Comparison of the specificity of PCR and the histopathological detection of leishmania for the diagnosis of American cutaneous leishmaniasis

More precise and rapid diagnostic methods for American cutaneous leishmaniasis (ACL) are necessary because of the growing number of cases observed in Brazil, including the northeastern region of the State of São Paulo. We applied PCR to 54 skin or mucosal biopsies from patients with a clinical and/or laboratory diagnosis of ACL using primers 13A and 13B, with positive results being obtained for 82% of the samples. When the PCR results were compared to those of histopathological leishmania detection, PCR showed superior results with 81.5% sensitivity and 95% CI of 68.0-95.1%. The Montenegro skin test (MST) was positive in 88.7% of patients. Since MST cannot be used as a diagnostic tool in endemic areas, the present results strongly suggest the use of PCR for the etiological confirmation of ACL, with emphasis on the mucosal form.

DNA-lipid systems: A physical chemistry study

It is well known that the interaction of polyelectrolytes with oppositely charged surfactants leads to an associative phase separation; however, the phase behavior of DNA and oppositely charged surfactants is more strongly associative than observed in other systems. A precipitate is formed with very low amounts of surfactant and DNA. DNA compaction is a general phenomenon in the presence of multivalent ions and positively charged surfaces; because of the high charge density there are strong attractive ion correlation effects. Techniques like phase diagram determinations, fluorescence microscopy, and ellipsometry were used to study these systems. The interaction between DNA and catanionic mixtures (i.e., mixtures of cationic and anionic surfactants) was also investigated. We observed that DNA compacts and adsorbs onto the surface of positively charged vesicles, and that the addition of an anionic surfactant can release DNA back into solution from a compact globular complex between DNA and the cationic surfactant. Finally, DNA interactions with polycations, chitosans with different chain lengths, were studied by fluorescence microscopy, in vivo transfection assays and cryogenic transmission electron microscopy. The general conclusion is that a chitosan effective in promoting compaction is also efficient in transfection.

Skin secretion of Siphonops paulensis (Gymnophiona, Amphibia) forms voltage-dependent ionic channels in lipid membranes

The effect of the skin secretion of the amphibian Siphonops paulensis was investigated by monitoring the changes in conductance of an artificial planar lipid bilayer. Skin secretion was obtained by exposure of the animals to ether-saturated air, and then rinsing the animals with distilled water. Artificial lipid bilayers were obtained by spreading a solution of azolectin over an aperture of a Delrin cup inserted into a cut-away polyvinyl chloride block. In 9 of 12 experiments, the addition of the skin secretion to lipid bilayers displayed voltage-dependent channels with average unitary conductance of 258 ± 41.67 pS, rather than nonspecific changes in bilayer conductance. These channels were not sensitive to 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid or tetraethylammonium ion, but the experimental protocol used does not permit us to specify their characteristics.

High specificity PCR screening for 22q11.2 microdeletion in three different ethnic groups

Congenital heart defects are the most common of all human birth defects. Numerous studies have shown that a deletion within chromosome 22q11 is associated with DiGeorge syndrome and certain forms of sporadic congenital cardiovascular disease. We have determined the value of a PCR assay using markers D22S941, D22S944 and D22S264 designed for the screening of 22q11.2 deletion through consecutive homozygosity in an ethnically admixed urban population. The study population comprised 149 unrelated men and women from three different ethnic groups (white, mulatto and black). Test specificity for the overall population was estimated at 98.3%. We found no significant difference when comparing heterozygosity indices and ethnicity (P value = 0.43 (D22S944), 0.22 (D22S264), and 0.58 (D22S941)). There was no significant difference regarding assay specificity between the three different ethnic groups studied. This assay could constitute a cost-effective way to screen a large number of patients at increased risk, since PCR techniques are easily available, are fast, can be automatized, and are significantly less expensive than fluorescence in situ hybridization.

Lipid peroxidation and total radical-trapping potential of the lungs of rats submitted to chronic and sub-chronic stress

