Role of hypothetical protein YicS in the pathogenicity of Avian Pathogenic Escherichia coli in vivo and in vitro.

Avian Pathogenic Escherichia coli (APEC) strains belong to the extra-intestinal pathogenic group of E. coli (ExPEC) that causes colibacillosis in poultry. A variety of putative virulence factors of APEC are recognized as potent causes of pathogenicity, the mechanisms underlying their pathogenicity are still not fully understood. The role of yicS in the virulence of pathogenic E. coli is still unclear. Thus, yicS may be related to biofilm formation, which in some bacteria plays a role in pathogenicity. Therefore, the fact that this gene appears to be under positive selection pressure suggests that yicS may be associated with the pathogenicity of APEC. To better understand the role of yicS protein in APEC biological characteristics and pathogenicity, we deleted yicS in an APEC Swollen Head Syndrome strain (APEC strain SCI-07) and studied its effects by comparing wild type and isogenic mutants through comprehensive in vitro and in vivo assays. We demonstrated that yicS plays a role in pathogenicity of APEC. We suggest that the yicS gene, which encodes an exporter protein, has a significant role in biofilm formation, motility, invasion of CEC-32 and Hep-2 cells and APEC pathogenicity in a day-old chick model.

Variants of astA gene among extra-intestinal Escherichia coli of human and avian origin.

Many Escherichia coli strains harbour astA, which is the gene encoding the enteroaggregative E. coli heat-stable enterotoxin (EAST1). This gene is embedded in a putative transposase (ORF1) and presents polymorphism in diarrheagenic strains. Although astA and orf1 are detected in extraintestinal strains, little is known about polymorphism and differential gene transcription in this pathotype. In the present work, extraintestinal E. coli from humans (ExPEC - Extraintestinal Pathogenic E. coli) and poultry (APEC - Avian Pathogenic E. coli) were assayed to verify the presence of astA/orf1 and possible polymorphisms in these genes. Three astA/orf1 patterns were detected via Sanger sequencing. Pattern 1 was novel and represented an astA pseudogene. Pattern 2 and pattern 3 presented distinct amino acids within the reading frame encoding astA and were identical to the sequences found in EAEC 17-2 and EAEC 042, respectively. Regarding the frame encoding ORF1, all mutations detected in the three patterns were neutral. The transcripts of astA/orf1 in vitro were underregulated in strains possessing the pattern 1 sequence. The results demonstrate that the same astA sequences may be detected in diarrheagenic and extra-intestinal E. coli. However, extraintestinal isolates may also present an astA pseudogene that has not been reported in diarrheagenic E. coli.

The virulence factor ychO has a pleiotropic action in an Avian Pathogenic Escherichia coli (APEC) strain.

BACKGROUND: Avian pathogenic Escherichia coli strains cause extraintestinal diseases in birds, leading to substantial economic losses to the poultry industry worldwide. Bacteria that invade cells can overcome the host humoral immune response, resulting in a higher pathogenicity potential. Invasins are members of a large family of outer membrane proteins that allow pathogen invasion into host cells by interacting with specific receptors on the cell surface. RESULTS: An in silico analysis of the genome of a septicemic APEC strain (SEPT362) demonstrated the presence of a putative invasin homologous to the ychO gene from E. coli str. K-12 substr. MG1655. In vitro and in vivo assays comparing a mutant strain carrying a null mutation of this gene, a complemented strain, and its counterpart wild-type strain showed that ychO plays a role in the pathogenicity of APEC strain SEPT362. In vitro assays demonstrated that the mutant strain exhibited significant decreases in bacterial adhesiveness and invasiveness in chicken cells and biofilm formation. In vivo assay indicated a decrease in pathogenicity of the mutant strain. Moreover, transcriptome analysis demonstrated that the ychO deletion affected the expression of 426 genes. Among the altered genes, 93.66% were downregulated in the mutant, including membrane proteins and metabolism genes. CONCLUSION: The results led us to propose that gene ychO contributes to the pathogenicity of APEC strain SEPT362 influencing, in a pleiotropic manner, many biological characteristics, such as adhesion and invasion of in vitro cultured cells, biofilm formation and motility, which could be due to the possible membrane location of this protein. All of these results suggest that the absence of gene ychO would influence the virulence of the APEC strain herein studied.

