Synergic effect of oral contraceptives, GSTP1 polymorphisms, and high-risk HPV infection in development of cervical lesions.

Human papillomavirus (HPV) infection is considered a risk factor for cervical cancer. Even if the high-risk HPV (HR-HPV) infection is necessary, environmental co-factors and genetic susceptibility also play an important role in cervical cancer development. In this study, a possible association of rs1695 GSTP1 polymorphisms, HR-HPV infection, and oral contraceptive use with cancer lesion development in women was investigated. The study population comprised 441 Brazilian women from the Northeast region including 98 HPV-infected women with high-grade squamous intraepithelial lesions, 77 HPV-infected women with low-grade squamous intraepithelial lesions, and 266 HPV-negative women with no lesion, used as a control. Our data did not show a significant association between the GSTP1 polymorphism A/G (rs1695) and any HPV-related cervical abnormalities. However, considering the use of oral contraceptives, the GSTP1 rs1695 polymorphism was associated with higher susceptibility to the development of cervical lesions in HR-HPV-infected women. Our study suggests a synergic effect of oral contraceptive use, GSTP1 polymorphisms, and HR-HPV infection in the development of cervical lesions. Together, these risk factors may induce neoplastic transformation of the cervical squamous epithelium, setting conditions for secondary genetic events leading to cervical cancer.

The synthetic peptide PnPP-19 induces peripheral antinociception via activation of NO/cGMP/KATP pathway: Role of eNOS and nNOS.

BACKGROUND: and purpose: The peptide PnPP-19, derived from the spider toxin PnTx2-6 (renamed as δ-CNTX-Pn1c), potentiates erectile function by activating the nitrergic system. Since NO has been studied as an antinociceptive molecule and PnPP-19 is known to induce peripheral antinociception, we intended to evaluate whether PnPP-19 could induce peripheral antinociception through activation of this pathway. EXPERIMENTAL APPROACH: Nociceptive thresholds were measured by paw pressure test. PGE2 (2 µg/paw) was administered intraplantarly together with PnPP-19 and inhibitors/blockers of NOS, guanylyl cyclase and KATP channels. The nitrite concentration was accessed by Griess test. The expression and phosphorylation of eNOS and nNOS were determined by western blot. KEY RESULTS: PnPP-19 (5, 10 and 20 µg/paw) induced peripheral antinociception in rats. Administration of NOS inhibitor (L-NOarg), selective nNOS inhibitor (L-NPA), guanylyl cyclase inhibitor (ODQ) and the blocker of KATP (glibenclamide) partially inhibited the antinociceptive effect of PnPP-19 (10 µg/paw). Tissue nitrite concentration increased after PnPP-19 (10 µg/paw) administration. Expression of eNOS and nNOS remained the same in all tested groups, however the phosphorylation of nNOS Ser852 (inactivation site) increased and phosphorylation of eNOS Ser1177 (activation site) decreased after PGE2 injection. Administration of PnPP-19 reverted this PGE2-induced effect. CONCLUSIONS AND IMPLICATIONS: The peripheral antinociceptive effect induced by PnPP-19 is resulting from activation of NO-cGMP-KATP pathway. Activation of eNOS and nNOS might be required for such effect. Our results suggest PnPP-19 as a new drug candidate to treat pain and reinforce the importance of nNOS and eNOS activation, as well as endogenous NO release, for induction of peripheral antinociception.

Transmission of visceral leishmaniasis in dogs in a risk area of the metropolitan region of Belo Horizonte, Minas Gerais, Brazil / Transmissão da leishmaniose visceral em cães em área de risco da Região Metropolitana de Belo Horizonte, Minas Gerais, Brasil

