IgA response in serum and gut secretion in sensitized mice fed with the dust mite Dermatophagoides pteronyssinus extract

Induced oral tolerance to mucosal-exposed antigens in immunized animals is of particular interest for the development of immunotherapeutic approaches to human allergic diseases. This is a unique feature of mucosal surfaces which represent the main contact interface with the external environment. However, the influence of oral tolerance on specific and natural polyreactive IgA antibodies, the major defense mechanism of the mucosa, is unknown. We have shown that oral administration of an extract of the dust mite Dermatophagoides pteronyssinus (Dp) to primed mice caused down-regulation of IgE responses and an increase in tumor growth factor-ß secretion. In the present study, we observed that primed inbred female A/Sn mice (8 to 10 weeks old) fed by gavage a total weight of 1.0-mg Dp extract on the 6th, 7th and 8th days post-immunization presented normal secretion of IL-4 and IL-10 in gut-associated lymphoid tissue and a decreased production of interferon gamma induced by Dp in the draining lymph nodes (13,340 ± 3,519 vs 29,280 ± 2,971 pg/ml). Mice fed the Dp extract also showed higher levels of serum anti-Dp IgA antibodies and an increase of IgA-secreting cells in mesenteric lymph nodes (N = 10), reflecting an increase in total fecal IgA antibodies (N = 10). The levels of secretory anti-Dp IgA antibodies increased after re-immunization regardless of Dp extract feeding. Oral tolerance did not interfere with serum or secretory IgA antibody reactivity related to self and non-self antigens. These results suggest that induction of oral tolerance to a Dp extract in sensitized mice triggered different regulatory mechanisms which inhibited the IgE response and stimulated systemic and secretory IgA responses, preserving the natural polyreactive IgA antibody production.

Quenching of the intrinsic fluorescence of bovine serum albumin by chlorpromazine and hemin

The binding of chlorpromazine (CPZ) and hemin to bovine serum albumin was studied by the fluorescence quenching technique. CPZ is a widely used anti-psychotic drug that interacts with blood components, influences bioavailability, and affects function of several biomolecules. Hemin is an important ferric residue of hemoglobin that binds within the hydrophobic region of albumin with high specificity. Quenching of the intrinsic fluorescence of bovine serum albumin (BSA) was observed by selectively exciting tryptophan residues at 290 nm. Emission spectra were recorded in the range from 300 to 450 nm for each quencher addition. Stern-Volmer graphs were plotted, and the quenching constant estimated for BSA solution titrated with hemin at 25ºC was 1.44 (± 0.05) x 10(5) M-1. Results showed that bovine albumin tryptophans are not equally accessible to CPZ, in agreement with the idea that polar or charged quenchers have more affinity for amino acid residues on the outer wall of the protein. Hemin added to albumin solution at a molar ratio of 1:1 quenched about 25% of their fluorescence. The quenching effect of CPZ on albumin-hemin solution was stronger than on pure BSA. This increase can be the result of combined conformational changes in the structure of albumin caused firstly by hemin and then by CPZ. Our results suggest that the primary binding site for hemin on bovine albumin may be located asymmetrically between the two tryptophans along the sequence formed by subdomains IB and IIA, closer to tryptophan residue 212.

Correlation between serum E-selectin levels and panoramic nailfold capillaroscopy in systemic sclerosis

