TCell Signaling Abnormalities in Human Systemic Lupus Erythematosus
Madhusoodana P. Nambiar, Sandeep Krishnan, and George C. Tsokos Abnormal expression of key signaling molecules and defective functions of T lymphocytes play a significant role in the pathogenesis of systemic lupus erythematosus SLE . T-cell receptor TCR CD3-mediated stimulation of SLE T cells shows increased protein tyrosine phos-phorylation of cellular proteins, with faster kinetics, heightened calcium response, and decreased interleukin IL -2 production. The molecular mechanism of T-cell...
Pathogenesis and Spectrum of Autoimmunity
The immune system specifically recognizes and eliminates foreign antigens and thus protects the integrity of the host. During maturation of the immune system, tolerance mechanisms develop that prevent or inhibit potentially harmful reactivities to self-antigens. Autoreactive B and T cells that are generated during immune responses are eliminated by apoptosis in the thymus, lymph nodes, or peripheral circulation or are actively suppressed by regulatory T cells. However, autoreactive cells may...
Measurement of ROI Production
Production of ROI can be assessed fluorometrically using oxidation-sensitive fluorescent probes DCFH-DA, DHR, and HE as described elsewhere 14 . Cells are stained in annexin-binding buffer or HBS with 0.1 M DHR for 2 min, 1 M DCFH-DA for 15 min, or 1 M HE for 15 min, and samples are analyzed using a Becton Dickinson FACStar Plus flow cytometer equipped with an argon ion laser delivering 200 mW of power at 488 nm. Fluorescence emission from 5,6-carboxy-2',7'-dichlorofluorescein DCF green or DHR...
Molecular Biology Studies of Signaling Molecule in SLE T Cells
3.2.1. Reverse Transcriptase Polymerase Chain Reaction 1. Lyse 5 x 106 cells in 600 L RLT buffer supplied with Qiagen RNeasy minikit Qiagen Inc. . If not used immediately, the sample can be frozen at -80 C for several months. 2. Homogenize the samples by centrifugation at 18,000g for 2 min in a Qiashredder. 3. Add 600 L of 70 ethanol to the homogenized lysate and mix well by pipetting. 4. Apply 700 L of the sample to RNeasy minicolumn placed in a 2-mL collection tube and spin at 8000g for 15 s....
Mitochondrial Hyperpolarization and ATP Depletion Predispose Lupus T Cells to
In response to treatment with exogenous H2O2, a precursor, or ROI, lupus T cells failed to undergo apoptosis, and cell death preferentially occurred via necrosis. As noted in ref. 24, H2O2 triggered a rapid increase of AWm and ROI production, which was followed by apoptosis of PBLs in healthy subjects. By contrast, H2O2 failed to elevate AWm, ROI production, and apoptosis, but rather elicited necrosis in patients with lupus. Both CD3 CD28-induced H2O2 production and H2O2-induced apoptosis...
Contents
vii xi 1 Pathogenesis and Spectrum of Autoimmunity Part i. Models of Pathogenesis in Human Autoimmune Disorders 2 Mapping the Systematic Lupus Erythematosus Susceptibility Genes Swapan K. Nath, Jennifer A. Kelly, John B. Harley, 3 T-Cell Signaling Abnormalities in Human Systemic Lupus Erythematosus Madhusoodana P. Nambiar, Sandeep Krishnan, and George C. 4 TCR -Chain Abnormalities in Human Systemic Lupus Erythematosus Madhusoodana P. Nambiar, Sandeep Krishnan, Vishal G. Warke, and George C. 5...
Info Stm
Immunoblots are developed using Western Lightning enhanced chemilumi-nescence ECL reagents PerkinElmer Life Sciences, Boston, MA . The blot is usually probed by PKA-RI MAb first this MAb detects both RIa and RI subunits. To detect the presence of RIIa and RII , the blot is first stripped with 20 mM glycine pH 2.0 solution at 50 C in a microhybridization oven for 30 min, followed by three washings with TBS-T. The remainder of the procedure is as described in Subheading 3.3. A representative blot...
Mitochondrial Dysfunction in Lupus T Cells
SLE T cells exhibit both defective AICD and enhanced spontaneous apoptosis Table 1 . Coordinate mitochondrial hyperpolarization and ATP depletion play key roles in abnormal T-cell death of patients with SLE 36 , AWm and ROI levels as well as cytoplasmic pH are elevated in patients with SLE in comparison to healthy or controls with rheumatoid arthritis 36,37 . Baseline mitochondrial hyperpolarization and ROI levels correlated with diminished GSH levels, suggesting increased utilization of...
Mitochondrial Checkpoints of TCell Activation and Apoptosis
Both cell proliferation and apoptosis are energy-dependent processes. Energy in the form of adenosine triphosphate ATP is provided through glycolysis and oxidative phosphorylation. The mitochondrion Fig. 1 , the site of oxidative phosphorylation, has long been identified as a source of energy and cell survival 8 . The synthesis of ATP is driven by an electrochemical gradient across the inner mitochondrial membrane maintained by an electron transport chain and the membrane potential negative...
GradiFrac Control Settings
1. Control unit UV-1 Knob set to 0.5, switch set to range AU. 2. Pump P-50 Flow rate adjustments will be made using the control panel on the fraction collector. 3. Chart recorder Chart speed set to 5 mm min, blue pen range set to 10 mV, and red pen range set to 1 V. For operation, the valve settings of the GradiFrac are shown in Fig. 1. Fig. 1. Valve settings of the GradiFrac. Fig. 1. Valve settings of the GradiFrac. Fig. 2. Schematic representation of the tubing connection on the main port of...
Contributors
Marina Afanasyeva Department of Medicine, University of Calgary, Calgary, Alberta, Canada Rajeev K. Agarwal Laboratory of Immunology, National Eye Institute, National Institutes of Health, Bethesda, MD Katalin Banki Department of Pathology, State University of New York, Upstate Medical University College of Medicine, Syracuse, NY David D. Brand Department of Medicine, University of Tennessee Health Sciences Center Veterans Affairs Medical Center, Memphis, TN Livia A. Casciola-Rosen Departments...
Info Ima
Liver antibody-mediated targeting cytochrome P450 Although the causes of autoimmune diseases have not been defined, independent lines of evidence have implicated environmental factors and genetic determinants of the host 2 . As shown in Table 1, polymorphisms of HLA molecules that regulate antigen presentation 3 and complement deficiency states 4 have been identified as inherited factors influencing disease susceptibility. Concordance rates for autoimmune diseases such as SLE, IDDM, RA, and MS...
Info Rsj
NF-kB, nuclear facotr k 3 ALPS, autoimmune lymphoproliferative syndrome. NF-kB, nuclear facotr k 3 ALPS, autoimmune lymphoproliferative syndrome. molecular mimicry i.e., presentation and recognition of cryptic epitopes of self-antigens . Further downstream, execution of immune responses depends on cooperation of many cell types, cytokines, and intracellular signaling networks. Therefore, autoimmunity represents the end result of the breakdown of one or more of the basic mechanisms of immune...