The major endocannabinoid anandamide (AEA) induces apoptosis of human granulosa cells.

The endocannabinoid system (ECS) plays a crucial role in human reproduction. Changes in anandamide (AEA) levels affect reproductive events and has already been suggested as biomarker of reproductive potential of male and female gametes. Although cannabinoid-receptor 1 (CB1) was already identified in human granulosa cells (hGCs) the ECS was not characterized on granulosa cells line COV434 nor the effects of AEA on GCs viability and function depicted. Therefore, the aim of this study was to characterize the ECS elements and explore the effects of AEA on both COV434 and hGCs. Our results revealed that hGCs express the full enzymatic machinery responsible for AEA metabolism as well as cannabinoid receptors. In addition, AEA induced a reduction in both COV434 and hGCs viability in a concentration and time-dependent manner. Nevertheless, the effects of AEA in cell viability was independent of either CB1 or CB2 receptors. There was no ROS release in both cell models; however, AEA induced morphological changes, presenting chromatin condensation at 72 h, and variation on mitochondrial membrane potential. Moreover, AEA induced an increase in caspase -3/-7 activities in both cell models, but in hGCs there was also an increase in caspase 8 activity. This study supports the idea that ECS balance is crucial for folliculogenesis and oocyte quality as dysregulated AEA levels may compromise female fertility.

Metabolic syndrome in human immunodeficiency virus-infected patients.

The objective of this study was to investigate the factors underlying the development of metabolic syndrome (MetS) in HIV-infected patients. Two hundred and sixty-six clinical cases were selected for a retrospective study. The sample was classified using the Adult Treatment Panel III guidelines and the identification of risk or protective factors associated with MetS evaluated via multivariate logistic or multinomial regressions. HIV-infected individuals diagnosed with MetS tend to be older, overweight, or obese (85% have a BMI ≥ 25), with a waist circumference > 90 cm (96.5 [88.8-105.5] cm, median [interquartile range]). Blood testing these individuals revealed high fasting levels of insulin (8.1 [5.8-21.6] pg/ml), glucose (98.0 [84.0-116.0] mg/dl), triglycerides (201.0 [142.0-267.3] mg/dl), and high-density lipoprotein cholesterol (36.5 [29.8-43.3] mg/dl) in addition with higher levels of inflammatory mediators such as high-sensitivity C-reactive protein (2.5 [1.0-4.9] mg/dl) and interleukin-6 (3.4 [2.8-3.8] pg/ml). The likelihood of HIV-infected individuals who are virally suppressed developing MetS is about 60% higher than those with acute infection. Treatment with nucleoside reverse transcriptase inhibitors (NRTIs) and protease inhibitors (PIs) increases the chance of developing MetS by around 2.4 times. Individuals with a lower antioxidant capacity (total antioxidant status [TAS] <1.33) have a 2.6 times higher risk of developing MetS. HIV-related chronic inflammation, a low TAS, and treatment with NRTIs in association with PIs are additional MetS risk factors.

Unravelling the relationship between protein sequence and low-complexity regions entropies: Interactome implications.

Low-complexity regions are sub-sequences of biased composition in a protein sequence. The influence of these regions over protein evolution, specific functions and highly interactive capacities is well known. Although protein sequence entropy has been largely studied, its relationship with low-complexity regions and the subsequent effects on protein function remains unclear. In this work we propose a theoretical and empirical model integrating the sequence entropy with local complexity parameters. Our results indicate that the protein sequence entropy is related with the protein length, the entropies inside and outside the low-complexity regions as well as their number and average size. We found a small but significant increment in the sequence entropy of hubs proteins. In agreement with our theoretical model, this increment is highly dependent of the balance between the increment of protein length and average size of the low-complexity regions. Finally, our models and proteins analysis provide evidence supporting that modifications in the average size is more relevant in hubs proteins than changes in the number of low-complexity regions.

Glycaemic profile changes by highly active antiretroviral therapy in human immunodeficiency virus-infected patients.

