Potential immunological markers for diagnosis of human strongyloidiasis using heterologous antigens.

Strongyloides venezuelensis is a parasitic nematode of rodents that is frequently used to obtain heterologous antigens for immunological diagnosis of human strongyloidiasis. The aim of this study was to identify antigens from filariform larvae of S. venezuelensis for immunodiagnosis of human strongyloidiasis. Soluble and membrane fractions from filariform larvae of S. venezuelensis were obtained in phosphate saline (SS and SM) and in Tris-HCl buffer (TS and TM), and were analysed by Western blotting. Different antigenic components were recognized by IgG antibodies from the sera of strongyloidiasis patients. Highest recognition was observed for a 30-40 kDa mass range present in all antigenic fractions. The band encompassing this mass range was then excised and subjected to mass spectrometry for protein identification. Immunoreactive proteins identified in the soluble fractions corresponded to metabolic enzymes, whereas cytoskeletal proteins and galectins were more abundant in the membrane fractions. These results represent the first approach towards identification of S. venezuelensis antigens for use in immunodiagnostic assays for human strongyloidiasis.

Uptake by breast carcinoma of a lipidic nanoemulsion after intralesional injection into the patients: a new strategy for neoadjuvant chemotherapy.

OBJECTIVE: Previously we showed that after intravenous injection a lipidic nanoemulsion concentrates in breast carcinoma tissue and other solid tumors and may carry drugs directed against neoplastic tissues. Use of the nanoemulsion decreases toxicity of the chemotherapeutic agents without decreasing the anticancer action. Currently, the hypothesis was tested whether the nanoemulsion concentrates in breast carcinoma tissue after locoregional injection. METHODS: Three different techniques of injection of the nanoemulsion were tested in patients scheduled for surgical treatment: G1 (n=4) into the mammary tissue 5 cm away from the tumor; G2 (n=4) into the peritumoral mammary tissue; G3 (n=6) into the tumoral tissue. The nanoemulsion labeled with radioactive cholesteryl oleate was injected 12 h before surgery; plasma decay of the label was determined from blood samples collected over 24 h and the tissue fragments excised during the surgery were analyzed for radioactivity uptake. RESULTS: Among the three nanoemulsion injection techniques, G3 showed the greatest uptake (data expressed in c.p.m/g of tissue) by the tumor (44,769+/-54,749) and by the lymph node (2356+/-2966), as well as the greatest concentration in tumor compared to normal tissue (844+/-1673). In G1 and G2, uptakes were, respectively, tumor: 60+/-71 and 843+/-1526; lymph node: 263+/-375 and 102+/-74; normal tissue: 139+/-102 and 217+/-413. CONCLUSIONS: Therefore, with intralesional injection of the nanoemulsion, a great concentration effect can be achieved. This injection technique may be thus a promising approach for drug-targeting in neoadjuvant chemotherapy in breast cancer treatment.

Presence of t(14;18) translocation in healthy individuals varies according to ethnic background in the Brazilian population.

Several groups have demonstrated that healthy individuals can present the t(14;18) translocation. In this report, the presence of the translocation was examined in healthy blood donors in Brazil, a country considered an ethnic melting pot. The translocation was detected by nested PCR in 227 peripheral blood samples from individuals with different ethnic backgrounds. The t(14;18) translocation was found in 45 of 85 White individuals (52.94%); in 57 of 72 Black individuals (79.17%); and in 68 of 70 individuals (97.14%) of Japanese-descent. In conclusion, the frequency of the t(14;18) translocation in the Brazilian population varies according to the ethnic background.

Hyperhomocystinemia in patients with coronary artery disease.

