First Description of a Cluster of Acute/Subacute Paracoccidioidomycosis Cases and Its Association with a Climatic Anomaly

Background: Identifying clusters of acute paracoccidioidomycosis cases could potentially help in identifying the environmental factors that influence the incidence of this mycosis. However, unlike other endemic mycoses, there are no published reports of clusters of paracoccidioidomycosis. Methodology/Principal Findings: A retrospective cluster detection test was applied to verify if an excess of acute form (AF) paracoccidioidomycosis cases in time and/or space occurred in Botucatu, an endemic area in Sao Paulo State. The scan-test SaTScan v7.0.3 was set to find clusters for the maximum temporal period of 1 year. The temporal test indicated a significant cluster in 1985 (P<0.005). This cluster comprised 10 cases, although 2.19 were expected for this year in this area. Age and clinical presentation of these cases were typical of AF paracccidioidomycosis. The space-time test confirmed the temporal cluster in 1985 and showed the localities where the risk was higher in that year. The cluster suggests that some particularities took place in the antecedent years in those localities. Analysis of climate variables showed that soil water storage was atypically high in 1982/83 (similar to 2.11/2.5 SD above mean), and the absolute air humidity in 1984, the year preceding the cluster, was much higher than normal (similar to 1.6 SD above mean), conditions that may have favored, respectively, antecedent fungal growth in the soil and conidia liberation in 1984, the probable year of exposure. These climatic anomalies in this area was due to the 1982/83 El Nino event, the strongest in the last 50 years. Conclusions/Significance: We describe the first cluster of AF paracoccidioidomycosis, which was potentially linked to a climatic anomaly caused by the 1982/83 El Nino Southern Oscillation. This finding is important because it may help to clarify the conditions that favor Paracoccidioides brasiliensis survival and growth in the environment and that enhance human exposure, thus allowing the development of preventive measures.

Association of alpha1a-adrenergic receptor polymorphism and blood pressure phenotypes in the Brazilian population

Background: The alpha1A-adrenergic receptor (alpha(1A)-AR) regulates the cardiac and peripheral vascular system through sympathetic activation. Due to its important role in the regulation of vascular tone and blood pressure, we aimed to investigate the association between the Arg347Cys polymorphism in the alpha(1A)-AR gene and blood pressure phenotypes, in a large sample of Brazilians from an urban population. Methods: A total of 1568 individuals were randomly selected from the general population of the Vitoria City metropolitan area. Genetic analysis of the Arg347Cys polymorphism was conducted by polymerase chain reaction/restriction fragment length polymorphism. We have compared cardiovascular risk variables and genotypes using ANOVA, and Chi-square test for univariate comparisons and logistic regression for multivariate comparisons. Results: Association analysis indicated a significant difference between genotype groups with respect to diastolic blood pressure (p = 0.04), but not systolic blood pressure (p = 0.12). In addition, presence of the Cys/Cys genotype was marginally associated with hypertension in our population (p = 0.06). Significant interaction effects were observed between the studied genetic variant, age and physical activity. Presence of the Cys/Cys genotype was associated with hypertension only in individuals with regular physical activity (odds ratio = 1.86; p = 0.03) or younger than 45 years (odds ratio = 1.27; p = 0.04). Conclusion: Physical activity and age may potentially play a role by disclosing the effects of the Cys allele on blood pressure. According to our data it is possible that the Arg347Cys polymorphism can be used as a biomarker to disease risk in a selected group of individuals.

