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Biomedical subjects

R D Nelson

Publications and source records attributed to R D Nelson.

At least 19 recordsLinked to original sources
PubMed1992

Selectively amplified expression of an isoform of the vacuolar H(+)-ATPase 56-kilodalton subunit in renal intercalated cells.

The intercalated cells of the kidney collecting duct are specialized for physiologically regulated proton transport. In these cells, a vacuolar H(+)-ATPase is expressed at enormous levels in a polarized distribution on the plasma membrane, enabling it to serve in transepithelial H+ transport. In contrast, in most eukaryotic cells, vacuolar H(+)-ATPases reside principally in intracellular compartments to effect vacuolar acidification. To investigate the basis for the selective amplification of the proton pump in intercalated cells, we isolated and sequenced cDNA clones for two isoforms of the approximately 56-kDa subunit of the H(+)-ATPase and examined their expression in various tissues. The predicted amino acid sequence of the isoforms was highly conserved in the internal region but diverged in the amino and carboxyl termini. mRNA hybridization to a cDNA probe for one isoform (the "kidney" isoform) was detected only in kidney cortex and medulla, whereas mRNA hybridization to the other isoform of the approximately 56-kDa subunit and to the H(+)-ATPase 31-kDa subunit was found in the kidney and other tissues. Immunocytochemistry of rat kidney with an antibody specific to the kidney isoform revealed intense staining only in the intercalated cells. Staining was absent from proximal tubule and thick ascending limb, where H(+)-ATPase was detected with a monoclonal antibody to the 31-kDa subunit of the H(+)-ATPase. This example of specific amplification of an isoform of one subunit of the vacuolar H(+)-ATPase being limited to a specific cell type suggests that the selective expression of the kidney isoform of the approximately 56-kDa subunit may confer the capacity for amplification and other specialized functions of the vacuolar H(+)-ATPase in the renal intercalated cell.

Adult↗
PubMed1992

Actin polymerization contributes to neutrophil chemotactic dysfunction following thermal injury.

The agent(s) and mechanism(s) responsible for suppression of neutrophil chemotaxis in association with major thermal injury have not been identified. We have proposed that the reduced random motility characterizing patients' cells may contribute to their generalized chemotactic dysfunction. Here we report that actin polymerization may be responsible for the loss of neutrophil motility associated with major thermal injury. Using a fluorescent ligand specific for polymerized or filamentous actin (NBD-phallacidin) in conjunction with flow cytometry, we have discovered that peripheral blood and exudate neutrophils from patients with major thermal injury contain increased levels of actin in a stably polymerized form. Because cyclic polymerization and depolymerization of actin is essential to cell motility, we suggest that actin polymerization may contribute in a major way to the attenuation of neutrophil random and chemotactic functions induced by major thermal injury.

Actin Cytoskeleton↗
PubMed1992

Early occurrence of end-stage renal disease in a patient with infantile nephropathic cystinosis.

We report the case of a patient with infantile nephropathic cystinosis who required renal transplantation at age 30 months. Exhaustive evaluation did not identify a cause of progressive renal failure other than cystinosis. The patient's genetic lesion was allelic with those of other patients with cystinosis; fusion of this patient's fibroblasts with fibroblasts from another patient with infantile nephropathic cystinosis did not demonstrate complementation of the biochemical defect.

Clone Cells↗
PubMed1992

Binding of Trichophyton rubrum mannan to human monocytes in vitro.

