Impact of thymidylate synthase promoter and DNA repair gene polymorphisms on susceptibility to childhood acute lymphoblastic leukemia

The aim of this study was to evaluate the frequency of polymorphisms in the TYMS, XRCC1, and ERCC2 DNA repair genes in pediatric patients with acute lymphoblastic leukemia using polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) approaches. The study was conducted in 206 patients and 364 controls from a Brazilian population. No significant differences were observed among the analyzed groups regarding XRCC1 codon 399 and codon 194 and ERCC2 codon 751 and codon 312 polymorphisms. The TYMS 3R variant allele was significantly associated with a reduced risk of childhood ALL, represented by the sum of heterozygous and polymorphic homozygous genotypes (odds ratio 0.60; 95% confidence interval 0.37-0.99). The results suggest that polymorphism in TYMS may play a protective role against the development of childhood ALL.

Isolation and genetic characterisation of Toxoplasma gondii from a red-handed howler monkey (Alouatta belzebul), a jaguarundi (Puma yagouaroundi), and a black-eared opossum (Didelphis aurita) from Brazil

Toxoplasma gondii isolates are highly diverse in domestic animals from Brazil. However, little is known about the genetics of this parasite from wild mammals in the same region. Reveal genetic similarity or difference of T. gondii among different animal populations is necessary for us to understand transmission of this parasite. Here we reported isolation and genetic characterisation of three T. gondii isolates from wild animals in Brazil. The parasite was isolated by bioassay in mice from tissues of a young male red handed howler monkey (Alouatta belzebul), an adult male jaguarundi (Puma yagouaroundi), and an adult female black-eared opossum (Didelphis aurita). The monkey and the jaguarundi had inhabited the Zoo of Parque Estadual Dois Irmaos, Pernambuco State, Northeastern Brazil, for 1 year and 8 years, respectively. The wild black-eared opossum was captured in Sao Paulo State, Southeastern Brazil, and euthanised for this study because it was seropositive for T. gondii (titre 1:100 by the modified agglutination test, MAT). Ten PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) markers, SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico, were used to genotype the isolates. T. gondii was isolated from the brain and heart homogenate of the monkey, the muscle homogenate of the jaguarundi, and the heart homogenate of the black-eared opossum. This was the first isolation of T. gondii from a neotropical fetid from Brazil. The isolate from the monkey (TgRhHmBr1) was not virulent in mice, whereas the isolates from the jaguarundi (TgJagBr1) and the black-eared opossum (TgOpBr1) were virulent in mice. The genotype of the isolate from the monkey has been identified in isolates from a goat and ten chickens in the same region of Brazil, suggesting that it may be a common lineage circulating in this region. The genotypes of the isolates from the jaguarundi and the black-eared opossum have not been previously reported. Although there are already 88 genotypes identified from a variety of animal hosts in Brazil, new genotypes are continuously being identified from different animal species, indicating an extremely high diversity of T. gondii in the population. (C) 2010 Elsevier B.V. All rights reserved.

Cytochrome-b variation in Apis mellifera samples and its association with COI-COII patterns

Five Mbo I (Mbo-A, Mbo-M, Mbo-C(1), Mbo-C(2) and Mbo-C(3)) and Hinf I (Hinf-1 to Hinf-5) patterns were observed in Apis mellifera samples after restriction of a 485 bp fragment of the mitochondrial cytochrome-b (cyt-b) gene. Associating the cyt-b Restriction fragment length polymorphism (RFLP) pattern of each sample to its respective previously established COI-COII (Dra I sites) pattern, five restriction patterns (Mbo-C(1), Mbo-C(2), Mbo-C(3), Hinf-1 and Hinf-4) were observed in samples of maternal origin associated to the evolutionary branch C. No deletions or insertions were observed and the nucleotide substitution rate was estimated at 5.4%. Higher nucleotide diversity was observed among the branch C-haplotypes when compared with A and M lineages. Further studies are needed to confirm if the cyt-b + COI-COII haplotypes help to assign certain phylogeographic patterns to the branch C and to clarify phylogenetic relationships among A. mellifera subspecies.

