B cells expressing IL-10 mRNA modulate memory T cells after DNA-Hsp65 immunization

In DNA vaccines, the gene of interest is cloned into a bacterial plasmid that is engineered to induce protein production for long periods in eukaryotic cells. Previous research has shown that the intramuscular immunization of BALB/c mice with a naked plasmid DNA fragment encoding the Mycobacterium leprae 65-kDa heat-shock protein (pcDNA3-Hsp65) induces protection against M. tuberculosis challenge. A key stage in the protective immune response after immunization is the generation of memory T cells. Previously, we have shown that B cells capture plasmid DNA-Hsp65 and thereby modulate the formation of CD8+ memory T cells after M. tuberculosis challenge in mice. Therefore, clarifying how B cells act as part of the protective immune response after DNA immunization is important for the development of more-effective vaccines. The aim of this study was to investigate the mechanisms by which B cells modulate memory T cells after DNA-Hsp65 immunization. C57BL/6 and BKO mice were injected three times, at 15-day intervals, with 100 µg naked pcDNA-Hsp65 per mouse. Thirty days after immunization, the percentages of effector memory T (TEM) cells (CD4+ and CD8+/CD44high/CD62Llow) and memory CD8+ T cells (CD8+/CD44high/CD62Llow/CD127+) were measured with flow cytometry. Interferon γ, interleukin 12 (IL-12), and IL-10 mRNAs were also quantified in whole spleen cells and purified B cells (CD43−) with real-time qPCR. Our data suggest that a B-cell subpopulation expressing IL-10 downregulated proinflammatory cytokine expression in the spleen, increasing the survival of CD4+ TEM cells and CD8+ TEM/CD127+ cells.

Partial purification, immunogenicity and putative new localization of a native leishmania heat shock protein 70

In this work we focused on a recombinant protein, containing approximately 230 aminoacids from the carboxy-terminal extremity of the Leishmania chagasi heat shock protein 70. The heat shock proteins are among the most abundant parasite antigens and conserved proteins in nature, and this family is one of the most immunogenic proteins present within pathogenic organisms. The recombinant protein has been partially purified by electroelution and further precipitation in acetone. The electroelution process did not modify its immunological and antigenic properties, as it continued to be recognized by visceral leishmaniasis positive sera and by the immunological system of rabbits during the immunization, both in ELISA and Western blots. The production of polyclonal sera with an antigen concentration that is far from the maximum dose, strengthens the idea that the proteins of this family are highly antigenic and immunogenic. Our results with these polyclonal sera in the Direct Agglutination Assay allow the conclusion that the Leishmania chagasi native heat shock protein 70 is distributed on the surface of the parasite.

Neste trabalho estudamos urna proteína recombinante (S7) contendo aproximadamente 230 aminoácidos da extremidade carboxi-terminal da proteína de choque térmico de 70 kDa (HSP70) de Leishmania chagasi. As proteínas de choque térmico estâo entre os antígenos parasitarios mais abundantes e mais conservados na natureza. Esta familia pertence a urna das classes de proteínas mais imunogênicas, presentes em organismos patogênicos. Aproteína S7 foi parcialmente purificada por eletroeluição, e em seguida precipitada em acetona. A eletroeluição não modificou suas propriedades imunológicas e antigênicas, pois a proteína continuou a ser reconhecida (tanto no ELISA como no Western blot) por soros positivos para leishmaniose visceral e pelo sistema imunológico de coelhos durante a imunização. Aproducção de soros policlonais com urna concentração antigênica muito inferior a dose máxima, reforca a idéia de que as proteínas desta familia sâo altamente antigênicas e imunogénicas. Nossos resultados com os soros policlonais no ensaio de aglutinação direta (DAT) permitem concluir que a HSP70 nativa de L. chagasi está presente na superficie do parásita.