We recently reported that the mannan component of Trichophyton rubrum cell wall (TRM) has an inhibitory influence on cell-mediated immune function in vitro. We now describe experiments designed to identify the target cell for this effect of TRM. T. rubrum mannan labeled with fluorescein (FITC-TRM) was incubated with peripheral blood mononuclear leukocytes, monocytes, or lymphocytes. Binding and uptake of the FITC-TRM were monitored by fluorescence microscopy and flow cytometry. Approximately 10% of mononuclear leukocytes were stained with this reagent and the fluorescent cells appeared to be monocytes by morphology. Virtually all purified monocytes and no purified lymphocytes stained with FITC-TRM. Flow cytometry to analyze FITC-TRM monocyte-specific binding of FITC-TRM involved the use of a phycoerythrin-labeled anti-CD14 antibody to identify monocytes. The only cells stained with FITC-TRM were those stained with the monocyte-specific antibody. The ability of monocytes to endocytose mannan was assessed by fluorescence microscopy. Cells were exposed to FITC-TRM and washed, and the staining pattern recorded periodically over a 48-h incubation period. After 15 min, staining was homogeneous and involved the entire cell surface; by 30 min, "patching" was observed; by 90 min, bright granules had formed along the cell border and a large number of small granules were present in the cytoplasm; by 8-12 h, the fluorescent granules were enlarged in size and reduced in number; by 24-36 h, the intensity of cytoplasmic fluorescence began to diminish; and, after 48 h, all fluorescent staining had disappeared. An additional feature of staining during the 8-12-h period was the appearance of a large round bright spot in the nuclear region of each cell, which may represent nucleolar staining. A role for "mannan receptors" is suggested by observations that FITC-TRM binding was prevented by unlabeled TRM or pretreatment of the monocytes with trypsin. Our finding that monocytes selectively and specifically bind TRM appears to identify the monocyte rather than the lymphocyte as the target cell for the inhibitory effect of mannan on cell-mediated immune function.

Adult↗
PubMed1992

Dapsone suppresses integrin-mediated neutrophil adherence function.

The anti-inflammatory influence of dapsone may involve suppression of neutrophil chemotaxis to selected attractants, but other actions of the drug are likely also involved. We have discovered that dapsone may suppress migration of neutrophils to extravascular sites through inhibition of adherence functions required for neutrophil recruitment. Neutrophil adherence mediated by integrins (CD11/CD18 or Mac-1 family receptors) was measured in vitro in terms of binding of stimulated cells to albumin-coated wells of microtiter plates, using phorbol myristate acetate (PMA) and N-formylmethionyl-leucyl-phenylalanine (FMLP) as stimuli. Adherence was assessed by staining attached cells with crystal violet dye and measuring the dye concentration at OD590 using an automated plate reader. The role of integrins in this assay was confirmed by the ability of anti-integrin antibody to suppress stimulated neutrophil adherence. The OD590 value for cells adhering to albumin in the absence of stimulus and dapsone averaged 0.2 +/- 0.04 (SEM) over five experiments. In the presence of 0.1 microM PMA or 10(-6) M FMLP, the OD590 values averaged 0.88 +/- 0.1 and 0.75 +/- 0.12, respectively. Dapsone did not affect unstimulated neutrophil adherence but, when present with stimulus, produced a dose-related inhibitory effect on adherence. Fifty percent inhibitory doses were approximately 150 micrograms/ml dapsone for both stimuli. Sulfapyridine reproduced the inhibitory effect of dapsone, but two structurally related compounds, hydrochlorothiazide and furosamide, did not. The observed ability of dapsone to inhibit neutrophil chemotaxis under agarose to FMLP and interleukin-8 may also be explained by interference with integrin-mediated adherence required for motility in this assay system. To consider if dapsone might have a similar inhibitory influence on neutrophil adherence in vivo, we tested the stimulated adherence function of neutrophils isolated from three individuals on dapsone therapy for dermatitis herpetiformis. Stimulated adherence of patients' cells averaged less than 40 percent of the control value. Suppression of leukocyte integrin function may therefore also contribute to the ability of dapsone to inhibit neutrophil infiltration in neutrophilic dermatoses.

Cell Adhesion↗
PubMed1992

Biochemistry of the renal V-ATPase.

In most eukaryotic cells, vacuolar H(+)-ATPases (V-ATPases) are present primarily or exclusively in intracellular membrane compartments, functioning in the acidification of the endocytic and secretory vacuolar apparatus necessary for constitutive cell function. V-ATPases also participate in renal hydrogen ion secretion in both the proximal and distal nephron, residing at high concentrations on the plasma membrane, where they are regulated physiologically to maintain the acid-base balance of the organism. Recent experiments have begun to reveal how the kidney controls transcellular proton transport while still maintaining acidification of intracellular compartments. Control may occur by recruitment of proton pumps to or away from the plasma membrane. The proton-transporting plasma membrane of intercalated cells is a specialized apparatus that translocates the enzyme between an intracellular membrane pool and the plasma membrane in response to physiological stimuli. Regulation may also occur by changes in the kinetics of the V-ATPase. V-ATPases are a family of structurally similar enzymes which differ in the composition of specific subunits. Cytosolic regulatory enzymes present in renal cells may preferentially affect V-ATPases in selective membrane compartments.

