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Biomedical subjects

G G Singer

Publications and source records attributed to G G Singer.

At least 37 records · Page 2Linked to original sources
PubMed1997

Effects of storage temperature and time on qualitative and quantitative detection of cytomegalovirus in blood specimens by shell vial culture and PCR.

Cytomegalovirus (CMV) infectious titers and DNA levels were determined by quantitative shell vial culture and quantitative-competitive PCR with blood samples from 10 renal transplant recipients with active CMV infection. Blood samples were stored at either room temperature or 4 degrees C and were processed at intervals of 0, 6, 24, 48, and 72 h. All samples were culture and PCR positive at baseline. Whereas the sensitivity of shell vial culture progressively declined, with only 55% positive at 24 h and 10% positive at 48 h, all samples remained PCR positive at all time points. Furthermore, the infectious titer diminished by 83 to 91% by 24 h compared to that at baseline (P < 0.0001), but quantitative DNA levels did not decline over time. Storage temperature had no significant effect on either infectious titer or DNA levels.

Cells, Cultured↗
PubMed1994

Apoptosis, Fas and systemic autoimmunity: the MRL-lpr/lpr model.

Proteins encoded by the fas and fas ligand (fasL) genes are involved in apoptotic cell death in lymphocytes. In this article we review the recent elucidation of the role of the Fas-FasL interactions in the maintenance of tolerance to self antigens and in the homeostatic regulation of lymphocyte clonal expansion, and discuss the mechanisms of autoimmunity in Fas- and FasL-deficient mutant mouse strains.

Animals↗
PubMed1994

The fas antigen is involved in peripheral but not thymic deletion of T lymphocytes in T cell receptor transgenic mice.

The role of a cell death-associated gene, fas, in T lymphocyte development and responses to antigen has been analyzed by breeding a transgenic T cell receptor specific for the 81-104 peptide of pigeon cytochrome c into fas-defective MRL-lpr/lpr and control MRL+/+ mice. Transgene-expressing T cells mature normally in both strains and populate peripheral lymphoid tissues in normal numbers. Mature CD4+ T cells from the lpr/lpr mice are resistant to suppression by high doses of antigen and to apoptotic cell death. In vivo administration of peptide antigen causes deletion of thymic T cells in both MRL-lpr/lpr and MRL+/+ strains. By contrast, antigen-induced deletion of peripheral T cells occurs in the MRL+/+ but not in the MRL-lpr/lpr strain. Therefore, the fas gene plays an essential role in activation-induced cell death in mature T lymphocytes, but not in the negative selection of immature cells in the thymus.

Animals↗
PubMed1993

Mature CD4+ T lymphocytes from MRL/lpr mice are resistant to receptor-mediated tolerance and apoptosis.

The goal of this study was to examine the functional responses and tolerance susceptibility of T lymphocytes from mice of the autoimmune strain, MRL/lpr. A population of autoreactive CD4+ T cells can be readily expanded from the lymphoid tissues of young lpr mice. Lines of IL-2-producing autoreactive and alloreactive lpr and alloreactive +/+ T cells were developed to study their responses to tolerance-inducing stimuli. Culture of +/+ T cells with high concentrations of immobilized anti-CD3 antibody induces both functional anergy and apoptosis. By contrast, lpr-derived T cell lines are relatively resistant to anergy and apoptosis. The implications of these findings for the development of autoimmunity, and the possible role of the Fas Ag in determining resistance or susceptibility to tolerance, are discussed.

Animals↗
PubMed1993

Colony stimulating factor-1 in the induction of lupus nephritis.