Visceral leishmaniasis (VL) has spread rapidly across cities in the metropolitan region of Belo Horizonte. The aim of this study was to investigate VL dynamics in a prospective cohort study of dogs in Juatuba, between 2010 and 2011, to confirm the incidence of Leishmania infantum, and to assess possible risk factors associated with infection. An observational and prospective closed cohort study was performed using serology testing in dogs, randomly selected from the whole municipality. All seronegative dogs, or dogs with inconclusive results were monitored using indirect immunofluorescence (IIF) at 6-month intervals. The dog's owners completed a semi-structured questionnaire to assess possible causal factors of seroconversion, and the responses were assessed using logistic regression. The canine incidence coefficient was 206/1,000 dogs per year (CI: 178-238), and a cluster was identified in an area with a high concentration of seropositive dogs, but a low overall canine population. Large dogs were identified as a risk factor and the following variables were identified as protection factors: dogs aged over 4 years, daily peridomicile cleaning, and better socioeconomic conditions. VL is spreading over a large area in Juatuba in a short period of time.(AU)

A leishmaniose visceral (LV) expandiu-se de forma rápida e extensa pelos municípios da Região Metropolitana de Belo Horizonte. Objetivou-se estudar a dinâmica da LV em uma coorte prospectiva de cães em Juatuba, entre 2010 e 2011, para verificar a incidência e fatores de risco associados à infecção por Leishmania infantum. Foi feito um estudo observacional e prospectivo de coorte fechada por meio de análise sorológica em cães selecionados aleatoriamente em todo o município, com acompanhamento semestral dos resultados soronegativos e indeterminados na imunofluorescência indireta (IFI). Usou-se questionário semiestruturado junto aos proprietários de cães para avaliação da soroconversão e dos fatores determinantes a essa, por meio da regressão logística. O coeficiente de incidência canina foi de 206/1000 cães.ano (IC: 178 - 238), e foi identificado cluster em área com elevada concentração de cães soropositivos, mas com baixa densidade populacional canina. A variável cão de porte grande foi identificada como fator de risco, e as variáveis idade do cão superior a quatro anos, limpeza diária do peridomicílio e melhores condições socioeconômicas como fatores de proteção. A infecção por LV está ocorrendo em curto período de tempo e com ampla distribuição em Juatuba.(AU)

Putative Mechanisms of Viral Transmission and Molecular Dysregulation of Mammary Epithelial Cells by Human Papillomavirus: Implications for Breast Cancer.

Breast cancer is the most common cancer in women worldwide. Several studies have demonstrated the presence of Human papillomavirus (HPV) DNA in breast cancer samples. However, the role of HPV in breast carcinogenesis is not clear, and the interaction mechanisms between this infectious agent and the breast cancer cell need to be more fully clarified. In this article, we discuss the putative roles of HPV infection in breast carcinogenesis.

Prevalence of human papillomavirus (HPV), distribution of HPV types, and risk factors for infection in HPV-positive women.

The aim of this study was to describe the prevalence of human papillomavirus (HPV), the distribution of different HPV types, and the putative risk factors for infection among HPV-positive women from the State of Alagoas, Northeast Brazil. We analyzed data from 515 patients attending public and private health centers. HPV DNA from cervical samples was extracted and HPV genotyping was performed by polymerase chain reaction using MY09/11 consensus primers followed by direct sequencing. The chi-squared test for independence was used to assess statistical differences between the HPV groups. HPV DNA was found in 111 (21.55%) cervical samples. Twenty genotypes were detected: HPV6, 11, 16, 31, 33, 35, 39, 52, 53, 54, 58, 61, 62, 66, 70, 72, 81, 82, 83, and 84. In addition, multiple sexual partners (P = 0.002) and the use of oral contraceptives (P = 0.015) were associated with the presence of HPV. These findings may be relevant to the design of screening and vaccination strategies targeting specific groups of women in Northeast Brazil.

PnPP-19, a spider toxin peptide, induces peripheral antinociception through opioid and cannabinoid receptors and inhibition of neutral endopeptidase.

