kappa-Opioid receptor in humans: cDNA and genomic cloning, chromosomal assignment, functional expression, pharmacology, and expression pattern in the central nervous system.

Using the mouse delta-opioid receptor cDNA as a probe, we have isolated genomic clones encoding the human mu- and kappa-opioid receptor genes. Their organization appears similar to that of the human delta receptor gene, with exon-intron boundaries located after putative transmembrane domains 1 and 4. The kappa gene was mapped at position q11-12 in human chromosome 8. A full-length cDNA encoding the human kappa-opioid receptor has been isolated. The cloned receptor expressed in COS cells presents a typical kappa 1 pharmacological profile and is negatively coupled to adenylate cyclase. The expression of kappa-opioid receptor mRNA in human brain, as estimated by reverse transcription-polymerase chain reaction, is consistent with the involvement of kappa-opioid receptors in pain perception, neuroendocrine physiology, affective behavior, and cognition. In situ hybridization studies performed on human fetal spinal cord demonstrate the presence of the transcript specifically in lamina II of the dorsal horn. Some divergences in structural, pharmacological, and anatomical properties are noted between the cloned human and rodent receptors.

T-cell receptor (TCR) usage in Lewis rat experimental autoimmune encephalomyelitis: TCR beta-chain-variable-region V beta 8.2-positive T cells are not essential for induction and course of disease.

Predominant usage of V beta 8.2 gene segments, encoding a T-cell receptor (TCR) beta chain variable region, has been reported for pathogenic Lewis rat T cells reactive to myelin basic protein (MBP). However, up to 75% of the alpha/beta T cells in a panel of MBP-specific T-cell lines did not display TCR V beta 8.2, V beta 8.5, V beta 10, or V beta 16 elements. To further investigate TCR usage, we sorted the T-cell lines for V beta 8.2- and V beta 10-positive T cells or depleted the lines of cells with these TCRs. V beta 8.2-positive T cells and one of the depleted T-cell lines strongly reacted against the MBP peptide MBP-(68-88). The depleted T-cell line caused marked experimental autoimmune encephalomyelitis (EAE) even in Lewis rats in which endogenous V beta 8.2-positive T cells had been eliminated by neonatal treatment with anti-V beta 8.2 monoclonal antibodies. T-cell hybridomas generated from this line predominantly used V beta 3 TCR genes coexpressed with TCR V alpha 2 transcripts, which were also used by V beta 8.2-positive T cells. Furthermore, V beta 10-positive T cells reactive to MBP-(44-67) were encephalitogenic when injected immediately after positive selection. After induction of EAE by sorted V beta 8.2- or V beta 10-positive T-cell lines, immunocytochemical analysis of the spinal cord tissue showed a predominance of the injected TCR or of nontypable alpha/beta T cells after injection of the depleted line. Our results demonstrate heterogeneity of TCR beta-chain usage even for a single autoantigen in an inbred strain. Moreover, V beta 8.2-positive T cells are not essential for the induction and progression of adoptive-transfer EAE.

Insulin-like growth factor I treatment reduces demyelination and up-regulates gene expression of myelin-related proteins in experimental autoimmune encephalomyelitis.

To compare effects of insulin-like growth factor I (IGF-I) and placebo treatment on lesions that resemble those seen during active demyelination in multiple sclerosis, we induced experimental autoimmune encephalomyelitis in Lewis rats with an emulsion containing guinea pig spinal cord and Freund's adjuvant. On day 12-13, pairs of rats with the same degree of weakness were given either IGF-I or placebo intravenously twice daily for 8 days. After 8 days of placebo or IGF-I (200 micrograms/day or 1 mg/day) treatment, the spinal cord lesions were studied by in situ hybridization and with immunocytochemical and morphological methods. IGF-I produced significant reductions in numbers and areas of demyelinating lesions. These lesions contained axons surrounded by regenerating myelin segments instead of demyelinated axons seen in the placebo-treated rats. Relative mRNA levels for myelin basic protein, proteolipid protein (PLP), and 2',3'-cyclic nucleotide 3'-phosphodiesterase in lesions of IGF-I-treated rats were significantly higher than they were in placebo-treated rats. PLP mRNA-containing oligodendroglia also were more numerous and relative PLP mRNA levels per oligodendrocyte were higher in lesions of IGF-I-treated rats. Finally, a significantly higher proportion of proliferating cells were oligodendroglia-like cells in lesions of IGF-I-treated rats. We think that IGF-I effects on oligodendrocytes, myelin protein synthesis, and myelin regeneration reduced lesion severity and promoted clinical recovery in this experimental autoimmune encephalomyelitis model. These IGF-I actions may also benefit patients with multiple sclerosis.

Regulatory region in choline acetyltransferase gene directs developmental and tissue-specific expression in transgenic mice.