Adenosine Triphosphatases↗
PubMed1992

Binding and uptake of Trichophyton rubrum mannan by human epidermal keratinocytes: a time-course study.

Trichophyton rubrum infects skin. This fungus or its products might affect the function of epidermal cells. We previously reported that T. rubrum mannan (TRM) exhibits a suppressive effect on proliferation of human lymphocytes. The goal of the present study was to investigate the possibility of direct interaction of TRM with cultured normal human epidermal keratinocytes (EK). Mannan, a cell wall glycoprotein, was extracted from T. rubrum by precipitation with cetyltrimethylammonium bromide and conjugated with fluorescein isothiocyanate (FITC-TRM). After incubation of EK with 50 micrograms/ml FITC-TRM for 30 min, the surface of EK showed bright fluorescent staining. EK cultures pretreated with non-labelled TRM remained unstained. The fate of TRM bound to EK surface was determined in a time-course study. After pulse exposure to FITC-TRM, EK cultures were washed and incubated for various time periods. The EK moved surface mannan to the one area of cell membrane, so that at 4-6 h, the homogeneous staining of the entire cell surface was replaced by staining in a "cap" pattern. By 12 h, FITC-TRM was taken up into the cell, brought to the nuclear area and concentrated in the EK nucleoli. During the next 3 days nucleolar and cytoplasmic staining of the cells was observed. The intensity of fluorescence gradually diminished. On the 4th day, the sharp staining of organelles disappeared; instead, a large number of small fluorescent granules were seen intra- and extracellularly. By the 6th day after exposure, no EK staining remained. Thus, EK specifically bound, internalized and apparently catabolized TRM.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Division↗
PubMed1991

An immunoinhibitory cell wall glycoprotein (mannan) from Trichophyton rubrum.

Trichophyton rubrum causes 90% of chronic dermatophyte infections. Most patients with widespread chronic T. rubrum infection fail to express a delayed hypersensitivity reaction to intradermally injected trichophytin. We propose that cell-mediated immunity to T. rubrum may be suppressed in chronic infections by the mannan cell wall component of the fungus. The proposed suppressive effect of T. rubrum mannan on cell-mediated immunity was tested by measuring the ability of extracted mannan to inhibit lymphoproliferative responses of human mononuclear leukocytes to antigens, mitogens, and an anti-T-cell receptor antibody (anti-CD3) in vitro. Mannan was found to be highly antigenic in two of five donors and weakly antigenic in the other three. Despite its antigenic property, mannan exhibited a dose-related ability to inhibit lymphoproliferation stimulated by other agents including 1) antigens from Candida albicans, T. rubrum, and tetanus toxoid (ID50 = 250 micrograms/ml); 2) anti-CD3 antibody (ID50 = 250 micrograms/ml); and 3) Phaseolus limensis mitogenic lectin (ID50 = 64 micrograms/ml). Mannan added to cultures later than 24 h after initiation had no inhibitory influence, but culture of cells with mannan for a period of 24 h prior to the addition of stimulus enhanced the inhibitory effect of the glycoprotein. Lymphoproliferation in response to recombinant interleukin-2 (IL-2) was not inhibited. The influence of time of addition of mannan and the failure of mannan to inhibit IL-2-stimulated lymphoproliferation demonstrate that the suppressive effect of mannan must be pharmacologic rather than cytotoxic. The observed ability of T. rubrum cell wall mannan to suppress cell-mediated immune function in vitro may provide an important clue to a mechanism enabling the fungus to avoid elimination in chronically infected patients.

Chronic Disease↗
PubMed1991

Candida mannan: chemistry, suppression of cell-mediated immunity, and possible mechanisms of action.