In this study we examine the role of colony stimulating factor-1 (CSF-1) in the induction of lupus nephritis. The purpose of the study was to establish the relationship of CSF-1 to the prominent influx of macrophages (M phi) in the glomeruli of MRL-lpr mice with autoimmune lupus nephritis. The kidneys of MRL-lpr mice were examined before (< 12 weeks of age) and after (> 12 weeks of age) renal injury for CSF-1 transcripts by in situ hybridization. CSF-1 mRNA was detected at four weeks of age within glomeruli and increased with disease severity. To examine whether glomerular M phi (glom M phi) required CSF-1 we isolated M phi from the kidneys of MRL-lpr mice. Two types of glom M phi (with morphological and growth characteristics which correlated with the presence or absence of proteinuria) were isolated. Under CSF-1-free culture conditions, where the viability of glom M phi from proteinuric mice was maintained, glom M phi from pre-proteinuric mice were unable to survive. Neutralization of CSF-1 in the media reduced viability of pre-proteinuric glom M phi (5 to 6 x), while viability of proteinuric glom M phi was diminished < 1.5 x. Additionally, CSF-1 supplementation induced a 10 x proliferation of pre-proteinuric glom M phi when compared to CSF-1-free medium. In contrast, proteinuric glom M phi did not proliferate in response to CSF-1. These studies suggest that CSF-1 induces macrophage proliferation and differentiation within glomeruli and, in turn, renal injury.

Animals↗
PubMed1993

Stimulated renal tubular epithelial cells induce anergy in CD4+ T cells.

Renal tubular epithelial cells (TEC) can express MHC class II molecules in vitro and in vivo. Their ability to also secrete cytokines and express adhesion molecules suggests a possible immune accessory role for TEC. We have previously documented that TEC process and present antigen to T cell hybridomas. However, engagement of the T cell receptor alone is sufficient to induce IL-2 secretion by T cell hybridomas. We now report that presentation of antigen by TEC to a CD4+ T cell clone results in functional inactivation of the T cells. Despite antigen-specific anergy, these T cells are viable and proliferate in response to IL-2. Furthermore, allogeneic antigen presenting cells were unable to restore the T cell proliferative response, suggesting that the mechanism(s) was not entirely costimulator-dependent.

Animals↗
PubMed1993

Transgenic tubular cell expression of class II is insufficient to initiate immune renal injury.

Autoimmune disease in mouse models of lupus nephritis is associated with enhanced renal tubular epithelial cell (TEC) expression of major histocompatibility complex (MHC) class II (Ia) molecules. It is unknown whether de novo TEC expression of syngeneic la alone can initiate immune attack or whether expression is secondary to cytokines released by infiltrating lymphocytes. To establish if the expression of high levels of self-MHC molecules alone can initiate immune renal injury in the adult animal, kidneys from transgenic C57BL/6 (B6) mice (Ins-I-E) bearing constitutively high levels of I-Eb on proximal TEC were transplanted into nephrectomized male B6 x C3H F1 hybrid mice (I-Eb/k). Control mice received kidneys from I-Eb negative, nontransgenic B6 mice, and all transplant recipient mice were evaluated for renal disease. At the end of the study (> 8.3 months mean survival), the transgenic transplant recipients did not become proteinuric (< 1+ urinary protein) and had normal serum creatinine levels (control = 95 +/- 8 versus transgenic transplants = 116 +/- 23 mumol/L; N = four/group), and the kidneys remained histologically normal. These results establish that the expression of high levels of transgenic MHC class II molecules on TEC is insufficient to initiate autoimmune injury in this model. It is suggested that, in addition to MHC class II molecules, other signals or accessory molecules are required from TEC to initiate immune renal injury.

Animals↗
PubMed1993

Renal tubular epithelial and T cell interactions in autoimmune renal disease.

