Increased expression of Interleukin-6 related to nephritis in chickens challenged with an Avian infectious bronchitis virus variant

A Brazilian field isolate (IBV/Brazil/PR05) of avian infectious bronchitis virus (IBV), associated with development of nephritis in chickens, was previously genotyped as IBV variant after S1 gene sequencing. The aim of this study was to evaluate the levels of IL-6 in kidneys and trachea of birds vaccinated and challenged with IBV/Brazil/PR05 strain, correlating these results with scores of microscopic lesions, specific IBV antigen detection and viral load. The up-regulation of IL-6 and the increased levels of viral load on renal and tracheal samples were significantly correlated with scores of microscopic lesions. Reduced levels of viral load were detected in kidneys of birds previously vaccinated and challenged, compared to non-vaccinated challenged group, although markedly microscopic lesions were observed for both groups. The expression of IL-6, present both in the kidney and in the tracheas, was dependent on the load of the virus present in the tissue, and the development of lesions was related with IL-6 present in the tissues. These data suggest that variant IBV/Brazil/PR05 can induce the expression of proinflammatory cytokines in a manner correlated with viral load and increased IL-6 is involved in the tissue with the influx of inflammatory cells and subsequent nephritis. This may contribute with a model to the development of immunosuppressive agents of IL-6 to prevent acute inflammatory processes against infection with IBV and perhaps other coronaviruses, as well as contribute to the understanding of the immunopathogenesis of IBV nephropatogenic strains.

Development of two potential diagnostic monoclonal antibodies against human cytomegalovirus glycoprotein B

Human cytomegalovirus glycoprotein B (gB) represents a target for diagnosis and treatment in view of the role it plays in virus entry and spread. Nevertheless, to our knowledge, rare detection of a gB antigen has been reported in transplant patients and limited information is available about diagnostic gB monoclonal antibodies (mAbs). Our aim was to develop gB mAbs with diagnostic potential. Hydrophilic gB peptides (ST: amino acids 27-40, SH: amino acids 81-94) of favorable immunogenicity were synthesized and used to immunize BALB/c mice. Two mAbs, named ZJU-FH6 and ZJU-FE6, were generated by the hybridoma technique and limited serial dilution and then characterized by indirect ELISA, Western blotting, immunoprecipitation, and immunohistochemical staining. The mAbs displayed high titers of specific binding affinities for the ST and SH synthetic peptides at an mAb dilution of 1:60,000 and 1:240,000, respectively. Western blotting and immunoprecipitation indicated that these mAbs recognized both denatured and native gB of the Towne and AD169 strains. The mAbs, when used as the primary antibody, showed positive staining in cells infected with both Towne and AD169 strains. The mAbs were then tested on patients submitted to allogeneic hematopoietic stem cell transplantation. The gB antigen positivity rates of the patients tested using ZJU-FH6 and ZJU-FE6 were 62.0 and 63.0%, respectively. The gB antigen showed a significant correlation with the level of pp65 antigen in peripheral blood leukocytes. In conclusion, two potential diagnostic gB mAbs were developed and were shown to be capable of recognizing gB in peripheral blood leukocytes in a reliable manner.

Analysis of testicular cell populations using flow cytometry

Studying testis is complex, because the tissue has a very heterogeneous cell composition and its structure changes dynamically during development. In reproductive field, the cell composition is traditionally studied by morphometric methods such as immunohistochemistry and immunofluorescence. These techniques provide accurate quantitative information about cell composition, cell-cell association and localization of the cells of interest. However, the sample preparation, processing, staining and data analysis are laborious and may take several working days. Flow cytometry protocols coupled with DNA stains have played an important role in providing quantitative information of testicular cells populations ex vivo and in vitro studies. Nevertheless, the addition of specific cells markers such as intracellular antibodies would allow the more specific identification of cells of crucial interest during spermatogenesis. For this study, adult rat Sprague-Dawley rats were used for optimization of the flow cytometry protocol. Specific steps within the protocol were optimized to obtain a singlecell suspension representative of the cell composition of the starting material. Fixation and permeabilization procedure were optimized to be compatible with DNA stains and fluorescent intracellular antibodies. Optimization was achieved by quantitative analysis of specific parameters such as recovery of meiotic cells, amount of debris and comparison of the proportions of the various cell populations with already published data. As a result, a new and fast flow cytometry method coupled with DNA stain and intracellular antigen detection was developed. This new technique is suitable for analysis of population behavior and specific cells during postnatal testis development and spermatogenesis in rodents. This rapid protocol recapitulated the known vimentin and γH2AX protein expression patterns during rodent testis ontogenesis. Moreover, the assay was applicable for phenotype characterization of SCRbKO and E2F1KO mouse models.

