Overexpression of GPC6 and TMEM132D in Early Stage Ovarian Cancer Correlates with CD8+ T-Lymphocyte Infiltration and Increased Patient Survival.

Infiltration of cytotoxic T-lymphocytes in ovarian cancer is a favorable prognostic factor. Employing a differential expression approach, we have recently identified a number of genes associated with CD8+ T-cell infiltration in early stage ovarian tumors. In the present study, we validated by qPCR the expression of two genes encoding the transmembrane proteins GPC6 and TMEM132D in a cohort of early stage ovarian cancer patients. The expression of both genes correlated positively with the mRNA levels of CD8A, a marker of T-lymphocyte infiltration [Pearson coefficient: 0.427 (p = 0.0067) and 0.861 (p < 0.0001), resp.]. GPC6 and TMEM132D expression was also documented in a variety of ovarian cancer cell lines. Importantly, Kaplan-Meier survival analysis revealed that high mRNA levels of GPC6 and/or TMEM132D correlated significantly with increased overall survival of early stage ovarian cancer patients (p = 0.032). Thus, GPC6 and TMEM132D may serve as predictors of CD8+ T-lymphocyte infiltration and as favorable prognostic markers in early stage ovarian cancer with important consequences for diagnosis, prognosis, and tumor immunobattling.

Sanitary quality of edible bivalve mollusks in Southeastern Brazil using an UV based depuration system

The increase in seafood production, especially in mariculture worldwide, has brought out the need of continued monitoring of shellfish production areas in order to ensure safety to human consumption. The purpose of this research was to evaluate pathogenic protozoa, viruses and bacteria contamination in oysters before and after UV depuration procedure, in brackish waters at all stages of cultivation and treatment steps and to enumerate microbiological indicators of fecal contamination from production site up to depuration site in an oyster cooperative located at the Southeastern estuarine area of Brazil. Oysters and brackish water were collected monthly from September 2009 to November 2010. Four sampling sites were selected for enteropathogens analysis: site 1- oyster growth, site 2- catchment water (before UV depuration procedure), site 3 - filtration stage of water treatment (only for protozoa analysis) and site 4- oyster's depuration tank. Three microbiological indicators ! were examined at sites 1, 2 and 4. The following pathogenic microorganisms were searched: Giardia cysts, Cryptosporidium oocysts, Human Adenovirus (HAdV), Hepatitis A virus (HAV), Human Norovirus (HnoV) (genogroups I and II), JC strain Polyomavirus (JCPyV) and Salmonella sp. Analysis consisted of molecular detection (qPCR) for viruses (oysters and water samples); immunomagnetic separation followed by direct immunofluorescence assay for Cryptosporidium oocysts and Giardia cysts and also molecular detection (PCR) for the latter (oysters and water samples); commercial kit (Reveal-Neogee (R)) for Salmonella analysis (oysters). Giardia was the most prevalent pathogen in all sites where it was detected: 36.3%, 18.1%, 36.3% and 27.2% of water from sites 1, 2, 3 and 4 respectively; 36.3% of oysters from site 1 and 54.5% of depurated oysters were harboring Giardia cysts. The huge majority of contaminated samples were classified as Giardia duodenalis. HAdv was detected in water and o! ysters from growth site and HnoV GI in two batches of oysters ! (site 1) in huge concentrations (2.11 x 10(13), 3.10 x 10(12) gc/g). In depuration tank site, Salmonella sp., HAV (4.84 x 10(3)) and HnoV GII (7.97 x 10(14)) were detected once in different batches of oysters. Cryptosporidium spp. oocysts were present in 9.0% of water samples from site four. These results reflect the contamination of oysters even when UV depuration procedures are employed in this shellfish treatment plant. Moreover, the molecular comprehension of the sources of contamination is necessary to develop an efficient management strategy allied to shellfish treatment improvement to prevent foodborne illnesses. (C) 2011 Elsevier Ltd. All rights reserved.

The High Prevalence and Diversity of Chlamydiales DNA within Ixodes ricinus Ticks Suggest a Role for Ticks as Reservoirs and Vectors of Chlamydia-Related Bacteria.

