Lack of Fas antagonism by Met in human fatty liver disease

Hepatocytes in fatty livers are hypersensitive to apoptosis and undergo escalated apoptotic activity via death receptor-mediated pathways, particularly that of Fas-FasL, causing hepatic injury that can eventually proceed to cirrhosis and end-stage liver disease. Here we report that the hepatocyte growth factor receptor, Met, plays an important part in preventing Fas-mediated apoptosis of hepatocytes by sequestering Fas. We also show that Fas antagonism by Met is abrogated in human fatty liver disease (FLD). Through structure-function studies, we found that a YLGA amino-acid motif located near the extracellular N terminus of the Met alpha-subunit is necessary and sufficient to specifically bind the extracellular portion of Fas and to act as a potent FasL antagonist and inhibitor of Fas trimerization. Using mouse models of FLD, we show that synthetic YLGA peptide tempers hepatocyte apoptosis and liver damage and therefore has therapeutic potential.     

Myelin-specific regulatory T cells accumulate in the CNS but fail to control autoimmune inflammation

Treatment with ex vivo-generated regulatory T cells (T-reg) has been regarded as a potentially attractive therapeutic approach for autoimmune diseases. However, the dynamics and function of T-reg in autoimmunity are not well understood. Thus, we developed Foxp3gfp knock-in (Foxp3gfp.KI) mice and myelin oligodendrocyte glycoprotein (MOG)(35-55)/IA(b) (MHC class II) tetramers to track autoantigen-specific effector T cells (T-eff) and T-reg in vivo during experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. MOG tetramer-reactive, Foxp3(+) T-reg expanded in the peripheral lymphoid compartment and readily accumulated in the central nervous system (CNS), but did not prevent the onset of disease. Foxp3(+) T cells isolated from the CNS were effective in suppressing naive MOG-specific T cells, but failed to control CNS-derived encephalitogenic T-eff that secreted interleukin (IL)-6 and tumor necrosis factor (TNF). Our data suggest that in order for CD4(+)Foxp3(+) T-reg to effectively control autoimmune reactions in the target organ, it may also be necessary to control tissue inflammation.     

Requirement for T-cell apoptosis in the induction of peripheral transplantation tolerance.

The mechanisms of allograft tolerance have been classified as deletion, anergy, ignorance and suppression/regulation. Deletion has been implicated in central tolerance, whereas peripheral tolerance has generally been ascribed to clonal anergy and/or active immunoregulatory states. Here, we used two distinct systems to assess the requirement for T-cell deletion in peripheral tolerance induction. In mice transgenic for Bcl-xL, T cells were resistant to passive cell death through cytokine withdrawal, whereas T cells from interleukin-2-deficient mice did not undergo activation-induced cell death. Using either agents that block co-stimulatory pathways or the immunosuppressive drug rapamycin, which we have shown here blocks the proliferative component of interleukin-2 signaling but does not inhibit priming for activation-induced cell death, we found that mice with defective passive or active T-cell apoptotic pathways were resistant to induction of transplantation tolerance. Thus, deletion of activated T cells through activation-induced cell death or growth factor withdrawal seems necessary to achieve peripheral tolerance across major histocompatibility complex barriers.     

Proteomic analysis of eccrine sweat: implications for the discovery of schizophrenia biomarker proteins

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and multiple reaction monitoring mass spectrometry (MRM-MS) proteomics analyses were performed on eccrine sweat of healthy controls, and the results were compared with those from individuals diagnosed with schizophrenia (SZ). This is the first large scale study of the sweat proteome. First, we performed LC-MS/MS on pooled SZ samples and pooled control samples for global proteomics analysis. Results revealed a high abundance of diverse proteins and peptides in eccrine sweat. Most of the proteins identified from sweat samples were found to be different than the most abundant proteins from serum, which indicates that eccrine sweat is not simply a plasma transudate and may thereby be a source of unique disease-associated biomolecules. A second independent set of patient and control sweat samples were analyzed by LC-MS/MS and spectral counting to determine qualitative protein differential abundances between the control and disease groups. Differential abundances of selected proteins, initially determined by spectral counting, were verified by MRM-MS analyses. Seventeen proteins showed a differential abundance of approximately 2-fold or greater between the SZ pooled sample and the control pooled sample. This study demonstrates the utility of LC-MS/MS and MRM-MS as a viable strategy for the discovery and verification of potential sweat protein disease biomarkers.     