E-selectin is expressed by the activated endothelium and its plasma levels are increased in patients with systemic sclerosis. Eighteen patients fulfilling the American Rheumatism Association criteria for systemic sclerosis, 15 females and 3 males, 42-70 years old, 9 with diffuse and 9 with limited forms, were sequentially recruited for this study. Serum E-selectin levels were determined by commercially available ELISA and their association with nailfold capillaroscopic abnormalities was investigated. Nailfold capillaries were analyzed by 16X magnification wide-field capillaroscopy. Two parameters on capillaroscopy were used to correlate to serum E-selectin: deletion and ectasia. Data were analyzed statistically by the Student t-test and Spearman correlation. Two-tailed P values below 0.05 were considered significant. E-selectin range was 38 to 200 ng/ml (80 ± 39.94). There was a correlation between serum E-selectin levels and the deletion capillaroscopic score (r = 0.50, P < 0.035). This correlation was even stronger within the first 48 months of diagnosis (r = 0.63, P < 0.048). On the other hand, no association was observed between selectin and ectasia. Patients with diffuse disease presented higher serum E-selectin levels than patients with limited disease, although the difference was not statistically significant (96.44 ± 48.04 vs 63.56 ± 21.77 ng/dl; P = 0.08). The present study is the first showing a correlation between soluble serum E-selectin levels and alterations in capillaroscopy. The stronger correlation of deletion score in capillaroscopy in early disease suggests that serum E-selectin levels might be a useful biochemical marker of disease activity in systemic sclerosis.

Serum laminin, type IV collagen and hyaluronan as fibrosis markers in non-alcoholic fatty liver disease

Hepatic fibrosis in patients with non-alcoholic fatty liver disease is associated with progression of the disease. In the present study, we analyzed the discriminative ability of serum laminin, type IV collagen and hyaluronan levels to predict the presence of fibrosis in these patients. In this preliminary report, we studied 30 overweight patients divided into two groups according to the absence (group I, N = 19) or presence (group II, N = 11) of fibrosis in a liver biopsy. Triglycerides, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltranspeptidade, hyaluronan (noncompetitive fluoroassay), type IV collagen, and laminin (ELISA) were determined. Group II presented significantly higher mean laminin, hyaluronan, type IV collagen, and aspartate aminotransferase values, which were due to the correlation between these parameters and the stage of fibrosis in the biopsy (Spearman's correlation coefficient, rS = 0.65, 0.62, 0.53, and 0.49, respectively). Analysis of the ROC curve showed that laminin values >282 ng/ml were those with the best diagnostic performance, with 87% accuracy. Association of laminin with type IV collagen showed improvement in the positive predictive value (100%), but with reduction in diagnostic sensitivity (64%). When compared with the criteria of Ratziu et al. [Gastroenterology (2000) 118: 1117-1123] for the diagnosis of septal fibrosis, laminin values presented a better diagnostic accuracy (83 vs 70%). Determination of extracellular matrix components in serum, especially of laminin, may identify patients with non-alcoholic fatty liver disease and fibrosis and these components may be used as indicators for liver biopsy in these patients.

High baseline serum total and LDL cholesterol levels are associated with MDR1 haplotypes in Brazilian hypercholesterolemic individuals of European descent

The MDR1 gene encodes the P-glycoprotein, an efflux transporter with broad substrate specificity. P-glycoprotein has raised great interest in pharmacogenetics because it transports a variety of structurally divergent drugs, including lipid-lowering drugs. The synonymous single-nucleotide polymorphism C3435T and the nonsynonymous single-nucleotide polymorphism G2677T/A in MDR1 have been indicated as potential determinants of variability in drug disposition and efficacy. In order to evaluate the effect of G2677T/A and C3435T MDR1 polymorphisms on serum levels of lipids before and after atorvastatin administration, 69 unrelated hypercholesterolemic individuals from São Paulo city, Brazil, were selected and treated with 10 mg atorvastatin orally once daily for four weeks. MDR1 polymorphisms were analyzed by PCR-RFLP. C3435T and G2677T polymorphisms were found to be linked. The allelic frequencies for C3435T polymorphism were 0.536 and 0.464 for the 3435C and 3435T alleles, respectively, while for G2677T/A polymorphism allele frequencies were 0.580 for the 2677G allele, 0.384 for the 2677T allele and 0.036 for the 2677A allele. There was no significant relation between atorvastatin response and MDR1 polymorphisms (repeated measures ANOVA; P > 0.05). However, haplotype analysis revealed an association between T/T carriers and higher basal serum total (TC) and LDL cholesterol levels (TC: 303 ± 56, LDL-C: 216 ± 57 mg/dl, respectively) compared with non-T/T carriers (TC: 278 ± 28, LDL-C: 189 ± 24 mg/dl; repeated measures ANOVA/Tukey test; P < 0.05). These data indicate that MDR1 polymorphism may have an important contribution to the control of basal serum cholesterol levels in Brazilian hypercholesterolemic individuals of European descent.