The Chlamydiales order is composed of nine families of strictly intracellular bacteria. Among them, Chlamydia trachomatis, C. pneumoniae, and C. psittaci are established human pathogens, whereas Waddlia chondrophila and Parachlamydia acanthamoebae have emerged as new pathogens in humans. However, despite their medical importance, their biodiversity and ecology remain to be studied. Even if arthropods and, particularly, ticks are well known to be vectors of numerous infectious agents such as viruses and bacteria, virtually nothing is known about ticks and chlamydia. This study investigated the prevalence of Chlamydiae in ticks. Specifically, 62,889 Ixodes ricinus ticks, consolidated into 8,534 pools, were sampled in 172 collection sites throughout Switzerland and were investigated using pan-Chlamydiales quantitative PCR (qPCR) for the presence of Chlamydiales DNA. Among the pools, 543 (6.4%) gave positive results and the estimated prevalence in individual ticks was 0.89%. Among those pools with positive results, we obtained 16S rRNA sequences for 359 samples, allowing classification of Chlamydiales DNA at the family level. A high level of biodiversity was observed, since six of the nine families belonging to the Chlamydiales order were detected. Those most common were Parachlamydiaceae (33.1%) and Rhabdochlamydiaceae (29.2%). "Unclassified Chlamydiales" (31.8%) were also often detected. Thanks to the huge amount of Chlamydiales DNA recovered from ticks, this report opens up new perspectives on further work focusing on whole-genome sequencing to increase our knowledge about Chlamydiales biodiversity. This report of an epidemiological study also demonstrates the presence of Chlamydia-related bacteria within Ixodes ricinus ticks and suggests a role for ticks in the transmission of and as a reservoir for these emerging pathogenic Chlamydia-related bacteria.

Comparing assays for the measurement of AhR ligand activity

In this thesis (TFG) the results of the comparison of three assays for the measurement of AhR ligand activity are exposed. This study was part of a collaborative project aiming at the characterization of the AhR signaling activities of known naturally occurring compounds to explore the potential of using non-toxic compounds to treat inflammatory diseases via oral administration. The first goal of this project was to find an assay able to measure AhR-activity, so the comparison of different assays has been done in order to find the most convenient one according to the efficiency, sensitivity and precision. Moreover, other elements with operational nature such as price, toxicity of components or ease of use has been considered. From the use of compounds known from the literature to be AhR ligands, three assays have been tested: (1) P450-GloTM CYP1A2 Induction/Inhibition assay, (2) quantitative Polymerase Chain Reaction (qPCR) and (3) DR. CALUX® Bioassay. Moreover, a different experiment using the last assay was performed for the study in vivo of the transport of the compounds tested. The results of the TFG suggested the DR. CALUX® Bioassay as the most promising assay to be used for the screening of samples as AhR-ligands because it is quicker, easier to handle and less expensive than qPCR and more reproducible than the CYP1A2 Induction/Inhibition assay. Moreover, the use of this assay allowed having a first idea of which compounds are uptaken by the epithelial barrier and in with direction the transport happens.

Mouse Model of Respiratory Tract Infection Induced by Waddlia chondrophila.

Waddlia chondrophila, an obligate intracellular bacterium belonging to the Chlamydiales order, is considered as an emerging pathogen. Some clinical studies highlighted a possible role of W. chondrophila in bronchiolitis, pneumonia and miscarriage. This pathogenic potential is further supported by the ability of W. chondrophila to infect and replicate within human pneumocytes, macrophages and endometrial cells. Considering that W. chondrophila might be a causative agent of respiratory tract infection, we developed a mouse model of respiratory tract infection to get insight into the pathogenesis of W. chondrophila. Following intranasal inoculation of 2 x 108 W. chondrophila, mice lost up to 40% of their body weight, and succumbed rapidly from infection with a death rate reaching 50% at day 4 post-inoculation. Bacterial loads, estimated by qPCR, increased from day 0 to day 3 post-infection and decreased thereafter in surviving mice. Bacterial growth was confirmed by detecting dividing bacteria using electron microscopy, and living bacteria were isolated from lungs 14 days post-infection. Immunohistochemistry and histopathology of infected lungs revealed the presence of bacteria associated with pneumonia characterized by an important multifocal inflammation. The high inflammatory score in the lungs was associated with the presence of pro-inflammatory cytokines in both serum and lungs at day 3 post-infection. This animal model supports the role of W. chondrophila as an agent of respiratory tract infection, and will help understanding the pathogenesis of this strict intracellular bacterium.