Exposure to stress induces a cluster of physiological and behavioral changes in an effort to maintain the homeostasis of the organism. Long-term exposure to stress, however, has detrimental effects on several cell functions such as the impairment of antioxidant defenses leading to oxidative damage. Oxidative stress is a central feature of many diseases. The lungs are particularly susceptible to lesions by free radicals and pulmonary antioxidant defenses are extensively distributed and include both enzymatic and non-enzymatic systems. The aim of the present study was to determine lipid peroxidation and total radical-trapping potential (TRAP) changes in lungs of rats submitted to different models of chronic stress. Adult male Wistar rats weighing 180-230 g were submitted to different stressors (variable stress, N = 7) or repeated restraint stress for 15 (N = 10) or 40 days (N = 6) and compared to control groups (N = 10 each). Lipid peroxidation levels were assessed by thiobarbituric acid reactive substances (TBARS), and TRAP was measured by the decrease in luminescence using the 2-2'-azo-bis(2-amidinopropane)-luminol system. Chronic variable stress induced a 51% increase in oxidative stress in lungs (control group: 0.037 ± 0.002; variable stress: 0.056 ± 0.007, P < 0.01). No difference in TBARS was observed after chronic restraint stress, but a significant 57% increase in TRAP was presented by the group repeatedly restrained for 15 days (control group: 2.48 ± 0.42; stressed: 3.65 ± 0.16, P < 0.05). We conclude that different stressors induce different effects on the oxidative status of the organism.

Effect of dietary fat saturation on lipid metabolism, arachidonic acid turnover and peritoneal macrophage oxidative stress in mice

We investigated the effects of a saturated fat diet on lipid metabolism and arachidonic acid (AA) turnover in mouse resident peritoneal macrophages. The pro-oxidative effect of this diet was also studied. Female C57BL/6 mice were weaned at 21 days of age and assigned to either the experimental diet containing coconut oil (COCO diet), or the control diet containing soybean oil as fat source (10 mice per group). The fat content of each diet was 15% (w/w). Mice were fed for 6 weeks and then sacrificed. The concentration of total lipids, triglycerides, (LDL + VLDL)-cholesterol, thiobarbituric acid-reactive substances (TBARS) and reduced glutathione were increased in the plasma of mice fed the COCO diet, without changes in phospholipid or total cholesterol concentrations compared to control. The concentrations of total cholesterol, free and esterified cholesterol, triglycerides, and TBARS were increased in the macrophages of COCO-fed mice, while the content of total phospholipids did not change. The phospholipid composition showed an increase of phosphatidylcholine and a decrease of phosphatidylethanolamine. The [³H]-AA distribution in the phospholipid classes showed an increase in phosphatidylcholine and phosphatidylethanolamine. Incorporation of [³H]-cholesterol into the macrophages of COCO-fed mice and into the cholesterol ester fraction was increased. The COCO diet did not affect [³H]-AA uptake but induced an increase in [³H]-AA release. The COCO diet also enhanced AA mobilization induced by lipopolysaccharide. These results indicate that the COCO diet, high in saturated fatty acids, alters the lipid metabolism and AA turnover of peritoneal macrophages in female mice and also produces a significant degree of oxidative stress.

Elevated levels of erythrocyte-conjugated dienes indicate increased lipid peroxidation in schistosomiasis mansoni patients

Schistosoma mansoni causes liver disease by inducing granulomatous inflammation. This favors formation of reactive oxygen species, including superoxide ions, hydrogen peroxide and hydroxyl radicals all of which may induce lipid peroxidation. We have evaluated lipid peroxidation in 18 patients with hepatosplenic schistosomiasis mansoni previously treated with oxamniquine followed by splenectomy, ligature of the left gastric vein and auto-implantation of spleen tissue, by measuring levels of erythrocyte-conjugated dienes and plasma malondialdehyde (MDA). Age-matched, healthy individuals (N = 18) formed the control group. Erythrocyte-conjugated dienes were extracted with dichloromethane/methanol and quantified by UV spectrophotometry, while plasma MDA was measured by reaction with thiobarbituric acid. Patient erythrocytes contained two times more conjugated dienes than control cells (584.5 ± 67.8 vs 271.7 ± 20.1 µmol/l, P < 0.001), whereas the increase in plasma MDA concentration (about 10%) was not statistically significant. These elevated conjugated dienes in patients infected by S. mansoni suggest increased lipid peroxidation in cell membranes, although this was not evident when a common marker of oxidative stress, plasma MDA, was measured. Nevertheless, these two markers of lipid peroxidation, circulating MDA and erythrocyte-conjugated dienes, correlated significantly in both patient (r = 0.62; P < 0.01) and control (r = 0.57; P < 0.05) groups. Our data show that patients with schistosomiasis have abnormal lipid peroxidation, with elevated erythrocyte-conjugated dienes implying dysfunctional cell membranes, and also imply that this may be attenuated by the redox capacity of antioxidant agents, which prevent accumulation of plasma MDA.

