Ethical challenges in medical care for the pregnant substance abuser.
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Child Welfare↗
Biomedical subjects
D E Campbell
Publications and source records attributed to D E Campbell.
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↗
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↗
Carcinoembryonic antigen enhances metastatic potential of human colorectal carcinoma.
Patients with human colorectal carcinoma have a poor prognosis when serum carcinoembryonic antigen level exceeds 5 ng/mL. The hypothesis that carcinoembryonic antigen enhances metastasis by promoting the attachment of tumor cells to Kupffer cells and hepatocytes was tested in an experimental metastasis model in which colorectal carcinoma cells were injected into the spleens of BALB/c athymic nude mice and liver colonies counted 5 weeks later. Pretreatment with systemic injections of carcinoembryonic antigen significantly increased the metastatic potential of a poorly metastatic colorectal carcinoma cell line KM-12c, but did not induce the nonmetastatic colorectal carcinoma cell line HC 2998 to produce metastases, nor did carcinoembryonic antigen make the highly metastatic colorectal carcinoma cell line mHC 1410 more metastatic. Carcinoembryonic antigen did not stimulate proliferation of colorectal carcinoma but appeared to be a cofactor for metastasis possibly as an adhesion factor.
Animals↗
Natural killer cell-mediated lysis of T cell lines chronically infected with HIV-1.
The susceptibility of HIV-1-infected CD4+ T cell lines to natural killer (NK) cell-mediated lysis was examined. Non-adherent peripheral blood mononuclear cells (PBMC) of healthy adults lysed HUT cells chronically infected with the IIIB or WMJ1 strains of HIV-1 to a significantly greater extent than uninfected HUT cells. In contrast, Sup-T1 cells chronically infected with these two strains of HIV-1 were not lysed to a greater extent than uninfected Sup-T1 cells. Clone A1.25-infected Sup-T1 (A1.25/Sup-T1), derived from IIIB-infected Sup-T1 cells (IIIB/Sup-T1), were susceptible to non-adherent PBMC-mediated lysis, as were A1.25-infected HUT cells (A1.25/HUT). When non-adherent PBMC were depleted of CD16 (Leu-11b)+ NK cells by treatment with anti-Leu-11b plus C, lysis of HIV-1-infected HUT or Sup-T1 cells was reduced to low levels, indicating that the lysis was mediated by NK cells. Expression of HIV antigens on these target cells did not correlate with their susceptibility to NK cell-mediated lysis. Depletion of interferon-alpha (IFN-alpha) producing HLA-DR+ cells from non-adherent PBMC had no effect on the magnitude of NK cell-mediated lysis of IIIB or WMJ1-infected HUT cells. In contrast, lysis of A1.25/Sup-T1 or A1.25/HUT cells required the presence of HLA-DR+ cells. IFN-alpha production appeared to be required for NK cell-mediated lysis of A1.25/Sup-T1 or A1.25/HUT cells, while lysis of HUT cells infected with the WMJ1 or IIIB strains of HIV-1 was IFN-alpha independent. These results indicate considerable variability in the susceptibility of different HIV-1 infected T cell lines to NK cell-mediated lysis and suggest the existence of alternative mechanisms of activation of NK cells for lysis of HIV-1-infected T cell lines.
Acquired Immunodeficiency Syndrome↗
Synthetic peptide homologous to the envelope proteins of retroviruses shares a cross-reacting epitope with the CD4 receptor.
A synthetic peptide (CKS-17) homologous to a highly conserved region of the retroviral transmembrane protein p15E was tested for its effect on receptor expression on monocytes. The CKS-17 amino acid sequence is present in several retroviruses including human T-cell lymphotropic virus types I and II and human immunodeficiency virus. The CKS-17 peptide has been previously shown to inhibit monocyte superoxide production, natural killer cell activity, polyclonal B-cell activation, and monocyte-mediated killing by inactivation of interleukin-1. In this study, we demonstrated that the anti-CD4 monoclonal antibody OKT4 binds strongly in vitro to CKS-17-treated human blood monocytes, whereas other antibodies tested were not reactive. This observed binding was the result of direct interaction of OKT4 antibody with the CKS-17 peptide. Moreover, a partial homology was found in amino acid sequence analysis of the CD4 epitope and the CKS-17 peptide.
Amino Acid Sequence↗
Expression of the complement receptors CR1 and CR3 and the type III Fc gamma receptor on neutrophils from newborn infants and from fetuses with Rh disease.