Spinal Cord T-Cell Infiltration in the Rat Spared Nerve Injury Model: A Time Course Study.

The immune system is involved in the development of neuropathic pain. In particular, the infiltration of T-lymphocytes into the spinal cord following peripheral nerve injury has been described as a contributor to sensory hypersensitivity. We used the spared nerve injury (SNI) model of neuropathic pain in Sprague Dawley adult male rats to assess proliferation, and/or protein/gene expression levels for microglia (Iba1), T-lymphocytes (CD2) and cytotoxic T-lymphocytes (CD8). In the dorsal horn ipsilateral to SNI, Iba1 and BrdU stainings revealed microglial reactivity and proliferation, respectively, with different durations. Iba1 expression peaked at D4 and D7 at the mRNA and protein level, respectively, and was long-lasting. Proliferation occurred almost exclusively in Iba1 positive cells and peaked at D2. Gene expression observation by RT-qPCR array suggested that T-lymphocytes attracting chemokines were upregulated after SNI in rat spinal cord but only a few CD2/CD8 positive cells were found. A pronounced infiltration of CD2/CD8 positive T-cells was seen in the spinal cord injury (SCI) model used as a positive control for lymphocyte infiltration. Under these experimental conditions, we show early and long-lasting microglia reactivity in the spinal cord after SNI, but no lymphocyte infiltration was found.

Analysis of TRIP expression in different types of skin tumor

TRAF-interacting protein (TRIP) is a ubiquitously expressed nucleolar E3 ubiquitin ligase. Ubiquitination of proteins is a post-translational modification, which decides on the cellular fate of the protein. TRIP in vivo substrate has not been yet identified. However, TRIP has been shown to play an important role in cellular proliferation, especially in keratinocytes. TRIP was found to be up-regulated in basal cell carcinoma (BCC) at the mRNA level. This prompted us to elucidate its role in skin proliferative diseases such as cancer by analyzing its expression in BCCs at protein level and in squamous cell carcinoma (SCC) at mRNA and protein level. To that purpose, we performed a real-time PCR (qPCR) analysis followed by an immunohistochemistry (IHC) on formalin-fixed, paraffin-embedded (FFPE) biopsies. The real-time PCR was performed on 12 RNA samples of which 6 were extracted from SCC biopsies and 6 from normal human skin. The results were statistically insignificant. Further analyses are needed on new RNA samples. The IHC assay was performed on 20 biopsies from BCCs, 21 biopsies from SCCs and on 5 tissues from normal human skin. The results obtained showed an extensive expression of TRIP in keratinocytes nuclei. Due to various limitations related to the technique and to doubts about preservation of the antigens in the tissues from normal human skin, we could not highlight a clear difference in TRIP expression between the different tissues. In conclusion, further analyses are needed on new RNA samples (qPCR) and on better preserved FFPE tissues from normal skin (IHC) to assess TRIP relative expression in BCCs and SCCs versus normal human skin.

Papel protetor do alelo G do polimorfismo no gene Interleucina 10 (-1082G/A) contra o desenvolvimento de pré-eclâmpsia