Epstein-Barr-viruksen antigeeniosoitustestin kehittäminen

Epstein-Barr-virus (EBV) aiheuttaa mononukleoosia eli pusutautia, joka ilmenee yleensä murrosiällä tai nuorella aikuisiällä. Mononukleoosissa on tyypilliset nielutulehduksen oireet, minkä takia sitä on vaikea erottaa muiden taudinaiheuttajien aiheuttamista nielutulehduksista. Erotusdiagnostiikan käyttö nielutulehduksessa on oleellista, koska vain Streptokokki-bakteerien aiheuttamat nielutulehdukset vaativat antibioottihoitoa. Akuutin EBV-infektion pikadiagnostiikka perustuu nykyisellään infektion seurauksena muodostuvien heterofiilisten vasta-aineiden mittaamiseen verestä. Niiden mittaamisessa on useita ongelmia, koska useilla lapsilla niitä ei muodostu lainkaan ja vanhemmillakin niitä muodostuu yleensä vasta viikon päästä mononukleoosin oireiden alkamisesta. Mittaamalla EBV:n antigeeneja saataisiin positiivinen testitulos vasta-ainetestiä nopeammin. EBV:lle ei ole kuitenkaan kehitetty antigeeniosoitustestiä todennäköisesti siksi, että EBV:n erittymisen terveiden viruksen kantajien limakalvoille uskotaan olevan ongelma antigeenitestauksessa. Diplomityön tavoitteena oli kehittää akuutin EBV-infektion pikadiagnostiikkaan soveltuva limakalvonäytteestä tehtävä immunometrinen antigeeniosoitustesti. Työssä kehitettiin uusia polyklonaalisia vasta-aineita sekä testattiin kaupallisia vasta-aineita. Vasta-aineiden toimintaa tutkittiin immunomäärityksissä kokonaista EBV:ta ja puhdistettuja proteiiniantigeeneja vastaan. Monoklonaalisten vasta-aineiden kehitys lopetettiin ennen varsinaista tuottoa, koska ensin kehitetyt polyklonaaliset vasta-aineet eivät tunnistaneet natiivia virusta vaan pelkästään immunisointeihin käytetyt rekombinanttiset kohdeproteiinit. Kaupallisista vasta-aineista yhdellä onnistuttiin kehittämään tiettävästi maailman ensimmäinen immunometrinen EBV-antigeeniosoitustesti, jossa saatiin tunnistus sekä natiivilla EBV:lla että puhdistetulla proteiiniantigeenilla. Testin herkkyys proteiiniantigeenilla oli hyvä (4 pM) ja puhdistetulla EBV-virusvalmisteellakin todennäköisesti riittävä (6,2×106 viruspartikkelia/ml) kliiniseen diagnostiikkaan. Testillä ei kuitenkaan saatu suppeasta mononukleoosipotilaiden nielunäyteaineistosta yhtään EBV-positiivista tulosta. Referenssiksi tilatut PCR-testit osoittivat näytteiden EBV-pitoisuuksien olevan liian alhaisia osoitettavaksi kehitetyllä EBV-antigeeniosoitustestillä. PCR-testauksessa mononukleoosipotilaiden nielunäytteistä osoitettujen alhaisten EBV-määrien perusteella spekuloitiin, että olisiko edustavin näytteenottopaikka akuutissa EBV-infektiossa sittenkin nielun sijaan nenänielu. Myös kirjallisuudesta löytyi tälle tukea. EBV-antigeeniosoitustestin osoitettiin toimivan hyvin standardinäyte-materiaalilla. Testikehitysprojektin jatkon kannalta oleellista on kuitenkin vielä selvittää laajemmalla kliinisellä näyteaineistolla, mistä EBV:n antigeeneja pitäisi osoittaa akuutissa EBV-infektiossa ja ovatko niiden pitoisuudet riittävän korkeita.