Serum and cerebrospinal fluid concentrations of E-selectin in patients with aneurysmal subarachnoid hemorrhage

The goal of the present study was to determine concentrations of E-selectin in both cerebrospinal fluid (CSF) and serum of patients with aneurysmal subarachnoid hemorrhage (SAH) and to evaluate the correlation between the clinical parameters and E-selectin levels. Both CSF and serum samples obtained from 12 patients with aneurysmal SAH and 8 patients with hydrocephalus (control group) without any other known central nervous system disease were assayed for E-selectin by quantitative enzyme-linked immunosorbent assay and the results were compared between the two groups. Mean levels of soluble forms of E-selectin within the first 3 days and on the 5th and 7th days of SAH were 4.0 ± 7.9, 2.8 ± 5.2, and 3.1 ± 4.9 ng/ml in the patient's CSF, and 33.7 ± 9.2, 35.1 ± 7.0, and 35.2 ± 8.7 ng/ml in serum, respectively. In contrast, mean E-selectin levels were 0.1 ± 0.2 ng/ml in CSF and 8.7 ± 5.0 ng/ml in serum of control patients. The difference between groups was statistically significant regarding both CSF and serum E-selectin levels (P < 0.05). Thus, we have demonstrated a marked increase of E-selectin concentration in both CSF and serum of patients with aneurysmal SAH compared with control and suggest that blocking the interaction between E-selectin and vascular endothelium may have a beneficial effect on vasospasms.

Genetic potential for an acute inflammatory response in IgA glomerulonephritis in mice

Mice selected on the basis of an acute inflammatory response (AIR) can provide information about the immunopathological mechanisms of glomerulonephritis. We studied the differences between mice selected for a maximal AIR (AIRmax that attract more polymorphonuclear cells to the site of injury) or a minimal AIR (AIRmin that attract more mononuclear cells) in an experimental model of IgA nephropathy in order to investigate the effect of genetic background on glomerular disease progression and the participation of the monocyte chemoattractant protein-1 (MCP-1) chemokine. IgA nephropathy was induced by intraperitoneal ovalbumin injection and bile duct ligation in AIRmax and AIRmin mice. Histological changes, urinary protein/creatinine ratio, serum IgA levels, immunofluorescence for IgA, IgG and complement C3 fraction, immunohistochemistry for macrophages and MCP-1, and MCP-1 levels in macerated kidney were determined. Mesangial IgA deposition was seen only in AIRmin mice, which presented more renal lesions. Increased serum IgA levels (1.5 ± 0.4 vs 0.3 ± 0.1 mg/mL, P < 0.001), high glomerular MCP-1 expression and decreased monocyte/macrophage infiltration in the interstitial area (0.3 ± 0.3 vs 1.1 ± 0.9 macrophages/field, P < 0.05) were detected in AIRmin mice compared to AIRmax mice. No glomerular monocyte/macrophage infiltration was detected in either strain. In spite of the absence of IgA deposition, AIRmax mice presented discrete or absent mesangial proliferation. The study showed that there are differences between mice selected for AIRmax and AIRmin with respect to serum IgA levels, histological damage and MCP-1 chemokine production after ovalbumin injection in combination with bile duct ligation.