Hyperhomocystinemia has been related to an increased risk of cardiovascular disease in several studies. The C677T polymorphism for the gene that encodes the methylenetetrahydrofolate reductase enzyme (MTHFR) and low plasma folate levels are common causes of hyperhomocystinemia. Due to differences in nutritional patterns and genetic background among different countries, we evaluated the role of hyperhomocystinemia as a coronary artery disease (CAD) risk factor in a Brazilian population. The relation between homocysteine (Hcy) and the extent of CAD, measured by an angiographic score, was determined. A total of 236 patients referred for coronary angiography for clinical reasons were included. CAD was found in 148 (62.7%) patients and 88 subjects had normal or near normal arteries. Patients with CAD had higher Hcy levels [mean (SD)] than those without disease (14 (6.8) vs 12.5 (4.0) microM; P = 0.04). Hyperhomocystinemia (Hcy >17.8 microM) prevalence was higher in the CAD group: 31.1 vs 12.2% (P = 0.01). After adjustment for major risk factors, we found an independent association between hyperhomocystinemia and CAD (OR = 2.48; 95% CI = 1.02-6.14). Patients with a more advanced coronary score had a higher frequency of hyperhomocystinemia and tended to have higher mean Hcy levels. An inverse relation between plasma folate and Hcy levels was found (r = -0.14; P = 0.04). Individuals with the MTHFR C677T polymorphism had a higher prevalence of hyperhomocystinemia than those without the mutated allele. We conclude that hyperhomocystinemia is independently associated with CAD, with a positive association between Hcy level and disease severity.

Decreased circulating thrombomodulin is improved by tadalafil therapy in hypoxemic patients with advanced pulmonary arterial hypertension.

INTRODUCTION: Advanced pulmonary arterial hypertension (PAH) in patients with congenital cardiac communications and right-to-left shunting (Eisenmenger syndrome - PAH-ES) is associated with hypoxemia and decreased circulating levels of thrombomodulin (TM), probably reflecting decreased endothelial TM production. The combination of these two factors has been shown to induce fibrin deposition, with increased risk of thrombosis, a well known complication in this syndrome. PATIENTS AND METHODS: We tested the hypothesis that vasodilator therapy with the phosphodiesterase-5 inhibitor tadalafil, an approved drug for management of PAH could improve endothelial dysfunction markers, in particular plasma TM, in addition to improving the physical capacity (expected effect of pulmonary vasodilatation) in PAH-ES patients. This was a prospective observational study of treatment-naïve patients subjected to specific PAH therapy. Fifteen patients aged 12 to 51years (median 30years) were treated for 6months with a single daily dose of 40mg oral tadalafil. The physical capacity (distance walked during the 6-min walk test - 6MWD), systemic oxygen saturation and laboratory parameters were measured at baseline, and 90days and 180days of treatment. RESULTS: Plasma TM, which was decreased at baseline compared to controls (p<0.001) increased at 90 and 180days (p=0.003), and this was directly related (r=0.57, p=0.026) to improvement of oxygen saturation (p=0.008). Heightened baseline tissue-type plasminogen activator decreased during treatment (p=0.010), while heightened von Willebrand factor antigen remained unchanged. The 6MWD improved significantly (p<0.001). CONCLUSION: Tadalafil therapy improved circulating TM and tissue-type plasminogen activator, in addition to improving the physical capacity and oxygen saturation in PAH-ES patients.

Human breast milk contains a factor which markedly stimulates aortic prostacyclin synthesis.

Human breast milk has been shown to contain a potent factor which markedly stimulates the synthesis of prostacyclin by rabbit aorta. The stimulatory effect of colostrum was modest when compared to mature milk. This factor appears to be protein in nature with a molecular weight of less than 10 000. The main site of action of the factor appears to be on the conversion of arachidonic acid into prostacyclin. This factor might have a role in the optimal development of prostacyclin synthetic pathways which are not fully developed at birth.

Lipid peroxidation in the peritoneal fluid of infertile women with peritoneal endometriosis.