Détection de protéines par diffusion Raman exaltée par effet de pointe (TERS)

La concentration locale des messagers chimiques sécrétés par les cellules peut être mesurée afin de mieux comprendre les mécanismes moléculaires liés à diverses maladies, dont les métastases du cancer. De nouvelles techniques analytiques sont requises pour effectuer ces mesures locales de marqueurs biologiques à proximité des cellules. Ce mémoire présentera le développement d’une nouvelle technique basée sur la réponse plasmonique sur des leviers AFM, permettant d’étudier les réactions chimiques et biologiques à la surface des leviers grâce au phénomène de résonance des plasmons de surface (SPR), ainsi qu’à la diffusion Raman exaltée par effet de pointe (TERS). En effet, il est possible de localiser l’amplification du signal Raman à la pointe d’un levier AFM, tout comme le principe de la diffusion Raman exaltée par effet de surface (SERS) basée sur la diffusion de la lumière par des nanoparticules métalliques, et permettant une large amplification du signal Raman. La surface du levier est recouverte d’une nano-couche métallique d’or, suivi par des réactions biologiques pour l’immobilisation d’un récepteur moléculaire, créant ainsi un biocapteur sur la pointe du levier. Une détection secondaire utilisant des nanoparticules d’or conjuguées à un anticorps secondaire permet également une amplification du signal SPR et Raman lors de la détection d’antigène. Ce mémoire démontrera le développement et la validation de la détection de l’immunoglobuline G (IgG) sur la pointe du levier AFM.Dans des projets futurs, cette nouvelle technique d’instrumentation et d’imagerie sera optimisée grâce à la création d’un micro-détecteur protéique généralement adapté pour l’étude de la communication cellulaire. En intégrant le signal SPR à la microscopie AFM, il sera alors possible de développer des biocapteurs SPR couplés à une sonde à balayage, ce qui permettra d’effectuer une analyse topographique et de l’environnement chimique d’échantillons cellulaires en temps réel, pour la mesure des messagers moléculaires sécrétés dans la matrice extracellulaire, lors de la communication cellulaire.

Construção de uma biblioteca de fragmentos de anticorpos monoclonais de galinhas com cadeia única (scFv) por phage display com reatividade cruzada para estirpes heterólogas do vírus da bronquite infecciosa aviária

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Sex determination of murine and bovine embryos using cytotoxicity and immunofluorescence assays

Murine and bovine embryos were sexed by using polyclonal and monoclonal anti-H-Y antibodies in cytotoxic and immunofluorescence assays. The specificity of 3 different sources of antibodies are compared. Sexing was accomplished by complement-mediated cytotoxicity or by H-Y antigen detection using a anti-mouse Ig fluorescein conjugate. Cytogenetic ascertainment for correct sex identification yielded a score of about 85% in both species, while the immunofluorescence assay yielded a score of 83%. © 1993.

Subcutaneous and testicular metastasis from prostatic adenocarcinoma with neuroendocrine differentiation

The pathological finding of testicular metastasis in cases of disseminated prostatic adenocarcinoma is rare, but was more frequently reported in the past, when bilateral castration was performed more often. The existence of skin and subcutaneous metastasis adds a worse prognosis, because generally it is sign of advanced disease with an average survival time of less than one year. The synchronous occurrence of such metastasis has not been described previously, neither their association to neuroendocrine differentiation. The presence of such differentiation of prostatic adenocarcinoma represents a very unfavorable prognostic factor, as suggested in recent literature. Herein, we discuss the case of a 53 year old man, who presented with macroscopic hematuria and frequency associated to several painless subcutaneous nodules in left axilla and shoulder, as well as in the lower abdominal wall. The right testis was painful, endured and on rectal examination, the prostate was diffusely enlarged. Serum PSA was elevated, reaching 1760 ng/ml and prostatic biopsy disclosed a Gleason 10 prostatic adenocarcinoma with neuroendocrine differentiation. The same pathological pattern was detected in the right testis and in all subcutaneous nodules, documented by positive staining of chromogranin, a marker of neuroendocrine cells. He was submitted to a prostate tunnelization and maximal androgen blockade plus adjuvant chemotherapy, nevertheless, he died 5 months latter.