Serum and cerebrospinal fluid S100B concentrations in patients with neurocysticercosis

The clinical manifestations of neurocysticercosis (NC) are varied and depend on the number and location of cysts, as well as on the host immune response. Symptoms usually occur in NC when cysticerci enter a degenerative course associated with an inflammatory response. The expression of brain damage markers may be expected to increase during this phase. S100B is a calcium-binding protein produced and released predominantly by astrocytes that has been used as a marker of reactive gliosis and astrocytic death in many pathological conditions. The aim of the present study was to investigate the levels of S100B in patients in different phases of NC evolution. Cerebrospinal fluid and serum S100B concentrations were measured in 25 patients with NC: 14 patients with degenerative cysts (D), 8 patients with viable cysts (V) and 3 patients with inactive cysts. All NC patients, except 1, had five or less cysts. In most of them, symptoms had been present for at least 1 month before sample collection. Samples from 8 normal controls (C) were also assayed. The albumin quotient was used to estimate the blood-brain barrier permeability. There were no significant differences in serum (P = 0.5) or cerebrospinal fluid (P = 0.91) S100B levels among the V, D, and C groups. These findings suggest that parenchymal changes associated with a relatively small number of degenerating cysts probably have a negligible impact on glial tissue.

A new model for the population pharmacokinetics of didanosine in healthy subjects

Didanosine (ddI) is a component of highly active antiretroviral therapy drug combinations, used especially in resource-limited settings and in zidovudine-resistant patients. The population pharmacokinetics of ddI was evaluated in 48 healthy volunteers enrolled in two bioequivalence studies. These data, along with a set of co-variates, were the subject of a nonlinear mixed-effect modeling analysis using the NONMEM program. A two-compartment model with first order absorption (ADVAN3 TRANS3) was fitted to the serum ddI concentration data. Final pharmacokinetic parameters, expressed as functions of the co-variates gender and creatinine clearance (CL CR), were: oral clearance (CL = 55.1 + 240 x CL CR + 16.6 L/h for males and CL = 55.1 + 240 x CL CR for females), central volume (V2 = 9.8 L), intercompartmental clearance (Q = 40.9 L/h), peripheral volume (V3 = 62.7 + 22.9 L for males and V3 = 62.7 L for females), absorption rate constant (Ka = 1.51/h), and dissolution time of the tablet (D = 0.43 h). The intraindividual (residual) variability expressed as coefficient of variation was 13.0%, whereas the interindividual variability of CL, Q, V3, Ka, and D was 20.1, 75.8, 20.6, 18.9, and 38.2%, respectively. The relatively high (>30%) interindividual variability for some of these parameters, observed under the controlled experimental settings of bioequivalence trials in healthy volunteers, may result from genetic variability of the processes involved in ddI absorption and disposition.

Lack of relationship between glycemic control and bone mineral density in type 2 diabetes mellitus

We assessed the effect of chronic hyperglycemia on bone mineral density (BMD) and bone remodeling in patients with type 2 diabetes mellitus. We investigated 42 patients with type 2 diabetes under stable control for at least 1 year, 22 of them with good metabolic control (GMC: mean age = 48.8 ± 1.5 years, 11 females) and 20 with poor metabolic control (PMC: mean age = 50.2 ± 1.2 years, 8 females), and 24 normal control individuals (CG: mean age = 46.5 ± 1.1 years, 14 females). We determined BMD in the femoral neck and at the L2-L4 level (DEXA) and serum levels of glucose, total glycated hemoglobin (HbA1), total and ionic calcium, phosphorus, alkaline phosphatase, follicle-stimulating hormone, intact parathyroid hormone (iPTH), 25-hydroxyvitamin D (25-OH-D), insulin-like growth factor I (IGFI), osteocalcin, procollagen type I C propeptide, as well as urinary levels of deoxypyridinoline and creatinine. HbA1 levels were significantly higher in PMC patients (12.5 ± 0.6 vs 7.45 ± 0.2% for GMC and 6.3 ± 0.9% for CG; P < 0.05). There was no difference in 25-OH-D, iPTH or IGFI levels between the three groups. BMD values at L2-L4 (CG = 1.068 ± 0.02 vs GMC = 1.170 ± 0.03 vs PMC = 1.084 ± 0.02 g/cm²) and in the femoral neck (CG = 0.898 ± 0.03 vs GMC = 0.929 ± 0.03 vs PMC = 0.914 ± 0.03 g/cm²) were similar for all groups. PMC presented significantly lower osteocalcin levels than the other two groups, whereas no significant difference in urinary deoxypyridine was observed between groups. The present results demonstrate that hyperglycemia is not associated with increased bone resorption in type 2 diabetes mellitus and that BMD is not altered in type 2 diabetes mellitus.

