An analysis of the outer membrane of desulfovibrio vulgaris (Woolwich)

D.vulgaris (Woolwich) OMs can be extracted successfully and reproducibly from the cell envelopes by the selective solubilisation of cell membranes by sarkosyl. Three Omps (Omps 1, 2 & 3) occur consistently in the OMs of C-Fe, NC & C+Fe cells. NC C+Fe OMs have, in addition, minor proteins absent from C-Fe OMs. Omp2 band is seen to be enhanced in C-Fe and NC OMs as indicated by PAGE. PAGE-immunoblotting of LPS indicates heterogeneity of these molecules. Immunological analyses of NC, C-Fe & C+Fe cells using specific antisera indicate no differences among these cell-types. Neither are antigenic differences revealed by further analyses of OM Western blots by specific antisera. However, pyrolysis analyses indicate that the cell-types analyses show variations in the cell ability of this technique to bacterial species cultured under different growth conditions indicates its potential as a tool for numerical taxonomy of the sulphate reducers. D.vulgaris (Woolwich) does not produce extra Omps in response to iron deprivation. Instead, it increases the synthesis of 0mp2 and LPS in its OM as reflected in the OM PAGE pattern and increased yields of LPS extracted from C-Fe cultures. HPLC sugar analyses indicate the LPS contain N-glucosamine, possibly rhamnose and an unidentified sugar. A change in HPLC sugar pattern of the C+Fe LPS samples indicates interactions between Fe(II) and LPS. This interaction is substantiated by X-ray microanalysis of the elemental content extracted LPS. The evidence indicates that LPS may be involved in Fe(II) uptake by the cells. LPS also takes part in the adhesion of D.vulgaris (Woolwich) to mild steel surfaces. This is demonstrated by the data from experiments using anti-LPS Fab fragments. The presence of these antibodies reduces the number of cells adhering to mild steel coupons. Fe-radiolabelling of OM components immobilised on nitrocellulose shows that Omps 1, 2 & 3 bind Fe(II). Blocking experiments using copper & magnesium indicate Omp1 to be Fe(II) specific while Omps 2 3 are not. The enhancement of Omp2 observed in C-Fe NC OMs in PAGE analyses indicates Omp2 to be principal cation binder, whose synthesis is increased under iron deprivation

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 β2 microglobulin (β2m) 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 β2m/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/β2m/CD3-ζ genes to a reporter T cell line and purified primary mouse CD8 T cells. The peptide/β2m/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/β2m/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/β2m/CD3-ζ or islet-specific glucose-6-phosphatase catalytic subunit-related protein, amino acids 206–214 (IGRP206–214)/β2m/CD3-ζ killed their respective autoreactive CD8 T cell targets in vitro. Furthermore, transfer of primary CD8 T cells transfected with InsB15–23/β2m/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

Activation of Insulin-Reactive CD8 T-Cells for Development of Autoimmune Diabetes

Objective: We have previously reported a highly diabetogenic CD8 T cell clone, G9C8, in the Non Obese Diabetic (NOD) mouse, specific to low avidity insulin peptide B15-23 and cells responsive to this antigen are among the earliest islet infiltrates. We aimed to study the selection, activation and development of diabetogenic capacity of these insulin-reactive T cells. Research Design and Methods: We generated a TCR transgenic mouse expressing the cloned TCR Vα18/Vβ6 receptor of the G9C8 insulin-reactive CD8 T cell clone. The mice were crossed to TCRCα−/− mice so that the majority of the T cells expressed the clonotypic TCR and the phenotype and function of the cells was investigated. Results: There was good selection of CD8 T cells with a predominance of CD8 single positive thymocytes, in spite of thymic insulin expression. Peripheral lymph node T cells had a naïve phenotype (CD44lo, CD62Lhi) and proliferated to insulin B15-23 peptide and to insulin. These cells produced interferon-γ and TNF-α in response to insulin peptide and were cytotoxic to insulin-peptide coated targets. In vivo, the TCR transgenic mice developed insulitis but not spontaneous diabetes. However, the mice developed diabetes on immunization, and the activated transgenic T cells were able to transfer diabetes to immunodeficient NOD.scid mice. Conclusion: Autoimmune CD8 T cells responding to a low affinity insulin B chain peptide escape from thymic negative selection, and require activation in vivo to cause diabetes

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

The autoimmune attack on pancreatic β-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 β-cell

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, ε4 was associated with lower concentrations of all three synaptic proteins and ε2 with significantly elevated PSD95 (p = 0.03). A combined measure of synaptic proteins showed a significant linear decrease from ε2 through ε3 to ε4 (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. © 2005 Elsevier Inc. All rights reserved

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-ζ) to target insulin-specific CD8 T cells. Insulin-reactive target cells were cultured with InsCD3-ζ CD8 T cells and cytotoxicity was assessed. Activated, but not naïve, InsCD3-ζ 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

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

The autoimmune attack on pancreatic β-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 β-cell

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 β2 microglobulin (β2m) 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 β2m/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/β2m/CD3-ζ genes to a reporter T cell line and purified primary mouse CD8 T cells. The peptide/β2m/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/β2m/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/β2m/CD3-ζ or islet-specific glucose-6-phosphatase catalytic subunit-related protein, amino acids 206–214 (IGRP206–214)/β2m/CD3-ζ killed their respective autoreactive CD8 T cell targets in vitro. Furthermore, transfer of primary CD8 T cells transfected with InsB15–23/β2m/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