OBJECTIVE: To assess the level of lipid peroxidation in the peritoneal fluid of infertile women with peritoneal endometriosis and of fertile disease-free controls. STUDY DESIGN: Level of lipid peroxidation (malondialdeyde, malondialdeyde with copper addition, and cholest-3,5-dien-7-one) was measured in the peritoneal fluid obtained from 21 women with endometriosis-related infertility and from 21 fertile women having tubal ligation. RESULTS: : The level of lipid peroxidation did not differ significantly (P > 0.05) according to the stage of endometriosis. The level of lipid peroxidation (malondialdeyde, malondialdeyde with the addition of copper, and cholest-3,5-dien-7-one) did not differ significantly (P > 0.05) between patients with endometriosis-related infertility (0.07 nmol/ml, 0.34 nmol/ml, 0.24 microg/ml, respectively) and disease-free controls (0.04 nmol/ml, 0.21 nmol/ml, 0.25 microg/ml, respectively). CONCLUSION: The level of lipid peroxidation did not differ between women with endometriosis-related infertility and fertile disease-free controls, suggesting that increased reactive oxygen species may not be one of the factors responsible for compromised fertility in patients with endometriosis.

Circulating endothelial cells are increased in chronic myeloid leukemia blast crisis.

We measured circulating endothelial precursor cells (EPCs), activated circulating endothelial cells (aCECs), and mature circulating endothelial cells (mCECs) using four-color multiparametric flow cytometry in the peripheral blood of 84 chronic myeloid leukemia (CML) patients and 65 healthy controls; and vascular endothelial growth factor (VEGF) by quantitative real-time PCR in 50 CML patients and 32 healthy controls. Because of an increase in mCECs, the median percentage of CECs in CML blast crisis (0.0146%) was significantly higher than in healthy subjects (0.0059%, P<0.01) and in the accelerated phase (0.0059%, P=0.01). There were no significant differences in the percentages of CECs in chronic- or active-phase patients and healthy subjects (P>0.05). In addition, VEGF gene expression was significantly higher in all phases of CML: 0.245 in blast crisis, 0.320 in the active phase, and 0.330 in chronic phase patients than it was in healthy subjects (0.145). In conclusion, CML in blast crisis had increased levels of CECs and VEGF gene expression, which may serve as markers of disease progression and may become targets for the management of CML.

Abnormalities in circulating von Willebrand factor and survival in pulmonary hypertension.

BACKGROUND: Changes in circulating von Willebrand factor (vWF) have been widely used for evaluating the severity of endothelial dysfunction in vascular disorders. In pulmonary hypertension, quantitative and structural abnormalities in circulating von Willebrand factor have been identified. We therefore hypothesized that these abnormalities could have prognostic implications. PATIENTS AND METHODS: We studied 30 consecutive medically treated patients with primary (n = 11) or secondary precapillary pulmonary hypertension associated with congenital heart disease (n = 16) or schistosomiasis (n = 3). Plasma antigenic activity of vWF (vWF:Ag) was measured by electroimmunodiffusion. The relative concentration of low molecular weight vWF multimers (vWF:LMW/Total) was determined by Western immunoblotting. Results of initial evaluation were analyzed at the end of the first and third years of follow-up. RESULTS: Baseline vWF:Ag activity (P <0.0002) and the vWF: LMW/Total ratio (P <0.005) were higher in patients who died during the first year than in survivors. All patients with vWF:Ag activity >250% or a vWF:LMW/Total ratio >70% died in the first year. All 7 patients with vWF:Ag activity <100% were alive at the end of 3 years of follow-up. A vWF:LMW/Total ratio >68% was 67% sensitive and 95% specific for 1-year mortality, with an overall predictive value of 80%. Both vWF:Ag levels and mortality were greater in the patients with primary pulmonary hypertension than in patients with secondary pulmonary hypertension. CONCLUSION: Patients with pulmonary hypertension who have abnormalities in circulating vWF have reduced 1-year survival. This might affect decisions such as patient assignment to lung transplantation.

Metabolism of an artificial emulsion resembling chylomicrons in patients with multiple myeloma.

