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Biomedical subjects

S D Douglas

Publications and source records attributed to S D Douglas.

At least 19 recordsLinked to original sources
PubMed1992

Alpha-1-antichymotrypsin inhibits the NADPH oxidase-enzyme complex in phorbol ester-stimulated neutrophil membranes.

The generation of superoxide anion and release of granule contents are essential to the bactericidal function of neutrophils, but may also contribute to host tissue damage during inflammation. In previous studies (J. Immunol. 146:2388), we have demonstrated that the acute phase reactant alpha-1-antichymotrypsin (ACT), a potent inhibitor of the serine protease cathepsin G, also suppresses superoxide anion generation. The inhibitory effect of ACT was not directly linked to its antiproteolytic activity and may reflect interaction at a site other than its reactive loop. To further characterize the mechanism of inhibition, we investigated the direct effects of ACT on the NADPH oxidase enzyme complex and the signaling pathways that regulate motivation of the respiratory burst. We present evidence that ACT does not intefer with agonist-stimulated calcium mobilization or translocation and activity of protein kinase C. ACT was an effective inhibitor of superoxide anion generation in membrane preparations isolated from PMA-activated cells. These results support the notion that ACT is acting on a component of the active assembled NADPH oxidase complex. Thus, ACT may have an important role in regulation of specific aspects of the inflammatory processes and the modulation of toxic oxygen-based host tissue damage.

Arachidonic Acid↗
PubMed1992

Antibody responses to bacteriophage phi X174 in patients with adenosine deaminase deficiency.

Adenosine deaminase (ADA) deficiency and its biochemical consequences cause severe combined immunodeficiency (SCID). Treatment strategies, designed to correct the biochemical abnormalities, include transplantation of matched bone marrow or haploidentical bone marrow stem cells, repeated partial exchange transfusions with frozen irradiated human red blood cells (RBC), or weekly injection of polyethylene glycol-modified bovine ADA (PEG-ADA). To evaluate the effect of these therapeutic options, we studied in vitro T-cell function and in vivo antibody responses to the T-cell-dependent neoantigen, bacteriophage phi X174, in 10 children with ADA-deficient SCID. In untreated patients, T-cell function was severely depressed, and only minute amounts of antibacteriophage antibody were produced. Transplantation of bone marrow from a matched sibling (one patient) or a phenotypically matched parent (one patient) resulted in a stable graft, normal T-cell function, and substantial but subnormal antibody titers to bacteriophage, with reduced memory and impaired switch from IgM to IgG. Patients receiving T-cell-depleted haploidentical bone marrow stem cells had markedly depressed antibody responses for as long as 3 years posttransplantation, despite rapidly improving T-cell function that became normal in two of four patients. Two methods of enzyme replacement were explored. During treatment with human RBC transfusions, antibody responses to bacteriophage were as severely depressed as in untreated ADA-deficient patients. Treatment with weekly injections of PEG-ADA resulted in normalization of T-cell numbers in all four patients, normal or near-normal T-cell function in two, and mildly but variably improved T-cell function in the other two patients. Quantitatively and qualitatively normal antibody responses to bacteriophage were observed in three of four patients. Assessment of antibody responses to immunization with bacteriophage phi X174 is a useful method to monitor humoral immune function in treated ADA-deficient patients and can be used to estimate when intravenous immunoglobulin (IVIG) prophylaxis may be safely discontinued.

Adenosine Deaminase↗
PubMed1992

Glutathione and N-acetylcysteine suppression of human immunodeficiency virus replication in human monocyte/macrophages in vitro.

Glutathione (GSH), its derivatives and N-acetylcysteine (NAC) inhibit the induction of HIV-1 expression in a chronically HIV-1-infected promonocytic cell line (U1/HIV) and peripheral blood mononuclear cells (PBMC). We have examined the effects of GSH and NAC on HIV-1 replication in human primary monocyte/macrophages cultured in vitro. Ficoll-gradient purified human monocytes were cultivated in vitro for 7-10 days and then infected with HIV-1 (Bal and Ada-M). Infection was blocked or substantially reduced by GSH or NAC (5-20 mM). Significant reduction (greater than or equal to 90%) in the amount of virus released, as determined by measuring supernatant reverse transcriptase activity and secreted p24 protein, was obtained when the cells were treated for 4 h with greater than or equal to 10 mM of GSH or NAC. The inhibitory effects of GSH and NAC were concentration dependent. This anti-HIV-1 effect persisted in these cultures for at least 35 days without evidence of significant increase in HIV-1 expression. Thus, a single pulse exposure of HIV-1-infected monocyte/macrophages with GSH or NAC led to a sustained, concentration-dependent decrease in HIV-1 p24 antigen levels, as well as, reverse transcriptase activity without producing detectable cellular toxicity in monocyte/macrophages.