TCF7L2 variant genotypes and type 2 diabetes risk in Brazil: significant association, but not a significant tool for risk stratification in the general population

Background: Genetic polymorphisms of the TCF7L2 gene are strongly associated with large increments in type 2 diabetes risk in different populations worldwide. In this study, we aimed to confirm the effect of the TCF7L2 polymorphism rs7903146 on diabetes risk in a Brazilian population and to assess the use of this genetic marker in improving diabetes risk prediction in the general population. Methods: We genotyped the single nucleotide polymorphisms (SNP) rs7903146 of the TCF7L2 gene in 560 patients with known coronary disease enrolled in the MASS II (Medicine, Angioplasty, or Surgery Study) Trial and in 1,449 residents of Vitoria, in Southeast Brazil. The associations of this gene variant to diabetes risk and metabolic characteristics in these two different populations were analyzed. To access the potential benefit of using this marker for diabetes risk prediction in the general population we analyzed the impact of this genetic variant on a validated diabetes risk prediction tool based on clinical characteristics developed for the Brazilian general population. Results: SNP rs7903146 of the TCF7L2 gene was significantly associated with type 2 diabetes in the MASS-II population (OR = 1.57 per T allele, p = 0.0032), confirming, in the Brazilian population, previous reports of the literature. Addition of this polymorphism to an established clinical risk prediction score did not increased model accuracy (both area under ROC curve equal to 0.776). Conclusion: TCF7L2 rs7903146 T allele is associated with a 1.57 increased risk for type 2 diabetes in a Brazilian cohort of patients with known coronary heart disease. However, the inclusion of this polymorphism in a risk prediction tool developed for the general population resulted in no improvement of performance. This is the first study, to our knowledge, that has confirmed this recent association in a South American population and adds to the great consistency of this finding in studies around the world. Finally, confirming the biological association of a genetic marker does not guarantee improvement on already established screening tools based solely on demographic variables.

An empirical evaluation of imputation accuracy for association statistics reveals increased type-I error rates in genome-wide associations

Background: Genome wide association studies (GWAS) are becoming the approach of choice to identify genetic determinants of complex phenotypes and common diseases. The astonishing amount of generated data and the use of distinct genotyping platforms with variable genomic coverage are still analytical challenges. Imputation algorithms combine directly genotyped markers information with haplotypic structure for the population of interest for the inference of a badly genotyped or missing marker and are considered a near zero cost approach to allow the comparison and combination of data generated in different studies. Several reports stated that imputed markers have an overall acceptable accuracy but no published report has performed a pair wise comparison of imputed and empiric association statistics of a complete set of GWAS markers. Results: In this report we identified a total of 73 imputed markers that yielded a nominally statistically significant association at P < 10(-5) for type 2 Diabetes Mellitus and compared them with results obtained based on empirical allelic frequencies. Interestingly, despite their overall high correlation, association statistics based on imputed frequencies were discordant in 35 of the 73 (47%) associated markers, considerably inflating the type I error rate of imputed markers. We comprehensively tested several quality thresholds, the haplotypic structure underlying imputed markers and the use of flanking markers as predictors of inaccurate association statistics derived from imputed markers. Conclusions: Our results suggest that association statistics from imputed markers showing specific MAF (Minor Allele Frequencies) range, located in weak linkage disequilibrium blocks or strongly deviating from local patterns of association are prone to have inflated false positive association signals. The present study highlights the potential of imputation procedures and proposes simple procedures for selecting the best imputed markers for follow-up genotyping studies.

Accelerating aging chemotherapy on aged animals: Protective effect from nutraceutical modulation

The aim of this study was to test a novel phytocompound in an experimental model of antitumor-induced immunosuppression. Five groups of mice were considered: young (Y) and aged (A) that were given intraperitoneally 10 doses of cyclophosphamide (CPX, 25mg/kg/bw) or CPX plus (150 mg/kg/bw) of the nutraceutical DTS (Denshichi-Tochiu-Sen), and control. After sacrifice, macrophage chemotaxis and serum levels of IFN-gamma, IL-2, and GM-CSF were determined. Liver and urinary bladder were examined histologically, as were the liver and kidney for redox enzymes. CPX significantly decreased macrophage chemotaxis and all cytokines (p < 0.05, A >> Y). DTS restored macrophage function and cytokine concentration (p < 0.001) and partly improved the necro-inflammatory score and substance P receptor expression in the bladder and the redox status in liver and kidney (p < 0.05). Such data suggest that DTS effectively prevents CPX-induced immune suppression and oxidative-inflammatory damage, which are particularly enhanced in aged organisms.

