Prokaryotic expression of a truncated form of bovine herpesvirus 1 glycoprotein E (gE) and its use in an ELISA for gE antibodies / Expressão procariota de uma forma truncada da glicoproteína E (gE) do herpesvírus bovino tipo 1 e uso em ELISA para anticorpos contra a gE

This article describes the expression of a truncated form of bovine herpesvirus 1 (BoHV-1) glycoprotein E (gE) for use as immunodiagnostic reagent. A 651 nucleotide fragment corresponding to the amino-terminal third (217 amino acids) of BoHV-1 gE - that shares a high identity with the homologous BoHV-5 counterpart - was cloned as a 6×His-tag fusion protein in an Escherichia coli expression vector. A soluble protein of approximately 25 kDa purified from lysates of transformed E. coli was recognized in Western blot (WB) by anti-6xHis-tag and anti-BoHV-1 gE monoclonal antibodies. In addition, the recombinant protein was specifically recognized in WB by antibodies present in the sera of cattle seropositive to BoHV-1 and BoHV-5. An indirect ELISA using the expressed protein as coating antigen performed comparably to a commercial anti-gE ELISA and was able to differentiate serologically calves vaccinated with a gE-deleted BoHV-5 strain from calves infected with BoHV-1. Thus, the truncated gE may be useful for serological tests designed to differentiate BoHV-1/BoHV-5 infected animals from those vaccinated with gE-negative marker vaccines.

Este trabalho relata a expressão de uma forma truncada da glicoproteína E (gE) do herpesvírus bovino tipo 1 (BoHV-1) para uso em imunodiagnóstico. Um fragmento de 651 pares de bases (pb) correspondente ao terço amino-terminal (217 aminoácidos) da gE do BoHV-1 - que compartilha uma alta identidade com a gE do BoHV-5 - foi clonada como proteína de fusão com cauda 6x de histidina em um vetor de expressão em Escherichia coli. Uma proteína solúvel de aproximadamente 25 kDa purificada de lisados de E.coli foi reconhecida em Western blot (WB) por anticorpos monoclonais anti-6xHis-tag e anti-gE. Além disso, a proteína recombinante purificada foi reconhecida em WB por anticorpos presentes no soro de animais soropositivos ao BoHV-1 e BoHV-5. Um ELISA indireto utilizando a proteína recombinante como antígeno apresentou performance comparável a um ELISA gE comercial e foi capaz e diferenciar sorologicamente animais vacinados com uma cepa gE-negativa de BoHV-5 de animais infectados com o BoHV-1. Portanto, a gE truncada pode ser útil em testes sorológicos diferenciais para uso conjunto com vacinas com marcador antigênico gE para o BoHV-1 e BoHV-5.

Detection and quantification of Duffy antigen on bovine red blood cell membranes using a polyclonal antibody / Detecção e quantificação do antigeno Duffy nos eritrócitos de bovinos empregando um anticorpo policlonal

Babesiosis is one of the most important diseases affecting livestock agriculture worldwide. Animals from the subspecies Bos taurus indicus are more resistant to babesiosis than those from Bos taurus taurus. The genera Babesia and Plasmodium are Apicomplexa hemoparasites and share features such as invasion of red blood cells (RBC). The glycoprotein Duffy is the only human erythrocyte receptor for Pasmodium vivax and a mutation which abolishes expression of this glycoprotein on erythrocyte surfaces is responsible for making the majority of people originating from the indigenous populations of West Africa resistant to P. vivax. The current work detected and quantified the Duffy antigen on Bos taurus indicus and Bos taurus taurus erythrocyte surfaces using a polyclonal antibody in order to investigate if differences in susceptibility to Babesia are due to different levels of Duffy antigen expression on the RBCs of these animals, as is known to be the case in human beings for interactions of Plasmodium vivax-Duffy antigen. ELISA tests showed that the antibody that was raised against Duffy antigens detected the presence of Duffy antigen in both subspecies and that the amount of this antigen on those erythrocyte membranes was similar. These results indicate that the greater resistance of B. taurus indicus to babesiosis cannot be explained by the absence or lower expression of Duffy antigen on RBC surfaces.

