholinergic nuclei over the last decades, their functional roles
remain cryptic.
ACKNOWLEDGEMENTS
Neuroscience 119
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PS 293
INDUCED EXPRESSION OF THE NEURONAL INJURY MARKER ATF3 IN PRI-
MARY AFFERENTS DURING MONOARTHRITIS: CHARACTERIZATION OF THE
NEURONAL POPULATIONS INVOLVED
D. NASCIMENTO, D.H. POZZA, J.M. CASTRO-LOPES, F. L. NETO
INSTITUTO DE HISTOLOGIA E EMBRIOLOGIA, FACULDADE DE MEDICINA DO PORTO E INSTITUTO DE BIOLOGIA MOLECULAR E
CELULAR (IBMC), UNIVERSIDADE DO PORTO, PORTUGAL
AIM
Recent studies on ATF-3 expression describe it as an adaptive response, behaving accordingly to the cellular
context and leading to a wide range of events. Using a well established model of chronic articular inflammatory
pain, monoarthritis (MA), we aimed to investigate if ATF-3 expression is also induced and how. We also proposed
to characterize the neuronal population including ATF-3 expressing cells and part of a possible signaling pathway
underneath this process.
INTRODUCTION
Elucidation of the molecular mechanisms underlying pain transmission processing is of great interest for the
development of better therapeutic approaches. Activating transcription factor 3 (ATF-3) is commonly accepted as
a neuronal injury marker since its expression is highly induced in a neuropathic pain condition. However, ATF-3
still vague; its implication in survival pathways, nerve elongation and neuroregeneration processes have also been
described. role in pain transmission is Since it has been described in several different cellular contexts we found very
interesting to analyze its expression in a model of chronic inflammatory pain, MA.
METHODS
After MA induction by complete Freunds adjuvant (CFA) injection into the tibiotarsal joint, triple immunolabeling
against ATF-3 and the well known neuronal populations markers calcitonin gene related-peptide (CGRP) and
isolectin (IB4) was performed to determine the most implicated neuronal populations in ATF-3 mediated signaling
pathway. Ipsilateral L5 ganglia from CFA vehicle-injected controls and inflamed animals sacrificed 2d, 4d, 7d and 14
days after CFA injection. Cell size distribution of ATF3 immunoreactive (IR) cells was also assessed. Double immu-
nolabbeling against ATF-3 and pAkt, as well as Western Blot, was also proceeded to investigate a possible survival
pathway activation.
RESULTS
Our results showed ATF-3 expression was significantly induced during MA particularly at 2 and 4 days, in the L5
ganglion. Cell size measurements revealed that ATF-3 IR cells were majorly small and medium ganglia neurons.
However ATF-3 colocalization with CGRP or IB4 was very low: double CGRP+ATF-3 positive cells were only observed
for the 2 and 4 days of MA, whereas no considerable double labeling for ATF-3 with IB4 was observed in any
experimental group. CGRP expression significantly decreased at 7 and 14 days of MA while IB4 expression remained
unchanged in L5 ganglia from MA rats.
CONCLUSION
Data suggest ATF-3 expression is somehow involved in articular inflammatory pain processing at the periphery,
mostly at early timepoints. Further studies are necessary to ensure ATF-3 expression is triggered due to a neuropath-
ic component in the origin of this disease or/and to the development of an inflammatory process. The prevalence of
ATF3 immunoreactivity mainly in small and medium neurons along with its lack of a noteworthy colocalization with
CGRP or IB4, may suggest that ATF3 expressing cells are possibly injured neurons. These cells probably loose their
ability to produce some molecules like neuropeptides as CGRP.
REFERENCES
120 Abstracts
YES Meeting 2010
PS 294
AVALIAO DA ATROFIA ENCEFLICA MEDIANTE TAC EM DOENTES COM
PROVVEL DEMNCIA NEURODEGENERATIVA. ESTUDO DE CASOS E CON-
TROLOS.
1 - SAMPAIO R., 2 - ALVAREZ F.
FACULTY OF MEDICINE, UNIVERSITY OF BEIRA INTERIOR
AIM
The main goal of this study is to test an eventual pattern of atrophy, at a radiological level, through craneal-ence-
phalic CT-scan, in groups of patients with diagnosis of Alzheimers disease and mixed dementia, and to compare
them with controls. The complementary goals are the following: assess correlations of clinical parameters (MMSE,
CDR, evolution time, cardiovascular risk factors) and measurements of encephalic structures between both groups.