The ability of Candida albicans to establish an infection involves multiple components of this fungal pathogen, but its ability to persist in host tissue may involve primarily the immunosuppressive property of a major cell wall glycoprotein, mannan. Mannan and oligosaccharide fragments of mannan are potent inhibitors of cell-mediated immunity and appear to reproduce the immune deficit of patients with the mucocutaneous form of candidiasis. However, neither the exact structures of these inhibitory species nor their mechanisms of action have yet been clearly defined. Different investigators have proposed that mannan or mannan catabolites act upon monocytes or suppressor T lymphocytes, but research from unrelated areas has provided still other possibilities for consideration. These include interference with cytokine activities, lymphocyte-monocyte interactions, and leukocyte homing. To stimulate further research of the immunosuppressive property of C. albicans mannan, we have reviewed (i) the relationship of mannan to other antigens and virulence factors of the fungus; (ii) the chemistry of mannan, together with methods for preparation of mannan and mannan fragments; and (iii) the historical evidence for immunosuppression by Candida mannan and the mechanisms currently proposed for this property; and (iv) we have speculated upon still other mechanisms by which mannan might influence host defense functions. It is possible that understanding the immunosuppressive effects of mannan will provide clues to novel therapies for candidiasis that will enhance the efficacy of both available and future anti-Candida agents.

Animals↗
PubMed1991

Down-regulation of homing receptors: a mechanism for impaired recruitment of human phagocytes in sepsis.

Receptors known as DREG adhesion molecules on human neutrophils and monocytes provide for homing of these phagocytic leukocytes to sites of inflammation. They mediate the initial adhesive interaction of the leukocytes to vascular endothelial cells and are then shed from the cell surface in response to chemotactic factors and inflammatory mediators. Systemic accumulation of these agents following major injury or sepsis may therefore promote shedding of DREG receptors from circulating leukocytes and impair their recruitment to sites of inflammation. To test this hypothesis, we have analyzed the expression of DREG receptors on neutrophils and monocytes from 25 patients admitted to the Surgical Intensive Care Unit. Receptor expression was measured by flow cytometry of cells stained with murine monoclonal DREG-56 anti-DREG antibody. For 14 nonseptic patients, mean monocyte positivity for DREG was reduced from 64% to 40%. For 11 septic patients, mean neutrophil and monocyte positivity for DREG was reduced from 94% to 82% and 64% to 34%, respectively. These results suggest that monocytes are more affected than neutrophils in vivo by conditions expected to stimulate shedding of DREG and that sepsis promotes shedding of these adherence receptors. Accumulation of DREG-negative monocytes in association with sepsis may be sufficient to impair their recruitment to inflammatory sites and limit their contribution to host defense against infection and tissue repair.

Adult↗
PubMed1990

Tumor necrosis factor alpha/cachectin stimulates eosinophil oxidant production and toxicity towards human endothelium.

Eosinophils (EOs) participate in a variety of inflammatory states characterized by endothelial cell damage, such as vasculitis, pneumonitis, and endocarditis. We find that 100 U/ml TNF-alpha/cachectin (TNF), a concentration attainable in the blood of humans with parasitic infestations, stimulates highly purified populations of EOs to damage human umbilical vein endothelial cells (HUVEC), a model of human endothelium. This TNF-dependent EO cytotoxicity is strongly inhibited by heparin and methyprednisolone but unaffected by the platelet-activating factor antagonist BN52012 or scavengers of superoxide anion and H2O2, superoxide dismutase and catalase. However, addition of a physiologically relevant concentration of Br- (100 microM) enhances EO/TNF damage to HUVEC, implicating the possible participation of EO peroxidase (EPO) in the killing mechanism. EOs adherent to FCS-coated plastic wells more than double their production of superoxide anion and the cytotoxic EPO-derived oxidant HOBr when exposed to TNF, showing that TNF activates the respiratory burst of EOs attached to a "physiologic" surface. Unlike PMNs, EOs were not irreversibly activated to kill unopsonized endothelium by previous exposure to TNF, and did not degranulate or upregulate CR3 expression as detected by Mo1 in the presence of 100 U/ml TNF. HUVEC exposed 18 h to TNF were considerably more susceptible to lysis by PMA-activated EOs and reagent H2O2, demonstrating a direct effect of TNF upon endothelium, perhaps through inhibition of antioxidant defenses. These findings suggest that abnormally elevated serum levels of TNF may provoke EOs to damage endothelial cells and thereby play a role in the pathogenesis of tissue damage in hypereosinophilic states.