BACKGROUND AND PURPOSE: The synthetic peptide PnPP-19 has been studied as a new drug candidate to treat erectile dysfunction. However, PnTx2-6, the spider toxin from which the peptide was designed, induces hyperalgesia. Therefore, we intended to investigate the role of PnPP-19 in the nociceptive pathway. EXPERIMENTAL APPROACH: Nociceptive thresholds were measured by paw pressure test. PnPP-19 was administered intraplantarly alone or with selective cannabinoid or opioid receptor antagonists. The hydrolysis of PnPP-19 by neutral endopeptidase (NEP) (EC 3.4.24.11), an enzyme that cleaves enkephalin, was monitored by HPLC and the cleavage sites were deduced by LC-MS. Inhibition by PnPP-19 and Leu-enkephalin of NEP enzyme activity was determined spectrofluorimetrically. KEY RESULTS: PnPP-19 (5, 10 and 20 µg per paw) induced peripheral antinociception in rats. Specific antagonists of µ opioid receptors (clocinnamox), δ opioid receptors (naltrindole) and CB1 receptors (AM251) partly inhibited the antinociceptive effect of PnPP-19. Inhibition of fatty acid amide hydrolase by MAFP or of anandamide uptake by VDM11 enhanced PnPP-19-induced antinociception. NEP cleaved PnPP-19 only after a long incubation, and Ki values of 35.6 ± 1.4 and 14.6 ± 0.44 µmol·L(-1) were determined for PnPP-19 and Leu-enkephalin respectively as inhibitors of NEP activity. CONCLUSIONS AND IMPLICATIONS: Antinociception induced by PnPP-19 appears to involve the inhibition of NEP and activation of CB1, µ and δ opioid receptors. Our data provide a greater understanding of the antinociceptive effects of PnPP-19. This peptide could be useful as a new antinociceptive drug candidate.

Detection of Different Bovine Papillomavirus Types and Co-infection in Bloodstream of Cattle.

Bovine papillomavirus (BPV) is a diverse group of double-stranded DNA oncogenic viruses. BPVs are classically described as epitheliotropic, however, they have been detected in body fluids, such as blood and semen. The presence of BPV in these sites can have implications for the dissemination of BPV. The aim of this study was to verify the prevalence of BPV types in cattle blood. A total of 57 blood samples were analyzed by PCR using BPV type-specific primers to BPVs 1-6 and 8-10, and subsequent sequencing. Sequencing quality was determined using Staden package with Phred 20. Similarity analysis was performed with BioEdit and BLAST programs to assess the identity with known BPV types. Statistical analysis was performed by Fisher's exact test. The results showed seven different types of BPVs in the blood, with the exception of BPV 5 and 9. This is the first study that demonstrates BPVs 3, 6, 8 and 10 DNA in cattle blood. BPVs 1 and 2 were the viral types most frequent in blood, while BPVs 4 and 10 were the least frequent types. All the samples showed co-infection by at least two BPV types. These data suggest that several BPV types may infect blood cells at the same time and demonstrate the possibility that the BPV infection in non-epithelial tissue can occur without restriction to one or two viral types. These results can contribute to future studies aimed at the control and prevention of papillomaviruses.

Development of an IP-Free Biotechnology Platform for Constitutive Production of HPV16 L1 Capsid Protein Using the Pichia pastoris PGK1 Promoter.

The human papillomavirus (HPV) L1 major capsid protein, which forms the basis of the currently available vaccines against cervical cancer, self-assembles into virus-like particles (VLPs) when expressed heterologously. We report the development of a biotechnology platform for HPV16 L1 protein expression based on the constitutive PGK1 promoter (PPGK1) from the methylotrophic yeast Pichia pastoris. The L1 gene was cloned under regulation of PPGK1 into pPGKΔ3 expression vector to achieve intracellular expression. In parallel, secretion of the L1 protein was obtained through the use of an alternative vector called pPGKΔ3α, in which a codon optimized α-factor signal sequence was inserted. We devised a work-flow based on the detection of the L1 protein by dot blot, colony blot, and western blot to classify the positive clones. Finally, intracellular HPV VLPs assembly was demonstrated for the first time in yeast cells. This study opens up perspectives for the establishment of an innovative platform for the production of HPV VLPs or other viral antigens for vaccination purposes, based on constitutive expression in P. pastoris.

Development of a DNA-based vaccine strategy against bovine papillomavirus infection, involving the E5 or L2 gene.