Shiga toxin-producing Escherichia coli and atypical enteropathogenic Escherichia coli strains isolated from healthy sheep of different populations in Sao Paulo, Brazil

Aims: Sheep are important carriers of Shiga toxin-producing Escherichia coli (STEC) in several countries. However, there are a few reports about ovine STEC in American continent. Methods and Results: About 86 E. coli strains previously isolated from 172 healthy sheep from different farms were studied. PCR was used for detection of stx(1), stx(2), eae, ehxA and saa genes and for the identification of intimin subtypes. Restriction fragment length polymorphism (RFLP)-PCR was performed to investigate the variants of stx(1) and stx(2), and the flagellar antigen (fliC) genes in nonmotile isolates. Five isolates were eae(+) and stx(-), and belonged to serotypes O128:H2/beta-intimin (2), O145:H2/gamma, O153:H7/beta and O178:H7/epsilon. Eighty-one STEC isolates were recovered, and the stx genotypes identified were stx(1c)stx(2d-O118) (46.9%), stx(1c) (27.2%), stx(2d-O118) (23.4%), and stx(1c)stx(2dOX3a) (2.5%). Pulsed-field gel electrophoresis (PFGE) revealed 27 profiles among 53 STEC and atypical enteropathogenic Escherichia coli (EPEC) isolates. Conclusions: This study demonstrated that healthy sheep in Sao Paulo, Brazil, can be carriers of potential human pathogenic STEC and atypical EPEC. Significance and Impact of the Study: As some of the STEC serotypes presently found have been involved with haemolytic uraemic syndrome (HUS) in other countries, the important role of sheep as sources of STEC infection in our settings should not be disregarded.

Species diversity of Leishmania (Viannia) parasites circulating in an endemic area for cutaneous leishmaniasis located in the Atlantic rainforest region of northeastern Brazil

OBJECTIVES To identify the aetiological agents of cutaneous leishmaniasis and to investigate the genetic polymorphism of Leishmania (Viannia) parasites circulating in an area with endemic cutaneous leishmaniasis (CL) in the Atlantic rainforest region of northeastern Brazil. METHODS Leishmania spp. isolates came from three sources: (i) patients diagnosed clinically and parasitologically with CL based on primary lesions, secondary lesions, clinical recidiva, mucocutaneous leishmaniasis and scars; (ii) sentinel hamsters, sylvatic or synanthropic small rodents; and (iii) the sand fly species Lutzomyia whitmani. Isolates were characterised using monoclonal antibodies, multilocus enzyme electrophoresis (MLEE) and polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region rDNA locus. RESULTS Seventy-seven isolates were obtained and characterised. All isolates were identified as Leishmania (Viannia) braziliensis serodeme 1 based on reactivity to monoclonal antibodies. MLEE identified 10 zymodemes circulating in the study region. Most isolates were classified as zymodemes closely related to L. (V.) braziliensis, but five isolates were classified as Leishmania (Viannia) shawi. All but three of the identified zymodemes have so far been observed only in the study region. Enzootic transmission and multiclonal infection were observed. CONCLUSIONS Our results confirm that transmission cycle complexity and the co-existence of two or more species in the same area can affect the level of genetic polymorphism in a natural Leishmania population. Although it is not possible to make inferences as to the modes of genetic exchange, one can speculate that some of the zymodemes specific to the region are hybrids of L. (V.) braziliensis and L. (V.) shawi.

Bacterial Community Composition in Brazilian Anthrosols and Adjacent Soils Characterized Using Culturing and Molecular Identification

Microbial community composition was examined in two soil types, Anthrosols and adjacent soils, sampled from three locations in the Brazilian Amazon. The Anthrosols, also known as Amazonian dark earths, are highly fertile soils that are a legacy of pre-Columbian settlement. Both Anthrosols and adjacent soils are derived from the same parent material and subject to the same environmental conditions, including rainfall and temperature; however, the Anthrosols contain high levels of charcoal-like black carbon from which they derive their dark color. The Anthrosols typically have higher cation exchange capacity, higher pH, and higher phosphorus and calcium contents. We used culture media prepared from soil extracts to isolate bacteria unique to the two soil types and then sequenced their 16S rRNA genes to determine their phylogenetic placement. Higher numbers of culturable bacteria, by over two orders of magnitude at the deepest sampling depths, were counted in the Anthrosols. Sequences of bacteria isolated on soil extract media yielded five possible new bacterial families. Also, a higher number of families in the bacteria were represented by isolates from the deeper soil depths in the Anthrosols. Higher bacterial populations and a greater diversity of isolates were found in all of the Anthrosols, to a depth of up to 1 m, compared to adjacent soils located within 50-500 m of their associated Anthrosols. Compared to standard culture media, soil extract media revealed diverse soil microbial populations adapted to the unique biochemistry and physiological ecology of these Anthrosols.