Acetylcysteine↗
PubMed1992

Infection of cord blood monocyte-derived macrophages with human immunodeficiency virus type 1.

We have investigated the susceptibility of cord blood monocyte-derived macrophages to human immunodeficiency virus type 1 (HIV-1) infection in vitro. Cord blood monocytes were maintained in vitro for 10 to 15 days and then infected with HIV-1. Syncytia were observed 14 days after infection by light microscopy. Viral proteins were detected by immunofluorescence assay. Electron microscopic examination demonstrated typical lentivirus particles within cytoplasmic vacuoles. The supernatants from the HIV-1-infected cultures also contained significant reverse transcriptase activity and p24 antigen. Like adult monocyte/macrophages, cord-derived monocyte/macrophages expressed the CD4 receptor molecule. Pretreatment with blocking antibody prior to infection with HIV-1 Bal significantly reduced or blocked infection of cord monocyte/macrophages. When cord and adult monocyte/macrophages were infected with HIV-1 Bal or Ada-M and directly compared, higher reverse transcriptase activities and p24 antigen expression were obtained with cord monocyte/macrophages. However, no significant difference was found between adult and cord monocyte/macrophages infected with HIV-1 IIIB. These observations suggest that cord monocyte-derived macrophages may be important in the pathogenesis of pediatric AIDS and that the increased susceptibility of cord monocyte/macrophages to HIV-1 infection in vitro may be relevant to the enhanced susceptibility of neonates to HIV-1 diseases in vivo.

Adult↗
PubMed1992

Staphylococcal infections in childhood dermatomyositis--association with the development of calcinosis, raised IgE concentrations and granulocyte chemotactic defect.

There is a high incidence of staphylococcal infection in children with dermatomyositis, which is limited to those children who either already have or subsequently develop calcinosis. Of 15 children followed up for 3-10 years after diagnosis, all nine who developed calcinosis had infections with Staphylococcus aureus compared with none of six without calcinosis. Of these nine, the occurrence of staphylococcal infections before calcinosis was observed in four, suggested by history in two, and unclear in three children. Granulocyte chemotaxis to Staphylococcus aureus was more severely depressed in those children with calcinosis, whereas those without calcinosis did not differ significantly from controls. The chemotactic defect was due to a serum factor (patients' serum depressed control chemotaxis and control serum corrected the patients' chemotaxis). The nine children with calcinosis also had significantly higher serum IgE concentrations than non-atopic age matched controls; the six without calcinosis did not differ from controls. The increased IgE concentrations appeared to develop after staphylococcal infection and before calcinosis. Two of five patients with calcinosis had increased antistaphylococcal IgE antibodies; neither of the two patients without calcinosis had such increased antibodies. This suggests preceding immunological differences in patients with dermatomyositis who do and do not subsequently develop calcinosis, either increasing susceptibility to Staphylococcus aureus infection or potentially resulting from such infections.

Antibodies, Bacterial↗
PubMed1991

Inhibition of human neutrophil superoxide generation by alpha 1-antichymotrypsin.

Reactive oxygen intermediates and serine proteases are important components of host defense systems but can produce host injury if not tightly regulated. To determine whether these components can be coordinately controlled, we investigated regulation of superoxide generation by physiologically relevant concentrations of a) highly purified serum-derived antichymotrypsin (ACT), b) recombinant, wild-type ACT, c) rACT in which amino acid substitutions were engineered into the reactive center, and d) chymotrypsin/ACT complexes. These proteins and protein complexes inhibited superoxide anion production in neutrophils stimulated by f-Met-Leu-Phe, Con A, or PMA. In contrast, ligand-stimulated degranulation was not inhibited. In addition, using the recombinants and complexes, the region of ACT involved in inhibiting superoxide anion production was shown to be structurally distinct from the reactive center of the protein. The results indicate that functional domains of ACT corresponding to different biological activities can be decoupled and suggest that three species of ACT (intact ACT, a complexed protease/ACT form, and a partially denatured or proteolyzed form of ACT) that can exist in the microenvironment of an activated neutrophil may play an important role in regulating neutrophil function.

Cell Degranulation↗
PubMed1991

Isolation and flow cytometric characterization of newborn mouse brain-derived microglia maintained in vitro.