Multiple myeloma, as other neoplastic diseases, is accompanied by alterations in lipid metabolism. The metabolism of chylomicrons is unexplored in this condition, despite the importance of these lipoproteins for the energy body supply. Chylomicron metabolism in the bloodstream consists of lipolysis by lipoprotein lipase and uptake of remnants by the liver. Triglyceride-rich emulsions can mimic chylomicron metabolism in man and are a useful tool to evaluate this metabolic pathway. A double-labeled chylomicron-resembling emulsion was injected into 20 patients with multiple myeloma and 30 normolipidemic healthy subjects. The plasma kinetic curves of the emulsion 3H-triglyceride and 14C-cholesteryl ester were determined in plasma samples collected over 60 minutes. The fractional clearance rate (FCR) of triglycerides in multiple myeloma was not changed compared to controls. However, FCR of cholesteryl esters was smaller in multiple myeloma (0.025 +/- 0.003 and 0.061 +/- 0.010 min(-1), respectively). These results indicate that chylomicron lipolysis is not affected in multiple myeloma, whereas remnant removal is diminished.

Abnormal multimeric and oligomeric composition is associated with enhanced endothelial expression of von Willebrand factor in pulmonary hypertension.

Abnormalities in endothelial von Willebrand factor (vWF) structure have been reported in pulmonary hypertension. These include loss of high molecular weight plasma multimers, resulting in decreased biologic activity. If endothelial processing of vWF is altered in this disorder, abnormalities in oligomeric composition may also be expected. We examined this possibility in ten adult patients with primary pulmonary hypertension. Enhanced endothelial vWF expression in these patients was indicated by increased plasma levels of vWF antigen (vWF:Ag) (214 +/- 91 vs 99 +/- 51 percent activity in controls, p < 0.001) and intense immunoperoxidase stain of pulmonary arterial endothelium for vWF (autopsy, 1 patient). Plasma from these patients also had a decreased capacity of inducing platelet aggregation in the presence of ristocetin, relative to vWF:Ag levels (57 +/- 20 percent activity). In addition to mild loss of the largest multimers, changes in oligomeric composition of plasma vWF were observed in most patients using both agarose and polyacrylamide gel electrophoresis. These included decreased concentration of dimeric (470 kDa) vWF in most patients, variable concentration of the 860-kDa fraction, and a relative decrease in subunit (223 kDa) density in subjects with elevated vWF:Ag. These findings provide additional information on the mechanisms responsible for endothelial production of dysfunctional vWF in patients with pulmonary hypertension.

Stimulation of human smooth muscle cell proliferation by thrombin involves increased synthesis of platelet-derived growth factor.

OBJECTIVES: Thrombin generated at sites of vascular injury not only participates in the coagulation cascade but can also signal other events related to cell mitogenesis and migration. In this report, we investigated the effects of thrombin on the proliferation of human arterial smooth muscle cells (SMCs) in culture and its interaction with platelet-derived growth factor (PDGF). MATERIAL AND METHODS: Human arterial SMCs originated from a renal artery were grown in cell culture. The effect of thrombin on DNA synthesis was evaluated by 3H-thymidine incorporation. The effect of thrombin on inositol-phosphate formation by SMCs was also analyzed as well as the binding of PDGF AA and BB to these cells. PDGF secretion was analyzed by radioimmunoassay (RIA). RESULTS: Exposure of cultured human SMCs to thrombin caused an increased rate of DNA synthesis in a dose-response manner, with a maximal stimulatory effect at a concentration of 2.0 U/mL. Thrombin was found to increase the accumulation of inositol phosphates and to increase the production of PDGF as measured by RIA. Exposure of cells to 2.0 U/mL thrombin resulted in a strong decrease in PDGF AA binding to PDGF receptors and did not change PDGF BB binding, probably indicating that PDGF alpha-receptors could be occupied by endogenously produced PDGF A. CONCLUSION: Thrombin stimulates human vascular SMC proliferation in a dose-response way, in part by the formation of inositol phosphates. The mechanism responsible for this effect involves, at least in part, an increased endogenous synthesis of PDGF.

Differential effects of enzymatic treatments on the storage and secretion of von Willebrand factor by human endothelial cells.