Antigens, Differentiation↗
PubMed1990

Different effects of native Candida albicans mannan and mannan-derived oligosaccharides on antigen-stimulated lymphoproliferation in vitro.

Yeast cell wall mannan polysaccharide has been proposed to contribute to immune dysfunction associated with chronic infections involving Candida albicans. This influence of mannan has been suggested based partially upon studies of the in vitro immunoinhibitory effects of mannans prepared from Saccharomyces cerevisiae or C. albicans by precipitation with Fehling's reagent, which provides a structurally modified product contaminated with copper. We have therefore evaluated the immunoinhibitory influence of a more native C. albicans mannan prepared by complexation with cetyltrimethylammonium bromide (CTAB). CTAB mannan was a potent stimulator of lymphoproliferation when added to human PMBC from donors responsive to Candida; it has no inhibitory influence on lympho-proliferation induced by Candida or other Ag. In contrast, members of a family of mannose oligosaccharides (disaccharide through hexasaccharide) derived from the CTAB mannan by weak alkaline degradation did not stimulate lymphoproliferation, but were potent inhibitors of lymphoproliferation stimulated by Candida and other Ag. Fifty percent inhibitory doses were 260 to 34 microM, respectively. Compositional analyses of these immunoinhibitory oligomers showed them to be composed of mannose and free of contaminating protein. We propose that mannan-derived oligosaccharides produced by catabolism of mannan in vivo are immunoinhibitory and contribute to the deficit in cell-mediated immune function associated with chronic candidiasis.

Antigens, Fungal↗
PubMed1990

Interactions of Escherichia coli and Proteus mirabilis with mouse mononuclear phagocytes.

Five strains of enterobacteria (three of Escherichia coli and two of Proteus mirabilis) were studied to assess and compare their phagocytic uptake and intracellular killing by mouse macrophages. Each strain was injected intraperitoneally into separate groups of mice and peritoneal exudate cells were harvested after 3 min for phagocytosis to occur in vivo. Acridine orange staining showed that there were approximately 10-fold fewer intracellular P. mirabilis than E. coli cells. The average numbers of viable intracellular bacteria per leucocyte were 0.03 and 0.02 for P. mirabilis strains M13 and H1, respectively, and 0.48, 0.45, and 0.28 for E. coli strains M14, A-D M5 and H40. Thus, both P. mirabilis strains were ingested less readily than any of the three E. coli strains (p less than 0.01). The rates of in-vitro intracellular killing were similar for all five strains of bacteria. The intracellular killing constants (Kk) for the three mouse isolates were 0.017, 0.016 and 0.020 min for E. coli M14 and A-D M5, and P. mirabilis M13, respectively; the Kks for the two human isolates were 0.026 and 0.029/min for E. coli H40 and P. mirabilis H1, respectively. The Kks for all five strains were not significantly different. Assuming that the numbers of viable intracellular bacteria at the beginning of the assay represented 100% viability, 6-17% of the intracellular bacteria remained viable after 2 h, reflecting log10 3.9-5.6 bacteria (6-8) x 10(6) peritoneal exudate cells. Intravenous injection of these five strains into separate groups of mice demonstrated that the P. mirabilis strains were more virulent than the E. coli strains. Injection of each P. mirabilis strain was associated with ruffled fur and death, whereas mice given any of the three E. coli strains remained visibly healthy and none died. Consistent with these observations, quantitation of viable bacteria in the liver and spleen showed that greater numbers of P. mirabilis M13 than of E. coli M14 or A-D M5 persisted in these organs; similarly greater numbers of P. mirabilis H1 than of E. coli H40 persisted in the liver and spleen. Because the rates of intracellular killing of these five strains were similar, the relative virulence of both strains of P. mirabilis appeared to be associated with decreased phagocytic uptake rather than differences in intracellular survival.

Animals↗
PubMed1990

Staphylococcal and streptococcal pyrogenic toxins involved in toxic shock syndrome and related illnesses.