INTRODUCTION
In Portugal, it is estimated that there are 153,000 people with dementia, which leads to a progressive loss of
autonomy, and capacity for the realization of daily activities, both professional and personal. Despite its various
etiologies, Alzheimers disease and vascular dementia are the most frequent forms in the elderly. Neurological im-
aging is currently the most important investigation used for the differential diagnosis of dementia and therapeutic
decision-making. Various studies have showed an association between the atrophy of the medial temporal lobe
and the dilatation of the temporal horns of the lateral ventricles, visualized by cranial-encephalic CT-scan, and the
diagnosis of Alzheimers disease with histological confirmation.
METHODS
In this retrospective study, data were obtained from hospital records of patients with Alzheimers disease, mixed
dementia and controls from the Neurology of CHCB, E.P.E. The maximum width of the occipital ventricles, never
before measured by CT-scan or MRI, was also evaluated.
RESULTS
The sample was integrated by 68 subjects, 38 of which had clinical diagnoses of dementia and 30 were controls. Of
the 38 patients, 25 had the diagnosis of Alzheimer and 13 had the clinical diagnosis of mixed dementia. All of the
measurements of studied cephalic structures, except the ratio of the interuncal distance with the cranial interbone
distance, were significant (p<0,001) between the groups of patients and controls but not between both groups of
patients. The width of the occipital horns of the lateral ventricles was significant between the Alzheimer disease
patients and controls (p=0.01), but not so between groups of mixed dementia and controls and between Alzheimer
disease patients and mixed dementia. Regarding the cardiovascular risk factors and other clinical factors studied,
only dyslipidemia was found to be significantly in mixed dementia patients when compared with Alzheimers
disease patients.
CONCLUSION
Despite the reduced size of the sample, this study confirmed the existence of an encephalic pattern of atrophy in
patients with Alzheimers disease, very similar to that also found in patients with mixed dementia. The width of
the occipital horns of the lateral ventricles was associated to this neurodegenerative disease. Regarding the variable
of time evolution, since it was not correlated with the measurements of the studied encephalic structures, its diag-
nostic utility can be distinguished in clinical practice for the detection of cortical-subcortical atrophy of the medial
temporal lobe in patients with Alzheimers disease.
REFERENCES
Neuroscience 121
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PS 330
DESCRIPTIVE STUDY OF THE USE OF VALPROATE AND TOPIRAMATE IN MI-
GRAINE PROPHYLAXIS IN THE MEXICAN CHILDREN HOSPITAL.
DRA. SALCEDO AC. DR. HERNNDEZ AJ. MC REYES LA.
FACULTY OF MEDICINE,UNUVERSITY OF MXICO
AIM
To analyze the experience in migraine prophylaxis treatment with Topiramate and Valproate and compare their
effectiveness in the Mexican Children Hospital neurology consultation service.
INTRODUCTION
Headache is one of the symptoms with highest prevalence among children and adolescent reaching 90% worldride
prevalence. Furthermore, it is the main cause for consultation. Headache is classified in primary and secondary
according to the International Headache Classification, migraine ensues to be the most important cause of primary
class headache. Migraine in children is a frequent pathology that can be disabling and patients need prophylactic
treatment to avoid these consequences .There are several treatments but have been mostly studied in adults.
METHODS
Sample: patients diagnosed with migraine based on the International Headache Classification in the Mexican
Children Hospital neurology consultation service. These patients received prophylactic treatment with magnesium
valproate or topiramate from 2004 2008. This is an observational, descriptive and retrospective study. Inclusion
Criteria: a) Age: 6 to 18 years .b) males and females .c) Migraine diagnose based on the International Headache
Classification. d)Patients under valproate and topiramate treatment within a two months period and two or more
neurology consultations . Exclusion Criteria: a) Incomplete information in medical records. b) Patient that have
droped treatment. Dependent Variables : Frequency (number of events per month), intensity (mild, moderate and
severe), events duration (minutes per day) and adverse effects. Independent Variables : Prophylactic Medication
(Valproate and Topiramate). Effectiveness criteria: Decreased frequency, intensity and duration of migraine events
with prophylactic treatment.
RESULTS
The selected sample of 76 patients was divided into two demographically comparable groups: the Group under
topiramate ( 48 patients), Valproate Group (28 patients) . Mann Whitney, Wilcoxon and chi Square tests were
used. After a three month treatment both medication showed a decrease in frequency, duration and intensity of
migraine events( statisctically significance p <0.05). There was no statistical difference between treatments . No
important side effects were observed under the doses used for migraine treatment., In Group under Topiramate
presented (18.8%) : cognitive impairment, insomnia, somnolence, parestesias, dizziness and anxiety .
CONCLUSION
Topiramate and Magnesium Valproate are equally effective in migraine prophylactic treatment in children when
administered during 3 months. There were no important side effects with the prescribed doses.
