Effect of metabolic control on interferon-gamma and interleukin-10 production by peripheral blood mononuclear cells from type 1 and type 2 diabetic patients

The objective of the present study was to evaluate the production of cytokines, interferon-g (INF-g) and interleukin-10 (IL-10), in cultures of peripheral blood mononuclear cells (PBMC) from type 1 and type 2 diabetic patients and to correlate it with inadequate and adequate metabolic control. We studied 11 type 1 and 13 type 2 diabetic patients and 21 healthy individuals divided into two groups (N = 11 and 10) paired by sex and age with type 1 and type 2 diabetic patients. The PBMC cultures were stimulated with concanavalin-A to measure INF-g and IL-10 supernatant concentration by ELISA. For patients with inadequate metabolic control, the cultures were performed on the first day of hospitalization and again after intensive treatment to achieve adequate control. INF-g levels in the supernatants of type 1 diabetic patient cultures were higher compared to type 2 diabetic patients with adequate metabolic control (P < 0.001). Additionally, INF-g and IL-10 tended to increase the liberation of PBMC from type 1 and 2 diabetic patients with adequate metabolic control (P = 0.009 and 0.09, respectively). The increased levels of INF-g and IL-10 released from PBMC of type 1 and 2 diabetic patients with adequate metabolic control suggest that diabetic control improves the capacity of activation and maintenance of the immune response, reducing the susceptibility to infections.

Lipolysis of emulsion models of triglyceride-rich lipoproteins is altered in male patients with abdominal aorta aneurysm

Disorders of the lipid metabolism may play a role in the genesis of abdominal aorta aneurysm. The present study examined the intravascular catabolism of chylomicrons, the lipoproteins that carry the dietary lipids absorbed by the intestine in the circulation in patients with abdominal aorta aneurysm. Thirteen male patients (72 ± 5 years) with abdominal aorta aneurysm with normal plasma lipid profile and 13 healthy male control subjects (73 ± 5 years) participated in the study. The method of chylomicron-like emulsions was used to evaluate this metabolism. The emulsion labeled with 14C-cholesteryl oleate and ³H-triolein was injected intravenously in both groups. Blood samples were taken at regular intervals over 60 min to determine the decay curves. The fractional clearance rate (FCR) of the radioactive labels was calculated by compartmental analysis. The FCR of the emulsion with ³H-triolein was smaller in the aortic aneurysm patients than in controls (0.025 ± 0.017 vs 0.039 ± 0.019 min-1; P < 0.05), but the FCR of14C-cholesteryl oleate of both groups did not differ. In conclusion, as indicated by the triglyceride FCR, chylomicron lipolysis is diminished in male patients with aortic aneurysm, whereas the remnant removal which is traced by the cholesteryl oleate FCR is not altered. The results suggest that defects in the chylomicron metabolism may represent a risk factor for development of abdominal aortic aneurysm.

ABCB1 C1236T, G2677T/A and C3435T polymorphisms in systemic lupus erythematosus patients

P-glycoprotein (Pgp), the ABCB1 gene product, acts as an efflux pump that transports a large variety of substrates and is a mechanism of cell protection against xenobiotics. An increasing number of studies have shown that some ABCB1 polymorphisms may affect Pgp expression and activity, as well as affecting the development and susceptibility to diseases and pharmacological response. High activity of Pgp has been detected in systemic lupus erythematosus (SLE) patients. The C1236T, G2677T/A, and C3435T are the most commonly studied single nucleotide polymorphisms in the ABCB1 gene. Therefore, their frequencies were determined in Brazilian individuals with European ancestry (N = 143) and in SLE patients (N = 137). Genotyping was performed by PCR-RFLP analysis using specific primers followed by incubation with the appropriate restriction enzymes. The resulting DNA fragments were visualized on agarose or polyacrylamide gels. No statistically significant differences were observed in allelic and genotypic frequencies between SLE and healthy subjects (Fisher exact test). Nevertheless, the 2677A allelic frequency was lower in SLE patients with malar rash (0.007) compared with patients without this feature (0.04; P = 0.0054), while the frequency of this variant was higher in SLE patients with pleuritis (0.07) compared with patients without this feature (0.01; P = 0.0156). We suggest that although the ABCB1 polymorphisms do not directly interfere in SLE susceptibility, their evaluation, especially the 2677A allele, in other immunological processes may be interesting since they can interfere in clinical features of this disease.