To study dysglycaemia in human immunodeficiency virus (HIV)-infected patients we conducted a retrospective cohort study of the glucose profile in HIV-infected patients. The fasting blood glucose was analysed taking into consideration conventional risk factors as well as HIV infection and highly active antiretroviral therapy (HAART). One hundred seventy-three cases were selected for this study. Five risk factors had significant effects (p < 0.05) on glucose levels: age, body mass index (BMI), hepatitis C virus/hepatitis B virus (HCV/HBV) co-infection, viral load (VL), and CD4(+) T-lymphocyte count. Fasting blood glucose levels increased with age (0.59 mg/dL/year), decreased with the VL (-4.1 × 10(-6 )mg/dL/number of viral RNA copies) and the CD4(+) T-lymphocyte count (-0.016 mg/dL/cell count). Furthermore, obese patients and those co-infected with HCV/HBV were more prone to develop dysglycaemia having, on average, 15.4 mg/dL and 13.8 mg/dL higher levels, respectively, of fasting blood glucose. Despite an increase of 1.0% and 8.4% in the glucose levels noticed among HIV patients treated with non-nucleotide inhibitors of reverse transcriptase and protease inhibitors, respectively, HAART did not prove to be a significant predictor of fasting glucose levels as well as lipodystrophy and male gender. Age, BMI, HCV/HBV co-infection and HIV-related (VL and CD4(+) T-lymphocyte count) factors seem to be the most influential on fasting blood glucose levels in HIV-infected individuals.

Lipid profile changes by high activity anti-retroviral therapy.

OBJECTIVE: Study of the lipid profile in patients infected with HIV treated with different combinations of high activity anti-retroviral therapy (HAART). DESIGN AND METHODS: A retrospective cohort study of the lipid profile in patients undergoing HAART. The study analyzes the evolution of concentrations of triglycerides (TG), total cholesterol (TC), LDL-cholesterol (LDLc) and HDL-cholesterol (HDLc) in a period of at least 3 years of treatment. From a total of 750 clinical cases analyzed in Hospital Joaquim Urbano (Oporto, Portugal) 124 patients were selected for this study. RESULTS: After 3 years of treatment, we observed the development of dyslipidaemia by increases in TG (17%), TC (29%) and LDLc (9%), particularly in patients treated with a combination of drugs which included protease inhibitors (PI). Moreover, the non-nucleoside reverse transcriptase inhibitors (NNRTI) were associated with better lipid profile. The increase of 46% in HDLc was the most surprising finding. CONCLUSIONS: The results indicate that patients with HAART have a more atherogenic lipidic profile with increased TC, LDLc and TG levels. Since the effectiveness of NNRTI is similar to that of PI, but with a smaller atherogenic profile, it should be the first choice drug to be selected in the HIV treatment.

PP159. In women with previous pregnancy hypertension, cardiovascular risk biomarkers may be modulated by haptoglobin genotype.