Structure/function analysis of interleukin-2-toxin (DAB486-IL-2). Fragment B sequences required for the delivery of fragment A to the cytosol of target cells

We have used cassette and deletion mutagenesis to analyze the structural features of fragment B-related sequences in the fusion toxin DAB486-IL-2 (where IL-2 represents interleukin-2) that are necessary for the efficient delivery of fragment A to the cytosol of target cells. We demonstrate that whereas an intact disulfide bond between Cys461 and Cys471 may be required for the cytotoxic action of native diphtheria toxin, this bond is not required for the cytotoxic action of DAB486-IL-2. The in-frame deletion of the 97 amino acids from Thr387 to His485 of DAB486-IL-2 increases both the potency and the apparent dissociation constant (Kd) of the resulting fusion toxin for high affinity interleukin-2 receptor-bearing target cells. In contrast, the inframe deletion of either the 191 amino acids between Asp291 and Gly483 or the 85 amino acids between Asn204 and Ile290 results in a 1000-fold loss in potency. These regions contain the putative membrane-spanning regions and the amphipathic membrane surface-associating regions of fragment B, respectively. These results indicate that the efficient delivery of the ADP-ribosyltransferase from DAB486-IL-2 to the cytosol requires the membrane-associating domains of fragment B. This function has been postulated to play a role in the diphtherial intoxication of eukaryotic cells. However, unlike native diphtheria toxin, fragment B sequences distal to Thr387 do not enhance the potency of DAB486-IL-2.     

Two phenotypically distinct populations of T cells have suppressor capabilities simultaneously in the maintenance phase of immunologic enhancement

The events leading to immunologic enhancement in LEW rats immunized actively with Brown Norway (BN) rat spleen cells and passively with LEW anti-BN hyperimmune serum 11 and 10 days before receiving (LEW X BN)F1 cardiac allografts, respectively, have been studied. Cellular suppressor mechanisms developing during the induction phase of this phenomenon have recently been shown to be mediated by W3/25+ T cells in an antigen-specific manner. The present study suggests that the late maintenance phase of immunologic enhancement is mediated in vivo by simultaneously present separate donor-specific T cell subpopulations of W3/25+ and OX8+ phenotypes. Splenocyte subsets from grafted recipients greater than 100 days after transplantation were adoptively transferred into unmodified syngeneic LEW rats that received a specific test allograft 24 hr later, or into B recipients bearing indefinitely surviving heart grafts. Test graft survival was prolonged significantly in the first group and not altered in the second. Indeed, nonoverlapping W3/25+ and OX8+ cell fractions were separately responsible for suppression. However, when suppressor activity was tested in vitro in a three-component coculture mixed lymphocyte reaction, no suppression by T cells was obtained; this lack of correlation between in vivo and in vitro results has also been noted by other investigators in different systems. Thus, in the maintenance phase of actively and passively induced immunologic enhancement, interplay between two phenotypically distinct T cells with suppressor characteristics, but not putative cell-surface blocking factors, seems to prevent development of an alloreactive response and mediate host unresponsiveness.     

Evidence for plasmid-mediated toxin and bacteriocin production in Clostridium botulinum type G

A single 81-megadalton plasmid was previously isolated from each of six toxigenic strains of Clostridium botulinum type G (M. S. Strom, M. W. Eklund, and F. T. Poysky, Appl. Environ. Microbiol. 48:956-963, 1984). In this study, nontoxigenic derivatives isolated from each of the toxigenic strains following consecutive daily transfers in Trypticase (BBL Microbiology Systems, Cockeysville, Md.)-yeast extract-glucose broth at 44 degrees C simultaneously ceased to produce type G neurotoxin and to harbor the resident 81-megadalton plasmid. The nontoxigenic derivatives also ceased to produce bacteriocin and lost their immunity to the bacteriocin produced by the toxigenic strains. In contrast, all of the toxigenic isolates continued to carry the resident plasmid and to produce both bacteriocin and type G neurotoxin. This is the first evidence suggesting that the production of neurotoxin and bacteriocin by C. botulinum is mediated by a plasmid.     

Rapid stereospecific stimulation of lymphocytic metabolism by interleukin 2

IL 2 maintains the viability of IL 2-dependent CTLL-2 cells. IL 2, a lymphocytotrophic factor, stimulates the cellular metabolism of IL 2 receptor-bearing CTLL-2 murine cytolytic T cells. Both aerobic (oxygen consumption) and anaerobic (lactic acid production) metabolism of CTLL-2 cell are stimulated by rIL 2. The effects of IL 2 upon murine T cells is blocked by an anti-IL 2 receptor monoclonal antibody but not by other monoclonal antibodies that bind to other proteins upon CTLL-2 cells. Changes in aerobic and anaerobic cellular metabolism occur rapidly after interaction with IL 2, whereas the effects on the cell cycle are relatively slow and may be dependent upon antecedent metabolic stimulation by IL 2. This effect of IL 2 on cell viability appears to be mediated by a direct effect on important aerobic and anaerobic metabolic pathways.