Distribution of the human leukocyte antigen class II alleles in Brazilian patients with chronic hepatitis C virus infection

Hepatitis C virus (HCV) infection is a global medical problem. The current standard of treatment consists of the combination of peginterferon plus ribavirin. This regimen eradicates HCV in 55% of cases. The immune response to HCV is an important determinant of disease evolution and can be influenced by various host factors. HLA class II may play an important role in immune response against HCV. The objective of the present study was to determine the distribution of HLA class II (DRB1 and DQB1) alleles, their association with chronic HCV infection and their response to interferon therapy. One hundred and two unrelated white Brazilian patients with chronic HCV infection, 52 responders (45 males and 7 females) and 50 non-responders (43 males and 7 females) to antiviral treatment, were included in the study. Healthy Brazilian bone marrow donors of Caucasian origin from the same geographic area constituted the control group (HLA-DRB1, N = 99 and HLA-DQB1, N = 222 individuals). HLA class II genotyping was performed using a low-resolution DRB1, DQB1 sequence-specific primer amplification. There were higher frequencies of HLA-DRB1*13 (26.5 vs 14.1%) and HLA-DQB1*02 (52.9 vs 38.7%) in patients compared with controls; however, these were not significantly different after P correction (Pc = 0.39 and Pc = 0.082, respectively). There was no significant difference between the phenotypic frequencies of HLA-DRB1 (17.3 vs 14.0%) and HLA-DQB1 alleles in responder and non-responder HCV patients. The HLA-DRB1*07 allele was significantly more common in HCV patients (33.3 vs 12.1%) than in controls (Pc = 0.0039), suggesting that the HLA-DRB1*07 allele is associated with chronic HCV infection.

Anti-cyclic citrullinated peptide antibodies and rheumatoid factor in leprosy patients with articular involvement

The objective of the present research was to evaluate the usefulness of anti-cyclic citrullinated peptide (anti-CCP) antibodies and the IgM rheumatoid factor (IgM RF) test for the differential diagnosis of leprosy with articular involvement and rheumatoid arthritis (RA). Anti-CCP antibodies and IgM RF were measured in the sera of 158 leprosy patients (76 with and 82 without articular involvement), 69 RA patients and 89 healthy controls. Leprosy diagnosis was performed according to Ridley and Jopling classification criteria and clinical and demographic characteristics of leprosy patients were collected by a standard questionnaire. Leprosy patients with any concomitant rheumatic disease were excluded. Serum samples were obtained from all participants and frozen at _20°C. Measurement of anti-CCP antibodies and IgM RF were performed by ELISA, using a commercial second-generation kit, and the latex agglutination test, respectively. Anti-CCP antibodies and IgM RF were detected in low frequencies (2.6 and 1.3%, respectively) in leprosy patients and were not associated with articular involvement. Among healthy individuals both anti-CCP antibodies and IgM RF were each detected in 3.4% of the subjects. In contrast, in the RA group, anti-CCP antibodies were present in 81.2% and IgM RF in 62.3%. In the present study, both anti-CCP antibodies and IgM RF showed good positive predictive value for RA, helping to discriminate between RA and leprosy patients with articular involvement. However, anti-CCP antibodies were more specific for RA diagnosis in the population under study.