APOA1/C3/A4 gene cluster variability and lipid levels in Brazilian children

Genetic studies have suggested that polymorphisms of genes coding for apolipoproteins are significant determinants of serum lipoprotein and lipid levels in adults. However, only a few studies have investigated the association of these polymorphisms in children. Therefore, in the present investigation we studied the distribution of APOA1 -75 G>A, +83 C>T, APOC3 -482 C>T, -455 T>C and 3238 C>G, and APOA4 Q360H and T347S polymorphisms and their influence on plasma lipoprotein levels in children from a Brazilian northeastern admixed population. The seven polymorphic sites were genotyped in 414 children aged 5 to 15 years (mean 8.9 ± 2.9). The genotypes of the seven polymorphic sites were assessed by PCR-RFLP methods. The frequencies of the less common alleles were, in general, intermediate among parental populations, as expected. Strong linkage disequilibrium was detected between polymorphisms at the APOA1, APOC3 and APOA4 loci in this admixed population sample. Overall the genotype effects seen in adults were weaker or absent in children. The APOC3/-455 and APOA4 T347S variants showed significant effects on HDL cholesterol in girls (P = 0.033 and P = 0.016, respectively). Significantly higher plasma total (P = 0.003) and LDL cholesterol (P = 0.004) levels were observed in boys who were carriers of the 3238G allele at the APOC3/3238 C>G site. These results disclosed an overall absence of associations between these polymorphisms and lipids in children. This finding is not unexpected because expression of the effect of these polymorphisms might depend on the interaction with environmental variables both internal and external to the individual.

Atherogenic lipid profile of Brazilian near-term newborns

Cardiovascular disease is the primary cause of death in Brazil. Recent studies have shown that low birth weight and preterm birth are linked to a higher prevalence of cardiovascular disease. The aim of the present study was to compare the levels of lipids and apolipoproteins and atherogenic indexes between term and near-term newborn infants. A sample of umbilical cord blood was obtained from 135 newborns (66 males) divided into two groups: 25 near-term neonates (35-36.6 weeks of gestational age) and 110 term neonates (37-42 weeks of gestational age). The total cholesterol concentrations were higher in the near-term neonates than in the term group (94.04 ± 8.02 vs 70.42 ± 1.63 mg/dl, P < 0.01), due to an increase in the LDL-cholesterol fraction in the near-term group (57.76 ± 6.39 vs 34.38 ± 1.29 mg/dl, P < 0.001). The atherogenic indexes (total cholesterol/HDL-cholesterol, LDL-cholesterol/HDL-cholesterol and apolipoprotein B/apolipoprotein A-I) were higher in the near-term group (P < 0.001, P < 0.001, and P < 0.05, respectively). The gestational age of the newborns was inversely correlated with total cholesterol and LDL-cholesterol, and also with the total cholesterol/HDL-cholesterol and LDL-cholesterol/HDL-cholesterol indexes. These findings demonstrate that the lipid profile is worse in the group of near-term neonates compared with the term group. Future studies are needed to determine if this atherogenic profile in near-term neonates can affect body metabolism, increasing the risk for cardiovascular diseases in adult life.

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.

Effects of metformin on the glycemic control, lipid profile, and arterial blood pressure of type 2 diabetic patients with metabolic syndrome already on insulin

Fifty-seven type 2 diabetic patients with metabolic syndrome and on insulin were assessed by a paired analysis before and 6 months after addition of metformin as combination therapy to evaluate the impact of the association on glycemic control, blood pressure, and lipid profile. This was a historical cohort study in which the files of type 2 diabetic patients with metabolic syndrome on insulin were reviewed. The body mass index (BMI), waist circumference, lipid profile, A1C level, fasting blood glucose level, daily dose of NPH insulin, systolic blood pressure, and diastolic blood pressure were assessed in each patient before the start of metformin and 6 months after the initiation of combination therapy. Glycemic control significantly improved (P < 0.001) after the addition of metformin (1404.4 ± 565.5 mg/day), with 14% of the 57 patients reaching A1C levels up to 7%, and 53% reaching values up to 8%. There was a statistically significant reduction (P < 0.05) of total cholesterol (229.0 ± 29.5 to 214.2 ± 25.0 mg/dL), BMI (30.7 ± 5.4 to 29.0 ± 4.0 kg/m²), waist circumference (124.6 ± 11.7 to 117.3 ± 9.3 cm), and daily necessity of insulin. The reduction of total cholesterol occurred independently of the reductions of A1C (9.65 ± 1.03 to 8.18 ± 1.01%) and BMI and the reduction of BMI and WC did not interfere with the improvement of A1C. In conclusion, our study showed the efficacy of the administration of metformin and insulin simultaneously without negative effects. No changes were detected in HDL-cholesterol or blood pressure.