Immunocytochemical study of Ki-67 as a prognostic marker in canine mammary neoplasia

Background: Canine mammary tumors are challenging for clinicians and pathologists because of complex histologic classification, low specificity of cytologic diagnosis, and unpredictable biological behavior. In histologic specimens, expression of tumor proliferation marker Ki-67, a nuclear nonhistone protein, has been shown to have prognostic value for canine mammary tumors and to correlate with malignancy and low survival rates. Objective: The objective of this study was to measure the proliferation index of canine mammary tumors by immunochemical detection of Ki-67 in cytologic specimens and to determine its relationship to clinical and pathologic variables and patient outcome. Methods: Spontaneous mammary tumors from 31 female dogs were surgically excised. Imprint specimens for cytologic evaluation were wet-fixed in ethanol; histologic specimens were prepared routinely. Immunostaining was performed with the PH 177 monoclonal antibody against Ki-67; proliferation index was graded from negative to +++. Dogs were followed for 18 months. Multivariate logistic regression analysis was used to determine correlations between immunocytochemical results, tumor and clinical variables, and patient outcome. Results: Ki-67 proliferation indices in cytologic specimens were significantly lower for nonmalignant tumors than for malignant tumors. High index values of Ki-67 were positively correlated with metastasis, death from neoplasia, low disease-free survival rates, and low overall survival rate. With the exception of 4 specimens for which cellularity was insufficient, positive expression of Ki-67 in cytologic specimens correlated with that of histologic specimens. Conclusions: The prognostic value of the Ki-67 index in canine mammary tumors by using wet-fixed cytology imprint specimens was similar to that observed previously for histologic specimens. Immunocytochemical detection of Ki-67 could improve the accuracy and value of cytology by providing safe and rapid information about malignancy and patient outcome. © 2004 American Society for Veterinary Clinical Pathology.

Immunohistochemistry assay to detect Turkey coronavirus (TCoV) from experimentally infected poults

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Sistema de coleta, análise e detecção de código malicioso baseado no sistema imunológico humano

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Produção de fragmentos de anticorpos monoclonais (scFv) contra isolados de campo do vírus da bronquite infecciosa das galinhas utilizando phage display

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Estudo epidemiológico da amebíase no Estado do Pará utilizando diferentes metodologias para diagnóstico

A epidemiologia da amebíase está sendo reavaliada desde que a E. histolytica (patogênica) foi considerada espécie distinta de E. dispar (não patogênica). Neste estudo, investigou-se a freqüência da amebíase em uma amostra de residentes do Pará por diferentes técnicas de diagnóstico e avaliou-se a patogenia do parasito. Os participantes (n = 845) forneceram material fecal e destes, 191 foram entrevistados quanto aos sintomas de diarréia, cólicas intestinais, constipação, náuseas e vômito. Foram também analisados 8 exsudatos de pacientes com suspeita de amebíase hepática. As amostras foram observadas sob microscopia de luz e a confirmação de E. histolytica feita a partir da pesquisa de antígenos. Um total de 98 amostras fecais e todos os exsudatos foram semeados em meio Pavlova para isolamento e posterior caracterização bioquímica e molecular (identificação de espécie e genotipagem). Isolados de outras regiões do Brasil foram também genotipados. A positividade obtida foi de 29,35% (248/845) e não houve correlação com a faixa etária. A microscopia revelou baixa sensibilidade (45,26%; 74/334), porém elevada especificidade (87,03%; 260/334) quando comparada ao ELISA. Houve relação significativa (OR 4,4026) entre a presença de sintomas e a positividade no ELISA, sendo a diarréia (58,82%) e a cólica intestinal (58,82%) os sintomas mais relatados. Nenhum exsudato foi positivo no exame a fresco, porém 7 foram positivos no ELISA. Obteve-se 22 isolados de material fecal e a caracterização da HE foi possível em 13, dos quais 7 E. histolytica e 6 E. dispar. O DNA de 22 isolados e dos exsudatos foram testados para identificação molecular de espécie e genotipagem. Do total, 16 cultivos (9 cepas mistas, 4 E. dispar e 3 E. histolytica) e 5 exsudatos (todos E. histolytica) amplificaram na PCR. A genotipagem identificou adicional positividade para E. histolytica em um exsudato e revelou diferentes polimorfismos de comprimento para o locus 1-2 de E. histolytica e E. dispar do Pará e de outras regiões do Brasil e um caso de co-infecção por diferentes genótipos de E. dispar. Nossos resultados revelam que a amebíase invasiva é um importante problema de saúde pública em nossa população e grande variedade de genótipos de E. histolytica contribuem para a doença no Brasil.

Streptococcal acute pharyngitis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Tissue apoptosis in mice infected with Leptospira interrogans serovar Icterohaemorrhagiae

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)