INTRODUCTION: Preeclampsia may affect the risk for future cardiovascular disease. Haptoglobin (Hp), an acute phase protein with genetic polymorphism (Hp1.1, 2.1 and 2.2) synthesized in the hepatocyte and in many tissues, may modulate the risk through the antioxidant and anti-inflammatory differential effects of their genotypes. OBJECTIVES: Evaluate some parameters of cardiovascular risk in association with genetic polymorphism of Hp in women with previous preeclampsia (HBP) compared to healthy women during gestation (NBP). METHODS: We studied 352 women aged 35±5.48 current year, taking these, 165 presented previous preeclampsia, 2-14 (±6.6) years ago. Genetic polymorphism of Hp was determined from plasma by electrophoresis. The different circulating biomarkers, myeloperoxidase ng/mL (MPO) and nitrates and nitrites (nmol/ml) by ELISA, transaminases - AST and ALT (U/L) and apolipoprotein A and B (mg/dL), uric acid (mg/dl) hs-CRP (mg/L) were determined by conventional methods as well as blood pressure (BP) and anthropometric parameters BMI and hip and waist circumference (WC).Statistical methods were Chi-square, ANOVA (Bonferroni) and t-Student. RESULTS: There were no differences in the distribution of phenotypes of Hp between NBP and those with previous hypertension in pregnancy. There were differences between NBP and HBP in the following parameters: Systolic BP (118.9±13.4 vs 135.0±16.5, p<0.001); Diastolic BP (72.2±10.1 vs 85.9±12.0 p<0.001); MPO (62.3±30.9 vs 85.7±39.4, p=0.020); Nitrite (10.1±3.8 vs 19.1±7.0, p<0.001); ALT (18.0±8.0 vs 22.5±12.9, p=0.016); BMI (25.4±4.1 vs 27.1±4.8, p=0.031); WC (82.8±9.9 vs 89.5±15.6, p=0.002); Apo B (0.6±0.1 vs 0.6±0.1, p=0.023). Those values vary with the genotypes of Hp and were observed Hp 1.1 and 2.1 in NBP and HBP respectively: [Systolic BP 119.2±13.7 vs 134.7±18.3 (p<0.001); Diastolic BP (72.8±11.9 vs 86.2±19.4 p<0.001); MPO (57.9±32.5 vs 94.2±42.1, p=0.008); Nitrites (9.6±3.2 vs 20.0±8.5, p<0.001); ALT(17.0±7.2 vs 20.4±8.0, p=0.033); WC (82.4±10.0 vs 90.8±17.6, p=0.004); Apo A (0.9±0.2 vs 1.0±0.2, p=0.011)] and Hp 2.2 in NBP and HBP respectively: [Systolic BP 118.2±13.1 vs 135.6±12.8 (p<0.001); Diastolic BP (70.9±7.2 vs 85.5±16.3 p<0.001); Nitrites (11.5±5.1 vs 18.0±4.4, p<0.001)]. CONCLUSION: Women with previous hypertension during pregnancy, presented significant differences in some classic cardiovascular risk biomarkers as well as in some others, associated with the inflammatory process, whose variation may be modulated by haptoglobin functional genetic polymorphism. The history of hypertensive disease in pregnancy may be relevant, in association with these biomarkers to the prevention of cardiovascular disease in particular of postmenopausal women.

Application of desirability-based multi(bi)-objective optimization in the design of selective arylpiperazine derivates for the 5-HT1A serotonin receptor.

The multiobjective optimization technique based on the desirability estimation of several interrelated responses (MOOP-DESIRE) has been recently applied to quantitative structure-activity relationship (QSAR) studies. However, the advantage of applying this new methodology to the study of selectivity and affinity to competitive targets has been little explored. We used the MOOP-DESIRE methodology and a variation of this, to study the arylpiperazine derivates that could interact with 5-HT(1A) and 5-HT(2A), serotonin receptor subtypes with the objective of designing more selective molecules for the 5-HT(1A) receptor. We did show that the model results are in agreement with the available pharmacophore descriptions, guaranteeing an appropriate structural correlation and proving the methodology, as a useful tool for the important problem of selective drug design.

Protein deficiency balance as a predictor of clinical outcome in hereditary spherocytosis.

Vertical and horizontal interactions between membrane constituents account for integrity, strength and deformability of the erythrocyte. Disruption of vertical interactions caused by membrane protein deficiencies in hereditary spherocytosis (HS), favor membrane vesiculation with development of spherocytic cells. Our aim was to evaluate the hematological and clinical presentation of HS according to the type and amount of protein deficiency. We studied 81 Portuguese individuals, 71 belonging to 21 families plus 10 unrelated subjects, and found that 51 of them were HS patients. Patients were classified as presenting mild, typical or severe HS, according to laboratory results and clinical follow-up. We performed screening tests and the standardized electrophoretic membrane protein analysis to identify and quantify protein deficiencies. We found band 3 and ankyrin deficiencies as the major causes for HS. The ratios between the value of the primary and/or secondary protein deficiencies showed significantly different values according to the severity of HS, and a significant inverse correlation with the severity of HS was observed. In mild HS, the ratios between protein deficiencies reflected equivalent protein deficiencies, while an unbalance was observed in typical HS, which was enhanced in severe HS. Our data suggest that the relative quantification of each major membrane protein and of the ratios between the values of protein deficiencies may be helpful in providing additional data about the clinical outcome of HS.