Microglia have been identified in the white matter of developing and adult mouse brain using different murine macrophage markers. While several techniques for the isolation of murine microglia have been described, the small cell yields and partial purification have limited the progress of these studies. We now describe the isolation of murine microglia using a modification of McCarthy and de Vellis method. Brain tissues from 1-2 day old newborn mice were mechanically and chemically dissociated and maintained in in vitro culture for 3 weeks. In primary dissociated brain cultures, microglia are observed after 10 days migrating from small colonies. After 16-20 days, brain-derived microglia were isolated with high cell yields by continuous shaking of the cultures for 16 hr. In contrast to resident murine peritoneal macrophages, microglia express less Class II (Ia) antigen and a small percentage express L3T4 (CD4) antigen by flow cytometry.

Animals↗
PubMed1991

Chondroosseous dysplasia in severe combined immunodeficiency due to adenosine deaminase deficiency (chondroosseous dysplasia in ADA deficiency SCID).

Adenosine deaminase (ADA) deficiency may manifest as severe combined immunodeficiency (SCID) in early infancy. Some of these children develop radiologic changes which may be in part related to effects of this enzyme deficiency on the bony epiphysis. We describe the radiologic changes in a neonate with ADA deficiency and their resolution with polyethylene glycol conjugated adenosine deaminase (PEG-ADA, ADAGEN: Enzon, Inc., South Plainfield, NJ) enzyme replacement therapy.

Adenosine Deaminase↗
PubMed1991

Isolation and characterization of newborn rabbit brain-derived microglia.

We isolated brain microglia from newborn rabbits and maintained these cells in in vitro culture. Enriched populations of rabbit microglia share several characteristics of mononuclear phagocytes including intracellular staining for nonspecific esterase and acid phosphatase. Microglia express Fc receptors, generate superoxide anion, and stain positive with the lectin Ricinus communis. Rabbit brain microglia develop multinucleated giant cells and small colonies in in vitro culture. The cells are highly phagocytic in culture. Other investigators have recently demonstrated that rabbits can be infected with HIV-1 in vivo and that neurological symptoms occur only when HIV-1 infection was carried out in HTLV-1-infected rabbits. Brain microglia most likely play a central role in HIV-1 encephalopathy. The availability of rabbit brain microglia in in vitro culture, offers a valuable potential cell model to study HIV-1 infection in the central nervous system.

Animals↗
PubMed1991

Minitransfection: a simple, fast technique for transfections.

A fast, simple and inexpensive minitransfection technique, using either a lipofection or a calcium phosphate coprecipitation method to introduce foreign DNA into living cells is presented. This technique is based on the use of 24-well or 96-well tissue culture plates and can be used for both transient and stable transfections. Because it is a microtechnique, only small amounts of DNA, cells and transfection reagents are necessary, and it is easy to handle multiple DNA transfections or cotransfections in different cell lines and in duplicates or triplicates. The technique can be used to study viral gene expression, virus replication and chloramphenicol acetyltransferase (CAT) assay in different cell lines, for example, in the large scale screening and testing of antiviral agents.

Animals↗
PubMed1991

Isolation and characterization of human fetal brain-derived microglia in in vitro culture.

Human brain microglia may play a central role in immunopathogenesis of CNS diseases including HIV infection, multiple sclerosis and Alzheimer's disease. In order to investigate the possible relationship between microglia and the mononuclear phagocyte system, human brain microglia were isolated from 14-18-week-old fetal brains, and maintained in in vitro culture. Enriched fetal brain microglia were stained for different monocyte/macrophage and glial cell markers. Fresh dissociated brain cells lacked macrophage surface markers. Isolated microglial cells stained positive for complement receptor C3bi, Class II [human leukocyte antigen-DR (HLA-DR)] antigen and with the lectin Ricinus communis. Microglia also share several functional properties with monocyte/macrophages, which include generation of superoxide anion and histochemically demonstrable intracellular acid phosphatase and non-specific esterase. Primary human dissociated brain cultures were maintained in culture for at least 28 weeks. Although microglia were not observed above the astrocyte cell layer after 5 weeks in culture, microglia-like cells appear below the astrocyte layer after 12 weeks in culture. These cells stained positive for non-specific esterase and displayed oxidative burst activity upon activation with phorbol myristate acetate. Thus, we have successfully isolated an enriched population of microglia from human fetal brain and have demonstrated that these cells possess markers and properties which are characteristics of mononuclear phagocytes.