Expresión de proteínas del choque térmico HSP72 y HSP73 en casos colombianos de Linfoma de Hodgkin positivos y negativos para virus de Epstein Barr / Expression of heat shock proteins HSP72 and HSP73 in Epstein-Barr positive or negative Hodgkin lymphoma

Background: The expression of heat shock proteins (HSP70) in tumor cells or virus infected cells is important for the induction of specific cellular immune response. They are implicated in transport of immunodominants peptides in the endoplasmic reticulum, activation of antigen presenting cells and cross priming of CD8 T cells. Aim: To analyze the expression of HSP70 protein in its constitutive (HSP73) and inducible forms (HSP72) in Hodgkin's lymphoma (HL), infected or not by Epstein Barr virus (EBV) and to assess its relationship with pathological subtype, clinical stages and treatment response. Material and methods: The analysis of HSP73 and HSP72 was done by immunoperoxidase on routinely processed paraffin sections with prior antigen retrieval. Results: Sixty seven cases were studied. The expression of HSP73 and HSP72 was detected in 19.4 and 17.9% of samples respectively. The infiltrating lymphocytes expressed HSP72 in 58% of cases. The pathological subtypes with the higher expression in lymphocytes were mixed cellularity and nodular sclerosis. No differences in HSP70 expression were observed, according to clinical stage, treatment response or the presence of EBV. Conclusions: The expression of HSP72 on lymphocytes suggests that this protein plays an important role in the induction and amplification of anti-tumor immune response (Rev Méd Chile 2003; 131: 1375-81).

Respuesta inmunológica de linfocitos T frente a diferentes fracciones antigénicas de mycobacterium bovis en poliartritis crónica juvenil / T lymphocyte immunological response to different antigenic fractions of mycobacterium bovis in juvenile chronic polyarthritis

Background: The possible relationship of stress or heat-shock proteins (hsp) with the pathogenesis of autoimmune disease has been intensely studied recently. In adult rheumatoid arthritis, a bacterial hsp (65 kDa hsp from Mycobacterium tuberculosis or bovis) would have a cross reactivity with a hsp of ARTICULAR cartilage. Aim: To assess the cellular immune response to the 65 kA hsp from M Bovis in children. Patients and methods: The proliferative response of peripheral mononuclear cells of 20 children with juvenile chronic polyarthritis and 20 healthy controls, against the 65 kDa hsp and other antigenic fractions from M bovis, was studied. Results: Patients with juvenile chronic polyarthritis had a intense reaction against 65 kDa fraction and a second fraction located between 32.5 and 27.5 kDa. Patients with a prolonged evolution of the disease (more than five years), reacted preferentially to an antigenic segment located between 32.5 and 27.5 kDa and those with a shorter evolution did so with an antigen of 27.5-18.5 kDa. Conclusions: These results support the hypothesis that 65 kDa hsp from M bovis is involved in the pathogenesis of chronic juvenile polyarthritis and suggest that patients with short or prolonged evolutions of the disease would react to different antigenic fractions

Resposta imune anti-proteínas de choque térmico na leishmaniose visceral / Anti-HSP immune response in visceral leishmaniasis

A leishmaniose visceral (calazar) é um importante problema de saúde pública na regiäo Nordeste do Brasil, onde cerca de 3 milhöes de pessoas estäo sob o risco de contrair a doença. O diagnóstico precoce ajuda a reduzir a mortalidade, estimada em 10 por cento. Os métodos diagnóticos parasitológicos säo invasivos e têm uma sensibilidade de aproximadamente 70 por cento. A sorologia pode ser uma ferramenta importante no estabelecimento de um diagnóstico definitivo, mas tem a desvantagem de apresentar reaçöes cruzadas com outras doenças, endêmicas nas mesmas regiöes onde ocorre o calazar. Para evitar a complexidade antigênica que acompanha qualquer teste sorológico que utilize extratos de parasitas inteiros como antígeno, procedemos à triagem de bibliotecas de cDNA de Leishmania com o soro de um paciente calazar. Nosso objetivo era encontrar antígenos específicos, e selecionamos um antígeno que foi reconhecido especificamente pelo soro de pacientes com calazar. Através da análise de sequências de DNA mostramos que os antígenos estudados representam a fraçäo carboxi-terminal da proteína de choque térmico de 70kDa de Leishmania (HSP70). As proteínas de fusäo foram parcialmente purificadas através da eletroforese, em géis preparativos de poliacrilamida-SDS, dos sonicados de E.coli transformadas. Estas proteínas foram em seguida utilizadas no ELISA para o diagnóstico do calazar humano e canino. O teste mostrou-se mais sensível e específico que o ELISA convencional e consideravelmente mais específico que a imunofluorescência indireta. Além disso, utilizando Western blots de antígenos de E.coli foi transformadas e soros imunoadsorvidos contra HSP70 e HSP90 recombinantes, foi possível demonstrar que a resposta imune humoral é fortemente dirigida contra ambas as HSP. Através do uso de cDNA progrssivamente mais longos, estendendo-se a partir da cauda poli-A para a extremidade 5` do referencial aberto de leitura HSP70, e da expansäo destes clones em E.coli, mostramos que os epitopos de linfócitos B estäo distribuidos na parte carboxi-terminal da HSP70. Säo descritos também alguns resultados preliminares do usoda HSP70 recombinante na vacinaçäo de cäes, assim como uma discussäo abrangente do papel das HSP na leishmaniose