Fimbria-Encoding Gene yadC Has a Pleiotropic Effect on Several Biological Characteristics and Plays a Role in Avian Pathogenic Escherichia coli Pathogenicity.

The extraintestinal pathogen termed avian pathogenic Escherichia coli (APEC) is known to cause colibacillosis in chickens. The molecular basis of APEC pathogenesis is not fully elucidated yet. In this work, we deleted a component of the Yad gene cluster (yadC) in order to understand the role of Yad in the pathogenicity of the APEC strain SCI-07. In vitro, the transcription level of yadC was upregulated at 41°C and downregulated at 22°C. The yadC expression in vivo was more pronounced in lungs than in spleen, suggesting a role in the early steps of the infection. Chicks infected with the wild-type and mutant strains presented, respectively, 80% and 50% mortality rates. The ΔyadC strain presented a slightly decreased ability to adhere to HeLa cells with or without the d-mannose analog compared with the wild type. Real-time PCR (RT-PCR) assays showed that fimH was downregulated (P < 0.05) and csgA and ecpA were slightly upregulated in the mutant strain, showing that yadC modulates expression of other fimbriae. Bacterial internalization studies showed that the ΔyadC strain had a lower number of intracellular bacteria recovered from Hep-2 cells and HD11 cells than the wild-type strain (P < 0.05). Motility assays in soft agar demonstrated that the ΔyadC strain was less motile than the wild type (P < 0.01). Curiously, flagellum-associated genes were not dramatically downregulated in the ΔyadC strain. Taken together, the results show that the fimbrial adhesin Yad contributes to the pathogenicity and modulates different biological characteristics of the APEC strain SCI-07.

Influence of the major nitrite transporter NirC on the virulence of a Swollen Head Syndrome avian pathogenic E. coli (APEC) strain.

Avian Pathogenic Escherichia coli (APEC) strains are extra-intestinal E. coli that infect poultry and cause diseases. Nitrite is a central branch-point in bacterial nitrogen metabolism and is used as a cytotoxin by macrophages. Unlike nitric oxide (NO), nitrite cannot diffuse across bacterial membrane cells. The NirC protein acts as a specific channel to facilitate the transport of nitrite into Salmonella and E. coli cells for nitrogen metabolism and cytoplasmic detoxification. NirC is also required for the pathogenicity of Salmonella by downregulating the production of NO by the host macrophages. Based on an in vitro microarray that revealed the overexpression of the nirC gene in APEC strain SCI-07, we constructed a nirC-deficient SCI-07 strain (ΔnirC) and evaluated its virulence potential using in vivo and in vitro assays. The final cumulative mortalities caused by mutant and wild-type (WT) were similar; while the ΔnirC caused a gradual increase in the mortality rate during the seven days recorded, the WT caused mortality up to 24h post-infection (hpi). Counts of the ΔnirC cells in the spleen, lung and liver were higher than those of the WT after 48 hpi but similar at 24 hpi. Although similar number of ΔnirC and WT cells was observed in macrophages at 3 hpi, there was higher number of ΔnirC cells at 16 hpi. The cell adhesion ability of the ΔnirC strain was about half the WT level in the presence and absence of alpha-D-mannopyranoside. These results indicate that the nirC gene influences the pathogenicity of SCI-07 strain.

Herpesvirus type 7 infection may play an important role in individuals with a genetic profile of susceptibility to Graves' disease.