Interaction between epithelial cells and T cells may initiate autoimmune tissue destruction. Renal tubular epithelial cells may participate in such immune interactions since they: (1) can be induced to express surface molecules which facilitate engagement with T cells; (2) secrete and express membrane bound cytokines; (3) are exposed to peptides from blood and the glomerular filtrate and are capable of processing these potentially immunogenic peptides. We have recently established T cell clones captured from the interstitium of MRL-lpr mice with lupus nephritis. These T cell clones are unique and are regulated by the lpr gene. They express the alpha/beta T cell receptor, and beta cell markers, but do not display CD4 or CD8 on their surface. These T cell clones proliferate to renal tubular cells but not to cells from other tissues and secrete IFN-gamma which induces class II and ICAM-1 on renal tubular epithelial cells. Expression of class II and ICAM-1 induced by IFN-gamma renders these epithelial cells capable of triggering T cell hybridomas to proliferate and secrete IL-2. Therefore, renal tubular epithelial cells are capable of processing and presenting antigen. This review will focus on the dynamic interaction of renal epithelial cells and T cells and discuss its importance in the initiation of autoimmune renal injury.

Animals↗
PubMed1992

Detection of intracellular interleukin-10 by flow cytometry.

Detection of cytokines is limited to measurements of the secreted molecule. To circumvent this problem we permeabilized the cell membrane with digitonin and localize cytokine expression using antibodies by flow cytometry. In this report we demonstrate that we can detect specific intracellular interleukin-10 (IL-10) in the HT-2 T cell line only after membrane permeabilization. With this technique intracellular cytokines are readily detectable.

Animals↗
PubMed1992

Cultured mesangial cells from autoimmune MRL-lpr mice have decreased secreted and surface M-CSF.

M-CSF has been implicated in the pathogenesis of lupus nephritis in MRL-lpr mice. We recently reported persistently high levels of serum M-CSF in MRL-lpr mice as early as one week of age, not present in normal mice including C3H mice. In addition, M-CSF transcripts in MRL-lpr renal cortex increased with an increase in the severity of nephritis. Because glomerular mesangial cells (MC) secrete M-CSF, we investigated whether cultured MRL-lpr MC secrete more M-CSF than C3H MC. Paradoxically, unstimulated MRL-lpr MC secreted substantially less M-CSF than C3H MC [26 +/- 11 vs. 109 +/- 7 colony forming units (CFU)]. We then explored whether MC could express membrane bound M-CSF. We detected a 31 kDa form of membrane M-CSF on both MRL-lpr and C3H MC. Fewer MRL-lpr MC than C3H MC (24 +/- 5% vs. 78 +/- 5%) expressed membrane M-CSF. Furthermore, the increase in the mean channel log fluorescence intensity on MRL-lpr MC was considerably less than in C3H MC, indicating a lower density of M-CSF on MRL-lpr MC. Because our prior studies established that MRL-lpr kidneys have enhanced expression of TNF alpha, we stimulated cultured MC with TNF alpha. TNF alpha increased M-CSF secretion by stimulated MRL-lpr by twofold over unstimulated MRL-lpr MC, but did not increase M-CSF in C3H MC. In addition, M-CSF secretion was modestly greater in stimulated MRL-lpr MC compared to stimulated C3H MC. In conclusion, this is the first report of membrane M-CSF detectable on cultured MC. These studies note that despite higher circulating M-CSF and renal M-CSF transcripts in MRL-lpr mice, cultured MRL-lpr MC have lower basal secreted and membrane bound M-CSF than cultured C3H MC.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Dexamethasone prevents autoimmune nephritis and reduces renal expression of Ia but not costimulatory signals.