Papillomaviruses are known to cause tumor lesions, generally benign, in epithelial tissues of diverse organisms; these lesions may progress to cancer under suitable conditions. Bovine papillomavirus (BPV) can cause urinary bladder cancer and cancer of the upper gastrointestinal tract. Furthermore, BPV1 and BPV2 are implicated in the development of tumors in equids. Many studies with animal models clearly demonstrate that DNA vaccines are very effective tools in controlling viral infections, providing strong humoral and cellular immune responses. In this study, we have described the development of two vaccine constructs for the control of diseases caused by BPV. The 1st strategy is prophylactic and is based on the L2 gene; the 2nd is therapeutic and is based on the E5 gene. Vaccine constructs were obtained and evaluated in vitro in mammalian cells. The results show the occurrence of E5 and L2 transcription and viral protein production. These results confirm the functionality of the vaccine constructs in mammalian cells. This is the 1st step in the development of a DNA-based vaccine strategy for the control and/or treatment of diseases caused by BPV.

Bovine papillomavirus E2 and E5 gene expression in sperm cells of healthy bulls.

Papillomaviruses are found in epithelial lesions and are linked to different carcinogenic processes in humans and other animals. Although BPV has been characterized as epitheliotropic, the presence of viral DNA has been detected in other tissues and fluids, such as fresh semen. The aim of this study was to evaluate the presence and expression of BPV in sperm cells of bulls (Bos taurus) asymptomatic for papillomatosis. A PCR assay was carried out with specific primers to test BPV2 in 26 semen samples. The presence of BPV transcripts was assessed by RT-PCR to E2 and E5 genes. BPV2 DNA was detected in nine out of 26 samples and the expression of E2 and E5 were detected in five out of nine BPV positive samples. This is the first record of BPV2 expression in bull sperm cells.

The presence and gene expression of bovine papillomavirus in the peripheral blood and semen of healthy horses.

Papillomavirus (PV) are double-stranded DNA viruses that can cause both benignant and malignant tumours in mammals. Twelve genotypes of bovine papillomavirus (BPV1-12) have been identified so far. The presence of BPV1 and 2 has been found in the body fluids of cattle and horses. The aim of this study is to investigate the presence of BPV DNA and the expression of viral genes in the blood and sperm cells of healthy horses using PCR and RT-PCR. BPV-1 or 2 was detected in 14 of 70 blood samples (20%) and in 11 of 31 semen samples (35%). In five of fourteen blood samples, the E5 expression tested positive, while no blood sample was positive for L1 expression. Four of 11 (36%) semen cell samples proved to be positive for E5 expression, while no gene expression in L1 could be detected. This is the first study that shows BPV1 gene expression in the blood and semen of healthy horses. Our data illustrate the need for a better understanding of the presence of BPV in non-epithelial tissues of horses and their role in the vertical and horizontal transmission of these viruses.

A high spatial resolution outdoor dose rate map of the Rio de Janeiro city, Brasil, risk assessment and urbanization effects.

Geophysical surveys have been widely used for geological mapping, and with appropriate ground calibration the database can be converted to radiometric surveys. Herein we present a case study of a high resolution map of the City of Rio de Janeiro, Brazil, constructed by integrating aerial and in situ (ground) measurements. This allowed assessing the influence of urbanization observed between 1978 and 2009 on radiation external exposure. Radiometric, geological and demographics databases were integrated in a Geographic Information System (GIS) facility. The average absorbed dose rate recorded for the city was 96 ± 0.3 nGy h(-1) and ranged from 22 to 152 nGy h(-1) among districts. The corresponding annual effective dose was determined as 0.09 ± 0.01 mSv. The number of people at risk per year according to the estimated natural radiation levels was 17 ± 1.4, among the 28,000 new cases estimated by the INCA (Brazilian National Cancer Institute) within a population of approximately 6.3 million people.

Antioxidative peptides: trends and perspectives for future research.

In recent years, much attention has been given to dietary antioxidants, especially polyphenols. Several peptides derived from protein molecules have also been found to show antioxidant capacity along with other biological properties and thus there is an increasing interest in these compounds as health promoters. This review summarizes and discusses the main sources of antioxidative peptides with focus on food-derived peptides (animal, plant and marine sources), methods of preparation, antioxidant capacity evaluation as well as their proposed mechanisms of action. A discussion of the potential health effects and comments on the different applications for these antioxidants and their potential research interest are also subject of this review.

