Adoptive Transfer of mRNA-Transfected T Cells Redirected against Diabetogenic CD8 T Cells Can Prevent Diabetes.

Chimeric major histocompatibility complex (MHC) molecules supplemented with T cell receptor (TCR) signaling motifs function as activation receptors and can redirect gene-modified T cells against pathogenic CD8 T cells. We have shown that ß2 microglobulin (ß2m) operates as a universal signaling component of MHC-I molecules when fused with the CD3-ζ chain. Linking the H-2Kd-binding insulin B chain peptide insulin B chain, amino acids 15-23 (InsB15-23) to the N terminus of ß2m/CD3-ζ, redirected polyclonal CD8 T cells against pathogenic CD8 T cells in a peptide-specific manner in the non-obese diabetic (NOD) mouse. Here, we describe mRNA electroporation for delivering peptide/ß2m/CD3-ζ genes to a reporter T cell line and purified primary mouse CD8 T cells. The peptide/ß2m/CD3-ζ products paired with endogenous MHC-I chains and transmitted strong activation signals upon MHC-I cross-linking. The reporter T cell line transfected with InsB15-23/ß2m/CD3-ζ mRNA was activated by an InsB15-23-H-2Kd-specific CD8 T cell hybrid only when the transfected T cells expressed H-2Kd. Primary NOD CD8 T cells expressing either InsB15-23/ß2m/CD3-ζ or islet-specific glucose-6-phosphatase catalytic subunit-related protein, amino acids 206-214 (IGRP206-214)/ß2m/CD3-ζ killed their respective autoreactive CD8 T cell targets in vitro. Furthermore, transfer of primary CD8 T cells transfected with InsB15-23/ß2m/CD3-ζ mRNA significantly reduced insulitis and protected NOD mice from diabetes. Our results demonstrate that mRNA encoding chimeric MHC-I receptors can redirect effector CD8 against diabetogenic CD8 T cells, offering a new approach for the treatment of type 1 diabetes.

Premorbid effects of APOE on synaptic proteins in human temporal neocortex.

APOE affects the risk of Alzheimer's disease (AD) and course of several other neurologic diseases. Experimental studies suggest that APOE influences synaptogenesis. We measured the concentration of two presynaptic proteins, synaptophysin and syntaxin 1, and also postsynaptic density-95 (PSD95), in superior temporal cortex from 42 AD and 160 normal brains, and determined the APOE genotypes. The concentration of both presynaptic proteins was approximately two-thirds lower in AD than normal brains and that of PSD95 one-third lower. No effect of APOE on synaptic proteins was found in advanced AD. However, in normal brain, epsilon4 was associated with lower concentrations of all three synaptic proteins and epsilon2 with significantly elevated PSD95 (p=0.03). A combined measure of synaptic proteins showed a significant linear decrease from epsilon2 through epsilon3 to varepsilon4 (p=0.01). APOE influences the concentration of synaptic proteins in normal superior temporal cortex and may thereby affect the response to injury, and the risk and outcome of a range of neurologic diseases.

CD8+ T-cells and their interaction with other cells in damage to islet beta-cells.

The autoimmune attack on pancreatic beta-cells is orchestrated by a variety of cells that produce cytokines and other toxic mediators. CD8(+) T-cells work together with other lymphocytes and antigen-presenting cells to mediate this damage and have been shown in animal models to be important both in the early stages of diabetes development and in the final effector stages. Recently, there has also been much interest in studying CD8(+) T-cells that may play a role in human Type 1 diabetes and identifying their antigenic targets. The present paper will focus on the activation of CD8(+) T-cells and their interaction with other cells of the immune system and discuss the target antigens and mechanisms of damage that the CD8(+) T-cells use in the attack on the islet beta-cell.

Inhibition of AMP-activated protein kinase protects pancreatic beta-cells from cytokine-mediated apoptosis and CD8+ T-cell-induced cytotoxicity.

OBJECTIVE: Apoptotic destruction of insulin-producing pancreatic beta-cells is involved in the etiology of both type 1 and type 2 diabetes. AMP-activated protein kinase (AMPK) is a sensor of cellular energy charge whose sustained activation has recently been implicated in pancreatic beta-cell apoptosis and in islet cell death posttransplantation. Here, we examine the importance of beta-cell AMPK in cytokine-induced apoptosis and in the cytotoxic action of CD8(+) T-cells. RESEARCH DESIGN AND METHODS: Clonal MIN6 beta-cells or CD1 mouse pancreatic islets were infected with recombinant adenoviruses encoding enhanced green fluorescent protein (eGFP/null), constitutively active AMPK (AMPK-CA), or dominant-negative AMPK (AMPK-DN) and exposed or not to tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma. Apoptosis was detected by monitoring the cleavage of caspase-3 and DNA fragmentation. The cytotoxic effect of CD8(+) purified T-cells was examined against pancreatic islets from NOD mice infected with either null or the AMPK-DN-expressing adenoviruses. RESULTS: Exposure to cytokines, or expression of AMPK-CA, induced apoptosis in clonal MIN6 beta-cells and CD1 mouse pancreatic islets. By contrast, overexpression of AMPK-DN protected against the proapoptotic effect of these agents, in part by preventing decreases in cellular ATP, and lowered the cytotoxic effect of CD8(+) T-cells toward NOD mouse islets. CONCLUSIONS: Inhibition of AMPK activity enhances islet survival in the face of assault by either cytokines or T-cells. AMPK may therefore represent an interesting therapeutic target to suppress immune-mediated beta-cell destruction and may increase the efficacy of islet allografts in type 1 diabetes.

Developing a novel model system to target insulin-reactive CD8 T cells.

CD8 T cells play an important role in autoimmune diabetes development, and therefore removing these cells may protect against disease. To test this, we designed a novel method using engineered cells (InsCD3-zeta) to target insulin-specific CD8 T cells. Insulin-reactive target cells were cultured with InsCD3-zeta CD8 T cells and cytotoxicity was assessed. Activated, but not naïve, InsCD3-zeta CD8 T cells readily killed insulin-reactive target CD8 T cells. This approach to immunotarget relevant pathogenic CD8 T cells may be a therapeutic option to delay or prevent type 1 diabetes.

Cationic interactions in the lipoplysaccharide of Desulfovibrio vulgaris

Foram utilizadas as técnicas de cromatografia à liquido de alto desempenho (HPLC), análise por raio-X EDAX) e eletroforese em gel de poliacrilamida (SDS-PAGE) na investigaçäo das interaçöes entre vários cátions e lipopolissacarídeo (LPS) extraído da espécie bacteriana Desulfovibrio vulgaris. O LPS demonstrou afinidades variáveis para diferentes íons divalentes. Eletrodiálise removeu a maioria dos íons Fe (II) do LPS e resultou num aumento dos íons Ca. A HPLC e SDS-PAGE demonstraram diferenças na estrutura de LPs isolado de células ricas ou pobres em Fe(II), que indicaram que o Fe(II)