OBJECTIVE: An inherited profile of genes related to the response to aggressive environmental factors such as viruses and chemicals may be related to an increased susceptibility to Graves' disease (GD). DESIGN AND METHODS: This prospective case-control study was designed to examine the relationship between human herpesviruses (HHV) infection, determined by circulating DNA; tumour protein p53 (TP53) apoptotic ability; and detoxification system genes, and GD. We studied 280 confirmed GD patients paired to 284 controls with respect to environmental exposure. Exclusion criteria included medications that could interfere with thyroid function evaluation and a recent history of viral and bacterial infections. RESULTS: A stepwise regression analysis adjusted for age, gender, and ethnicity established the inheritance of glutathione S-transferase pi 1 (GSTP1) (odds ratio (OR)=3.423; 95% confidence interval (CI)=2.120-5.527; P<0.001) and cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) variants (OR=1.649; 95% CI=1.012-2.686; P=0.0445) as significant risk factors for the disease. HHV-7 infection was much more common in GD patients (64.64%) than in controls (38.73%; chi(2), P<0.0001), and it increased the risk for GD more than three times (OR=3.133; 95% CI=1.959-5.011; P<0.0001). The inheritance of less efficient Pro/Pro TP53 gene variants significantly increased the risk of GD development (OR=5.196; 95% CI=2.112-12.783; P<0.0001) and also favored HHV-7 infection (OR=2.835; 95% CI=1.100-7.310; P=0.0275). In addition, 72TP53 variants augmented the risk of GD relapse (OR=1.860; 95% CI=1.015-3.410; P=0.0446). CONCLUSIONS: We suggest that an inherited genetic profile involving TP53 may favor HHV-7 infection and maintenance, which, in turn, may initiate and perpetuate GD autoimmune process.

Alguns agentes virais se relacionam com neoplasias ou doenças autoimunes: [revisão] / Viral agents and their correlation with the genetic profile of predisposition to human neoplastic and autoimmune diseases: [review]

Os herpesvírus têm sido vistos como potenciais agentes carcinogênicos e identificados em diversas malignidades. Acometem indivíduos imunossuprimidos e também indivíduos saudáveis e possuem elevada prevalência. A capacidade de permanecerem latentes nas células do hospedeiro garante aos vírus sua sobrevivência até serem reativados. Células infectadas por herpes supostamente não seriam destruídas por apoptose em portadores de alterações no gene TP53. Nossos estudos comprovam uma maior prevalência de herpesvírus tipo 6 em pacientes transplantados renais do que numa população controle e mostram que polimorfismos no gene TP53 poderiam influenciar na suscetibilidade à infecção por este vírus. Observamos que os herpesvírus também podem aumentar o risco para o desenvolvimento de carcinomas da pele e isso se associa ao perfil genotípico GSTM1-GSTT1+. Mais recentemente, estudando doenças auto-imunes, observamos que a infecção pelo herpesvírus 6 aumentou a susceptibilidade para o desenvolvimento da doença de Graves. Estes estudos poderão ter utilidade na prevenção de doenças. Por exemplo, pacientes em imunodepressão que tenham infecção por herpesvírus devem ser particularmente mais cuidadosos em relação à exposição solar.

Alguns agentes virais se relacionam com neoplasias ou doenþas autoimunes: [revisÒo] / Viral agents and their correlation with the genetic profile of predisposition to human neoplastic and autoimmune diseases: [review]

Os herpesvírus tÛm sido vistos como potenciais agentes carcinogÛnicos e identificados em diversas malignidades. Acometem indivíduos imunossuprimidos e também indivíduos saudáveis e possuem elevada prevalÛncia. A capacidade de permanecerem latentes nas células do hospedeiro garante aos vírus sua sobrevivÛncia até serem reativados. Células infectadas por herpes supostamente nÒo seriam destruídas por apoptose em portadores de alteraþ§es no gene TP53. Nossos estudos comprovam uma maior prevalÛncia de herpesvírus tipo 6 em pacientes transplantados renais do que numa populaþÒo controle e mostram que polimorfismos no gene TP53 poderiam influenciar na suscetibilidade O infecþÒo por este vírus. Observamos que os herpesvírus também podem aumentar o risco para o desenvolvimento de carcinomas da pele e isso se associa ao perfil genotípico GSTM1-GSTT1+. Mais recentemente, estudando doenþas auto-imunes, observamos que a infecþÒo pelo herpesvírus 6 aumentou a susceptibilidade para o desenvolvimento da doenþa de Graves. Estes estudos poderÒo ter utilidade na prevenþÒo de doenþas. Por exemplo, pacientes em imunodepressÒo que tenham infecþÒo por herpesvírus devem ser particularmente mais cuidadosos em relaþÒo O exposiþÒo solar.(AU)

Lack of influence of glutathione S-transferase genotype profile on cancer susceptibility in smokers and nonsmokers.