Acid Phosphatase↗
PubMed1991

Ca2+ responses to interleukin 1 and tumor necrosis factor in cultured human skin fibroblasts. Possible implications for Reye syndrome.

Elevated concentrations of cytokines were found in the plasma of patients acutely ill with Reye syndrome (RS) but not in control subjects or recovered RS patients. To determine whether this disorder involves a genetically determined abnormal response to cytokines, the effects of tumor necrosis factor (TNF) and IL-1 on intracellular free Ca2+ were compared in cultured skin fibroblasts from control subjects and patients with RS. IL-1 and TNF caused rapid, transient, and concentration-dependent increases in cytosolic free Ca2+. The peak cytosolic free Ca2+ was greater and occurred at higher concentrations of IL-1 and TNF in patient cells than in cells from age-matched controls. In control cells, the Ca2+ transient diminished sharply with increasing amounts of IL-1 or TNF above the maximum stimulatory concentration. In contrast, in patient fibroblast this bell-shaped curve of concentration dependency was much less apparent. Bradykinin-stimulated Ca2+ transients were similar in the two groups and did not exhibit the bell-shaped concentration dependency. Thus, plasma cytokine levels are elevated in RS patients and the Ca2+ response to cytokines is increased in cells derived from these patients. We propose that the increased response reflects a genetic defect in cytokine receptor-modulated signal transduction.

Adolescent↗
PubMed1991

Human cytomegalovirus infection and trans-activation of HIV-1 LTR in human brain-derived cells.

In order to investigate the hypothesis that human cytomegalovirus (HCMV) influences HIV-1 infection of brain cells, we studied primary astrocytes derived from human fetal brains and a neuronal cell line (SK-N-MC). Infection of these cells with two strains of HCMV resulted in expression of immediate early, early, and late antigens and production of infectious virus. HCMV infection of primary astrocytes also led to cytopathic effects and cell death. SK-N-MC cells were infected with HIV-1 strains with or without HCMV. HIV LTR-directed CAT activities and the expression of HIV p24 antigen from the SK-N-MC culture coinfected with both HIV-1 and HCMV were higher than those from the cells infected with HIV-1 alone. The primary astrocytes were cotransfected with HIV-1 proviral DNAs and HIV LTR-CAT with or without HCMV infection. HCMV-infected astrocytes produced greater amounts of CAT activity and higher p24 than the cells transfected with HIV-1 proviral DNAs alone. When both primary astrocytes and SK-N-MC cells were transfected with (a) HIV LTR-CAT alone, (b) HIV LTR-CAT plus HCMV-IE gene, or (c) HIV LTR-CAT plus HCMV infection 2 days before the transfection, both HCMV infection and its IE gene trans-activated the HIV LTR promoter. HCMV-IE gene 2 may play a critical role in trans-activation of HIV-1 LTR.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Effects of indomethacin and prostaglandin E1 on the production of fibronectin and lysozyme by monocyte-derived macrophages in vitro.

Fibronectin secreted by macrophages may contribute to the development of pulmonary fibrosis. Prostaglandins are important regulators of macrophage metabolism whose role in the regulation of fibronectin production is not known. In this study, we examined the effects of PGE1 and indomethacin on human monocyte-derived macrophages exposed to these agents in culture for 10 to 14 days. Indomethacin (10 micrograms/ml) reduced the ratio of supernatant fibronectin to adherent cell DNA by 32%, p < 0.01, and reduced lysozyme/DNA by 29%, p < 0.0001. Exogenous PGE1 (1 ng/ml) did not affect fibronectin, but increased lysozyme/DNA by 27%, p < 0.01. In additional experiments, supernatant fibronectin and total protein synthesized in the presence of 3H-leucine were measured. Indomethacin (10 micrograms/ml) had no effect on total supernatant protein radioactivity, but reduced fibronectin/DNA by 33%, p < 0.001, and reduced fibronectin/total protein by 19%, p < 0.01. Since indomethacin increases macrophage secretion of plasminogen activator and interleukin-1, these experiments add to the evidence that specific secretory products of macrophages are regulated independently. We conclude that indomethacin at 10 micrograms/ml decreases the production of fibronectin and lysozyme by monocyte-derived macrophages. The modest size of the effect, and its absence at lower doses of indomethacin, indicate that prostaglandins are unlikely to have a major role in the regulation of macrophage production of fibronectin.

Adult↗
PubMed1991

Homozygosity for a newly identified missense mutation in a patient with very severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID).