As doenças infecciosas e parasitárias causam perdas importantes em vários setores da produção da pecuária mundial. Estima-se que mais de 600 milhões de bovinos de países tropicais e subtropicais estejam expostos à infecção por Babesia sp. gerando grande prejuízo econômico. Os gêneros Babesia e Plasmodium são hemoparasitas pertencentes ao filo Apicomplexa e apresentam características comuns no processo de invasão eritrocitária. A babesiose bovina causada por Babesia bigemina e Babesia bovis apresenta sinais clínicos similares a malária humana causada por Plasmodium vivax e Plasmodium falciparum. A glicoproteína Duffy é a única receptora para o P. vivax em humanos. A maioria dos indivíduos negros africanos é resistente a este parasita devido a uma mutação que provoca a ausência de expressão desta glicoproteína na superfície das hemácias. Tendo em vista este fato, e que animais da subespécie Bos taurus taurus são mais susceptíveis à babesiose quando comparados à animais Bos taurus indicus, objetivou-se neste trabalho a detecção e quantificação do antígeno Duffy na superfície dos eritrócitos de bovinos empregando para tal, anticorpo policlonal que permitisse investigar se as diferenças na susceptibilidade são devido a diferentes níveis de expressão do antígeno Duffy nas hemácias. Ensaios de ELISA mostraram que o anticorpo produzido foi capaz de reconhecer o antígeno Duffy presente nas hemácias bovinas e a análise quantitativa não demonstrou diferença significativa na presença do mesmo. Estes resultados sugerem que a resistência maior dos zebuínos à babesiose não se deve à ausência de expressão, ou à presença em menor quantidade do antígeno Duffy na superfície de suas hemácias.

Efficacy of a gE-deleted, bovine herpesvirus 1 (BoHV-1) inactivated vaccine / Eficácia de uma vacina inativada, gE-deletada, contra o herpesvírus bovino tipo 1 (BoHV-1)

Bovine herpesvirus type 1 (BoHV-1) is recognized as a major cause of economic losses in cattle. Vaccination has been widely applied to minimize losses induced by BoHV-1 infections. We have previously reported the development of a differential BoHV-1 vaccine, based on a recombinant glycoprotein E (gE)-deleted virus (265gE-). In present paper the efficacy of such recombinant was evaluated as an inactivated vaccine. Five BoHV-1 seronegative calves were vaccinated intramuscularly on day 0 and boostered 30 days later with an inactivated, oil adjuvanted vaccine containing an antigenic mass equivalent to 10(7.0) fifty per cent cell culture infectious doses (CCID50) of 265gE-. Three calves were kept as non vaccinated controls. On day 60 post vaccination both vaccinated and controls were challenged with the virulent parental strain. No clinical signs or adverse effects were seen after or during vaccination. After challenge, 2/5 vaccinated calves showed mild clinical signs of infection, whereas all non vaccinated controls displayed intense rhinotracheitis and shed virus for longer and to higher titres than vaccinated calves. Serological responses were detected in all vaccinated animals after the second dose of vaccine, but not on control calves. Following corticosteroid administration in attempting to induce reactivation of the latent infection, no clinical signs were observed in vaccinated calves, whereas non vaccinated controls showed clinical signs of respiratory disease. In view of its immunogenicity and protective effect upon challenge with a virulent BoHV-1, the oil adjuvanted preparation with the inactivated 265gE- recombinant was shown to be suitable for use as a vaccine.

O Herpesvírus bovino tipo 1 (BoHV-1) é reconhecido como um importante agente de perdas econômicas em bovinos. Vacinação tem sido amplamente empregada para minimizar as perdas conseqüentes a infecções com o BoHV-1. Reportamos previamente o desenvolvimento de uma vacina diferencial para BoHV-1, baseada em um recombinante do qual a glicoproteína gE (gE) foi deletada (265gE-). No presente trabalho foi realizada a avaliação da eficácia de tal recombinante como vacina inativada. Cinco bovinos soronegativos para BoHV-1 foram vacinadas por via intramuscular no dia 0 e revacinadas 30 dias após com uma vacina inativada com adjuvante oleoso, contendo massa antigênica equivalente a 10(7.0) doses infectantes para 50 por cento dos cultivos celulares (DICC50) de 265gE-. Três animais foram mantidos como controles não vacinados. No dia 60 pós-vacinação, os animais vacinados e controles foram desafiados com a amostra virulenta parental. Nenhum sinal clínico ou efeito adverso foi observado após ou durante a vacinação. Após o desafio, 2 dos 5 animais vacinados apresentaram sinais leves de infecção, enquanto que todos os animais não vacinados apresentaram intensa rinotraqueíte e disseminaram vírus por mais tempo e em títulos mais elevados do que os animais vacinados. Respostas sorológicas foram detectadas em todos os animais vacinados depois da segunda dose de vacina, mas não nos animais do grupo controle. Após a administração de corticosteróide visando a reativação de infecções latentes, não foram observados sinais clínicos em nenhum dos 5 animais vacinados, enquanto os animais não vacinados apresentaram sinais leves de doença respiratória. Em vista de sua imunogenicidade e efeito protetor frente ao desafio com BoHV-1 virulento, a preparação oleosa com o recombinante 265gE- inativado foi demonstrada ser adequada para uso como vacina.