The inflammatory response in mild and in severe psoriasis.

BACKGROUND: Psoriasis is a chronic and recurrent inflammatory skin disease. The inflammatory response represents a fundamental ability of the organism to protect itself from infectious agents and from injury. OBJECTIVES: To evaluate the inflammatory response in mild and in severe psoriasis, to evaluate the endogenous systems counterbalancing the deleterious effects of the inflammation products, and to establish values of prognostic significance. METHODS: The study was performed in a control group (n = 40) and in 60 patients with psoriasis vulgaris, half presenting with mild psoriasis, and the other half with severe psoriasis. We evaluated total and differential leucocyte count; elastase, lactoferrin and lipid peroxidation as markers of neutrophil activation; total plasma antioxidant capacity (TAS), transferrin, ceruloplasmin, alpha(1)-antitrypsin and alpha(2)-macroglobulin as markers of the endogenous antioxidant and antiprotease systems; and fibrinogen, erythrocyte sedimentation rate, C-reactive protein (CRP), haptoglobin, C3 and C4 complement proteins as markers of inflammation. RESULTS: Our data suggested that psoriasis is an inflammatory condition in which neutrophils seem to play a crucial role by contributing to the development of oxidative and proteolytic stress. The worsening of the disease seemed to be linked to the enhancement of the inflammatory response and of the imbalance between neutrophil activation products and their inhibitors. CONCLUSIONS: We propose values for elastase, CRP, elastase/alpha(2)-macroglobulin, elastase/alpha(1)-antitrypsin, thiobarbituric acid/TAS and elastase/neutrophil ratios with prognostic significance for the worsening of psoriasis.

Erythrocyte damage in mild and severe psoriasis.

BACKGROUND: Psoriasis is a common chronic and recurrent inflammatory skin disorder. Oxygen metabolites and proteases released by activated inflammatory cells may induce oxidative and proteolytic damage to plasma constituents and red blood cells (RBCs). RBCs have a limited biosynthesis capacity and poor repair mechanisms. OBJECTIVES: To study RBCs as a potential cumulative marker of oxidative and proteolytic stress in psoriasis, and as a marker of worsening of the disease. METHODS: The study was performed in 70 patients with mild or severe psoriasis and in 40 control individuals. We evaluated total and differential leucocyte count and, as markers of leucocyte activation, plasma elastase and lactoferrin. Besides the basic RBC study (RBC count, haematocrit, haemoglobin concentration and haematimetric indices) we evaluated antioxidant defences (catalase, superoxide dismutase, glutathione peroxidase and selenium), osmotic fragility and reticulocyte count; in the RBC membrane we evaluated lipid peroxidation and susceptibility to lipid peroxidation, membrane fluidity, levels of cholesterol and phospholipids, membrane-bound haemoglobin, band 3 profile and levels of vitamin E; serum levels of bilirubin, total plasma antioxidant capacity, lipid profile and lipid peroxidation were also evaluated. RESULTS: Psoriasis patients showed a rise in leucocytes, mainly neutrophils, which was associated with a rise in elastase and lactoferrin. Patients had a reduced RBC count, antioxidant defences and membrane fluidity, elevated membrane lipid peroxidation, membrane-bound haemoglobin, osmotic fragility and reticulocyte count, and a different band 3 profile. Most of these modifications were enhanced in severe psoriasis. CONCLUSIONS: In summary, our data show that the RBCs are at a lower number in psoriasis patients, and present several changes denoting an enhanced damage and/or ageing process, which seem to be strongly connected with neutrophil activation, oxidative stress and worsening of psoriasis.

Dislipidemia and oxidative stress in mild and in severe psoriasis as a risk for cardiovascular disease.