Acetylcholine, one of the main neurotransmitters in the nervous system, is synthesized by the enzyme choline acetyltransferase (ChAT; acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6). The molecular mechanisms controlling the establishment, maintenance, and plasticity of the cholinergic phenotype in vivo are largely unknown. A previous report showed that a 3800-bp, but not a 1450-bp, 5' flanking segment from the rat ChAT gene promoter directed cell type-specific expression of a reporter gene in cholinergic cells in vitro. Now we have characterized a distal regulatory region of the ChAT gene that confers cholinergic specificity on a heterologous downstream promoter in a cholinergic cell line and in transgenic mice. A 2342-bp segment from the 5' flanking region of the ChAT gene behaved as an enhancer in cholinergic cells but as a repressor in noncholinergic cells in an orientation-independent manner. Combined with a heterologous basal promoter, this fragment targeted transgene expression to several cholinergic regions of the central nervous system of transgenic mice, including basal forebrain, cortex, pons, and spinal cord. In eight independent transgenic lines, the pattern of transgene expression paralleled qualitatively and quantitatively that displayed by endogenous ChAT mRNA in various regions of the rat central nervous system. In the lumbar enlargement of the spinal cord, 85-90% of the transgene expression was targeted to the ventral part of the cord, where cholinergic alpha-motor neurons are located. Transgene expression in the spinal cord was developmentally regulated and responded to nerve injury in a similar way as the endogenous ChAT gene, indicating that the 2342-bp regulatory sequence contains elements controlling the plasticity of the cholinergic phenotype in developing and injured neurons.

Desenvolvimento de video educativo para a aprendizagem do autocateterismo vesical intermitente

A bexiga neurogênica é uma disfunção vesical decorrente principalmente da lesão medular. O cateterismo vesical intermitente é o tratamento mais indicado na atualidade, deve ser realizado de 4 a 6 vezes ao dia, durante toda a vida, visando a proteção do trato urinário superior e a aquisição da continência urinária. Na reabilitação desses indivíduos, a autocateterização vesical é um desafio enfrentado na busca pela autonomia, privacidade, inserção social e participação. Os vídeos educativos são utilizados para o aprendizado do autocateterismo em vários países, por serem de fácil utilização e acesso via internet. Apesar disso, não existem vídeos realizados para o contexto brasileiro, levando em consideração os cateteres urinários e a técnica utilizada no Brasil. Este estudo teve como objetivo desenvolver e validar um vídeo educativo para a realização do autocateterismo vesical intermitente limpo. Trata-se de um estudo quantitativo, desenvolvido em duas fases: 1ª fase com a avaliação de vídeos educativos públicos direcionados para a aprendizagem do autocateterismo vesical intermitente com a técnica limpa; e 2ª Fase, com o desenvolvimento e validação de um vídeo educativo voltado para aprendizagem do autocateterismo. O levantamento dos vídeos utilizou um site de compartilhamento de vídeos utilizando o descritor \"autocateterismo\". Os vídeos foram avaliados por três juízes da área de saúde. O processo de desenvolvimento e validação do roteiro do vídeo educativo utilizou questionários previamente ratificados. Participaram dessas etapas, respectivamente, 18 e 17 juízes experts em reabilitação e/ou no ensino em saúde. O levantamento mostrou que apenas 3,5% (172) do total de vídeos disponíveis no site pesquisado eram voltados para o aprendizado do autocateterismo no contexto brasileiro. Seis vídeos eram específicos para o autocateterismo, dos quais quatro tinham informações desatualizadas ou incorretas, apenas dois atingiram a pontuação aceitável. Na validação do roteiro observou-se um predomínio de participantes do sexo feminino (94,44%), com idade de 30 a 60 anos, dos quais 72,22% possuíam mestrado e 50% atuavam há mais de cinco anos na área de reabilitação. O roteiro foi considerado validado com 96,29% das respostas dos juízes \"concordo\" ou \"concordo totalmente\" nas questões referentes ao quesito objetivo, 91,09% para quesito conteúdo, 98,12% em relação ao quesito relevância, 75% quanto ao quesito ambiente, 71,11% no quesito linguagem verbal e 92,70% referente à inclusão de tópicos. A produção do vídeo contou com uso de tecnologia 3D e apoio de uma equipe técnica especializada. No que se refere à validação do conteúdo do vídeo educativo, o conteúdo do vídeo foi considerado validado com 100% dos juízes que responderam \"concordo\" ou \"concordo totalmente\" nas questões referentes à funcionalidade, 86,27% referentes à usabilidade, 97,06% no quesito eficiência, 100% para técnica audiovisual, 94,11% quanto ao ambiente e 97,05% procedimento. O vídeo educativo foi avaliado positivamente tanto pela qualidade das informações quanto pela didática do ensino, mostrando a relevância da validação de materiais educativos. A expectativa é disseminar o vídeo educativo em diferentes centros de reabilitação e Universidades, visando propagar e tornar o conhecimento sobre a temática mais acessível à sociedade e aos profissionais de saúde, em especial os de reabilitação. Além de incentivar e embasar metodologicamente o desenvolvimento de outros vídeos educativos na área da saúde