Proton magnetic resonance spectroscopy of the frontal, cingulate and perirolandic cortices and its relationship to skin conductance in patients with schizophrenia

The aim of the present study was to determine whether specific subgroups of schizophrenic patients, grouped according to electrodermal characteristics, show differences in the N-acetylaspartate/creatine plus choline (NAA / (Cr + Cho)) ratios in the frontal, cingulate and perirolandic cortices. Skin conductance levels (SCL) and skin conductance responses to auditory stimulation were measured in 38 patients with schizophrenia and in the same number of matched healthy volunteers (control). All subjects were submitted to multivoxel proton magnetic resonance spectroscopic imaging. When compared to the control group, patients presented significantly lower NAA / (Cr + Cho) ratios in the right dorsolateral prefrontal cortex (schizophrenia = 0.95 ± 0.03; control = 1.12 ± 0.04) and in the right (schizophrenia = 0.88 ± 0.02; control = 0.94 ± 0.03) and left (schizophrenia = 0.84 ± 0.03; control = 0.94 ± 0.03) cingulates. These ratios did not differ between electrodermally responsive and non-responsive patients. When patients were divided into two groups: lower SCL (less than the mean SCL of the control group minus two standard deviations) and normal SCL (similar to the control group), the subgroup with a lower level of SCL showed a lower NAA / (Cr + Cho) ratio in the left cingulate (0.78 ± 0.05) than the controls (0.95 ± 0.02, P < 0.05) and the subgroup with normal SCL (0.88 ± 0.03, P < 0.05). There was a negative correlation between the NAA / (Cr + Cho) ratio in the left cingulate of patients with schizophrenia and the duration of the disease and years under medication. These data suggest the existence of a schizophrenic subgroup characterized by low SCL that could be a consequence of the lower neuronal viability observed in the left cingulate of these patients.

Minor sperm abnormalities in young male post-pubertal patients with juvenile dermatomyositis

The objective of the present study was to identify sperm abnormalities in young male patients with juvenile dermatomyositis (JDM). In 2005, 18 male JDM patients, diagnosed according to the criteria of Bohan and Peter, were followed at the Pediatric Rheumatology Unit and Rheumatology Division, of our Institution. Of the 18 males, 11 were pre-pubertal and 7 were post-pubertal. Two of 7 post-pubertal JDM male patients were excluded: one for orchidopexy for cryptorchidism and the other for testicular ectopia in the left testis. The remaining 5 post-pubertal JDM patients were prospectively evaluated on the basis of two semen analyses, according to the World Health Organization (WHO), urologic evaluation, testicular Doppler ultrasound hormone profile. The data of the JDM patients were compared with those of 5 age-matched healthy controls. The median age 18, was similar in JDM patients and controls. All JDM patients had teratozoospermia (abnormal sperm morphology), as did 4 (80%) of the controls. One of JDM patients had previous oligoasthenoteratozoospermia treated with intravenous cyclophosphamide with normalization of the number and concentration of the sperm after 5 years. All sperm parameters (sperm concentration, total sperm count and total motile sperm count by WHO, and sperm morphology by Kruger strict criteria), testicular volumes by Prader orchidometer and ultrasound, and hormones were similar in JDM patients compared with controls. The frequency of anti-sperm antibodies was similar in both groups. All JDM patients had minor sperm abnormalities in the head, midpiece, and/or tail of spermatozoids. Serial semen analyses in larger study populations are necessary to identify the extent and duration of sperm abnormalities in male patients with idiopathic inflammatory myopathies.