Psoriasis is a common chronic and recurrent inflammatory skin disorder that has been associated with oxidative stress, abnormal plasma lipid metabolism and with high frequency of cardiovascular events. This prevalence seems to be related to the severity of psoriasis, as it occurs more frequently in patients presenting large areas of the body affected with psoriasis lesions. The aim of our work was to evaluate the development of oxidative stress and of dislipidemia in psoriasis, and to look for a correlation between their levels and worsening of psoriasis. We evaluated lipid profile, total antioxidant capacity, antioxidant vitamins A and E, and lipoperoxidation products. The study was performed in controls and in patients presenting mild and severe psoriasis. Patients presented risk changes in lipid profile (a rise in cholesterol (P<0.01), triglycerides (P<0.001), low density lipoprotein cholesterol (P<0.01), very low density lipoprotein cholesterol (P<0.01), apolipoprotein B (P<0.001) and lipoprotein(a) (P<0.001); and a reduction in high density lipoprotein cholesterol (P<0.001)), a rise in lipoperoxidation products (P<0.001) and a reduction in total antioxidant capacity (P<0.001) and in antioxidant vitamins A (P<0.001) and E (P<0.05). Moreover, we found that the worsening of psoriasis was associated with the enhancement of oxidative stress and of the lipid risk changes. Our data suggest that psoriasis patients must be considered as a group at risk for cardiovascular disease and that this risk seems to be higher in severe psoriasis. In addition, a possible benefit of an enriched diet or of a supplement of vitamins A and E in psoriasis patients should be further studied.

[A retrospective assessment of spinocellular carcinomas of the lip treated by roentgenotherapy]. / Avaliação retrospectiva dos carcinomas espinho-celulares do lábio tratados por roentgenterapia.

From December 1965 to December 1987 (22 years), 279 patients with histologically confirmed squamous cell carcinoma of the lip were treated in this Department with roentgenotherapy. All the patients that had completed the treatment were evaluated with a follow-up time of 5 years (n = 255). Distribution by age, sex, occupation, residence, evolution time of the lesion, localization (upper lip, lower lip and commissure), tumor size and histological grade were analysed. In what concerns technical conditions and fractionation, we evaluated the type of acute reactions and the dose of radiation which elicited them as well as the results of radiotherapy (local and cosmetic control). A relation is made between the size of the lesion and outcome after treatment. The survival rate at 3 years was 76.6% and at 5 years (the usual follow-up period for these patients) 66%. Disease-free survival rate at 3 years was 79% and at 5 years 77.6%.

[A retrospective assessment of spinocellular carcinomas of the lip treated by roentgenotherapy]. / Avaliação retrospectiva dos carcinomas espinho-celulares do lábio tratados por roentgenterapia.

From December 1965 to December 1987 (22 years), 279 patients with histologically confirmed squamous cell carcinoma of the lip were treated in this Department with roentgenotherapy. All the patients that had completed the treatment were evaluated with a follow-up time of 5 years (n=255). Distribution by age, sex, occupation, residence, evolution time of the lesion, localization (upper lip, lower lip and commissure), tumoral size and histological grade were analysed. Concerning technical conditions and fractionation, we evaluated the type and which dose of radiation elicit acute reactions as well as the results of radiotherapy (local and cosmetic control). A relation is made between the size of the lesion and outcome after treatment. The survival rate at 3 years was 76,6% and at 5 years (the usual follow-up period for these patients) was 66%. Disease-free survival rate at 3 years was 79% and at 5 years was 77,6%.

Comparative study of lactoferrin and other blood markers of inflammatory stress between preeclamptic and normal pregnancies.

OBJECTIVE: To test lactoferrin as a blood discriminator of neutrophil activation between normal and preeclamptic pregnancy. DESIGN: Comparative study between normal (n = 40) and preeclamptic women receiving treatment (n = 42) in the third trimester of pregnancy and in the post partum period (30 women with normal pregnancy and 22 with preeclampsia). METHODS: Blood, serum or plasma measurements of neutrophils, lactoferrin, vitamin C, vitamin E, lipid peroxidation products, elastase, C-reactive protein (CRP), gamma-glutamyltranspeptidase (gamma GT), haptoglobin, osmotic fragility, urea, creatinine, uric acid, transaminases (ASAT, ALAT), lactic dehydrogenase (LDH), platelets, red and white blood cells. RESULTS: In preeclamptic women the ratios of lactoferrin per neutrophil or per erythrocyte are higher before delivery than in normal women but decrease after delivery. Delivery induces a greater inflammatory response in normal pregnancy as detected by blood concentrations of inflammatory markers and hepatic and renal parameters. CONCLUSION: Whereas in normal pregnant women neutrophil activation increases with delivery, in preeclamptic women the opposite occurs.