Lipoprotein lipase PvuII polymorphism is associated with variations in serum lipid levels in non-diabetic pregnant women

The aim of the present study was to determine if there is an association between the single nucleotide polymorphisms (SNPs) of the lipoprotein lipase (LPL) and apolipoprotein E (apo E) genes and the serum lipid profile in pregnancy and puerperium. Non-diabetic women of European descent in the third semester of pregnancy (N = 120) were selected. Those with diseases or other condition that could modify their lipid profile were excluded from the study (N = 32). Serum lipids were measured by routine laboratory procedures and genomic DNA was extracted by a salting out method. LPL (PvuII and HindIII) and apo E (HhaI) SNPs were detected by the polymerase chain reaction and restriction fragment length polymorphism. Categorical and continuous variables were compared by the chi-square test and Student t-test or ANOVA, respectively. Women carrying the LPL P1P1 genotype had higher serum LDL cholesterol (N = 21; 155 ± 45 mg/dL) than women carrying the P1P2/P2P2 genotypes (N = 67; 133 ± 45 mg/dL; P = 0.032). During the puerperium period, serum levels of triglycerides and VLDL cholesterol were significantly reduced in women carrying the P1P1 (73%, P = 0.006) and P1P2 (51%, P = 0.002) genotypes but not in women carrying the P2P2 genotype (23%, P > 0.05). On the other hand, serum concentrations of lipids did not differ between the LPL HindIII and apo E genotypes during pregnancy and after delivery. We conclude that LPL PvuII SNP is associated with variations in serum lipids during pregnancy and the puerperal period in non-diabetic women.

Biodistribution of 99m technetium- labeled creatinine in healthy rats

The distribution of creatinine, one of the toxic guanidine compounds, in various tissues has not been studied in detail by using radiolabeled creatinine. Our objective was to investigate the biodistribution of creatinine labeled with 99m technetium (99mTc) by the stannous (II) chloride method in healthy male Wistar rats. Quality controls were carried out by radio thin layer chromatography, high-performance liquid chromatography, and paper electrophoresis. The labeling yield was 85 ± 2% under optimum conditions (pH 7 and 100 µg stannous chloride). Rats (N = 12) were injected intravenously with 99mTc-creatinine and their blood and visceral organs were evaluated for 99mTc-creatinine uptake as percent of the injected dose per gram wet weight of each tissue (%ID/g). The lowest amount of uptake was detected in the brain and testis. When the rate of uptake was evaluated, only the kidney showed increasing rates of uptake of 99mTc-creatinine throughout the study. Kidneys showed the highest amount of uptake throughout the study (P < 0.001 compared to all other organs), followed by liver, spleen and lung tissue.

Early life, current socioeconomic position and serum lipids in young adulthood of participants in a cohort study initiated in 1978/1979

The association between socioeconomic position (SEP) and serum lipids has been little studied and the results have been controversial. A total of 2063 young adults born in 1978/79 were evaluated at 23-25 years of age in the fourth follow-up of a cohort study carried out in Ribeirão Preto, SP, Brazil, corresponding to 31.8% of the original sample. Total serum cholesterol (TC), triglycerides, high-density cholesterol (HDL cholesterol) and low-density cholesterol (LDL cholesterol) were analyzed according to SEP at birth and during young adulthood. SEP was classified into tertiles of family income and a cumulative score of socioeconomic disadvantage was created. TC was 11.85 mg/100 mL lower among men of lower SEP in childhood (P < 0.01) but no difference was found in women, whereas it was 8.46 lower among men (P < 0.01) and 8.21 lower among women of lower SEP in adulthood (P < 0.05). Individuals of lower SEP had lower LDL and HDL cholesterol, with small differences between sexes and between the two times in life. There was no association between SEP and triglyceride levels. After adjustment of income at one time point in relation to the other, some associations lost significance. The greater the socioeconomic disadvantage accumulated along life, the lower the levels of TC, LDL and HDL cholesterol (P < 0.05). The socioeconomic gradient of TC and LDL cholesterol was inverse, representing a lower cardiovascular risk for individuals of lower SEP, while the socioeconomic gradient of HDL cholesterol indicated a lower cardiovascular risk for individuals of higher SEP.