Enzymatic treatment used for passaging of endothelial cells may induce release of von Willebrand factor (vWF). Decreased ability to replenish intracellular stores results in decreased secretion of vWF in later passages of cells. Since both trypsin and pancreatin complex have been used for passaging endothelial cells, we analyzed the effects of successive passaging with these two enzyme preparations on the storage and secretion of vWF by human umbilical vein endothelial cells (HUVECs). Measurements were performed after the second to fifth passages. Cytoplasmic vWF was analyzed by indirect immunofluorescence and secreted vWF was measured in the supernatant of cultured HUVECs by ELISA. In trypsin-passaged cells, secreted vWF decreased progressively from passages 2 to 5. Respective concentrations were 355.0 +/- 30.4, 201.0 +/- 84.5, 150.0 +/- 1.4 and 120.5 +/- 38.9 ng vWF/10(5) cells. Comparatively, pancreatin-passaged cells secreted even less vWF protein (P = .001) at passages 4 and 5 (108.5 +/- 12.0 and 100.0 +/- 4.2 ng/10(5) cells, respectively) and had less vWF-positive cytoplasmic granules per cell. Thus, in experiments involving measurements of endothelial vWF, the use of low passage cells is recommendable and passaging with a pure trypsin preparation appears to be more appropriate.

Effect of lipid-free milk on prostaglandin synthesis and lipid esterification in the young rabbit aorta.

The effect of lipid-free human milk on prostaglandin synthesis and lipid esterification by [14C]-arachidonic acid was examined in one week-old rabbit aortas, in vitro. In the presence of albumin, lipid-free milk increased arachidonic acid incorporation into aortic phospholipids, cholesterol esters and triglycerides but not into mono- and diglycerides. Lipid-free milk also increased the conversion of arachidonic acid into aortic 6-keto-PGF1 alpha, but not into PGF2 alpha or PGE2.

A novel property of an aqueous guaraná extract (Paullinia cupana): inhibition of platelet aggregation in vitro and in vivo.

Aqueous extracts of guaraná were studied in terms of effects on the aggregation of human and rabbit platelets. Guaraná extracts have anti-aggregatory and de-aggregatory actions on platelet aggregation induced by ADP or arachidonate but not by collagen. The active material was shown to be water soluble and heat resistant and appeared to be different from salicylates, nicotinic acid or known xanthines. Guaraná extracts inhibited platelet aggregation in rabbits following either intravenous or oral administration.

Papaverine inhibits human platelet aggregation induced by ADP.

1. The in vitro and ex vivo effect of therapeutic levels of papaverine on human platelet aggregation induced by 3-5 microM adenosine-5'-diphosphate (ADP) was evaluated in platelet-rich plasma (PRP) by photometric and impedance aggregometry, and in whole blood by impedance aggregometry. 2. Platelet aggregation induced by 3-5 microM ADP in whole blood was significantly inhibited by 5.32 and 10.64 microM papaverine in vitro. This effect was also observed in PRP enriched with erythrocytes but not in PRP alone or enriched with leukocytes. 3. Papaverine (5.32 microM) significantly enhanced the antiplatelet activity of adenosine (0.75 microM) in human whole blood, an effect that was not observed in PRP. 4. A single oral dose of 100 mg papaverine hydrochloride, given to eight healthy human volunteers 1 h before the platelet aggregation evaluation, significantly inhibited the platelet aggregation induced by 3-5 microM ADP in whole blood. This effect was not observed in PRP. 5. Oral administration of the same dose at 8-h intervals (10 times) to seven additional healthy human volunteers led to a significant negative correlation (r = -0.55, P < 0.01) between the slope of platelet aggregation in whole blood and plasma papaverine levels (0.12-0.75 microM). 6. Papaverine and adenosine, alone or together, had no in vitro effect on whole blood platelet aggregation of male Wistar rats measured by impedance aggregometry. 7. These results suggest that papaverine inhibits human platelet aggregation in whole blood by an interaction with red blood cells.

An aqueous extract of guaraná (Paullinia cupana) decreases platelet thromboxane synthesis.

The effects of an aqueous extract of guaraná (Paullinia cupana) on rabbit platelet aggregation and thromboxane synthesis were examined. The guaraná extract (100 mg/ml) and fractions separated by TLC (origin and xanthines) decreased platelet aggregation (37, 27 and 31% of control values, respectively) and platelet thromboxane formation from [14C]-arachidonic acid (78, 70 and 50% of control values, respectively). The decreased thromboxane synthesis could be responsible, at least in part, for the antiaggregatory action of guaraná.