Real-Time PCR in HIV/Trypanosoma cruzi Coinfection with and without Chagas Disease Reactivation: Association with HIV Viral Load and CD4(+) Level

Background: Reactivation of chronic Chagas disease, which occurs in approximately 20% of patients coinfected with HIV/Trypanosoma cruzi (T. cruzi), is commonly characterized by severe meningoencephalitis and myocarditis. The use of quantitative molecular tests to monitor Chagas disease reactivation was analyzed. Methodology: Polymerase chain reaction (PCR) of kDNA sequences, competitive (C-) PCR and real-time quantitative (q) PCR were compared with blood cultures and xenodiagnosis in samples from 91 patients (57 patients with chronic Chagas disease and 34 with HIV/T. cruzi coinfection), of whom 5 had reactivation of Chagas disease and 29 did not. Principal Findings: qRT-PCR showed significant differences between groups; the highest parasitemia was observed in patients infected with HIV/T. cruzi with Chagas disease reactivation (median 1428.90 T. cruzi/mL), followed by patients with HIV/T. cruzi infection without reactivation (median 1.57 T. cruzi/mL) and patients with Chagas disease without HIV (median 0.00 T. cruzi/mL). Spearman's correlation coefficient showed that xenodiagnosis was correlated with blood culture, C-PCR and qRT-PCR. A stronger Spearman correlation index was found between C-PCR and qRT-PCR, the number of parasites and the HIV viral load, expressed as the number of CD4(+) cells or the CD4(+)/CD8(+) ratio. Conclusions: qRT-PCR distinguished the groups of HIV/T. cruzi coinfected patients with and without reactivation. Therefore, this new method of qRT-PCR is proposed as a tool for prospective studies to analyze the importance of parasitemia (persistent and/or increased) as a criterion for recommending pre-emptive therapy in patients with chronic Chagas disease with HIV infection or immunosuppression. As seen in this study, an increase in HIV viral load and decreases in the number of CD4(+) cells/mm(3) and the CD4(+)/CD8(+) ratio were identified as cofactors for increased parasitemia that can be used to target the introduction of early, pre-emptive therapy.

Association between intratumoral lymphatic microvessel density (LMVD) and clinicopathologic features in endometrial cancer: a retrospective cohort study

Background: Lymph node metastasis in endometrial cancer significantly decreases survival rate. Few data on the influence of intratumoral lymphatic microvessel density (LMVD) on survival in endometrial cancer are available. Our aim was to assess the intratumoral LMVD of endometrial carcinomas and to investigate its association with classical pathological factors, lymph node metastasis and survival. Methods: Fifty-seven patients with endometrial carcinoma diagnosed between 2000 and 2008 underwent complete surgical staging and evaluation of intratumoral LMVD and other histologic variables. Lymphatic microvessels were identified by immunohistochemical staining using monoclonal antibody against human podoplanin (clone D2-40) and evaluated by counting the number of immunostained lymphatic vessels in 10 hot spot areas at 400x magnification. The LMVD was expressed by the mean number of vessels in these 10 hot spot microscopic fields. We next investigated the association of LMVD with the clinicopathologic findings and prognosis. Results: The mean number of lymphatic vessels counted in all cases ranged between 0 and 4.7. The median value of mean LMVD was 0.5, and defined the cut-off for low and high LMVD. We identified low intratumoral LMVD in 27 (47.4%) patients and high LMVD in 30 (52.6%) patients. High intratumoral LMVD was associated with lesser miometrial and adnaexal infiltration, lesser cervical and peritoneal involvement, and fewer fatal cases. Although there was lower lymph node involvement among cases with high LMVD, the difference did not reach significance. No association was seen between LMVD and FIGO staging, histological type, or vascular invasion. On the other hand, low intratumoral LMVD was associated with poor outcome. Seventy-five percent of deaths occurred in patients with low intratumoral LMVD. Conclusion: Our results show association of high intratumoral LMVD with features related to more localized disease and better outcome. We discuss the role of lymphangiogenesis as an early event in the endometrial carcinogenesis.