Host parasite response in nerve in leprosy.

Nerve granulomas occur at all points across the leprosy spectrum. Studies have been made using experimental models in which mycobacteria were injected directly in the sciatic or posterior tibial nerve of the guinea pig. Clinical and electrophysiological studies demonstrated axonal damage which was confirmed by morphometric studies showing disrupted myelin sheaths and in places complete demyelination. Further immunohistological studies showed a complete disappearance of staining for certain neuropeptides. The role of Schwann cells has also been investigated. Schwann cells in nerves affected by mycobacterial granulomas, both experimental and in leprosy patients were not demonstrated to be MHC class II positive suggesting that they did not play a role in antigen presentation. Macrophages in leprosy granulomas were shown to contain TNF alpha, suggesting that this cytokine played a role in axonal damage. The role of mycobacterial heat-shock protein in nerve granulomas has not as yet been determined. The localized nature of granulomas in leprosy nerves and nerves with experimental mycobacterial granulomas has been studied by a process of excision and repair with muscle grafts. Marked recovery has been demonstrated by clinical, electrophysiological, morphometric and immuno-histochemical techniques, the latter demonstrating a return of neuropeptide production.

Avances en el conocimiento de las proteínas de estrés (HSP) en reumatología / Advances in the knowledge of the heat-shock proteins in rheumatology

Las proteÝnas de estrÚs, o tambiÚn llamadas de shock tÚrmico (hsp), estßn constituidas por un grupo de proteÝnas que son sintetizadas por las cÚlulas contra diferentes estÝmulos, entre los cuales se cuenta el calor. La función de estas hsp pareciera estar estrechamente relacionada con los mecanismos protectores que tiene la cÚlula frente a distintos agentes agresores. De los mecanismos descritos, el relacionado con la neutralización de los productos oxidativos pareciera ser uno de los mßs relevantes. Desde el punto de vista clÝnico, las hsp han adquirido relevancia debido a que poseen una elevada inmunogenicidad y porque presentan un alto grado de homologÝa con agentes bacterianos, entre los cuales se cuentan las micobacterias. Esta similitud antigÚnica entre hsp exógenas (bacterias) y algunas propias de nuestro organismo, podrÝa dar origen a respuestas autoinmunes, como se aprecia en las artritis reactivas o artritis reumatoidea (AR). Estudios efectuados con diversas tÚcnicas de laboratorio con el fin de medir anticuerpos contra la hsp de Micobacterium tuberculosis o bovis (hsp 65 kDa) en pacientes con AR, han dado cifras elevadas en comparación a los controles. En paÝses donde existe una incidencia mayor de TBC, como ocurre con algunas zonas de Africa, esta diferencia es menor. La respuesta mediada por linfocitos T en enfermos portadores de AR, especialmente los procedentes de lÝquido sinovial, exhibe tambiÚn una gran reactividad a la hsp 65 kDa de micobßcterio. El conocimiento de las hsp constituye un nuevo mecanismo patogÚnico que podrÝa relacionar a los agentes microbianos y ciertas fonnas de artritis, especialmente AR y algunas variedades de artritis reactivas