Janaína Luisa Leite

Smoking and susceptibility to thyroid cancer: an inverse association with CYP1A1 allelic variants

In contrast to most human malignancies, epidemiologic studies have frequently reported a reduced risk of differentiated thyroid cancer in tobacco consumers. Cytochrome P4501A1 (CYP1A1) gene variants may be related to an increased capacity to activate polycyclic aromatic hydrocarbons, producing highly reactive electrophilic intermediates that might damage DNA. Hence, the germline inheritance of a wild-type CYP1A1 gene may decrease the susceptibility for thyroid cancer. The present study was designed to investigate CYP1A1 (m1 and m2) role in thyroid tumorigenesis and its connection with GSTM1, GSTT1, GSTP1, GSTO1, and codon 72 of p53 genotypes. A total of 248 patients with thyroid nodules, including 67 benign goiters, 13 follicular adenomas, 136 papillary carcinomas, and 32 follicular carcinomas, and 277 controls with similar ethnic backgrounds were interviewed on their lifetime dietary and occupational histories, smoking habit, previous diseases, and other anamnestic data. DNA was extracted from a blood sample and submitted to PCR-restriction fragment length polymorphism assays. The wild-type CYP1A1m1 genotype was more frequent among papillary carcinoma patients (74.26%) than in the control population (62.45%; P=0.0147), reducing the risk for this type of cancer (odds ratio=0.564; 95% confidence interval=0.357-0.894). A multiple logistic regression analysis showed an inverse correlation between cigarette smoking (P=0.0385) and CYP1A1 germline inheritance (P=0.0237) with the susceptibility to papillary carcinomas. We were not able to find any correlation between smoking, clinical features, parameters of aggressiveness at diagnosis or during follow-up, and any of the GST or CYP genotypes considered separately or in different combinations. We suggest that CYP1A1 genotype might be associated with the reported reduced risk to papillary carcinomas among smokers.

Genetic polymorphisms associated with cigarette smoking and the risk of Graves’ disease

Objective  Cigarette smoking is a well‐recognized risk factor of Graves’ disease and, particularly, Graves’ ophthalmopathy. Hence, germline polymorphisms of detoxification genes and genes belonging to the major DNA repair–apoptosis pathways might have an important role in disease susceptibility. In addition, as some of these genes are regulated by thyroid hormones, they may affect the patients’ outcomes. We aimed to assess the influence of the GST, CYP and TP53 gene polymorphisms in the risk of Graves’ disease and its outcome.

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.

Role of the N-Acetyltransferase 2 Detoxification System in Thyroid Cancer Susceptibility

Purpose: Genetic polymorphisms in genes encoding for enzymes involved in the biotransformation of carcinogens have been shown to be relevant as risk for cancer and may be of considerable importance from a public health point of view. Considering that N-acetyltransferase 2 (NAT2) polymorphisms modulate the response to ionizing radiation, the strongest risk factor recognized to cause differentiated thyroid cancer (DTC) thus far, we sought to determine the influence of NAT2 detoxification system on thyroid cancer susceptibility. Experimental Design: We conducted a prospective case-control study, comparing 195 patients presenting with DTC that were previously genotyped for GSTT1, GSTM1, GSTP1, and CYP1A1, comprising 164 papillary carcinomas and 31 follicular carcinomas, with 196 control individuals paired for gender, age, ethnicity, diet routine, lifetime occupational history, smoking history, general health conditions, and previous diseases. We used PCR-RFLP assays and the combination of 6 variant alleles to define 18 NAT2 haplotypes that characterized slow, intermediate, or rapid phenotypes. Results: A multivariate logistic regression analysis identified the presence of *12A and the absence of *12B, *13, *14B, *14D, *6A, and *7A NAT2 haplotypes as risk factors for DTC. The inheritance of a rapid acetylation phenotype doubled the risk for a papillary carcinoma (odds ratio, 2.024; 95% confidence interval, 1.252-3.272). We found no relationship between genotypes and clinical, pathologic, or laboratory features of patients or between genotypes and outcome. Conclusions: We showed that NAT2 genotypes and the NAT2 rapid acetylation phenotype are important susceptibility factors for DTC, suggesting that NAT2 detoxification system is involved in this tumor pathogenesis.

Polymorphisms at exon 4 of p53 and the susceptibility to herpesvirus types 6 and 1 infection in renal transplant recipients

In order to replicate their own genome in the host nucleus, herpesviruses have to overcome the barrier presented by p53 gene. Variants of codon 72 and codon 47 of exon four decrease the ability of p53 to induce apoptosis. In order to investigate the influence of this germline inheritance on the susceptibility to herpesvirus type 6 (HHV6) and 1 (HHV1) infection, we examined 78 renal transplant recipients and 151 controls. HHV6 infection was more frequent among the renal transplant patients (35.89%) than in the control population (11.25%) (P < 0.001). HHV1 infection rate was similar in renal transplant patients (7.28%) and controls (2.56%). HHV6‐positive cases were more frequent among patients with codon 72 of p53 variants (60.71%) than among wild‐type p53 patients (28.20%) (P = 0.001) despite the higher frequency of codon 72 of p53 wild‐type variant in renal transplant patients compared with controls (64.1% vs. 36.4%; P < 0.001). The presence of a codon 72 of p53 germline variant genotype increased the risk for HHV6 infection more than five times (OR = 5.479; 95% CI = 1.992–15.069). Our data suggest that codon 72 of p53 polymorphism genotyping may be useful to screen for patients at higher risk for post‐transplant infections hence identifying individuals that could benefit from preventive treatment.

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.

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

ABSTRACT 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.