The Influence of Different Land Uses on the Structure of Archaeal Communities in Amazonian Anthrosols Based on 16S rRNA and amoA Genes

Soil from the Amazonian region is usually regarded as unsuitable for agriculture because of its low organic matter content and low pH; however, this region also contains extremely rich soil, the Terra Preta Anthrosol. A diverse archaeal community usually inhabits acidic soils, such as those found in the Amazon. Therefore, we hypothesized that this community should be sensitive to changes in the environment. Here, the archaeal community composition of Terra Preta and adjacent soil was examined in four different sites in the Brazilian Amazon under different anthropic activities. The canonical correspondence analysis of terminal restriction fragment length polymorphisms has shown that the archaeal community structure was mostly influenced by soil attributes that differentiate the Terra Preta from the adjacent soil (i.e., pH, sulfur, and organic matter). Archaeal 16S rRNA gene clone libraries indicated that the two most abundant genera in both soils were Candidatus nitrosphaera and Canditatus nitrosocaldus. An ammonia monoxygenase gene (amoA) clone library analysis indicated that, within each site, there was no significant difference between the clone libraries of Terra Preta and adjacent soils. However, these clone libraries indicated there were significant differences between sites. Quantitative PCR has shown that Terra Preta soils subjected to agriculture displayed a higher number of amoA gene copy numbers than in adjacent soils. On the other hand, soils that were not subjected to agriculture did not display significant differences on amoA gene copy numbers between Terra Preta and adjacent soils. Taken together, our findings indicate that the overall archaeal community structure in these Amazonian soils is determined by the soil type and the current land use.

Assessment of how pregnancy modifies plasma lead and plasma/whole blood lead ratio in ALAD 1-1 genotype women

Pregnant women are one of the most sensitive populations to the toxic effects associated with lead (Pb) exposure. These effects are primarily associated with plasma Pb (Pb-P), which reflects the most rapidly exchangeable fraction of Pb in the bloodstream, and elevated maternal Pb-P may be more relevant to foetal Pb exposure than whole blood Pb (Pb-B). We investigated how pregnancy affects Pb-B, Pb-P and %Pb-P/Pb-B ratios without the influence of the 6-aminolevulinic acid dehydratase (ALAD) G177C polymorphism, which is a major genetic factor influencing Pb-B, Pb-P and %Pb-P/Pb-B ratios. Genotypes for the ALAD G177C polymorphism were determined by PCR and restriction fragment length digestion in nine pregnant and 20 non-pregnant women, aged 18-33, environmentally exposed to Pb. Here, we included only women with ALAD 1-1 genotype. Pb-P and Pb-B were determined by inductively coupled plasma mass spectrometry and by graphite furnace atomic absorption spectrometry, respectively. We found no differences in Pb-B (P > 0.05). However, pregnant women had a 2-fold increase in Pb-P and a 3-fold increase in %Pb-P/Pb-B (both P < 0.01) compared to nonpregnant women. These alterations in Pb concentrations associated with pregnancy are similar to those associated with different ALAD gene variants. We can now better appreciate how pregnancy affects foetal exposure to Pb without the influence of this important genetic factor.

Analysis of vaginal lactobacilli from healthy and infected Brazilian women

Culture-dependent PCR-amplified rRNA gene restriction analysis and culture-independent (PCR-denaturing gradient gel electrophoresis) methodologies were used to examine vaginal lactobacilli from Brazilian women who were healthy or had been diagnosed with vulvovaginal candidiasis (VVC) or bacterial vaginosis. Only Lactobacillus crispatus was detected accordingly by both methods, and H2O2-producing lactobacilli were not associated with protection against VVC.