BACKGROUND: The glutathione-S-transferase (GST) gene family has an important role in the biotransformation and detoxification of different xenobiotics and endogenous carcinogens. GST profile has been associated to an increased risk for several types of tumors in different populations, but ethnic stratification makes data interpretation difficult. The Brazilian population represents a unique model in which the types and frequencies of GST gene polymorphisms are less influenced by ethnicity. MATERIAL/METHODS: To evaluate the influence of GST profile in different age and gender groups regarding the risk of developing cancer and its relationship to smoking habit, the GSTT1, GSTM1, and GSTP1 genotypes of 785 Brazilian patients with cancer and 873 cancer-free controls paired on the basis of sex, age, ethnicity, diet and exercise routine, lifetime occupational history, smoking history, general health conditions, and previous diseases were compared. RESULTS: A univariate logistic regression analysis demonstrated that age over 45 years (p=0.0417) and smoking (p=0.0015) were related to cancer. Multivariate analysis confirmed the importance of advanced age in susceptibility to cancer (p=0.0001). It was also observed that smoking significantly increased the risk of cancer among individuals over 45 years old (OR: 1.825, 95%CI: 1.241-2.682). However, no correlation between risk of cancer, smoking habit, age, or gender and any of the studied GST polymorphisms was found. CONCLUSIONS: It is suggested that GST profile does not exert an important impact on the influence of tobacco smoking on cancer risk.

Peroxisome proliferator-activated receptor y2 Pro115Gln and G972R insulin substrate receptor-1 variants in obesity and insulin resistance

Objective: The association among IRS-IG972R and PPAR-gama2Pro1l5Gln gene variants and insulin resistance is controversial. This study aimed to investigate the relationship between PPAR-gama2Pro115Gln and IRS-IG972R variants to in­sulin resistance. Design and Setting: This prospective study was developed in the University Hospital of Unicamp. Methods: We studied the prevalence of these mutations in 67 lean and 64 obese subjects (91 women and 40 men, 18 to 67 years old) evaluating metabolic and obesity parameters. Both genetic variants were detected by restriction fragment length polymorphism assays. Insulin sensitivity was estimated through the insulin resistance index; Body Mass Index (BMI), waist, fat and fat-free mass, indirect calorimetry, blood pressure, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, fasting plasma glucose, insulin and serum uric acid were also measured. Results: Genetic analysis showed that 5 (3.8%) individuals presented mutations in the PPAR-gama2 gene, all of them homozygotes, whereas polymorphism of the IRS-l gene was found in 12 (9.1 %) cases, all in heterozygosis. There was no correla­tion between the genetic profile and insulin resistance or any ofthe anthropometric, hemodynamic and biochemical parame­ters measured in the obese group. The rate of PPAR-gama2 and IRS-l variants was similar in lean and obese subjects. Among the PPAR-gama2Pro1l5Gln carriers, 3 were insulin resistant (p equal 0.05 HOMA-IR greater that 75th). Conclusion: We suggest that there is a trend to the asso­ciation between the PPAR -gama2Pro 115, but not the IRS-l G972R gene mutation to insulin resistance in the Brazilian population, that needs to be confirmed in larger samples.

Identifying a risk profile for thyroid cancer.