Comparison of two PCR strategies for the detection of bovine papillomavirus.

Bovine papillomavirus (BPV) is a diverse group of double-stranded DNA oncogenic viruses, which have been detected in epithelial lesions and body fluids. Most studies of BPV infection rely on a single method for DNA detection; however the use of any single method or technique may underestimate the true prevalence of this virus. The purpose of this study was to compare two PCR strategies for the detection of BPV in skin lesions and fluids: these involve the use of BPV type-specific and consensus primers. Seventy-two cutaneous lesions, 57 blood samples and 59 semen samples were collected. PCR was used with the FAP consensus primers and BPV type-specific primers (for BPVs 2, 3, 4, 5, 8, 9 and 10), along with sequencing assays, to detect the BPV types. Phylogenetic analysis was carried out by means of the maximum likelihood method. It was found that both FAP and BPV type-specific primer sets could amplify BPV types of DNA in skin lesions, blood and semen samples. However, the BPV type-specific primers were more sensitive than the consensus primers and were able to detect co-infection of BPV in the samples. The consensus primers amplified five BPV types and were more suitable for detecting new putative BPV types. Thus, account should be taken of both PCR primer systems to identify co-infection, the presence of novel viruses, and avoid false-negative results.

Detection and expression of bovine papillomavirus in blood of healthy and papillomatosis-affected cattle.

Papillomaviruses (PV) are double-stranded DNA viruses that can cause benignant and malignant tumors in amniotes. There are 13 types of bovine papillomavirus (BPV-1 to -13); they have been found in reproductive tissues and body fluids. Normally these viruses are detected in epithelial tissue. We looked for BPV in the blood of healthy cattle and cattle with papillomatosis, using PCR and RT-PCR. BPV types 1 and 2 were detected in 8/12 blood samples of asymptomatic bovines and in 8/9 samples from cattle with papillomatosis. Six of 8 asymptomatic samples positive for BPV also showed expression for BPV. Five of 6 samples were positive for E2 expression, while 3/6 samples were positive for E5 expression. Five of 8 symptomatic samples positive for BPV also showed BPV expression. Five of 5 were positive for E2 expression, while 1/5 was positive for E5 expression. Two of 6 blood samples of asymptomatic cattle and 1/5 symptomatic blood samples scored positive for both E2 and E5 expression. This is the first study showing expression of BPV genes in the blood of asymptomatic and papillomatosis-affected animals.

Human papillomavirus vertical transmission: review of current data.

Despite the increasing evidence of human papillomavirus (HPV) vertical transmission, this route is regarded as less clinically important because of the detections of transient HPV DNA. However, recent studies have provided clear evidence of papillomavirus productive infection in lymphocytes, placenta, and bovine fetal tissue. Furthermore, a model of papillomavirus latency has been recently proposed that could explain the failure or transience in HPV detection observed in some infected infants. This new evidence of hematogeneous and vertical spread of HPV suggests that these modes of transmission should be investigated in greater detail to obtain a better understanding of the infection and a fuller awareness of the preventive measures that can be taken against HPV-related diseases.

Entropy-based approach for selecting informative regions in the L1 gene of bovine papillomavirus for phylogenetic inference and primer design.

Bovine papillomaviruses (BPVs) cause many benign and malignant lesions in cattle and other animals. Twelve BPV types have been identified so far, and several putative novel BPV types have been detected based on the analysis of L1 gene fragments, generated by FAP59/64 and MY11/09 primers. Phylogenetic trees are important in studies that describe novel BPV types. However, topological mistakes could be a problem in such studies. Therefore, we made use of entropy to find phylogenetic informative regions in the BPV L1 gene sequences from all 12 BPVs. Six data sets were created and phylogenetically compared to each other using neighbor-joining and maximum likelihood methods of phylogenetic tree reconstruction. We found two major regions in the L1 gene, using an entropy-based approach, which selects regions with low information complexity. More robust phylogenetic trees were obtained with these regions, when compared to the ones obtained with FAP59/64 and MY11/09 primers. More robust phylogenetic trees are important to accurately position novel BPV types, subtypes and variants. We conclude that an entropy-based approach is a good methodology for selecting regions of the L1 gene of BPVs that could be used to design more specific and sensitive degenerate primers, for the development of improved diagnostic methods.