We have identified a previously unrecognized missense mutation in a patient with severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID). The mutation is a G646-to-A transition at a CG dinucleotide and predicts a glycine-to-arginine substitution at codon 216. Computer analysis of secondary structure predicts a major alteration with loss of a beta-pleated sheet in a highly conserved region of the protein. The basepair substitution also generates a new site for the restriction enzyme BstXI in exon 7 of the genomic DNA. Digestion of genomic DNA from the patient and from his parents revealed that he was homozygous for the mutation and that his mother and father were carriers. This mutation in homozygous form appears to be associated with very severe disease, since the patient had perinatal onset of clinical manifestations of SCID, the highest concentration of the toxic metabolite deoxyATP in nine patients studied, and a relatively poor immunologic response during the initial 2 years of therapy with polyethylene glycol-adenosine deaminase. Analysis of DNA from 21 additional patients with ADA-SCID and from 19 unrelated normals revealed that, while none of the normal individuals showed the abnormal restriction fragment, two of the 21 patients studied were heterozygous for the G646-to-A mutation.

Adenosine Deaminase↗
PubMed1991

Pulmonary functions in children with progressive systemic sclerosis.

The patterns of pulmonary involvement in 13 children with progressive systemic sclerosis were investigated. Eight patients (61%) had respiratory symptoms or signs and 7 patients (55%) had abnormalities on chest roentgenogram. Twelve patients (92%) had abnormal pulmonary function tests: 7 had restrictive disease, 2 had obstructive disease, 2 had small airway disease, and 1 had an isolated reduction in the diffusing capacity of carbon monoxide. Nine patients had the test performed during the first year of illness, 3 during the second year, and 1 at 5 years. All patients had abnormal pulmonary function tests when first studied. Subsequent pulmonary function tests over a period of 3 to 10 years (mean 6.2 years) showed substantial changes in only 2 patients (1 patient had initial worsening of diffusing capacity of carbon monoxide followed by normalization and another patient showed improvement of obstructive disease). Two patients died during follow-up, 1 of pulmonary hypertension, the other of severe restrictive lung disease and myocardial fibrosis. The major findings of this study were (1) high frequency of pulmonary disease in children with progressive systemic sclerosis, (2) early involvement of the lungs, (3) relatively indolent progression of lung disease, and (4) the prognostic importance of the severity of pulmonary disease. Pulmonary manifestations of progressive systemic sclerosis in children appear to be similar to those of affected adults.

Adolescent↗
PubMed1990

Monocyte-derived macrophage and alveolar macrophage fibronectin production and cathepsin D activity.

Alveolar macrophages are thought to play an important role in ongoing tissue breakdown and repair processes in the normal lung. The secretion and regulation of cathepsin D (important for the final breakdown of collagen) and fibronectin (involved in the healing process) in human peripheral blood monocytes (PBM) and pulmonary alveolar macrophages (PAM) were investigated. Cathepsin D enzyme activity was measured by quantitating the TCA-soluble fragments of [3H]hemoglobin. Freshly isolated PBM contained less cell-associated cathepsin D activity than did freshly isolated PAM (314 +/- 35 micrograms/10(6) cells vs 381 +/- 35 micrograms/10(6) cells, respectively). After 7-10 days in culture, cell-associated enzyme levels in both PBM and PAM were significantly increased (P less than 0.001 for PBM; P less than 0.0001 for PAM). In addition, freshly isolated PAM secreted more cathepsin D than did freshly isolated PBM (5.8 +/- 3.2 micrograms/10(6) cells vs 0.83 +/- 0.83 micrograms/10(6) cells, P less than 0.02). In the presence of LPS (10 micrograms/ml), cell-associated cathepsin D was inhibited in both PBM and PAM. With the addition of gamma-IFN (500 U/ml), both cell-associated and secreted enzyme were increased in freshly isolated and 10-day-cultured PBM and PAM. In parallel studies, fibronectin secretion (by ELISA assay) in both PBM and PAM increased over time in culture. LPS had no effect on PBM or PAM secretion of human fibronectin while gamma-IFN increased PBM and PAM fibronectin levels. Thus, both macrophage cathepsin D activity and fibronectin secretion are increased by gamma-interferon while macrophage cathepsin D activity, but not fibronectin secretion, is decreased by LPS. These studies demonstrate that human macrophage cathepsin D activity is actively modulated by inflammatory mediators and that macrophage mediators of tissue breakdown and repair are not modulated synchronously.

Adult↗