The low efficiency of dendritic cells and macrophages from mice susceptible to Paracoccidioides brasiliensis in inducing a Th1 response

In the present study we evaluated T cell proliferation and Th lymphokine patterns in response to gp43 from Paracoccidioides brasiliensis presented by isolated dendritic cells from susceptible and resistant mice. T cell proliferation assays showed that dendritic cells from susceptible mice were less efficient than those from resistant mice. The pattern of T cell lymphokines stimulated by dendritic cells was always Th1, although the levels of IL-2 and IFN-gamma were lower in T cell cultures from susceptible mice. To determie whether different antigen-presenting cells such as macrophages and dendritic cells stimulated different concentrations of Th1 lymphokines, the production of IFN-gamma and IL-2 was measured. It was observed that dendritic cells were more efficient than macrophages in stimulating lymphoproliferation in resistant mice. However, no significant difference was observed for IFN-gamma or IL-2 production. When cells from susceptible mice were used, macrophages were more efficient in stimulating lymphoproliferation than dendritic cells, but no difference was observed in the production of Th1 cytokine. Taken together, these results suggest the lower efficiency of dendritic cells and macrophages from B10.A mice in stimulating T cells that secrete Th1 lymphokines in vitro, an effect that may be involved in the progression of the disease in vivo

Disminucion del CD6 en linfocitos perifericos y en ganglios linfaticos en pacientes con cancer de mama / Reduction of CDG in peripheral blood lymphocytes and lymph nodes in patients presenting with breast cancer

Se estudio la expresion de la molecula CD6 en celulas mononucleares perifericas y en ganglios linfaticos axilares de 30 pacientes con cancer de mama, sin tratamiento oncoespecifico previo, mediante las tecnicas de inmunofluorescencia indirecta e inmunohistoquimica respectivamente. Se utilizo el anticuerpo monoclonal (AcM) IOR-TI (anti-CD6). Los resultados obtenidos en celulas mononucleares mostraron una disminucion de la significacion estadistica (p < 0,01) de las celulas CD6+ de pacientes con relacion a los controles, estas variaciones fueron mas evidentes en estadios avanzados. En el estudio de ganglios el resultado mas relevante fue el aumento del por ciento de casos con baja expresion de celulas CD6+ y la correlacion (p < 0,01) entre los niveles de celulas CD6+ en sangre periferica y ganglios

Trypanosoma cruzi binds to laminin in a carbohydrate-independent way

The bindings of 125I-laminin to trypomastigotes is specific and 2-5 x 10**3 laminin-binding sites were calculated to be presented on the surface of a live trypomastigote. Anti-laminin antibodies were able to inhibit the invasion of cultured cells by trypomastigotes (62-75 per cent), suggesting that laminin may be involved in the adhesion of the parasite to host cells. By affinity chromatography, an 85-KDa glycoprotein was isolated (laminin-bindign glycoprotein, LBG) from trypomastigote lysates, but not from epimastigote lysates. It is suggested that at least fragment E8 (but not E1) from laminin could be involbed in the reaction which is independent of the carbohydrate moieties from both ligand and recepto. It is also shown that LBG is member of the Tc-85 family, previously shown to be related to the invasion process of the parasite

Different pattern of glycoproteins with unusual O-linked oligosaccharides in two strains of Trypanosoma cruzi epimastigotes

The glycoconjugate profiles of epimastigotes of the Y strain and of two stocks of the tulahuen strain which differ in their infectivity have been compared. The surface location of the glycoconjugates was evidenced by labelling with glactose oxidase/NaB3H4. Fluorography of SDS-polyacrylamide gel electrophoresis showed a different pattern in the glycoprotein range for both strains and also for two lines (T0 and T2) of the Tulahuen strain. While T0 showed only one glycoprotein with Mr 45 kDa, T2 revealed three glycoproteins in the range of 25-57 kDa. Two fast components, corresponding to glycolipids were also shown. The glycoproteins were isolated with 44 percent phenol and they were purified from the aqueous phase. Alkaline borohydride treatment of the labelled glycoproteins under the conditions of beta-elimination released strongly labelled O-glycosidically linked oligosaccharides. These O-linked glycans are not of the usual type found in glycoproteins. N-acetylgalactosamine which links O-glycans to the protein in the known mucin type glycoproteins has not been detected in the T. cruzi glycoproteins (1).

