Human mesenchymal cells from adipose tissue deposit laminin and promote regeneration of injured spinal cord in rats.

Cell therapy is a promising strategy to pursue the unmet need for treatment of spinal cord injury (SCI). Although several studies have shown that adult mesenchymal cells contribute to improve the outcomes of SCI, a description of the pro-regenerative events triggered by these cells is still lacking. Here we investigated the regenerative properties of human adipose tissue derived stromal cells (hADSCs) in a rat model of spinal cord compression. Cells were delivered directly into the spinal parenchyma immediately after injury. Human ADSCs promoted functional recovery, tissue preservation, and axonal regeneration. Analysis of the cord tissue showed an abundant deposition of laminin of human origin at the lesion site and spinal midline; the appearance of cell clusters composed of neural precursors in the areas of laminin deposition, and the appearance of blood vessels with separated basement membranes along the spinal axis. These effects were also observed after injection of hADSCs into non-injured spinal cord. Considering that laminin is a well-known inducer of axonal growth, as well a component of the extracellular matrix associated to neural progenitors, we propose that it can be the paracrine factor mediating the pro-regenerative effects of hADSCs in spinal cord injury.

An efficient low cost method for gene transfer to T lymphocytes.

UNLABELLED: Gene transfer to T lymphocytes has historically relied on retro and lentivirus, but recently transposon-based gene transfer is rising as a simpler and straight forward approach to achieve stable transgene expression. Transfer of expression cassettes to T lymphocytes remains challenging, being based mainly on commercial kits. AIMS: We herein report a convenient and affordable method based on in house made buffers, generic cuvettes and utilization of the widely available Lonza nucleofector II device to promote efficient gene transfer to T lymphocytes. RESULTS: This approach renders high transgene expression levels in primary human T lymphocytes (mean 45%, 41-59%), the hard to transfect murine T cells (mean 38%, 36-42% for C57/BL6 strain) and human Jurkat T cell line. Cell viability levels after electroporation allowed further manipulations such as in vitro expansion and Chimeric Antigen Receptor (CAR) mediated gain of function for target cell lysis. CONCLUSIONS: We describe here an efficient general protocol for electroporation based modification of T lymphocytes. By opening access to this protocol, we expect that efficient gene transfer to T lymphocytes, for transient or stable expression, may be achieved by an increased number of laboratories at lower and affordable costs.

Silenciamento de CD55 e CD59 para aumento da lise de linfócitos T CD20 +mediada por Rituximabe / CD55 and CD59 silencing for augmentation of CD20+ T lymphocytes lysis mediated by Rituximabe

O transplante alogeneico de precursores hematopoiéticos é indicado para o tratamento de doenças malignas e não malignas e apresenta como benefícios a reconstituição hematopoiética e o efeito do enxerto contra a leucemia. No entanto, as mesmas células T que exercem o efeito do enxerto contra a leucemia exercem também um efeito não desejado: a doença do enxertocontra o hospedeiro (DECH). Quando o transplante é realizado para o tratamento de doenças malignas, não é recomendável a retirada dos linfócitos T do enxerto, pois o efeito benéfico do enxerto contra a leucemia é perdido.Os tratamentos disponíveis atualmente para a DECH consistem em sua maioria na administração de imunossupressores que nem sempre surtem o efeito desejado, sendo somente parcialmente bem sucedidos. Como alternativaforam propostos sistemas baseados em genes suicidas que consistem na modificação dos linfócitos T do doador para que passem a expressar ransgenes que permitam a eliminação destas células através da administração de uma droga. Um destes sistemas consiste na inserção da proteína CD20 nos linfócitos T do doador para que os mesmos passem a ser alvo do anticorpo anti -CD20 Rituximabe. Determinamos as doses sub-ótimas de lise de linfócitos B por Rituximabe in vitro com o objetivo de estabelecer uma dose de anticorpo para a eliminação dos linfócitos T CD20+capaz de poupar linfócitos B. A lise desencadeada por antígenos de membrana mediada por anticorpos e proteínas do sistema complemento é modulada por proteínas reguladoras do complemento como CD46, CD55 e CD59. Avaliamos a expressão dasproteínas reguladoras nas células B e T de doadores saudáveis e verificamos que a expressão de CD59 e de CD46 é maior em linfócitos T do que em B, e que a expressão de CD55 é maior em linfócitos B do que em T. Além disso, verificamos que os níveis de expressão de CD55, CD59 e CD46 são semelhantes nos linfócitos T e nas células Jurkat CD20+. A expressão destas proteína...

The hematopoietic stem cell transplantation is used to treat malignant and non-malignant diseases and provides as benefits the hematopoietic reconstitution and graft-versus-leukemia effect(GVL). However, the same T cells which cause the graft-versus-leukemia effect also cause an undesired effect: the graft-versus-host-disease (GVHD). When the transplantation is aimedto treat malignant diseases, it is not recommended to deplete T cells from the graft,as the benefic effect, the GVL, would be lost. The currently available treatments for GVHD are mainly based on the administration of immunosuppressors, which sometimes doesn’t provide the expected outcome, being only partly successful. As an alternative to the GVHDtreatment,suicide gene systems were proposed. These consist in the modification of donor’s T cells so that they express transgenes,which allow the efficient elimination of these cells by the administration of a drug. One of these systems consists in the insertion of CD20 protein in donor’s T lymphocytes,so that they become targets of the monoclonal antibody anti-CD20(Rituximabe). We have determined suboptimal in vitrolysis conditions for B cells by Rituximabe, in orderto establish concentrations of Rituximab which depletes CD20+T lymphocytes and spares at least a fraction of B lymphocytes. The lysis triggered by membrane antigens, mediated by antibodies and complement system proteins, is modulated by complement regulatory proteins such as CD46, CD55,and CD59. We assessed the expression of these regulatory proteins in B and T cells from healthy donors and our results showed that CD46 and CD59 expression is higher in T lymphocytes than in B lymphocytes,and that the expression of CD55 is higher in B lymphocytes than in T l