Prevalence and clinical implications of low-risk human papillomavirus among patients with recurrent respiratory papillomatosis in Rio de Janeiro, Brazil.

OBJECTIVE: The aim of this study was to investigate the presence of human papillomavirus (HPV) in biopsy specimens from juvenile and adult patients with histopathological diagnosis of recurrent respiratory papillomatosis (RRP) treated in two public hospitals in Rio de Janeiro, Brazil. METHODS: We performed the detection and genotyping of HPV by PCR technique for the types 6, 11, 16, and 18 in biopsy specimens from 41 RRP patients. RESULTS: The juvenile onset RRP (JoRRP) corresponded to 61% and the adult onset RRP (AoRRP) corresponded to 39% of the study group. Prevalence of males was observed in both the adult (81.3%) and the juvenile (56%) groups. Lesions in the larynx were more frequent in the glottis (46%). Genotyping analysis only revealed patients with HPV-6 (34.1%), HPV-11(17.1%), and co-infection HPV-6 and -11 (48.8%). RRP severity was significantly associated with the JoRRP (p<0.001), with extralaryngeal disease and more surgeries. However, no significant association between RRP severity and HPV types was found. One co-infected patient in the JoRRP died due to the evolution of the disease with lung involvement. CONCLUSION: These results show the strong association of HPV-6 and/or HPV-11 types with RRP and could complement the diagnosis, prognosis, and therapies for these patients. In addition, the HPV vaccination should be encouraged to prevent the disease.

Green Tobacco Sickness among Brazilian farm workers and genetic polymorphisms.

OBJECTIVE: Green Tobacco Sickness (GTS) is an occupational illness caused by dermal absorption of nicotine from tobacco leaves. It affects thousands of farm workers worldwide. Brazil is the second tobacco producer in the world; despite this, there are few studies on GTS among Brazilian harvesters. This study aimed to determine the prevalence of GTS among a population of tobacco workers from a producing area in northeastern Brazil and investigate whether the occurrence of the disease was influenced by factors such age, gender and smoking status. In addition, it was investigated if there was association between the onset of GTS and genetic polymorphisms in genes that encode some detoxification enzymes. A semi-structured questionnaire was used to collect demographic, behavioral and occupational data from the referred workers. Polymorphisms were tested through the Polymerase Chain Reaction technique. RESULTS: The total prevalence of GTS found was 56.9%, with a significant difference between genders (71.7% for women and 35.3% for men, p < 0.0001). No association was identified between the investigated polymorphisms and GTS. This study confirms the occurrence of GTS among tobacco harvesters in Brazil with high prevalence. The investigation suggests the need to take preventive measures to protect tobacco workers against this disease.

DNA detection of JC and BK virus in archival urine cytospin slides.

To identify decoy cells, cytological examination was performed in urine cytospin slides. Decoy cells are related to Polyomaviruses (JC virus [JCV] and BK virus [BKV]), which are recognized worldwide due to potential infection and morbidity in kidney transplant recipients. Cytologically, it is difficult to evaluate the cytopathic effect of JCV and BKV in urine of patients with urothelial neoplasia. For this reason, there is a need for molecular approaches. To evaluate the incidence of BKV and JCV DNA in archival slides of urine cytospin material with benign and malignant characteristics. A total of 176 urine specimens were used for cytological examination of neoplastic or decoy cells. The samples were analyzed for the presence of JCV and BKV, by polymerase chain reaction (PCR) in DNA Isolated from archival slides of urine cytospin material. A typical samples (n = 48) were compared with the remaining 128 samples without atypia/neoplasia for the presence of JCV or BKV DNA. A statistically nonsignificant result was observed correlating the presence of JCV or BKV. The results show that DNA Isolated from archival slides of urine cytospin material can be used for detection of BKV and JCV.

Comparing intestinal versus diffuse gastric cancer using a PEFF-oriented proteomic pipeline.