The large use of simple and effective diagnostic tools has significantly contributed to the increase in diagnosis of thyroid cancer over the past years. However, there is compelling evidence that most micropapillary carcinomas have an indolent behavior and may never evolve into clinical cancers. Therefore, there is an urgent need for new tools able to predict which thyroid cancers will remain silent, and which thyroid cancers will present an aggressive behavior. There are a number of well-established clinical predictors of malignancy and recent studies have suggested that some of the patients laboratory data and image methods may be useful. Molecular markers have also been increasingly tested and some of them appear to be very promising, such as BRAF, a few GST genes and p53 polymorphisms. In addition, modern tools, such as immunocytochemical markers, and the measure of the fractal nature of chromatin organization may increase the specificity of the pathological diagnosis of malignancy and help ascertain the prognosis. Guidelines designed to select nodules for further evaluation, as well as new methods aimed at distinguishing carcinomas of higher aggressiveness among the usually indolent thyroid tumors are an utmost necessity.

Identifying a risk profile for thyroid cancer

The large use of simple and effective diagnostic tools has significantly contributed to the increase in diagnosis of thyroid cancer over the past years. However, there is compelling evidence that most micropapillary carcinomas have an indolent behavior and may never evolve into clinical cancers. Therefore, there is an urgent need for new tools able to predict which thyroid cancers will remain silent, and which thyroid cancers will present an aggressive behavior. There are a number of well-established clinical predictors of malignancy and recent studies have suggested that some of the patientÆs laboratory data and image methods may be useful. Molecular markers have also been increasingly tested and some of them appear to be very promising, such as BRAF, a few GST genes and p53 polymorphisms. In addition, modern tools, such as immunocytochemical markers, and the measure of the fractal nature of chromatin organization may increase the specificity of the pathological diagnosis of malignancy and help ascertain the prognosis. Guidelines designed to select nodules for further evaluation, as well as new methods aimed at distinguishing carcinomas of higher aggressiveness among the usually indolent thyroid tumors are an utmost necessity.

O uso cada vez mais freqüente de métodos diagnósticos simples e efetivos tem contribuído significativamente para um aumento no diagnóstico de câncer da tiróide nos últimos anos. Entretanto, existem importantes evidências de que muitos dos microcarcinomas papilíferos têm um comportamento indolente e podem nunca evoluir para cânceres clínicos. Existe, portanto, uma necessidade urgente de desenvolver novas ferramentas capazes de predizer quais os tumores tiroidianos que permanecerão silenciosos e quais desenvolverão comportamento agressivo. Há uma série de marcadores clínicos de evolução bem estabelecidos e alguns estudos recentes sugerem que dados laboratoriais e métodos de imagem podem ser úteis. Marcadores moleculares também vêm sendo ativamente investigados e alguns, como BRAF, os genes GST e polimorfismos de p53, parecem promissores. Além disso, marcadores imunocitoquímicos e a medida da natureza fractal da cromatina podem aumentar a especificidade do diagnóstico anatomopatológico e ajudar a predizer o prognóstico. Existe uma necessidade imperiosa de elaborarmos diretrizes destinadas a selecionar os nódulos que merecem prosseguimento em sua avaliação, assim como novos métodos capazes de identificar lesões mais agressivas entre os geralmente indolentes tumores tiroidianos.

Role of glutathione-S-transferase and codon 72 of P53 genotypes in epithelial ovarian cancer patients.

PURPOSE: A series of polymorphisms in germ-line DNA have been investigated in an effort to delineate polygenic models of cancer susceptibility and prognosis. As low-penetrance susceptibility genes may combine additively or multiplicatively and contribute to cancer incidence and to the response to chemotherapy, we studied GSTT1, GSTM1, GSTO2, GSTP1 and codon 72 of p53 genotype profiles in ovarian cancer patients. METHODS: We compared 69 ovarian cancer patients with 222 control healthy women paired for ethnic and life-style characteristics. Outcome was evaluated in 29 stage III and IV patients submitted to a platinum-based chemotherapy followed-up for 6-29 months (17 +/- 9 months). RESULTS: GSTT1, GSTM1, GSTO2 and GSTP1 genes presented a similar genotype distribution, but codon 72 of p53 gene wild-type variant was less frequent in ovarian cancer patients than in controls (chi(2); P = 0.0004). CONCLUSIONS: We were unable to demonstrate any association between the GST genotypes studied and the risk of ovarian cancer but the inheritance of a heterozygous Arg/Pro genotype of p53 increased the risk of ovarian cancer more than 2.5 times (OR = 2.571; 95% CI = 1.453-4.550). There was no association of the studied genes to any clinical or pathological feature of the patients or to their response to chemotherapy.