Expression of Monocarboxylate Transporters 1, 2, and 4 in Human Tumours and Their Association with CD147 and CD44

Monocarboxylate transporters (MCTs) are important cellular pH regulators in cancer cells; however, the value of MCT expression in cancer is still poorly understood. In the present study, we analysed MCT1, MCT2, and MCT4 protein expression in breast, colon, lung, and ovary neoplasms, as well as CD147 and CD44. MCT expression frequency was high and heterogeneous among the different tumours. Comparing with normal tissues, there was an increase in MCT1 and MCT4 expressions in breast carcinoma and a decrease in MCT4 plasma membrane expression in lung cancer. There were associations between CD147 and MCT1 expressions in ovarian cancer as well as between CD147 and MCT4 in both breast and lung cancers. CD44 was only associated with MCT1 plasma membrane expression in lung cancer. An important number of MCT1 positive cases are negative for both chaperones, suggesting that MCT plasma membrane expression in tumours may depend on a yet nonidentified regulatory protein.

Relatedness and HLA-DRB1 typing may discriminate the magnitude of the genetic susceptibility to tuberculosis using a household contact model

Background Tuberculosis clusters in families may be due to increased household exposure, shared genetic factors, or both. Household contact studies are useful to control exposure because socioeconomic and environmental conditions are similar to all subjects, allowing the evaluation of the contribution of relatedness to disease development. Methods In this study, the familial aggregation of tuberculosis using relatedness and a specific inherited marker (HLA-DRB1) was evaluated. Fifty families, which had at least two cases of tuberculosis diagnosed within the past 5 years, were selected from a cohort of tuberculosis carried out in Recife, Brazil. The first case diagnosed was considered to be a primary case. The secondary attack rate of tuberculosis in household contacts was estimated according to the degree of relatedness. The relative risk of having tuberculosis based on the degree of relatedness household and the population attributable fraction to relatedness were also estimated. HLA-DRB1 typing and attributable etiologic/preventive fractions were calculated among sick and healthy household contacts. Results Compared to unrelated contacts, the relative risk for tuberculosis adjusted for age was 1.38 (95% CI 0.86 to 2.21). Relatedness contributed 23% to the development of tuberculosis at the population levels. The HLA-DRB1*04 allele group (OR = 2.44; p =0.0324; etiologic fraction =0.15) was overrepresented and the DRB1*15 allele group (OR=0.48; p=0.0488; protective fraction=0.19) was underrepresented among household contacts exhibiting tuberculosis. The presence of DRB1 shared alleles between primary cases and their contacts was a risk factor for tuberculosis (p=0.0281). Conclusion This household contact model together with the utilisation of two genetic variables permitted the evaluation of genetic factors contributing towards tuberculosis development.

Variability in Estrogen-Metabolizing Genes and Their Association with Genomic Instability in Untreated Breast Cancer Patients and Healthy Women

In the present study, we investigated the relationship between polymorphisms in the estrogen-metabolizing genes CYP17, CYP1B1, CYP1A1, and COMT and genomic instability in the peripheral blood lymphocytes of 62 BC patients and 62 controls considering that increased or prolonged exposure to estrogen can damage the DNA molecule and increase the genomic instability process in breast tissue. Our data demonstrated increased genomic instability in BC patients and that individuals with higher frequencies of MN exhibited higher risk to BC when belonging Val/Met genotype of the COMT gene. We also observed that CYP17 and CYP1A1 polymorphisms can modify the risk to BC depending on the menopause status. We can conclude that the genetic background in estrogen metabolism pathway can modulate chromosome damage in healthy controls and patients and thereby influence the risk to BC. These findings suggest the importance to ally biomarkers of susceptibility and effects to estimate risk groups.