Gastric cancer is the fifth most common malignant neoplasia and the third leading cause of cancer death worldwide. Mac-Cormick et al. recently showed the importance of considering the anatomical region of the tumor in proteomic gastric cancer studies; more differences were found between distinct anatomical regions than when comparing healthy versus diseased tissue. Thus, failing to consider the anatomical region could lead to differential proteins that are not disease specific. With this as motivation, we compared the proteomic profiles of intestinal and diffuse adenocarcinoma from the same anatomical region, the corpus. To achieve this, we used isobaric labeling (iTRAQ) of peptides, a 10-step HILIC fractionation, and reversed-phase nano-chromatography coupled online with a Q-Exactive Plus mass spectrometer. We updated PatternLab to take advantage of the new Comet-PEFF search engine that enables identifying post-translational modifications and mutations included in neXtProt's PSI Extended FASTA Format (PEFF) metadata. Our pipeline then uses a text-mining tool that automatically extracts PubMed IDs from the proteomic result metadata and drills down keywords from manuscripts related with the biological processes at hand. Our results disclose important proteins such as apolipoprotein B-100, S100 and 14-3-3 proteins, among many others, highlighting the different pathways enriched by each cancer type. SIGNIFICANCE: Gastric cancer is a heterogeneous and multifactorial disease responsible for a significant number of deaths every year. Despite the constant improvement of surgical techniques and multimodal treatments, survival rates are low, mostly due to limited diagnostic techniques and late symptoms. Intestinal and diffuse types of gastric cancer have distinct clinical and pathological characteristics; yet little is known about the molecular mechanisms regulating these two types of gastric tumors. Here we compared the proteomic profile of diffuse and intestinal types of gastric cancer from the same anatomical location, the corpus, from four male patients. This methodological design aimed to eliminate proteomic variations resulting from comparison of tumors from distinct anatomical regions. Our PEFF-tailored proteomic pipeline significantly increased the identifications as when compared to previous versions of PatternLab.

Colorectal cancer DNA methylation patterns from patients in Manaus, Brazil.

BACKGROUND: DNA methylation is commonly linked with the silencing of the gene expression for many tumor suppressor genes. As such, determining DNA methylation patterns should aid, in times to come, in the diagnosis and personal treatment for various types of cancers. Here, we analyzed the methylation pattern from five colorectal cancer patients from the Amazon state in Brazil for four tumor suppressor genes, viz.: DAPK, CDH1, CDKN2A, and TIMP2 by employing a polymerase chain reaction (PCR) specific to methylation. Efforts in the study of colorectal cancer are fundamental as it is the third most of highest incidence in the world. RESULTS: Tumor biopsies were methylated in 1/5 (20%), 2/5 (40%), 4/5 (80%), and 4/5 (80%) for CDH1, CDKN2A, DAPK, and TIMP2 genes, respectively. The margin biopsies were methylated in 3/7 (43%), 2/7 (28%), 7/7 (100%), and 6/7 (86%) for CDH1, CDKN2A, DAPK, and TIMP2, respectively. CONCLUSIONS: Our findings showed DAPK and TIMP2 to be methylated in most samples from both tumor tissues and adjacent non-neoplastic margins; thus presenting distinct methylation patterns. This emphasizes the importance of better understanding of the relation of these patterns with cancer in the context of different populations.

Chemo-resistant protein expression pattern of glioblastoma cells (A172) to perillyl alcohol.

Glioblastoma multiform (GBM) is by far the most malignant glioma. We have introduced a new treatment for GBMs that comprises the inhalation of a naturally occurring terpene with chemotherapeutic properties known as perillyl alcohol (POH). Clinical trial results on recurrent GBM patients showed that POH extends the average life by more than eight months, temporarily slows tumor growth, and in some cases even decreases tumor size. After approximately seven months, the tumor continues to grow and leads to a dismal prognosis. To investigate how these tumors become resistant to POH, we generated an A172 human glioblastoma cell culture tolerant to 0.06 mM of POH (A172r). We used Multidimensional Protein Identification Technology (MudPIT) to compare the protein expression profile of A172r cells to the established glioblastoma A172 cell line. Our results include a list of identified proteins unique to either the resistant or the nonresistant cell line. These proteins are related to cellular growth, negative apoptosis regulation, Ras pathway, and other key cellular functions that could be connected to the underlying mechanisms of resistance.