FANCD2 Western blot as a diagnostic tool for Brazilian patients with Fanconi anemia

Fanconi anemia is a rare hereditary disease showing genetic heterogeneity due to a variety of mutations in genes involved in DNA repair pathways, which may lead to different clinical manifestations. Phenotypic variability makes diagnosis difficult based only on clinical manifestations, therefore laboratory tests are necessary. New advances in molecular pathogenesis of this disease led researchers to develop a diagnostic test based on Western blot for FANCD2. The objective of the present study was to determine the efficacy of this method for the diagnosis of 84 Brazilian patients with Fanconi anemia, all of whom tested positive for the diepoxybutane test, and 98 healthy controls. The FANCD2 monoubiquitinated isoform (FANCDS+/FANCD2L-) was not detected in 77 patients (91.7%). In 2 patients (2.4%), there was an absence of both the monoubiquitinated and the non-ubiquitinated proteins (FANCD2S-/FANCD2L-) and 5 patients (5.9%) had both isoforms (FANCD2S+/FANCD2L+). This last phenotype suggests downstream subtypes or mosaicism. All controls were diepoxybutane negative and were also negative on the FANCD2 Western blot. The Western blot for FANCD2 presented a sensitivity of 94% (79/84) and specificity of 100% (98/98). This method was confirmed as an efficient approach to screen Brazilian patients with deleterious mutations on FANCD2 (FANCD2S-/FANCD2L-) or other upstream genes of the FA/BRCA pathway (FANCDS+/FANCD2L-), to confirm the chromosome breakage test and to classify patients according to the level of FA/BRCA pathway defects. However, patients showing both FANCD2 isoforms (FANCD2S+/FANCD2L+) require additional studies to confirm mutations on downstream Fanconi anemia genes or the presence of mosaicism.

The six-minute walk test and body weight-walk distance product in healthy Brazilian subjects

We assessed the 6-min walk distance (6MWD) and body weight x distance product (6MWw) in healthy Brazilian subjects and compared measured 6MWD with values predicted in five reference equations developed for other populations. Anthropometry, spirometry, reported physical activity, and two walk tests in a 30-m corridor were evaluated in 134 subjects (73 females, 13-84 years). Mean 6MWD and 6MWw were significantly greater in males than in females (622 ± 80 m, 46,322 ± 10,539 kg.m vs 551 ± 71 m, 36,356 ± 8,289 kg.m, P < 0.05). Four equations significantly overestimated measured 6MWD (range, 32 ± 71 to 137 ± 74 m; P < 0.001), and one significantly underestimated it (-36 ± 86 m; P < 0.001). 6MWD significantly correlated with age (r = -0.39), height (r = 0.44), body mass index (r = -0.24), and reported physical activity (r = 0.25). 6MWw significantly correlated with age (r = -0.21), height (r = 0.66) and reported physical activity (r = 0.25). The reference equation devised for walk distance was 6MWDm = 622.461 - (1.846 x Ageyears) + (61.503 x Gendermales = 1; females = 0); r2 = 0.300. In an additional group of 85 subjects prospectively studied, the difference between measured and the 6MWD predicted with the equation proposed here was not significant (-3 ± 68 m; P = 0.938). The measured 6MWD represented 99.6 ± 11.9% of the predicted value. We conclude that 6MWD and 6MWw variances were adequately explained by demographic and anthropometric attributes. This reference equation is probably most appropriate for evaluating the exercise capacity of Brazilian patients with chronic diseases.

Glomerular filtration is reduced by high tidal volume ventilation in an in vivo healthy rat model

Mechanical ventilation has been associated with organ failure in patients with acute respiratory distress syndrome. The present study examines the effects of tidal volume (V T) on renal function using two V T values (8 and 27 mL/kg) in anesthetized, paralyzed and mechanically ventilated male Wistar rats. Animals were randomized into two groups of 6 rats each: V T8 (V T, 8 mL/kg; 61.50 ± 0.92 breaths/min; positive end-expiratory pressure, 3.0 cmH2O; peak airway pressure (PAW), 11.8 ± 2.0 cmH2O), and V T27 (V T, 27 mL/kg; 33.60 ± 1.56 breaths/min; positive end-expiratory pressure, none, and PAW, 22.7 ± 4.0 cmH2O). Throughout the experiment, mean PAW remained comparable between the two groups (6.33 ± 0.21 vs 6.50 ± 0.22 cmH2O). For rats in the V T27 group, inulin clearance (mL·min-1·body weight-1) decreased acutely after 60 min of mechanical ventilation and even more significantly after 90 min, compared with baseline values (0.60 ± 0.05 and 0.45 ± 0.05 vs 0.95 ± 0.07; P < 0.001), although there were no differences between groups in mean arterial pressure or gasometric variables. In the V T8 group, inulin clearance at 120 min of mechanical ventilation remained unchanged in relation to baseline values (0.72 ± 0.03 vs 0.80 ± 0.05). The V T8 and V T27 groups did not differ in terms of serum thiobarbituric acid reactive substances (3.97 ± 0.27 vs 4.02 ± 0.45 nmol/mL) or endothelial nitric oxide synthase expression (94.25 ± 2.75 vs 96.25 ± 2.39%). Our results show that glomerular filtration is acutely affected by high tidal volume ventilation but do not provide information about the mechanism.