Genetic polymorphism in an inflammasome component, cervical mycoplasma detection and female infertility in women undergoing in vitro fertilization

The inflammasome is an inducible cytoplasmic structure that is responsible for production and release of biologically active interleukin-1 (IL-1). A polymorphism in the inflammasome component NALP3 has been associated with decreased IL-1 levels and increased occurrence of vaginal Candida infection. We hypothesized that this polymorphism-induced variation would influence susceptibility to infertility. DNA was obtained from 243 women who were undergoing in vitro fertilization (IVF) and tested for a length polymorphism in intron 2 of the gene coding for NALP3 (gene symbol CIAS1). At the conclusion of testing the findings were analyzed in relation to clinical parameters and IVF outcome. The frequency of the 12 unit repeat allele, associated with maximal inflammasome activity, was 62.3% in cases of female infertility vs. 75.6% in cases where only the male partner had a detectable fertility problem (p = 0.0095). Conversely, the frequency of the 7 unit repeat allele was 28.9% in those with a female fertility problem, 17.0% in women with infertile males and 18.4% in idiopathic infertility (p = 0.0124). Among the women who were cervical culture-positive for mycoplasma the frequency of the 7 unit repeat was 53.7% as opposed to 19.5% in those negative for this infection (p < 0.0001). We conclude that the CIAS1 7 unit repeat polymorphism increases the likelihood of mycoplasma infection-associated female infertility. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Molecular evidence of human T-cell lymphotropic virus types 1 and 2 (HTLV-1 and HTLV-2) infections in HTLV seroindeterminate individuals from Sao Paulo, Brazil

Background: Using enzyme immunoassays and Western blot (Wb) tests, HTLV serodiagnosis yields indeterminate results in a significant number of cases. Objective: To determine the prevalence of HTLV infection among HTLV-seroindeterminate individuals. Study design: We studied peripheral blood mononuclear cells from 65 anti-HTLV Wb-seroindeterminate individuals by attempting to amplify proviral DNA sequences (tax and pot) to identify HTLV-I and HTLV-2 infections. Results: These 65 specimens exhibited predominantly (43%) anti-HTLV antibodies to gag-coded antigens in the absence of anti-p24 on Wb analysis. Tax proviral sequences were detected in 6 (9.2%) samples. According to restricted fragment polymorphism analysis (RFLP), we identified HTLV-1 proviral DNA in 4 samples. HTLV-2 in one and sequences from both in another. Nested PCR for the pot region was positive in 3 (4.6%) specimens, which were also positive for tax sequences. After hybridization HTLV-1 infection was confirmed in 2 samples (3.1%) and HTLV-2 in another (1.5%). Detection of a single HTLV DNA sequence may be due to infection by defective provirus, but its significance remains undefined. In this cohort, no Wb reactivity pattern was predictive of proviral detection. HTLV-I infection was demonstrated in an individual who had Wb reactivity to gag-coded antigens only. Conclusions: This emphasizes the importance of clinical and laboratory follow-up of HTLV-seroindeterminate individuals from endemic areas. (c) 2009 Elsevier B.V. All rights reserved.

Two-stage transvaginal cervical length screening for preterm birth in twin pregnancies

Aim: To compare cervical length (CL) at 18-21 and 22-25 weeks` gestation in twin pregnancies in prediction of spontaneous preterm delivery and to examine cervical shortening. Methods: Retrospective cohort study of CL measured at 18-21 and 22-25 weeks` gestation in twin pregnancies. Results: Receiver operating characteristics (ROC) curve revealed area of 0.64 (95% CI 0.53-0.75) and 0.80 (95% CI 0.72-0.88) for measurements at 18-21 and 22-25 weeks, respectively (P <= 0.001). Sensitivities of 33.3% and 23% and negative predicting value (NPV) of 97.3% and 86.8% for delivery at <28 and <34 weeks gestation were reached for measurements at 18-21 weeks. Sensitivities of 71.4% and 38.2% and NPV of 99.1% and 91.4% for delivery at <28 and <34 weeks` gestation were reached for measurements at 22-25 weeks. Cervical length shortening analysis showed an area under ROC curve of 0.81 (95% CI 0.73-0.89) and best cut-off was at >= 2 mm/week. Sensitivities of 80% and 60.8% and NPV of 98.9% and 90.6% for delivery at <28 and <34 weeks gestation were reached. Conclusions: In twin gestations, assessment of CL at 22-25 weeks is better than assessment at 18-21 weeks to predict preterm delivery before 34 weeks. Cervical shortening at a rate of >= 2 mm/weeks between 18 and 25 weeks gestation was a good predictor of spontaneous preterm birth in this high-risk population.