Epstein-Barr virus and human papillomavirus infection in vulvar lichen sclerosus.

OBJECTIVE: We investigated the presence of the Epstein-Barr virus (EBV) and human papillomavirus (HPV) in patients with vulvar lichen sclerosus (LS). MATERIALS AND METHODS: We investigated the presence of HPV and EBV from 34 vulvar biopsies of patients with LS who had had no previous treatment and from 17 normal vulvar brushings used as controls. We used polymerase chain reaction to amplify DNA sequences of these viruses. Human papillomavirus and EBV DNA detection was carried out using MY09/MY11 and TC67/TC69 consensus primers, respectively. The amplified polymerase chain reaction products were analyzed by 10% polyacrylamide gel. RESULTS: The mean age of the patients was 57 years old, with the majority postmenopausal. Human papillomavirus DNA was not found in the LS samples studied, but it was found in 23.2% (4/17) of the controls. However, EBV DNA was found in 26.5% (9/34) of the LS samples analyzed, and it was not found in the controls. CONCLUSIONS: Our results showed no relationship between HPV and LS. This result is in accordance with the literature. We have found 26.5% of EBV in our samples. This is a preliminary study, and the follow-up of these patients will elucidate whether EBV could play a role in cases of LS.

Evaluation of DAPK gene methylation and HPV and EBV infection in cervical cells from patients with normal cytology and colposcopy.

OBJECTIVE: Evaluation of promoter methylation of the death-associated protein kinase (DAPK) gene and HPV and EBV infections in cervical cells from patients with normal cytology and colposcopy. STUDY DESIGN: Twenty women, who had been patients at the Institute of Gynecology of the Federal University of Rio de Janeiro (UFRJ) for routine examinations and who showed normal cytology and colposcopy, were selected for this work. Cervical brushings were used for DNA extraction, and the analysis of methylation patterns of the DAPK gene was done through chemical modification with sodium bisulfite. Analysis of viral infection was done using polymerase chain reaction (PCR). RESULTS: Of the 20 patients studied, six (30%) presented methylation of the DAPK gene, five (25%) presented infection with EBV and three (15%) presented coinfection with HPV/EBV. Associating methylation with viral infection, we found methylated DAPK in one patient (16%) with EBV, in two patients (33%) with co-infection and in three patients (50%) with no viral infection. CONCLUSIONS: In the present study, we verified, for the first time, the methylation pattern of the DAPK gene in cervical smears from patients with normal cytology and colposcopy. The results also showed the presence of viral infections in these patients. EBV infection, irrespective of whether associated with HPV or not, may contribute to cervical carcinogenesis as a cofactor. Methylation of the DAPK gene is associated with cell transformation, suggesting that DAPK methylation might be an important marker for the development of cervical epithelial neoplasias.

Recent advances in the molecular genetics of malignant gliomas disclose targets for antitumor agent perillyl alcohol.

Tumors of glial origin such as glioblastoma multiforme (GBM) comprise the majority of human brain tumors. Despite advances in surgery, radiation, and chemotherapy, the prognosis for patients with malignant glioma has not improved, emphasizing the need for a search for new chemotherapeutic drugs. Deregulated p21-Ras function, as a result of mutation, overexpression, or growth factor-induced overactivation, contributes to the growth of GBM. The monoterpene perillyl alcohol (POH) has preventive and therapeutic effects in a wide variety of preclinical tumor models and is currently under phase I and phase II clinical trials. As inhibition of posttranslational isoprenylation of Ras, a family of proteins that are involved in signal transduction is among the drug-related activities observed in this compound; POH may be a potential chemotherapeutic agent for GBM. Intranasal delivery is a practical and noninvasive approach that allows therapeutic agents that do not cross the blood-brain barrier to enter the central nervous system, reducing unwanted systemic side effects. This article describes the effect of intranasal delivery of POH in a patient with relapsed GBM.

