Decreased production of TNF-alpha by lymph node cells indicates experimental autoimmune encephalomyelitis remission in Lewis rats

Experimental autoimmune encephalomyelitis (EAE) is mediated by CD4+ Th1 cells that mainly secrete IFN-γ and TNF-α, important cytokines in the pathophysiology of the disease. Spontaneous remission is, in part, attributed to the down regulation of IFN-γ and TNF-α by TGF-β. In the current paper, we compared weight, histopathology and immunological parameters during the acute and recovery phases of EAE to establish the best biomarker for clinical remission. Female Lewis rats were immunised with myelin basic protein (MBP) emulsified with complete Freund's adjuvant. Animals were evaluated daily for clinical score and weight prior to euthanisation. All immunised animals developed the expected characteristics of EAE during the acute phase, including significant weight loss and high clinical scores. Disease remission was associated with a significant reduction in clinical scores, although immunised rats did not regain their initial weight values. Brain inflammatory infiltrates were higher during the acute phase. During the remission phase, anti-myelin antibody levels increased, whereas TNF-α and IFN-γ production by lymph node cells cultured with MBP or concanavalin A, respectively, decreased. The most significant difference observed between the acute and recovery phases was in the induction of TNF-α levels in MBP-stimulated cultures. Therefore, the in vitro production of this cytokine could be used as a biomarker for EAE remission.

Resistance to experimental autoimmune encephalomyelitis development in Lewis rats from a conventional animal facility

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the brain and spinal cord that is mediated by CD4+ T lymphocytes specific to myelin components. In this study we compared development of EAE in Lewis rats from two colonies, one kept in pathogen-free conditions (CEMIB colony) and the other (Botucatu colony) kept in a conventional animal facility. Female Lewis rats were immunized with 100 µl of an emulsion containing 50 µg of myelin, associated with incomplete Freund's adjuvant plus Mycobacterium butyricum. Animals were daily evaluated for clinical score and weight. CEMIB colony presented high EAE incidence with clinical scores that varied from three to four along with significant weight losses. A variable disease incidence was observed in the Botucatu colony with clinical scores not higher than one and no weight loss. Immunological and histopathological characteristics were also compared after 20 days of immunization. Significant amounts of IFN-gamma, TNF-alpha and IL-10 were induced by myelin in cultures from CEMIB animals but not from the Botucatu colony. Significantly higher levels of anti-myelin IgG1 were detected in the CEMIB colony. Clear histopathological differences were also found. Cervical spinal cord sections from CEMIB animals showed typical perivascular inflammatory foci whereas samples from the Botucatu colony showed a scanty inflammatory infiltration. Helminths were found in animals from Botucatu colony but not, as expected, in the CEMIB pathogen-free animals. As the animals maintained in a conventional animal facility developed a very discrete clinical, and histopathological EAE in comparison to the rats kept in pathogen-free conditions, we believe that environmental factors such as intestinal parasites could underlie this resistance to EAE development, supporting the applicability of the hygiene hypothesis to EAE.

Vacina gênica (DNAhsp65) para tuberculose em camundongos neonatos / DNA vacine (DNA hsp65) to tuberculoses in newborn mice

O presente estudo foi proposto para investigar as características e a eficácia da resposta imune induzida pela vacina gênica para tuberculose em camundongos neonatos. A construção genética empregada continha o gene da hsp65 de M.Leprae inserida no vetor pVax. Foram objetivos específicos deste trabalho: avaliar a imunização no período neonatal induz tolerância; determinar a presença de mensagem para hsp65, através de PCR, nos vários tecidos; caracterizar a resposta imune induzida por vacinação integral com DNAhsp65 no período neonatal avaliar as características da resposta imune induzida pela estratégia BCG no período neonatal seguida de boosters com a vacina gênica no período adulto e avaliar a eficácia protetora desta estratégia. Os protocolos experimentais incluíram imunização integral (3 doses) no período neonatal (5, 12 e 19 dias de idade) com 50miug de DNA/ dose por via IM; 1 dose de DNA no quinto dia seguida de 1 ou 2 doses no período adulto (100miug/ via IM) ou 1 dose de BCG no quinto dia seguida de 2 doses de DNA no período adulto. As avaliações foram feitas com células dos camundongos normais, injetados com PBS ou previamente imunizados e estimuladas ex vivo com S. aureus, LPS ou ConA. Os sobrenadantes das culturas foram coletados após 48 h de incubação e as concentrações de IFN-gama, IL-4, IL-5 e IL-10 foram determinadas por ELlSA. A produção de anticorpos (lgG1 e IgG2a anti-hsp65) foi avaliada no soro dos animais também pelo método de ELlSA. A detecção de mensagem para hsp65 nos diferentes tecidos foi feita por RT -PCR. Trinta dias após a infecção por via intratraqueal, os pulmões dos animais previamente imunizados com o protocolo BCG/DNA, foram analisados quanto ao número de unidades formadoras de colônias (UFC), quantidade de IFN-gama e aspectos histopatológicos...