OBJETIVOS:Identificar a frequência do polimorfismo no gene IL-10, rs1800896 (-1082 A/G), em mulheres com pré-eclâmpsia (PE) e em mulheres do grupo controle e associar a presença deste polimorfismo com a proteção contra o desenvolvimento da PE.MÉTODOS:Estudo do tipo caso-controle, no qual foram selecionadas 54 mulheres com PE, classificadas de acordo com os critérios da National High Blood Pressure Education Program e 172 mulheres do grupo controle, com pelo menos duas gestações saudáveis. O polimorfismo proposto foi estudado utilizando-se a técnica de reação em cadeia da polimerase em tempo real (qPCR), com sondas de hidrólise. A análise estatística foi realizada utilizando-se o teste de associação do χ2. Odds ratio e seu intervalo de confiança de 95% foram usados para medir a força de associação entre o polimorfismo estudado e o desenvolvimento da PE.RESULTADOS:Foi observado aumento significativo da frequência do genótipo AG entre mulheres do grupo controle (85 versus 15% nas mulheres com PE). O alelo G é significativamente mais frequente entre as mulheres do grupo controle do que nas com PE (Teste χ2; p=0,01). Oodds ratio para as portadoras do alelo G foi de 2, 13, indicando que apresentam menor risco de desenvolver PE do que as não portadoras.CONCLUSÕES:Sugere-se associação entre a presença do alelo G do polimorfismo no geneIL-10, rs1800896 (-1082 A/G), e a proteção contra o desenvolvimento da PE. Mais estudos sobre a contribuição dessas variações e os mecanismos pelos quais afetam o risco de desenvolver PE ainda necessitam de ser realizadas.

A thymidine kinase-deleted bovine herpesvirus 5 establishes latent infection but reactivates poorly in a sheep model

The ability of thymidine kinase (tk)-deleted recombinant bovine herpesvirus 5 (BoHV-5tkΔ) to establish and reactivate latent infection was investigated in lambs. During acute infection, the recombinant virus replicated moderately in the nasal mucosa, yet to lower titers than the parental strain. At day 40 post-infection (pi), latent viral DNA was detected in trigeminal ganglia (TG) of all lambs in both groups. However, the amount of recombinant viral DNA in TGs was lower (9.7-fold less) than that of the parental virus as determined by quantitative real time PCR. Thus, tk deletion had no apparent effect on the frequency of latent infection but reduced colonization of TG. Upon dexamethasone (Dx) administration at day 40 pi, lambs inoculated with parental virus shed infectious virus in nasal secretions, contrasting with lack of infectivity in secretions of lambs inoculated with the recombinant virus. Nevertheless, some nasal swabs from the recombinant virus group were positive for viral DNA by PCR, indicating low levels of reactivation. Thus, BoHV-5 TK activity is not required for establishment of latency, but seems critical for efficient virus reactivation upon Dx treatment.

Prevalência sorológica e molecular de Babesia bovis e Babesia bigemina em búfalos (Bubalus bubalis) na Ilha de Marajó, Pará

O objetivo do estudo foi testar a prevalência sorológica e molecular de Babesia bovis e Babesia bigemina em búfalos da Ilha de Marajó, Pará. Foi utilizado ensaio de imunoadsorção enzimático indireto (iELISA) com antígeno total contendo proteínas de superfície externa e reação em cadeia da polimerase (qPCR), envolvendo o uso de SYBR Green com base na amplificação de um pequeno fragmento de gene do citocromo b. A prevalência de animais positivos no ELISA para B. bovis, B. bigemina e para infecção mista foi de 24.87% (199/800), 20.75% (166/800) e 18.75% (150/800), respectivamente. Na PCR foi detectado a presença de B. bovis em 15% (18/199) e de B. bigemina em 16% (19/199) dos animais, sendo que destes, 58% (11/19) apresentavam-se co-infectados pelos dois agentes. Os resultados mostram uma baixa prevalência de anticorpos anti-B. bovis e anti-B. bigemina em búfalos da Ilha do Marajó. Porém, observou-se que os agentes da babesiose bovina circulam em búfalos, podendo estes atuar como reservatórios.