Maturation-dependent goat epididymal sperm autoagglutination and its inhibition by a glycoprotein factor.

The maturing goat epididymal spermatozoa were isolated from different segments of epididymis and these cells dispersed in a modified Ringer's solution, were incubated at 37 degrees C for 60 min to evaluate their autoagglutination efficacy. Distal corpus-epididymal spermatozoa specifically showed high order of head-to-head autoagglutination property whereas all other sperm cells did not show any detectable aggregation. The goat epididymal plasma has been shown to possess an anti-agglutinin that markedly inhibits sperm agglutination phenomenon and also dissociates the cells from the sperm clusters. Epididymal plasma is the most potent source of the anti-agglutinin which is a heat-stable specific glycoprotein. Like the autoagglutination phenomenon, the initiation of sperm forward progression also starts in the distal-corpus epididymis. The temporal correlation of these two events suggests that sperm autoagglutination may be a prerequisite for the induction of flagellar motility during the epididymal maturity of male gametes.

Estudio histoquímico ultraestructural con lecitinas del cáncer gástrico y mucosa adyacente / Lectins in gastric cancer. A histochemical and ultraestructural study

Hemos realizado un estudio ultraestructural con 5 tipows de lecitinas (UEA-1, PNA, WGA, SBA and DBA) en 25 piezas de gastrecromía con adenocarcinoma, con el objeto de establecer diferencias entre la composición del moco secretado por la mucosa aparentemente normal y la neoplásica. Todas las muestras fueron incluidas en resina Sprurr y LR White, con diferentes condiciones de fijación y deshidratación; además, las lecitinas fueron incubadas siguiendo dos métodos diferentes: directo e indirecto. Los mejores resultados fueron obtenidoscon el método indirecto en el tejido incluido en LR White. Nuestros resultados muestran una gran hetereogenicidad en la composición azucarada, especialmente en la secreción de la mucoso neoplásica. La reacción con WGA fue negativa en el moco extracelular secretado en normalidad, mientras que resultó positiva en el secretado por las células neoplásicas. En marcaje con SBA sobre el aparato de Golgi sugiere que el inicio de la glicosilación sucede a nivel de la cara cis, aunque no podemos excluir que en ocaciones se realice en el retículo endoplásmatico rugoso. La intensidad en el marcaje con lecitinas aumentó con la diferenciación tumoral. La presencia de reacción con WGA en el moco extracelular en los carcinomas, así como la distribución azarosa de la reacción sobre el aparato de Golgi, podría estar relacionadas con glicosilaciones aberrantes y/o elongaciones anormales de la cadena de carbohidratos.

Glycocalyx positive bacteria isolated from chronic osteomyelitis and septic arthritis.

Bacterial cultures isolated from cases of chronic osteomyelitis and septic arthritis were screened for the production of glycocalyx. The presence of glycocalyx was noted in 76.3% of Staphylococcus aureus, 57.14% of Staphylococcus epidermidis, 50% of Pseudomonas aeruginosa, and 75% of Escherichia coli isolates.

A study on the amphiphilic proteins of three Trypanosoma cruzi populations

Amphiphilic proteins were partially purified from culture-derived metacyclic trypomastigotes of the CL and Colombian strains and of the Dm 28c clone of T. cruzi by the use of Triton X-114. These proteins were subjected to one-and two-dimensional polyacrylamide electrophoresis in the presence of sodium dodecyl sulphate. Relatively simple protein profiles with only 5 to 6 major bands were obtained. The CL and Colombian strains produced at least one additional major protein band (86 kDa) compared to the Dm 28c proteins. Trypomastigote amphiphilic proteins displayed both electrophoretic mobilities and isoelectric points identical to those of two polypeptides precipitated by a rabbit antiserum which recognized metacyclic trypomastigote-specific surface antigens. The partial purification of the T. cruzi amphiphilic proteins with Triton X-114 may provide a simple preparative step for the study of these differentiation-related polypeptides, as well as for the study of strain-specific (glyco)proteins and of their possible biological role