Evaluation of Protective Effect of a Water-In-Oil Microemulsion Incorporating Quercetin Against UVB-Induced Damage in Hairless Mice Skin

Purpose. Histological aspects were considered in order to evaluate the in vivo photoprotective effect of a w/o microemulsion containing quercetin against UVB irradiation-induced dermal damages. The toxicity in cell culture and the potential skin irritation resulting from topical application of this formulation were investigated. Methods. Mouse dorsal surfaces were treated topically with 300 mg of the unloaded and quercetin-loaded (0.3%, w/w) microemulsions before and after exposure to UVB (2.87 J/cm(2)) irradiation. The untreated control groups irradiated and non-irradiated were also evaluated. UVB-induced histopathological changes as well as the photoprotective effect of this formulation were evaluated considering the parameters of infiltration of inflammatory cells, epidermis thickening (basale and spinosum layers) and collagen and elastic fiber contents. The cytotoxicity of the reported formulation was evaluated in L929 mice fibroblasts by MTT assay and the skin irritation was investigated after topical application of both unloaded and quercetin-loaded microemulsions once a day for 15 days. Results. The results demonstrated that the w/o microemulsion containing quercetin reduced the incidence of histological skin alterations, mainly the connective-tissue damage, induced by exposure to UVB irradiation. This suggests that protective effects of this formulation against UV-induced responses are not secondary to the interference of UV transmission (i.e., blocking the UVB radiation from being absorbed by the skin), as is usually implied with UVB absorbers and sunscreens, but is instead due to different biological effects of this flavonoid. Furthermore, by evaluating the cytotoxic effect on L929 cells and histological aspects such as infiltration of inflammatory cells and epidermis thickness of hairless mice, the present study also demonstrated the lack of toxicity of the proposed system. Conclusion. Based on these mice models, a detailed characterization of the w/o microemulsion incorporating quercetin effects as a photochemoprotective agent on human skin is presented.

A Genome-Wide Association Study of Upper Aerodigestive Tract Cancers Conducted within the INHANCE Consortium

Genome-wide association studies (GWAS) have been successful in identifying common genetic variation involved in susceptibility to etiologically complex disease. We conducted a GWAS to identify common genetic variation involved in susceptibility to upper aero-digestive tract (UADT) cancers. Genome-wide genotyping was carried out using the Illumina HumanHap300 beadchips in 2,091 UADT cancer cases and 3,513 controls from two large European multi-centre UADT cancer studies, as well as 4,821 generic controls. The 19 top-ranked variants were investigated further in an additional 6,514 UADT cancer cases and 7,892 controls of European descent from an additional 13 UADT cancer studies participating in the INHANCE consortium. Five common variants presented evidence for significant association in the combined analysis (p <= 5 x 10(-7)). Two novel variants were identified, a 4q21 variant (rs1494961, p = 1 x 10(-8)) located near DNA repair related genes HEL308 and FAM175A (or Abraxas) and a 12q24 variant (rs4767364, p = 2 x 10(-8)) located in an extended linkage disequilibrium region that contains multiple genes including the aldehyde dehydrogenase 2 (ALDH2) gene. Three remaining variants are located in the ADH gene cluster and were identified previously in a candidate gene study involving some of these samples. The association between these three variants and UADT cancers was independently replicated in 5,092 UADT cancer cases and 6,794 controls non-overlapping samples presented here (rs1573496-ADH7, p = 5 x 10(-8); rs1229984-ADH1B, p = 7 x 10(-9); and rs698-ADH1C, p = 0.02). These results implicate two variants at 4q21 and 12q24 and further highlight three ADH variants in UADT cancer susceptibility.

Association of SNPs on CAPN1 and CAST genes with tenderness in Nellore cattle

We examined whether single-nucleotide polymorphisms (SNPs) in the calpain (CAPN) and calpastatin (CAST) genes, described from Bos primigenius taurus, are polymorphic in Nellore cattle. We also looked for a possible association of linkage disequilibrium of this polymorphism with tenderness of the longissimus dorsi muscle after 7, 14 and 21 days of postmortem aging in 638 purebred Nellore bulls. Meat tenderness was measured as Warner-Bratzler shear force. Additive and dominance effects were tested for SNPs of the three genotypic classes; the substitution effect was tested for SNPs with missing genotypic classes. Genotypic and gene frequencies were also calculated for the different SNPs. An increase in tenderness was observed from 7 to 21 days; the average values for shear force at 7, 14 and 21 days of aging were 5.92 +/- 0.06, 4.92 +/- 0.05, and 4.38 +/- 0.04 kg, respectively. All markers showed polymorphism, but there was no CC genotype for CAPN316, and few animals showed the AA genotype for CAPN530. The alleles CAPN4751, UOGCAST1, and WSUCAST were found to have additive and dominance effects for shear force at 7, 14 and 21 days, while CAPN316 showed a substitution effect for shear force at 7 and 21 days. An additive-by-additive epistatic interaction was observed between CAPN4751 and markers on the CAST gene. In conclusion, these markers should be considered for use in breeding programs.