Freund's adjuvant-induced inflammation: clinical findings and its effect on hepcidin mRNA expression in horses

Hypoferremia observed during systemic inflammatory disorders is regulated by hepcidin. Hepcidin up-regulation is particularly important during acute inflammation, as it restricts the availability of iron, which is necessary for pathogenic microorganism growth before adaptive immunity occurs. The aim of this study was to evaluate the clinical findings and hepatic hepcidin mRNA expression in horses using a Freund's complete adjuvant (FCA) model of inflammation. The expression of hepcidin mRNA in the liver was determined in healthy horses following two intramuscular injections of FCA at 0 h and 12 h. Plasma iron and fibrinogen concentrations were measured at multiple time points between 0 h and 240 h post-FCA injection (PI). Hepcidin mRNA expression was determined by RT-qPCR using liver biopsy samples performed at 0 h (control), 6 h and 18 h PI. The mean plasma fibrinogen level was significantly different from the control values only between 120 and 216 h PI. The mean plasma iron level was significantly lower than the control between 16 and 72 h PI, reaching the lowest levels at 30 h PI (33 % of the initial value), and returned to the reference value from 96 h PI to the end of the experiment. Hepcidin mRNA expression increased at 6 h PI and remained high at 18 h PI. The iron plasma concentration was an earlier indicator of inflammatory processes in horses when compared with fibrinogen and might be useful for the early detection of inflammation in the horse. FCA administration caused the rapid onset of hypoferremia, and this effect was likely the result of up-regulated hepatic hepcidin gene expression. This study emphasizes the importance of hepcidin and iron metabolism during inflammation in horses.

Diagnóstico de paratuberculose por biópsia retal em búfalos

Resumo: Foram realizadas biópsias retais de 140 búfalos, machos e fêmeas, das raças Murrah e mestiços de Murrah com Mediterrâneo, com idade acima de três anos, em uma propriedade no município de São Mateus, Maranhão, Brasil. Adicionalmente foram realizadas necropsias de 11 búfalos, para realizar um estudo comparativo entre os achados das biópsias retais e de tecidos de íleo e linfonodo mesentérico. A propriedade apresentava histórico de animais com emagrecimento progressivo e diarreia não responsiva a antimicrobianos. Os búfalos apresentavam sinais clínicos caracterizados por diarreia, estado nutricional regular a ruim, desidratação e edema submandibular. Nas biópsias retais seis búfalos apresentaram lesões sugestivas da paratuberculose na Hematoxilina-Eosina (HE), sendo estas caracterizadas por inflamação granulomatosa multifocal moderada na lâmina própria com macrófagos epitelioides. Em quatro animais foram observadas adicionalmente células gigantes do tipo Langhans. Em 15 búfalos foi observado infiltrado linfocitário multifocal leve na lâmina própria. Pela coloração de Ziehl-Neelsen (ZN), 4,3% (6/140) apresentaram bacilos álcool-ácido resistentes (BAAR) e na PCR em tempo real (qPCR), 5,71% (7/140) tiveram amplificação do material genético. Foram necropsiados 11 búfalos, à necropsia foram observados aumento de linfonodos mesentéricos com áreas esbranquiçadas na superfície de corte; intestino delgado e grosso com dobras transversais evidentes, mucosa espessada e irregular, de aspecto reticulado, placas de Peyer evidentes e conteúdo líquido e marrom. Ainda se viam áreas espessadas em torno da válvula ileocecal e vasos linfáticos evidentes. As lesões histológicas localizadas no intestino delgado e linfonodos mesentéricos de quatro búfalos foram compatíveis com lesões já descritas na literatura, e apresentaram BAAR e amplificação de material genético na qPCR. A concordância entre a biópsia retal e a análise dos tecidos de íleo e linfonodo mesentérico, segundo o teste Kappa (K=0,792), foi alta. A biópsia retal realizada demonstrou ser promissora e pode ser empregada, juntamente com outras técnicas, para auxiliar no diagnóstico ante mortem em búfalos de rebanhos com suspeita de paratuberculose; pela mesma foi possível detectar animais positivos através da coloração de ZN e qPCR. Os resultados obtidos podem ser utilizados no controle da enfermidade para selecionar e eliminar animais positivos do rebanho, diminuindo gradualmente, a disseminação do agente no ambiente, e a consequente contaminação de outros animais.