TP53 codon 72 polymorphism as a risk factor for cardiovascular disease in a Brazilian population

TP53, a tumor suppressor gene, has a critical role in cell cycle, apoptosis and cell senescence and participates in many crucial physiological and pathological processes. Identification of TP53 polymorphism in older people and age-related diseases may provide an understanding of its physiology and pathophysiological role as well as risk factors for complex diseases. TP53 codon 72 (TP53:72) polymorphism was investigated in 383 individuals aged 66 to 97 years in a cohort from a Brazilian Elderly Longitudinal Study. We investigated allele frequency, genotype distribution and allele association with morbidities such as cardiovascular disease, type II diabetes, obesity, neoplasia, low cognitive level (dementia), and depression. We also determined the association of this polymorphism with serum lipid fractions and urea, creatinine, albumin, fasting glucose, and glycated hemoglobin levels. DNA was isolated from blood cells, amplified by PCR using sense 5'-TTGCCGTCCCAAGCAATGGATGA-3' and antisense 5'-TCTGGGAAGGGACAGAAGATGAC-3' primers and digested with the BstUI enzyme. This polymorphism is within exon 4 at nucleotide residue 347. Descriptive statistics, logistic regression analysis and Student t-test using the multiple comparison test were used. Allele frequencies, R (Arg) = 0.69 and P (Pro) = 0.31, were similar to other populations. Genotype distributions were within Hardy-Weinberg equilibrium. This polymorphism did not show significant association with any age-related disease or serum variables. However, R allele carriers showed lower HDL levels and a higher frequency of cardiovascular disease than P allele subjects. These findings may help to elucidate the physiopathological role of TP53:72 polymorphism in Brazilian elderly people.

Coronary fat content evaluated by morphometry in patients with severe atherosclerosis has no relation with serum lipid levels

The relationship between lipid serum levels and coronary atherosclerotic plaque fat content was studied in 51 necropsy patients. Serum lipids were measured by standard techniques, during life, in the absence of lipid-lowering drugs. Intima, intimal fat and media areas were measured using a computerized system in cryosections of the odd segments of the right, anterior descending and circumflex coronary arteries stained with Sudan-IV. Mean intimal and lipid areas were 5.74 ± 1.98 and 1.22 ± 0.55 mm² (22.12 ± 8.48%) in 26 cases with high cholesterol (³200 mg/dL) and 4.98 ± 1.94 and 1.16 ± 0.66 mm² (22.75 ± 9.06%) in 25 cases with normal cholesterol (<200 mg/dL; P > 0.05). Patients with high levels of low-density lipoprotein (³130 mg/dL, N = 15) had a higher intima/media area ratio than those with normal levels of low-density lipoprotein (<130 mg/dL, N = 13, P < 0.01). No significant difference in the morphometrical variables was found in groups with high or low serum levels of triglycerides (³200 mg/dL, N = 13 vs <200 mg/dL, N = 36) or high-density lipoprotein (³35 mg/dL, N = 11 vs <35 mg/dL, N = 17). The association between the morphological measurements and serum levels of cholesterol, its fractions, and triglycerides was also tested and the correlation coefficients were low. Although high cholesterol is a risk factor, we show here that in patients with severe atherosclerosis blood cholesterol and triglyceride levels seem to have little influence on coronary lipid content, indicating that other factors may contribute to arterial lipid deposition and plaque formation.