Association of Trypanosoma vivax in extracellular sites with central nervous system lesions and changes in cerebrospinal fluid in experimentally infected goats

Changes in cerebrospinal fluid (CSF) and anatomical and histopathological central nervous system (CNS) lesions were evaluated, and the presence of Trypanosoma vivax in CNS tissues was investigated through PCR. Twelve adult male goats were divided into three groups (G): G1, infected with T. vivax and evaluated during the acute phase; G2, infected goats evaluated during the chronic phase; and G3, consisting of non-infected goats. Each goat from G1 and G2 was infected with 1.25 x 10(5) trypomastigotes. Cerebrospinal fluid (CSF) analysis and investigation of T. vivax was performed at the 15(th) day post-infection (dpi) in G1 goats and on the fifth day after the manifestation of nervous system infection signs in G2 goats. All goats were necropsied, and CNS fragments from G1 and G2 goats were evaluated by PCR for the determination of T. vivax. Hyperthermia, anemia and parasitemia were observed from the fifth dpi for G1 and G2, with the highest parasitemia peak between the seventh and 21(st) dpi. Nervous system infection signs were observed in three G2 goats between the 30(th) and 35(th) dpi. CSF analysis revealed the presence of T. vivax for G2. Meningitis and meningoencephalitis were diagnosed in G2. PCR were positive for T. vivax in all the samples tested. In conclusion, T. vivax may reach the nervous tissue resulting in immune response from the host, which is the cause of progressive clinical and pathological manifestations of the CNS in experimentally infected goats.

Single-nucleotide polymorphism, linkage disequilibrium and geographic structure in the malaria parasite Plasmodium vivax: prospects for genome-wide association studies

Background: The ideal malaria parasite populations for initial mapping of genomic regions contributing to phenotypes such as drug resistance and virulence, through genome-wide association studies, are those with high genetic diversity, allowing for numerous informative markers, and rare meiotic recombination, allowing for strong linkage disequilibrium (LD) between markers and phenotype-determining loci. However, levels of genetic diversity and LD in field populations of the major human malaria parasite P. vivax remain little characterized. Results: We examined single-nucleotide polymorphisms (SNPs) and LD patterns across a 100-kb chromosome segment of P. vivax in 238 field isolates from areas of low to moderate malaria endemicity in South America and Asia, where LD tends to be more extensive than in holoendemic populations, and in two monkey-adapted strains (Salvador-I, from El Salvador, and Belem, from Brazil). We found varying levels of SNP diversity and LD across populations, with the highest diversity and strongest LD in the area of lowest malaria transmission. We found several clusters of contiguous markers with rare meiotic recombination and characterized a relatively conserved haplotype structure among populations, suggesting the existence of recombination hotspots in the genome region analyzed. Both silent and nonsynonymous SNPs revealed substantial between-population differentiation, which accounted for similar to 40% of the overall genetic diversity observed. Although parasites clustered according to their continental origin, we found evidence for substructure within the Brazilian population of P. vivax. We also explored between-population differentiation patterns revealed by loci putatively affected by natural selection and found marked geographic variation in frequencies of nucleotide substitutions at the pvmdr-1 locus, putatively associated with drug resistance. Conclusion: These findings support the feasibility of genome-wide association studies in carefully selected populations of P. vivax, using relatively low densities of markers, but underscore the risk of false positives caused by population structure at both local and regional levels.