Relevance of apolipoprotein E4 for the lipid profile of Brazilian patients with coronary artery disease

Apolipoprotein E (apoE - e2, e3, e4 alleles) plays a role in the regulation of lipid metabolism, with the e4 considered to be a risk factor for coronary artery disease (CAD). We aimed to evaluate the apoE polymorphisms in Brazilians with CAD and their influence on the lipid profile and other risk factors (hypertension, diabetes mellitus, smoking). Two hundred individuals were examined: 100 patients with atherosclerosis confirmed by coronary angiography and 100 controls. Blood samples were drawn to determine apoE polymorphisms and lipid profile. As expected, the e3 allele was prevalent in the CAD (0.87) and non-CAD groups (0.81; P = 0.099), followed by the e4 allele (0.09 and 0.14, respectively; P = 0.158). The e3/3 (76 and 78%) and e3/4 (16 and 23%) were the most common genotypes for patients and controls, respectively. The lipid profile was altered in patients compared to controls (P < 0.05), independently of the e4 allele. However, in the controls this allele was prevalent in individuals with elevated LDL-cholesterol levels only (odds ratio = 2.531; 95% CI = 1.028-6.232). The frequency of risk factors was higher in the CAD group (P < 0.05), but their association with the lipid profile was not demonstrable in e4 carriers. In conclusion, the e4 allele is not associated with CAD or lipid profile in patients with atherosclerosis. However, its frequency in the non-CAD group is associated with increased levels of LDL-cholesterol, suggesting an independent effect of the e4 allele on lipid profile when the low frequency of other risk factors in this group is taken into account.

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.

The role of autolysis loop in determining the specificity of coagulation proteases

We recently demonstrated that the substitution of the autolysis loop (residues 143 to 154 in the chymotrypsin numbering system) of activated protein C (APC) with the corresponding loop of factor Xa (fXa) renders the APC mutant (APC/fX143-154) susceptible to inhibition by antithrombin (AT) in the presence of pentasaccharide. Our recent results further indicated, that in addition to an improvement in the reactivity of APC/fX143-154 with AT, both the amidolytic and anti-factor Va activities of the mutant APC have also been significantly increased. Since the autolysis loop of APC is five residues longer than the autolysis loop of fXa, it could not be ascertained whether this loop in the mutant APC specifically interacts with the activated conformation of AT or if a shorter autolysis loop is responsible for a global improvement in the catalytic activity of the mutant protease. To answer this question, we prepared another APC mutant in which the autolysis loop of the protease was replaced with the corresponding loop of trypsin (APC/Tryp143-154). Unlike an ~500-fold improvement in the reactivity of APC/fX143-154 with AT in the presence of pentasaccharide, the reactivity of APC/Tryp143-154 with the serpin was improved ~10-fold. These results suggest that both the length and structure of residues of the autolysis loop are critical for the specificity of the coagulation protease interaction with AT. Further factor Va inactivation studies with the APC mutants revealed a similar role for the autolysis loop of APC in the interaction with its natural substrate.

Ambient particulate air pollution from vehicles promotes lipid peroxidation and inflammatory responses in rat lung

Oxidative stress plays a major role in the pathogenesis of particle-dependent lung injury. Ambient particle levels from vehicles have not been previously shown to cause oxidative stress to the lungs. The present study was conducted to a) determine whether short-term exposure to ambient levels of particulate air pollution from vehicles elicits inflammatory responses and lipid peroxidation in rat lungs, and b) determine if intermittent short-term exposures (every 4 days) induce some degree of tolerance. Three-month-old male Wistar rats were exposed to ambient particulate matter (PM) from vehicles (N = 30) for 6 or 20 continuous hours, or for intermittent (5 h) periods during 20 h for 4 consecutive days or to filtered air (PM <10 µm; N = 30). Rats continuously breathing polluted air for 20 h (P-20) showed a significant increase in the total number of leukocytes in bronchoalveolar lavage compared to control (C-20: 2.61 x 105 ± 0.51;P-20: 5.01 x 105 ± 0.81; P < 0.05) and in lipid peroxidation ([MDA] nmol/mg protein: C-20: 0.148 ± 0.01; P-20: 0.226 ± 0.02; P < 0.05). Shorter exposure (6 h) and intermittent 5-h exposures over a period of 4 days did not cause significant changes in leukocytes. Lipid damage resulting from 20-h exposure to particulate air pollution did not cause a significant increase in lung water content. These data suggest oxidative stress as one of the mechanisms responsible for the acute adverse respiratory effects of particles, and suggest that short-term inhalation of ambient particulate air pollution from street with high automobile traffic represents a biological hazard.