Toxic-shock syndrome (TSS) is an acute onset, multiorgan illness which resembles severe scarlet fever. The illness is caused by Staphylococcus aureus strains that express TSS toxin-1 (TSST-1), enterotoxin B, or enterotoxin C. TSST-1 is associated with menstrual TSS and approximately one-half of nonmenstrual cases; the other two toxins cause nonmenstrual cases, 47% and 3%, respectively. The three toxins are expressed in culture media under similar environmental conditions. These conditions may explain the association of certain tampons with menstrual TSS. Biochemically, the toxins are all relatively low molecular weight and fairly heat and protease stable. Enterotoxins B and C, share nearly 50% sequence homology with streptococcal scarlet fever toxin A; they share no homology with TSST-1 despite sharing numerous biological properties. Numerous animal models for development of TSS have suggested mechanisms of toxin action, though the exact molecular action is not known. The toxins are all potent pyrogens, induce T lymphocyte proliferation, requiring interleukin 1 release from macrophages, suppress immunoglobulin production, enhance endotoxin shock, and enhance hypersensitivity skin reactions. The genetic control of the toxins has been studied and suggests the exotoxins are variable traits. Some additional properties of TSS S. aureus which facilitate disease causation have been clarified.

Amino Acid Sequence↗
PubMed1990

Identification of an IgA inhibitor of neutrophil chemotaxis and its membrane target for the metabolic burst.

Affinity-purified IgA from the serum of an 8-year-old boy with a 5-year history of recurrent facial nodules, intermittent neutropenia and elevated immunoglobulin levels, inhibited the chemotaxis of polymorphonuclear neutrophils (PMN) from both patient and normal adults. Preincubation of normal PMN with IgA from the patient's serum (0.5 mg/ml) inhibited chemotaxis to C5a and to the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) by 80%, while IgA or IgG from pooled human serum and IgG from the patient were without effect. Normal PMN chemotaxis was restored after IgA depletion of the patient's serum by affinity chromatography. The patient's IgA, but not IgA from pooled human serum, bound specifically to normal PMN by its antigen-binding sites and recognized a 62,000 MW membrane protein on normal neutrophils, which was distinct from the FMLP receptor, the C5a receptor, or the Fca receptor. Attachment of the patient's IgA to the 62,000 MW protein activated intracellular oxidative metabolism on a parity with phorbol myristate acetate (PMA) and resulted in a significant up-regulation of membrane receptors for FMLP. After the binding of patient (Pt) IgA, normal neutrophils were rendered significantly less responsive to subsequent stimulation with phorbol esters. These results characterize a novel mechanism of chemotactic inhibition by serum IgA and also identify a neutrophil membrane protein that is linked to intracellular oxidative metabolism.

Chemotaxis, Leukocyte↗
PubMed1989

Pathogenesis of candidiasis. Immunosuppression by cell wall mannan catabolites.

Candida albicans cell wall mannan polysaccharide has an ability to negatively influence cell-mediated immune function. We have attempted to identify the mechanism of this phenomenon by testing the modulatory effects of isolated mannan and the chemical catabolites of mannan on cell-mediated immune function in vitro. We have determined that mannan isolated by complexation with cetyltrimethylammonium bromide (CTAB) is more antigenic than mannan isolated by precipitation with copper and that CTAB mannan does not inhibit lymphoproliferation stimulated by another antigen. We have also determined that oligosaccharides of three sizes, derived by chemical catabolism of CTAB mannan, are not antigenic, but instead are immunoinhibitory. Immunoinhibition does not involve interference with the mitogenic activity of interleukin 2. A similar occurrence of oligosaccharides may be produced by catabolism of mannan in vivo as evidenced by the presence of oligosaccharides of similar size in cell-free supernatant fluids derived from mononuclear leukocytes incubated with tritiated mannan. We propose that catabolites of fungal mannan may contribute significantly to suppression of cell-mediated immunity in candidiasis.

Antigens, Fungal↗
PubMed1989

Toxic shock syndrome-associated staphylococcal and streptococcal pyrogenic toxins are potent inducers of tumor necrosis factor production.

Toxic shock syndrome-associated staphylococcal and streptococcal exotoxins were tested for an ability to induce the production of tumor necrosis factor (TNF). Staphylococcal enterotoxins B and C1, along with streptococcal pyrogenic exotoxin A, all induced TNF production in a dose-dependent manner, with production peaking on the average at 3 days but continuing over the 6 days tested. This time course of exotoxin-induced TNF production contrasts with the 1-day peak-2-day duration observed with endotoxin as the stimulus and may be significant to development of toxic shock syndrome.

Animals↗