Detection of patients with functional dyspepsia using wavelet transform applied to their electrogastrogram

The aim of the present study was to develop a classifier able to discriminate between healthy controls and dyspeptic patients by analysis of their electrogastrograms. Fifty-six electrogastrograms were analyzed, corresponding to 42 dyspeptic patients and 14 healthy controls. The original signals were subsampled, filtered and divided into the pre-, post-, and prandial stages. A time-frequency transformation based on wavelets was used to extract the signal characteristics, and a special selection procedure based on correlation was used to reduce their number. The analysis was carried out by evaluating different neural network structures to classify the wavelet coefficients into two groups (healthy subjects and dyspeptic patients). The optimization process of the classifier led to a linear model. A dimension reduction that resulted in only 25% of uncorrelated electrogastrogram characteristics gave 24 inputs for the classifier. The prandial stage gave the most significant results. Under these conditions, the classifier achieved 78.6% sensitivity, 92.9% specificity, and an error of 17.9 ± 6% (with a 95% confidence level). These data show that it is possible to establish significant differences between patients and normal controls when time-frequency characteristics are extracted from an electrogastrogram, with an adequate component reduction, outperforming the results obtained with classical Fourier analysis. These findings can contribute to increasing our understanding of the pathophysiological mechanisms involved in functional dyspepsia and perhaps to improving the pharmacological treatment of functional dyspeptic patients.

Promoter region sequence differences in the A and G gamma globin genes of Brazilian sickle cell anemia patients

Fetal hemoglobin (HbF), encoded by the HBG2 and HBG1 genes, is the best-known genetic modulator of sickle cell anemia, varying dramatically in concentration in the blood of these patients. This variation is partially associated with polymorphisms located in the promoter region of the HBG2 and HBG1 genes. In order to explore known and unknown polymorphisms in these genes, the sequences of their promoter regions were screened in sickle cell anemia patients and correlated with both their HbF levels and their βS-globin haplotypes. Additionally, the sequences were compared with genes from 2 healthy groups, a reference one (N = 104) and an Afro-descendant one (N = 98), to identify polymorphisms linked to the ethnic background.The reference group was composed by healthy individuals from the general population. Four polymorphisms were identified in the promoter region of HBG2 and 8 in the promoter region of HBG1 among the studied groups. Four novel single nucleotide polymorphisms (SNP) located at positions -324, -317, -309 and -307 were identified in the reference group. A deletion located between -396 and -391 in the HBG2 promoter region and the SNP -271 C→T in the HBG1 promoter region were associated with the Central African Republic βS-globin haplotype. In contrast, the -369 C→G and 309 A→G SNPs in the HBG2 promoter region were correlated to the Benin haplotype. The polymorphisms -396_-391 del HBG2, -369 SNP HBG2 and -271 SNP HBG1 correlated with HbF levels. Hence, we suggest an important role of HBG2 and HBG1 gene polymorphisms on the HbF synthesis.