Demonstration of the lapachol as a potential drug for reducing cancer metastasis.

Metastasis is the major process responsible for the death in cancer patients. In the search for more effective antineoplasic drugs, many substances are under investigation, among them lapachol. This study aims to examine the molecular and morphological alterations caused by lapachol treatment, as well as its effects on the intrinsic tissue invasive property of this cell line. HeLa cells were exposed to different concentrations of lapachol, and the resulting alterations on cellular protein profile, morphology and invasiveness property were studied. At 400 microg/ml, cellular viability remains unchanged, but lapachol induces alterations in the protein profile and inhibits the invasiveness of HeLa cells in CAM model. With these results, we can conclude that lapachol has a great potential of application in fighting metastasis.

HLM/OSBP2 is expressed in chronic myeloid leukemia.

Oxysterols are oxygenated derivatives of cholesterol that have been shown to influence a wide variety of cellular processes including sterol metabolism, lipid trafficking, apoptosis and more recently, cell differentiation. The oxysterol binding proteins (OSBPs) comprise a large conserved family of proteins in eukaryotes with high affinity for oxysterols, but their precise function has not been defined yet. One member of this family in humans, HLM/OSBP2 protein, has recently been reported as a potential marker for solid tumor dissemination and worse prognosis in these cases. In this study we focused on the evaluation of HLM/OSBP2 expression in malignant cell lines from different origins (blood and solid tumors) and we also evaluated its expression in chronic myeloid leukemia patients, correlating the molecular findings with clinical outcome. Our results showed that HLM/OSBP2 was expressed in 80% of the analysed CML patients, suggesting that this protein could constitute a helpful tool for disease monitoring and reinforces recent findings that HLM/OSBP2 protein could be involved in the maintenance of the undifferentiated state necessary for leukemogenesis.

Sepsis: a follow-up of cytokine production in different phases of septic patients.

Sepsis and its sequelae are still a major cause of morbidity and mortality on today's intensive care units. The evidence that primary responses in sepsis are mediated by cytokines has led to various approaches to evaluate the potential of these mediators as markers of disease progression, prognosis or treatment. This study evaluated variations of plasma levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), interleukin-1 (IL-1) and nitric oxide (NO) in different phases of sepsis and compared the relation of these data with disease evaluation and outcome. No difference in interleukin production in different phases of septic patients or between septic and polytrauma group was found. The only parameter that showed correlation with disease severity was the increase in interleukin-6 in final phase of sepsis, which corresponds to septic shock. No significant difference in plasma cytokine levels was found between survival or non-survival septic or polytraumatic patients and the use of carbapenem and cephalosporin. Taken together, the data indicate that, with the exception of interleukin-6, cytokine determination does not serve as marker of infectious disease nor can it be used to predict the prognosis of sepsis.

Effects of perillyl alcohol in glial C6 cell line in vitro and anti-metastatic activity in chorioallantoic membrane model.

The search for new chemotherapeutic drugs has increased, especially for those that have a natural origin. The monoterpene perillyl alcohol (POH) has been shown to exert chemopreventive activity in mammary, liver, and lung tumor models. It has also been used to treat a variety of rodent cancers, including pancreatic and breast carcinomas. In vitro data suggest that it may be effective against neuroblastomas and leukemias. This work evaluates the effects of the treatment of murine glial C6 cells with perillyl alcohol. In vitro, our studies have indicated that POH inhibits proliferation of the C6 glial cell line. POH was logarithmically diluted in concentrations of 30% through 0.0003% and showed inhibition cell proliferation of 78.36% in concentration of 30%; 69.87% in concentration of 3%; and 67.04% in concentration of 0.03%. In addition, the anti-metastatic activity of POH against these cells was evaluated using chick embryos as an in vivo model. The experiments have shown anti-metastatic activity of POH when the C6 murine glial cells were exposed to a concentration of 0.3 to 0.003% POH for 2 h, prior to its inoculation in chick embryo chorioallantoic membrane model. This phenomenon shows the possible role of POH as an in vivo anti-metastatic drug.