Transferability and characterization of nine microsatellite markers for the tropical tree species Tabebuia roseo-alba

Microsatellite loci that were previously developed in the tropical tree Tabebuia aurea were used for the genetic analysis of Tabebuia roseo-alba populations. Nine of 10 simple sequence repeat markers were amplified, and the polymorphism was assessed in 58 individuals sampled from two stands in southeastern Brazil. All loci were polymorphic with Mendelian inheritance. The allele numbers were high, ranging from 5 to 13 in population I and 3 to 7 in population II, with means of 8.9 and 5.5, respectively. We conclude that these markers can be efficiently used for parentage and gene-flow studies.

Vitamin D receptor haplotypes affect lead levels during pregnancy

Pregnant women are particularly susceptible to toxic effects associated with lead (Pb) exposure. Pb accumulates in bone tissue and is rapidly mobilized from bones during pregnancy, thus resulting in fetal contamination. While vitamin D receptor (VDR) polymorphisms modify bone mineralization and affect Pb biomarkers including blood (Pb-B) and serum (Pb-S) Pb concentrations, and %Pb-S/Pb-B ratio, the effects of these polymorphisms on Pb levels in pregnant women are unknown. This study aimed at examining the effects of three (Fokl, Bsml and Apal) VDR polymorphisms (and VDR haplotypes) on Pb levels in pregnant women. Pb-B and Pb-S were determined by inductively coupled plasma mass spectrometry in samples from 256 healthy pregnant women and their respective umbilical cords. Genotypes for the VDR polymorphisms were determined by PCR and restriction fragment length digestion. While the three VDR polymorphisms had no significant effects on Pb-B, Pb-S or %Pb-S/Pb-B ratio, the haplotype combining the f, a, and b alleles for the Fokl, Apal and Bsml polymorphisms, respectively, was associated with significantly lower Pb-S and %Pb-S/Pb-B (P<0.05). However, maternal VDR haplotypes had no effects on Pb levels in the umbilical cords. To our knowledge, this is the first study showing that a combination of genetic polymorphisms (haplotype) commonly found in the VDR gene affects Pb-S and %Pb-S/Pb-B ratios in pregnant women. These findings may have major implications for Pb toxicity because they may help to predict the existence of a group of subjects that is genetically less prone to Pb toxicity during pregnancy. (C) 2010 Elsevier B.V. All rights reserved.

Expression of TLR-4 and -2 in peripheral mononuclear cells in renal transplant patients with TLR-4 gene polymorphism

Introduction: TLR-4 has also been identified as a receptor for endogenous alarmins, which are increased post transplantation. TLR-4 has also been associated with a polymorphism that could impact graft outcome. Objective: To assess the expression of TLR-4 in kidney transplant patients carrying or not a polymorphism. Methods: TLR-4 polymorphism (A299G/T399I) was studied in 200 renal transplant patients. Healthy volunteers were also enrolled as control group. The polymorphism analysis was performed using restriction enzymes technique (RFLP). Functionality of TLR-4 polymorphism was assessed in samples from controls by quantification of TNF-alpha after LPS stimulus. TLR-4 and -2 expressions were also analyzed by flow cytometry. Results: TLR-4 polymorphism was present in 8.5% of renal transplant patients. This polymorphism was associated with impairment in TNF-alpha secretion. In general, in renal transplant patients, TLR-4 expression in monocytes and in neutrophils was lower than in health volunteers. TLR-2 and TLR-4 expressions in healthy volunteers with A299G/T399I TLR-4 polymorphism was higher than in wild-type genotype healthy volunteers (p<0.01 and p<0.05, respectively), and also higher than A299G/T399I TLR-4 polymorphism renal transplant patients (p<0.05). TLR-2 expression on neutrophils in wild-type genotype renal transplant patients was higher compared to wild-type genotype healthy volunteers, and was also higher in relation to A299G/T399I kidney transplanted patients (p<0.01). Conclusion: Stable renal transplant patients with TLR-4 polymorphism have a lower expression of TLR-4 and TLR-2 receptors in peripheral mononuclear cells, which ultimately indicate a less responsiveness for alarmins. (C) 2010 Elsevier B.V. All rights reserved.