REFERENCES
122 Abstracts
YES Meeting 2010
PS 343
UNMASKING THE ROLE OF ALTERNATIVE SPLICING ON MACHADO-JOSEPH
DISEASE GENE (ATXN3): THE EXPERIENCE OF A MEDICAL STUDENT
RIBEIRO DOS SANTOS L1,2, BETTENCOURT C2,3,4, LIMA M2,3
1THIRD-YEAR MEDICAL STUDENT, FACULTY OF MEDICINE, UNIVERSITY OF COIMBRA, COIMBRA, PORTUGAL 2CENTER OF
RESEARCH IN NATURAL RESOURCES (CIRN), UNIVERSITY OF THE AZORES, PONTA DELGADA, PORTUGAL 3MOLECULAR AND CEL-
LULAR BIOLOGY INSTITUTE (IBMC), UNIVERSITY OF PORTO, PORTO, PORTUGAL 4LABORATORIO DE DIAGNOSTICO MOLECULAR
DEL BANCO DE TEJIDOS PARA INVESTIGACIONES NEUROLGICAS (BTIN), FACULTAD DE MEDICINA, UNIVERSIDAD COMPLUTENSE
DE MADRID, MADRID, SPAIN
AIM
Although the size of the CAG tract can be related with phenotypic variation (e.g. age at onset) it does not fully ex-
plains the MJDs pleomorphism, indicating that other factors must be also involved. The identification of such factors
is of extreme pertinence in a medical context, both on what concerns the ability to establish an adequate prognosis
as well as the possibility to identify therapeutic targets.
INTRODUCTION
Alternative splicing of pre-mRNA is a regulatory mechanism that enables a gene to produce multiple mature
transcripts. Since alternative forms of the correspondent protein may have different implications, the analyses of
the consequences of alternative splicing are essential, especially on what concerns genes associated with disorders
presenting clinical heterogeneity. Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3
(SCA3), is one of such disorders. This is a neurodegenerative disease caused by a CAG repeat expansion in the coding
region of the ATXN3 gene (14q32.1).
METHODS
In order to detect alternative splicing in the ATXN3 gene, a total of 415 cDNA clones (from 20 MJD patients and 14
controls) were analyzed using a protocol that involved the collection of blood samples, RNA extraction, RT-PCR,
clone screening and sequencing. A medical student (LRS) participated in this study, allowing her to practice several
molecular biology techniques as well as to closely examine the consequences of alternative splicing during ATXN3
mRNA maturation, reflecting on possible implications in terms of clinical variability.
RESULTS
. Fifty-six alternative transcript variants were detected for this gene, generated by four types of splicing events: a)
exon skipping; b) novel exons; c) usage of alternative 5 splice sites (ss); and d) usage of alternative 3 ss. As a conse-
quence of alternative splicing, some of those variants may constitute good targets for nonsense mediated decay (i.e.
degradation) and only a part would be translated into different protein isoforms.
CONCLUSION
These results confirm the role of alternative splicing as an important mechanism regulating protein diversity.
Furthermore, they point to the involvement of the variability generated by this mechanism in pathogenesis, and
that, in this particular case, may somehow contribute to MJDs clinical heterogeneity.
Neuroscience 123
 YES Guide YES Meeting 2010
PS 380
STUDY OF C-FOS EXPRESSION IN PREFRONTAL REAS AFTER A REVERSAL
LEARNING TASK ON RATS WITH PERIPHERAL NEUROPATHY
LUS ARAJO(A), HUGO LEITE-ALMEIDA(A), ARMANDO ALMEIDA(A)
A LIFE AND HEALTH RESEARCH INSTITUTE (ICVS), SCHOOL OF HEALTH SHCIENCES, UNIVERSITY OF MINHO, CAMPUS GUALTAR,
4710-057, BRAGA

INTRODUCTION
Neuropathic pain arrises from a lesion of the central or peripheral nervous system. This is manifested by sensorial,
emotional and cognitive changes. Recent studies have shown that animals with right peripheral neuropathic pain
shows a worst performance in reversal learning tasks relatively with animals with the same type of lesion on the left
side. Reversal learning task depends on structural integrity and cortex prefrontal functionality. Being functionally
lateralized, it was hypothesized that a lateral peripheral lesion would selectively affect each cortical hemisphere.
METHODS
To test this hypothesis it was measured the c-fos proto-oncogene expression level, after a reversal learning task,
Attentional set-shifting task on pre-frontal cortex.
RESULTS
Our results indicates a lesser c-fos expression in animals with right peripheral neuropathic lesion, relatively with
the same type of lesion on left side on ventral orbital cortex. This area, specifically the ventral orbital cortex in left
hemisphere seems to be involved in this task.
CONCLUSION
These results are accordingly with the worst performance watched on learning reversal task in animals with right
peripheral neuropathic lesion and are congruent with the contralateral projection of anatomical pathway affected.

124 Abstracts Neuroscience 125