Prevalence of integrons in Shigella sonnei from Brazil

Diarrhea contributes to high morbidity and mortality rates in developing countries. Diarrhea from all causes is ranked as the fourth cause of death and the second cause of loss of years of productive life.1 Infection by Shigella spp. is characterized by a watery diarrhea that may progress to mucoid bloody diarrhea, also known as dysentery. Each year, over 163 million episodes of endemic shigellosis occur in DCs, whereas only 1.5 million occur in developed ones.1 The increasing number of travelers to DCs, which have become attractive tourist destinations, has also increased the number of diarrhea episodes among millions of persons who travel each year from industrialized countries to DCs.2 Shigella dysenteriae and Shigella flexneri are the predominant species in tropical areas, whereas Shigella sonnei is predominantly isolated in industrialized countries. Although the disease is often self-limiting, effective antimicrobial therapy reduces the duration and severity of the dysentery and can also prevent potentially lethal complications. Concomitantly, the excretion of the pathogen in stools is shortened significantly by antibiotic therapy, reducing the spread of infection.3 New research suggests that gene cassettes and integrons may be one mechanism involved in transmission of multidrug resistance genes in many Gram negative bacteria.4,5 In Shigella spp., the dissemination of resistance-inducing genes is mostly facilitated by the ability of the bacteria to acquire transposons or plasmids that might contain integrons and DNA sequences that consist of two conserved segments (5¢-CS and 3¢-CS) separated by a variable region that usually comprises one or more gene cassettes. The 5¢-CS region contains the integrase gene (intI), the integration site and a promoter region that allows expression of any number of gene cassettes inserted at the integration site site in a suitable orientation. The 3¢-CS region usually comprises qacE D1 (encoding resistance to quaternary ammonium compounds), sul (resistance to sulfonamides), followed by ORF5 (of unknown function) and/or tni genes (transposition functions).6 Detailed studies have been conducted on three main classes of integrons, namely integron 1 (Int1), integron 2 (Int2) and integron 3 (Int3), each one characterised by the presence of a distinct and specific intI.7 In the present study, 25 different strains of S. sonnei, obtained from sporadic cases of shigellosis, were isolated in hospitals from Brazil’s south eastern district of São Paulo (Campinas region), from six different Brazilian cities namely Campinas, Bragança Paulista, Vinhedo, Mogi Guacu, Limeria and Cosmopolis from 1997 to 20028 (Table 1). The research aim is to investigate whether the resistance for two or more antibiotics encountered in these strains is associated with the presence of Int1, Int2 and Int3. The strains were tested with seven antibiotics (chloramphenicol, trimethoprim-sulfamethoxazole, streptomycin, sulfamethoxazole (SUT), cephalothin, ampicillin and tetracycline (TT)) to determine the antibiotic resistance profile of each strain according to the Manual of the Clinical and Laboratory Standards Institute.9 For the cell DNA extraction, only the bacterial colonies of virulent strains of Shigella were selected for analysis, on the basis of the uptake of Congo red from growth media. DNA was extracted according to the Spin Column Protocol (DNeasy Blood and Tissue Hand Book, Qiagen, USA, 2006) using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA, 2006). The primers specific to Int1 (5¢-ATGCCCGTTCCATACAGAAG-3¢/ 5¢-CGGCCTTGCTGTTCTTCTAC-3¢), Int2 (5¢-AATGCGTTGCACTT CATTTG-3¢/5¢-ATGGGCAGTGAAGAGGTCAG-3¢) and Int3 (5¢-CCG GTTCAGTCTTTCCTCAA-3¢/5¢-GAGGCGTGTATCTGCCTCAT-3¢) were designed using Primer3 software (http://frodo.wi.mit.edu/). The National Center for Biotechnology Information database (http:// www.ncbi.nlm.nih.gov/nuccore/) for Int1, Int2 and Int3 are, respectively, Locus FJ501977.1, Locus EF560799.1 and Locus AY219651.1. The sequences obtained were used to design the primers, for which real-time PCR analysis was carried out on the samples for the amplification of the genes Int1, Int2 and Int3 and was on the basis of the work of Maguire et al.10 with a few adaptations. The DNA from PCR was purified using a DNA purification kit (Qiagen, USA, 2006) and later it was sent for sequencing at MWG (Eurofins MWG operon, www.operon.com) to verify and confirm the presence of Int1, Int2 and Int3. The amplification signals for Int1 were registered between cycles 20–25 and showed a clear consistent signal until the 50th cycle for all strains except strains CS7 and CS16C, which failed to register any

No evidence for mutations in exons 1, 8 and 18 of the patched gene in sporadic skin lesions of Brazilian patients

There is strong evidence that the patched (PTCH) gene is a gene for susceptibility to the nevoid basal cell carcinoma syndrome. PTCH has also been shown to mutate in both familial and sporadic basal cell carcinomas. However, mutations of the gene seem to be rare in squamous cell carcinomas. In order to characterize the role of the gene in the broader spectrum of sporadic skin malignant and pre-malignant lesions, we performed a polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis of genomic DNA extracted from 105 adult patients (46 females and 59 males). There were 66 patients with basal cell carcinomas, 30 with squamous cell carcinomas, 2 with malignant melanomas and 7 patients with precancerous lesions. Two tissue samples were collected from each patient, one from the central portion of the tumor and another from normal skin. Using primers that encompass the entire exon 1, exon 8 and exon 18, where most of the mutations have been detected, we were unable to demonstrate any band shift. Three samples suspected to present aberrant migrating bands were excised from the gel and sequenced directly. In addition, we sequenced 12 other cases, including tumors and corresponding normal samples. A wild-type sequence was found in all 15 cases. Although our results do not exclude the presence of clonal alterations of the PTCH gene in skin cancers or mutations in other exons that were not screened, the present data do not support the presence of frequent mutations reported for non-melanoma skin cancer of other populations.

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.