Anatomical arrangement and distributionof the cerebral arterial circle in rats

The cerebral arterial circle is a polygonal shape-like arterial anastomosis placed in the brain base, whereit rounds the optic quiasm and the tuber cinereum, and also related to the interpeduncular fossa and theanterior perfurated substance. It is formed by the proximal parts of the anterior, middle and posterior cerebralarteries, and the right and left posterior communicating arteries. In order to describe the cerebral arterial circledisposition we investigated the brains of twenty rats. For each animal, the heart left ventricle was probed andacetone, distilled water at 37 °C and a solution of Neoprene Latex “450” stained with a specific red pigmentwere injected in sequence into it. To fix the brain in a better way, we isolated the head and made an apertureat the dorsal wall of the cranium and the whole specimen was fixed in a 15% formaldehyde solution. We tookoff the brain from the skull with the aid of a cold light source monocular magnifier. To take the photographicdata we used a semi-professional camera. The results showed that the cerebral arterial circle in rats is formedby branches of both internal carotid arteries and of the basilar artery, and is closed rostrally by the rostralcommunicating artery, and caudally by the right and left terminal branches of the basilar artery. We concludedfrom our data that the cerebral arterial circle in rats is compound by branches of both internal carotid arteriesand basilar artery, and that it is rostrally and caudally closed.

Expression of the bovine papillomavirus type 1, 2 and 4 L1 genes in the yeast Pichia pastoris.

Papillomaviruses are known to cause benign or malignant lesions in various animals. In cattle, bovine papillomavirus (BPV) is the etiologic agent of papillomatosis and neoplasia of the upper gastrointestinal tract and urinary bladder. Currently, there are no standard diagnostic tests or prophylactic vaccines. Protection against papillomavirus infection is conferred by neutralizing antibodies directed towards the major structural protein L1. These antibodies can be efficiently induced by immunization with virus-like particles that are formed spontaneously after L1 gene expression in recombinant systems. The yeast Pichia pastoris is known to provide an efficient system for expression of proteins due to reduced cost and high levels of protein production. We evaluated P. pastoris for expression of the L1 gene from BPV1, BPV2 and BPV4. After methanol induction, the recombinants were able to produce L1 proteins of the three different BPV types. To increase heterologous L1 protein levels, a codon optimization strategy was used for production under bioreactor conditions. The BPV1 L1 protein was identified by monoclonal antibody anti-6xHis. This is the first report of BPV L1 expression in yeast.

Biological Activities of Libidibia (Caesalpinia) ferrea var. parvifolia (Mart. ex Tul.) L. P. Queiroz Pod Preparations.

Libidibia ferrea has been used in folk medicine throughout Brazil, and this study evaluated the biological activities of crude extract (CE) as well as a partially purified fraction (F80) obtained from its pods. Results from the MTT assay revealed that only F80 inhibited NCI-H292 cell growth; however, neither CE nor F80 reduced HEp-2 cell growth or sarcoma 180 tumor weight with the in vivo assay. Acute oral toxicity of the extract and fraction was evaluated following the steps of Guideline 423, using female mice; LD(50) for both preparations was determined as 2,500 mg/kg body weight. CE and F80 promoted a reduction of the leukocyte number and nitrite level in inflammatory exudates when the anti-inflammatory assay (carrageenan-induced peritonitis) was performed. CE and F80 inhibited writhing regarding antinociceptive activity (acetic acid-induced writhing response in mice). In conclusion, CE and F80 have no significant cytotoxic or antitumor activities in cell lines showing low toxicity and no action against tumors in vivo. Both preparations revealed anti-inflammatory and antinociceptive activities, corroborating the pharmacological basis of L. ferrea for ethnomedical use.