Evaluation of the immune humoral response of Brazilian patients with Rubinstein-Taybi syndrome

Rubinstein-Taybi syndrome (RTS) is a rare developmental disorder characterized by craniofacial dysmorphisms, broad thumbs and toes, mental and growth deficiency, and recurrent respiratory infections. RTS has been associated with CREBBP gene mutations, but EP300 gene mutations have recently been reported in 6 individuals. In the present study, the humoral immune response in 16 RTS patients with recurrent respiratory infections of possible bacterial etiology was evaluated. No significant differences between patients and 16 healthy controls were detected to explain the high susceptibility to respiratory infections: normal or elevated serum immunoglobulin levels, normal salivary IgA levels, and a good antibody response to both polysaccharide and protein antigens were observed. However, most patients presented high serum IgM levels, a high number of total B cell and B subsets, and also high percentiles of apoptosis, suggesting that they could present B dysregulation. The CREBBP/p300 family gene is extremely important for B-cell regulation, and RTS may represent an interesting human model for studying the molecular mechanisms involved in B-cell development.

Gender differences in serum CK-MB mass levels in healthy Braziliansubjects

The creatine kinase-isoenzyme MB (CK-MB) mass assay is one of the laboratory tests used for the diagnosis of myocardial infarction. It is recommended, however, that reference limits should take gender and race into account. In the present study, we analyzed the plasma CK-MB mass and troponin levels of 244 healthy volunteers without a personal history of coronary artery disease and with no chronic diseases, muscular trauma or hypothyroidism, and not taking statins. The tests were performed with commercial kits, CK-MB mass turbo kit and Troponin I turbo kit, using the Immulite 1000 analyzer from Siemens Healthcare Diagnostic. The values were separated according to gender and showed significant differences by the Mann-Whitney test. Mean (± SD) CK-MB mass values were 2.55 ± 1.09 for women (N = 121; age = 41.20 ± 10.13 years) and 3.49 ± 1.41 ng/mL for men (N = 123; age = 38.16 ± 11.12 years). Gender-specific reference values at the 99th percentile level, according to the Medicalc statistical software, were 5.40 ng/mL for women and 7.13 ng/mL for men. The influence of race was not considered because of the high miscegenation of the Brazilian population. The CK-MB values obtained were higher than the 5.10 mg/mL proposed by the manufacturer of the laboratory kit. Therefore, decision limits should be related to population and gender in order to improve the specificity of this diagnostic tool, avoiding misclassification of patients

Expression of the chemokine receptor CXCR4 on lymphocytes of leprosy patients

Leprosy is caused by Mycobacterium leprae, which induces chronic granulomatous infection of the skin and peripheral nerves. The disease ranges from the tuberculoid to the lepromatous forms, depending on the cellular immune response of the host. Chemokines are thought to be involved in the immunopathogenesis of leprosy, but few studies have investigated the expression of chemokine receptors on leukocytes of leprosy patients. In the present study, we evaluated 21 leprosy patients (M/F: 16/5) with a new diagnosis from the Dermatology Outpatient Clinic of the University Hospital, Federal University of Minas Gerais. The control group was composed of 20 healthy members (M/F: 15/5) of the community recruited by means of announcements. The expression of CCR2, CCR3, CCR5, and CXCR4 was investigated by flow cytometry on the surface of peripheral blood lymphocytes. There was a decrease in percentage of CD3+CXCR4+ and CD4+CXCR4+ lymphocytes in the peripheral blood of leprosy patients (median [range], 17.6 [2.7-41.9] and 65.3 [3.9-91.9], respectively) compared to the control group (median [range], 43.0 [3.7-61.3] and 77.2 [43.6-93.5], respectively). The percentage of CD4+CXCR4+ was significantly lower in patients with the tuberculoid form (median [range], 45.7 [0.0-83.1]) of the disease, but not in lepromatous patients (median [range], 81.5 [44.9-91.9]). The CXCR4 chemokine receptor may play a role in leprosy immunopathogenesis, probably directing cell migration to tissue lesions in tuberculoid leprosy patients.