DNA polymorphisms of apolipoprotein B and AI-CII-AIV genes in a Brazilian population: a preliminary report.

Possible associations between coronary heart disease (CHD) and restriction fragment length polymorphisms (RFLPs) in the apo AI-CII-AIV cluster and the apo B gene were investigated in a Brazilian population consisting of 46 patients with CHD and 24 individuals without evidence of CHD. A preliminary genetic analysis of SstI RFLP in the apo AI-CII-AIV cluster showed a significantly higher frequency of the rare SstI allele (S2) in CHD patients as compared with controls. No significant differences were found in the frequencies of PstI RFLP in the apo AI-CII-AIV cluster or XbaI and EcoRI RFLPs in the apo B gene between CHD patients and controls. Moreover, no association was seen between the RFLPs studied and myocardial infarction or plasma cholesterol or triglyceride levels.

Association of hepatic nuclear factor-4 in the apolipoprotein B promoter: a preliminary report.

Previous studies have examined the arrangement of regulatory elements along the apolipoprotein B (apoB) promoter region (-3067 to +940) and a promoter fragment extending from nucleotides -150 to +124 has been demonstrated to be essential for transcriptional activation of the apoB gene in hepatic and intestinal cells. It has also been shown that transcriptional activation of apoB requires a synergistic interaction between hepatic nuclear factor-4 (HNF-4) and CCAAT/enhancer-binding protein alpha (C/EBP alpha) transcription factors. Here, we have examined the hypothesis that HNF-4 factor binding to DNA may induce a DNA helix bend, thus facilitating the communication with a C/EBP alpha factor located one helix turn from this HNF-4 factor in the apoB promoter. A gel electrophoretic mobility shift assay using wild type double-stranded oligonucleotides or modified wild type duplex oligonucleotides with 10 nucleotides inserted between HNF-4 and C/EBP alpha factor motifs showed similar retarded complexes, indicating that HNF-4 and C/EBP alpha factors interact independently of the distance between binding sites. However, when only one base, a thymidine, was inserted at the -71 position of the apoB promoter, the complex shift was completely abolished. In conclusion, these results regarding the study of the mechanisms involving the interaction between HNF-4 and C/EBP alpha factors in the apoB promoter suggest that the perfect 5'-CCCTTTGGA-3' motif is needed in order to facilitate the interaction between the two factors.

Ethnicity and glutathione S-transferase (GSTM1/GSTT1) polymorphisms in a Brazilian population.

The distribution of polymorphisms related to glutathione S-transferases (GST) has been described in different populations, mainly for white individuals. We evaluated the distribution of GST mu (GSTM1) and theta (GSTT1) genotypes in 594 individuals, by multiplex PCR-based methods, using amplification of the exon 7 of CYP1A1 gene as an internal control. In São Paulo, 233 whites, 87 mulattos, and 137 blacks, all healthy blood-donor volunteers, were tested. In Bahia, where black and mulatto populations are more numerous, 137 subjects were evaluated. The frequency of the GSTM1 null genotype was significantly higher among whites (55.4%) than among mulattos (41.4%; P = 0.03) and blacks (32.8%; P < 0.0001) from São Paulo, or Bahian subjects in general (35.7%; P = 0.0003). There was no statistically different distribution among any non-white groups. The distribution of GSTT1 null genotype among groups did not differ significantly. The agreement between self-reported and interviewer classification of skin color in the Bahian group was low. The interviewer classification indicated a gradient of distribution of the GSTM1 null genotype from whites (55.6%) to light mulattos (40.4%), dark mulattos (32.0%) and blacks (28.6%). However, any information about race or ethnicity should be considered with caution regarding the bias introduced by different data collection techniques, specially in countries where racial admixture is intense, and ethnic definition boundaries are loose. Because homozygous deletions of GST gene might be associated with cancer risk, a better understanding of chemical metabolizing gene distribution can contribute to risk assessment of humans exposed to environmental carcinogens.