Human herpesvirus type 6 and type 1 infection increases susceptibility to nonmelanoma skin tumors.

In order to investigate herpesvirus (HHV) role in the susceptibility to skin cancer, we compared HHV6 and HHV1 incidence in DNA samples extracted from 120 lesions and 41 normal skin tissues. HHV6 (31.7%) and HHV1 (23.8%) were detected more frequently in skin cancer than in control individuals (14.6 and 5%, respectively) (P=0.0391 and P=0.00094, respectively). The risk of presenting basal cell carcinomas (BCC) was more than 3 times higher for HHV-6 infected patients (OR=3.182; 95% CI: 1.125-8.997). The risk for HHV-1 infected individuals of presenting BCC and squamous cell carcinomas was increased 8 and 6 times, respectively (OR=8.125; 95% CI: 1.735-38.043 and OR=6.290; 95% CI: 1.283-30.856, respectively).

Lack of mutation in exon 10 of p53 gene in thyroid tumors

Background: p53 is a nuclear protein that exerts an important role in the negative control of cellular proliferation, as well as in masterminding signaling cascades important in DNA repair and/or apoptosis. Mutations of p53 have been reported with high frequency in many cancer types and are highly prevalent in poorly differentiated and undifferentiated thyroid carcinomas, but they are not found in benign tumors and are infrequent in well-differentiated cancer. Most mutations are located in exons 5-8 of the gene. Recently, a germline mutation in the seldom investigated exon 10, on codon 337 of p53 was described in Brazilian children who had adrenocortical tumors. Aim: To study codon 337 of exon 10 of p53 mutation in thyroid tumors. Material and methods: Seventy four thyroid tumors were studied (5 follicular carcinomas including 3 widely invasive, 22 papillary carcinomas including 6 tall cell variants, 11 follicular adenomas, 1 medullary carcinoma and 35 benign goiters). DNA was extracted from a central part of all tumors and contralateral normal thyroid tissue samples or blood from 38 of these patients. The products of PCR for exon 10 of p53 were examined by single strand conformation polymorphism (SSCP) analysis. We sequenced 2 samples suspected of presenting aberrant migrating bands and 3 additional PCR products from tumor samples with normal SSCP patterns but all were wild type. Results: In all samples studied, a wild type sequence was found. Conclusions: Exon 10 of p53 gene does not present mutations in thyroid tumors, suggesting that this mutation is specific of adrenocortical cancers.

The null genotype of glutathione s-transferase M1 and T1 locus increases the risk for thyroid cancer.

Susceptibility to chemical carcinogens plays an important role in the development of most cancers. Several polymorphisms of human drug-metabolizing enzymes influence this individual susceptibility. The genes that encode the isoenzymes of the glutathione s-transferase (GST) system present a polymorphic inheritance. The GST mu 1 (GSTM1) and GST theta 1 (GSTT1) genes have a null allele variant in which the entire gene is absent. The null genotype for both enzymes has been associated with many different types of tumors. To look for the influence of the inheritance pattern of these enzymes on thyroid cancer risk, we used a triplex PCR that included beta-globin gene as a DNA quality control to compare 300 normal individuals of our population to 116 goiter patients. There were 49 cases of benign and 67 cases of malignant nodules: 50 papillary and 17 follicular carcinomas. Comparison between thyroid tumor specimens and normal corresponding samples of 35 cancer patients demonstrated identical patterns, suggesting that the GST system is not involved in the process of follicular dedifferentiation. There was no statistical difference between the prevalence of the deleted alleles in the normal individuals and in the goiter patients. However, papillary carcinoma patients (10%) and follicular carcinoma patients (17%) presented a higher prevalence of the null genotype than the normal population individuals (5%; P < 0.05). We found a 2.6 increased risk of thyroid cancer in individuals with the GSTT1 and GSTM1 combined null inheritance, suggesting that this genotype may be associated with an increased susceptibility to thyroid cancer.