Although glucocorticoids are a conventional treatment for lupus nephritis, the cellular and molecular mechanisms responsible for preventing renal injury are unknown. MRL-lpr mice develop an aggressive autoimmune nephritis. As these mice become nephritic, there is an increase in the renal expression of molecules that permit or facilitate immune interactions, including MHC class II (Ia) antigens, intercellular adhesion molecule-1 (ICAM-1), and pro-inflammatory cytokines. Because dexamethasone (Dex) alters Ia antigen expression and suppresses cytokine generation, the authors prophylactically treated MRL-lpr mice and investigated the relative importance of these molecules in inducing renal injury. MRL-lpr mice given Dex (0.4 mg/kg/d) from age 6 weeks were killed 4, 8, and 16 weeks after the initiation of therapy, and tissue was removed for histology and extraction of total RNA. Dex prevented lymphadenopathy and renal injury. DEX eliminated the marked Thy 1.2+ lymphocytic infiltrates within the kidney and preserved normal renal histology and urinary protein levels. Northern blot analysis of steady-state mRNA transcripts indicated Dex suppressed a four-fold increase in kidney major histo-compatibility complex class II (Ia) molecule antigen mRNA seen by age 22 weeks (Ia/beta-actin ratios = 0.64 +/- 0.50 versus 2.32 +/- 0.48, P less than 0.01), but did not alter the costimulatory molecules ICAM-1 or tumor necrosis factor alpha (TNF alpha). Although all of these molecules are important mediators of inflammation, autoimmune nephritis was ameliorated without alteration of TNF alpha gene transcription or ICAM-1 transcription and surface expression. This study suggests that the benefit of steroids in nephritis stems from preventing lymphocyte infiltration into the kidney and decreasing immune interactions by limiting Ia expression.

Animals↗
PubMed1991

Stimulated kidney tubular epithelial cells express membrane associated and secreted TNF alpha.

Tumor necrosis factor-alpha (TNF alpha) is a pleiotropic, pro-inflammatory peptide cytokine which promotes immune renal injury, and participates in T cell activation. It is produced by macrophages, T cells, and some non-hematopoietic cells, and is cytotoxic in picogram quantities. As renal tubular epithelial cells (TEC) bearing MHC class II (Ia) antigens and adhesion molecules (ICAM-1) can act as immune accessory cells, the ability of TEC to produce costimulatory cytokines could augment TEC accessory capacity in vivo. We report that transformed TEC express low levels of TNF alpha in response to LPS or IL-1 alpha as a secreted product and as a cytotoxic membrane associated molecule displayed on the cell surface. Surface labelling and immunoprecipitation studies of TEC detect a number of bands including a prominent 26 kD protein, which is the predicted size of TNF alpha precursor. TNF alpha mRNA transcripts were also detected by in situ hybridization in cortical tubules of C3H/FeJ mice injected with LPS, demonstrating the capacity of normal tubular epithelial cells to express TNF alpha in vivo. This report demonstrates for the first time the ability of kidney tubular cells to express TNF alpha protein and that membrane associated TNF alpha is not limited to hematopoietic cells. The function of small amounts of TNF displayed on the surface of tubular cells may be amplified by the abundance of these cells within the renal cortex, and may allow TEC to modulate immune responses within the kidney during inflammation.

Animals↗
PubMed1986

Development of a test to evaluate the transtubular potassium concentration gradient in the cortical collecting duct in vivo.

The purpose of these investigations was to develop a noninvasive test to estimate the transtubular potassium concentration gradient (TTKG) and thereby aldosterone action in the late distal convoluted tubule and the cortical collecting duct in patients with disorders of potassium excretion. Experiments were performed in rats under conditions where the ratio of urine to renal venous potassium concentration could reflect this TTKG. A large furosemide-induced diuresis ensured that sodium delivery was adequate and minimized the change in water content during transit through the medullary collecting duct (equal osmolality and TF/P inulin at the base and the tip of the medullary collecting duct). There was no significant potassium reabsorption nor secretion during transit through the medulla as shown by micropuncture and microcatheterization. Thus the potassium concentration in the urine should mirror that in the lumen at the major nephron sites of potassium secretion. The potassium concentration in the renal vein provides the simplest estimate of the cortical peritubular potassium concentration (the mean renal A-V difference for potassium was 1.2 mM); with a very high fractional excretion of potassium, an adjustment can be made to the arterial potassium concentration to correct for the potassium extracted. If the urine/plasma potassium concentration ratio were a quantitative reflection, then the transepithelial potential difference (TEPD) would be close to -40 mV in normal rats. The TTKG fell to unity when amiloride was given, consistent with an abolition of the apparent TEPD in vivo by this drug. Similar results were obtained in non-diuretic rats. The clinical implications of these findings are discussed.

Aldosterone↗