J Immunoassay Immunochem
January 2021
Objective: To investigate neurodegenerative and inflammatory biomarkers in people with amyotrophic lateral sclerosis (PALS), evaluate their predictive value for ALS progression rates, and assess their utility as pharmacodynamic biomarkers for monitoring treatment effects.
Methods: De-identified, longitudinal plasma, and cerebrospinal fluid (CSF) samples from PALS (n = 108; 85 with samples from ≥2 visits) and controls without neurological disease (n = 41) were obtained from the Northeast ALS Consortium (NEALS) Biofluid Repository. Seventeen of 108 PALS had familial ALS, of whom 10 had C9orf72 mutations.
Objective: We measured the levels of mutant huntingtin (mtHtt) and total huntingtin (tHtt) in blood leukocytes from Prospective Huntington At-Risk Observational Study (PHAROS) subjects at 50% risk of carrying the Huntington disease mutation using a homogeneous time-resolved fluorescence (HTRF) assay to assess its potential as a biomarker.
Methods: Peripheral blood mononuclear cells from consenting PHAROS subjects were analyzed by HTRF using antibodies that simultaneously measured mtHtt and tHtt. mtHtt levels were normalized to tHtt, double-stranded DNA, or protein and analyzed according to cytosine-adenine-guanine repeat length (CAGn), demographics, predicted time to clinical onset or known time since clinical onset, and available clinical measures.
A means for measuring levels of soluble huntingtin proteins in clinical samples is essential for assessing the biological effects of potential mutant huntingtin (mtHtt) modifying treatments being developed for Huntington's disease (HD). We have optimized a previously described cell-based Homogeneous Time Resolved Fluorescence method that can measure soluble mtHtt and its ratio to the total Htt (tHtt) in blood buffy coats [1]. The results of the optimization and assay qualification indicate the assay to be specific for mtHtt in HD compared to Control subjects, highly sensitive, and technically and biologically reproducible.
View Article and Find Full Text PDFThe IL-2 pathway is portrayed often as central to allograft rejection. To test this hypothesis, we studied IL-2-deficient mice as allograft recipients. IL-2 gene knockout (KO) mice reject islet allografts and demonstrate a classical mononuclear leukocytic infiltrate, containing CD4+ and CD8+ T cells, surrounding and invading the islet allografts.
View Article and Find Full Text PDFThe antiinflammatory agent sulfasalazine (SS) is prescribed to treat Crohn's disease, ulcerative colitis and rheumatoid arthritis. Activated T cells are present within diseased mucosal and synovial sites. We tested whether SS or its metabolites 5-aminosalicylic acid (5-ASA) and sulfapyridine (SP) inhibited the T-cell activation products interleukin 2 (IL-2) and interleukin 2 receptor alpha-chain (IL-2R alpha).
View Article and Find Full Text PDFThe mechanism of peripheral immunological tolerance has not been fully established. While anergic T cells have been noted in tolerant hosts, the mechanism by which they contribute to the induction and maintenance of tolerance has not been defined. As we previously reported, an accelerated form of diabetogenic autoimmunity in nonobese diabetic mice can be blocked by passive transfer of a CD3+, CD8+, beta-chain variable region 11-positive islet-infiltrating T-cell clone (IS-2.
View Article and Find Full Text PDFCholera toxin irreversibly activates a 43-kDa guanosine triphosphate (GTP)-binding protein by adenosine diphosphate ribosylation, resulting in activation of adenylate cyclase and increased intracellular levels of cyclic adenosine monophosphate (cAMP). Because increases in intracellular cAMP inhibit interleukin 2 (IL 2) expression and cytotoxic T lymphocyte (CTL) generation and function in vitro and in vivo, we hypothesized that IL 2 may counteract the inhibition of CTL by cholera toxin. Activated CTL treated with IL 2 were protected from the inhibitory effects of cholera toxin.
View Article and Find Full Text PDFSince calcium channel blocking agents and CsA exert an antiproliferative effect upon T cell mitogenesis, we have compared and characterized their immunosuppressive properties at the level of gene activation. Verapamil (greater than or equal to 30 microM), which blocks T cell mitogenesis and a rise in cytosolic calcium, was added to cultures of peripheral blood mononuclear cells stimulated with phytohemagglutinin (5 micrograms/ml) and phorbol myristate acetate (5 ng/ml). Northern blot analysis was performed using cDNA probes for the p55 interleukin 2 receptor (Tac; IL-2R), interleukin 2 and c-myc at 20 hr of culture.
View Article and Find Full Text PDFThis report provides direct evidence that protein kinase C (PKC) is activated in isolated, rigorously accessory cell (AC)-depleted T cells when the T cell antigen recognition complex is stimulated by divalent anti-CD3 monoclonal antibody. Anti-CD3 monoclonal antibody-stimulated PKC activation alone does not, however, directly stimulate T cell proliferation in the absence of AC. A rise in cytosolic calcium is the second signal believed to be of paramount importance in T cell activation.
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