Lactoferrin as a sensitive blood marker of neutrophil activation in normal pregnancies.

OBJECTIVE: To test lactoferrin as a marker of neutrophil activation during pregnancy. DESIGN: Cross-sectional study in normal pregnant (n = 100), nonpregnant (n = 11) and post partum women (n = 30). METHODS: Serum or plasma measurements of neutrophils, lactoferrin, vitamin C, vitamin E, lipid peroxide, elastase, C-reactive protein gamma-glutamyltranspeptidase, haptoglobin and osmotic fragility. RESULTS: During normal pregnancy all markers of neutrophil-activation increase. CONCLUSION: Neutrophil-activation compromises the antioxidant defense mechanism during normal pregnancy.

[Locally advanced carcinoma of the breast. Initially treated with radiotherapy]. / Carcinoma localmente avançado da mama. Tratado inicialmente por radioterapia.

From January 1981 to December 1985, 133 patients with locally advanced breast cancer (stage III) were treated with teleradiotherapy using cobalt as the first modality of treatment aiming at appropriate conditions for surgery. Consequently, 58.6% of the patients achieved the required conditions for surgery after radiotherapy. A correlation is made between the final results and tumoral size and location, nodal invasion, histological grade and hormonal status. Local regional recurrence and systemic metastases are assessed according to the different treatment modalities used: Radiotherapy followed by surgery and or sequential systemic therapy (chemotherapy or hormonal therapy). We verified that the survival rate was higher in patients submitted to surgery. The global survival rate of the patients who underwent surgery at 5 and 8 years was 60% and 48% respectively. The same period of 5 and 8 years, the percentages decreased from 60% to 27% and from 48% to 10% in patients not submitted to surgery. The global disease-free survival was 46.6% at 5 years and 40% at 8 years.

[Pubic hair in children and late onset congenital adrenal hyperplasia]. / Pilosidade púbica na criança e hiperplasia congénita supra-renal não clássica.

Before the age of 8 in females and 9 in males, the presence of pubic hair can be the first sign of a gonadal or adrenal disease. In order to identify the late onset type of congenital adrenal hyperplasia in premature pubescence, we did the adrenal short stimulation test with Synacthen, on 30 children. Five girls and one boy showed a 21-hydroxylase deficiency, in which the seric 17-OH Progesterone at 60 minutes is 28.8 +/- 15.2 ng/ml, being in a control group 2.30 +/- 0.8 ng/ml [p less than 0.01]. The 20% incidence we have found, justifies the testing of every premature pubescent child.

Characterization of beta-lactamases encoded by pathogenic strains of Escherichia coli from Portugal.

A survey of 2036 strains of Escherichia coli was conducted in Oporto, Portugal, to establish the prevalence of resistance to beta-lactam antibiotics. Isolates were from hospital and clinical practice patients, and were further divided into urinary and non-urinary pathogens. A high level of resistance to aminopenicillins (55.3%) was observed. Further examination of the resistant strains showed that the frequencies of occurrence of known beta-lactamases were as follows: TEM-1 (78.2% of resistant strains); SHV-1 (7.9%); TEM-2 (0.45%); OXA-1 (1.5%); HMS-1 (0.18%); TEM-1 + SHV-1 (4.2%). Other pairs of beta-lactamases were also identified in less than 2% of the resistant strains. In addition, 4.7% of the resistant strains produced elevated levels of a presumed chromosomal cephalosporinase, while 0.9% produced a ceftazimidase of pI 5.8-5.9 which was similar to TEM-6.