Infecção transplacentária e intrauterina por Brucella abortus em búfalos (Bubalus bubalis)

RESUMO: O objetivo deste trabalho foi verificar a presença de Brucella abortus e as lesões causadas por esse agente nos anexos fetais e nos fetos de búfalas. Para isso, 20 búfalas em diversos meses de gestação, sorologicamente positivas para brucelose, foram submetidas ao abate sanitário. A idade fetal foi determinada através de exames ultrassonográficos associados à mensuração dos fetos durante a necropsia. Do útero fechado desses animais foram coletadas amostras para histopatologia e qPCR. A partir do segundo mês de gestação foi possível detectar a presença de DNA de B. abortus em líquido amniótico, líquido alantoide e em útero e, a partir do quinto mês, na placenta, coração, baço, rim, pulmão, intestino, fígado e linfonodos dos fetos. Os principais achados anatomopatológicos foram placentite fibrinopurulenta necrótica e endometrite supurativa crônica.

Detecção de Brucella abortus em linfonodos de búfalas (Bubalus bubalis) em diferentes fases da gestação

Resumo: O objetivo do presente trabalho foi verificar a presença do DNA de Brucella abortus e caracterizar as lesões causadas por esse agente em linfonodos de búfalas. Foram utilizadas 19 búfalas em diversos estágios de gestação, sorologicamente positivas para brucelose, submetidas ao abate sanitário, das quais se coletou fragmentos de diversos linfonodos. A idade fetal foi determinada através de exames ultrassonográficos associados à mensuração dos fetos durante a necropsia. Amostras foram coletadas e submetidas à qPCR e histopatologia. A detecção de DNA de B. abortus nos linfonodos das búfalas avaliadas foi verificada a partir do quarto mês de gestação em sete búfalas e em uma búfala pós-parição. Os achados histológicos foram linfadenite aguda a crônica. A presença de DNA de B. abortus foi detectada em todos os grupos de linfonodos avaliados, sendo que os linfonodos mais acometidos foram os mamários.

Teores de cobre, zinco e ferro no fígado de búfalos (Bubalus bubalis) com paratuberculose

Resumo: Com o objetivo de realizar um estudo dos teores de cobre (Cu), zinco (Zn) e ferro (Fe) em búfalas com paratuberculose (PTB) foram utilizadas 13 búfalas, das raças Murrah, Mediterrâneo e seus mestiços acima de três anos de idade, pertencentes a duas propriedades localizadas nos municípios de São Luiz e São Mateus, no Estado do Maranhão. Os animais foram selecionados de acordo com a presença de sinais clínicos sugestivos de paratuberculose, caracterizados por estado nutricional regular a ruim, diarreia crônica líquida a semi-líquida, desidratação, edema submandibular, anestro prolongado, mastites e verminose gastrintestinal. Foi realizada biópsia retal em todos os animais, para detecção de Mycobacterium avium subsp. paratuberculosis (Map) por meio da qPCR, e exames histopatológicos (HE e Ziehl-Neelsen). No Grupo1 sete animais foram positivos para presença de Map, e no Grupo 2 seis foram negativos. Todos os búfalos foram eutanasiados e necropsiados para coleta de diversos tecidos. Parte dos fragmentos foram fixados em formol a 10% para histopatologia e fragmentos de tecido hepático foram congelados para as dosagens dos microminerais (Cu, Zn e Fe). À necropsia todos os animais positivos para PTB apresentavam linfonodos mesentéricos de coloração castanha sugestiva de hemossiderose. Adicionalmente, em um animal foram observados pequenos pontos de cor marrom distribuídos difusamente na mucosa do intestino delgado. Na histopatologia foi observada hemossiderose moderada a acentuada no baço dos animais do Grupo 1. Na dosagem dos microminerais todos os animais com PTB apresentaram níveis abaixo dos valores de referência para Cu e Zn. Observou-se que a média dos teores de Cu dos búfalos com PTB foi 18,0ppm e de Zn 68,6ppm. No Grupo 2 a média dos teores de Cu foi 113,7ppm e de Zn 110,0ppm. Os teores de Fe em ambos os grupos foram elevados (>670ppm). Baseado nos achados clínico-patológicos e nas dosagens de minerais realizadas neste estudo, conclui-se que na região estudada, a PTB agravou o quadro clínico de animais com deficiência de Cu e Zn. Em áreas menos deficientes desses minerais sugere-se que a doença seja capaz de induzir quadros de deficiência mineral secundária.