Developmental defects in neutrophil function, including diminished expression of plasma membrane receptors, may play an important role in the susceptibility of the newborn infant to infection. We used monoclonal antibodies and flow cytometry to study the expression of complement receptor type one (CR1), complement receptor type three (CR3), and Fc gamma receptor type three (FcRIII) on neutrophils from six fetuses with Rh disease, 10 preterm infants, nine term infants, and nine adults. Expression of the complement receptors on unstimulated cells was similar for all groups, but significant differences in complement receptor expression were observed after stimulation with N-formyl-methionyl-leucyl-phenylalanine (FMLP). Fetal, preterm, and term infant neutrophils expressed less CR3 than FMLP-stimulated neutrophils of adults [61 +/- 2, 48 +/- 4, and 66 +/- 4% (mean +/- SEM) of the mean for adults, p less than 0.05]. FMLP-stimulated CR1 expression for these groups was 61 +/- 6, 73 +/- 6, and 91 +/- 9% of the adult mean (p less than 0.05, fetal versus term infant and adult). Expression of both CR3 and CR1 increased with postconceptional age in the infants (r2 = 0.49, p less than 0.001 for CR3; r2 = 0.23, p less than 0.05 for CR1). Neutrophils of the preterm and term infants expressed less FcRIII than adult neutrophils (68 +/- 10 and 77 +/- 7% of the adult mean, p less than 0.05, for FMLP-stimulated cells), whereas fetal neutrophil FcRIII expression did not differ from that of the adult.(ABSTRACT TRUNCATED AT 250 WORDS)
Antigens, Differentiation↗
Interaction between human peripheral blood monocytes and tumor promoters: effect on growth differentiation and function in vitro.
Studies on the differentiation and activation of human monocytes in tissue cultures have usually been limited by the deterioration of human monocytes and macrophages in long-term cultures. In this study, we attempted to establish long-term human monocyte/macrophage cultures using the phorbol ester 12-0 tetradecanoyl-phorbol-13-acetate (TPA), and we studied the morphology, function, and biochemical properties of such treated human blood monocytes. Enriched suspensions of monocytes were obtained using Ficoll-Hypaque gradient and cultured in the absence or presence of various concentrations of TPA. Samples were removed at different times and processed for scanning electron microscopy. Parallel samples were examined for numbers of adherent cells, phagocytosis, oxidative burst, beta-galactosidase assays, and lectin-mediated erythrolysis. TPA-treated monocytes survived in larger numbers in culture for up to 7 weeks and were more pleomorphic and exhibited higher beta-galactosidase activities after 14 days in culture than untreated monocytes. TPA-treated cells and untreated cells in long-term cultures showed a decrease in their oxidative burst activity while their phagocytic activity was not affected, and the TPA treatment augmented the lysis of wheat germ agglutinin-opsonized erythrocytes by the cultured monocytes. TPA treatment of adherent human monocytes resulted in cell cultures with increased numbers of viable and functionally adherent cells for extended periods of time and does not seem to interfere with the differentiation and maturation of the cells in culture.
Cell Count↗
Ontogeny of the antibody response to cow milk proteins.
Twenty-nine infants were followed prospectively for the development of immunoglobulin G (IgG) and IgE antibodies to the cow milk proteins (CMPs) alpha-lactalbumin, alpha-casein, and beta-lactoglobulin. Thirteen infants were fed formula, eight were exclusively breast fed, and eight were breast fed with formula supplementation. By 4 months of life, infants fed formula or breast milk with formula supplementation had elevated IgE and IgG antibody levels to CMPs, when compared with values before 1 month of age. Further, breast-fed infants demonstrated significantly lower levels of IgE and IgG antibodies to CMPs than infants who received formula or breast milk with formula supplementation. Clinical symptoms of cow milk allergy were more common in infants fed formula or breast fed with supplementation, but were not significantly related to the presence of specific antibody. Our data suggest that the formation of CMP antibodies may reflect a normal humoral response following exposure to CMP antigens in this age group.
Animals↗
Phorbol myristate acetate induced oxidation of 2',7'-dichlorofluorescin by neutrophils from patients with chronic granulomatous disease.
The oxidative metabolic burst of stimulated human polymorphonuclear leukocytes (PMNs) has been evaluated by the measurement of oxygen consumption, chemiluminescence, and oxygen radicals (O2-, H2O2, OH-) derived from activation of the hexose monophosphate shunt (HMPS). PMNs from patients with chronic granulomatous disease (CGD) are shown to lack functional NADPH oxidase and undetectable oxygen radical generation. However, using single cell analysis by flow cytometry and 2',7'-dichlorofluorescin (DCFH) oxidation by H2O2, significant DCFH oxidation by the PMA stimulated CGD PMNs was observed. Furthermore, 1mM potassium cyanide enhanced DCFH oxidation by control and CGD PMNs. DCFH oxidation by cells from an obligate heterozygous mother of an X-linked CGD patient was intermediate. These observations suggest that a PMA induced oxidase enzyme is present in CGD cells.
Cell Line↗
Flow cytometric analysis of oxidase activity of neutrophils from chronic granulomatous disease patients.
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Flow Cytometry↗
Marrow transplantation in chronic granulomatous disease: an update, with 6-year follow-up.
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Bone Marrow Transplantation↗
Pharmacology of olsalazine.
In the treatment of inflammatory bowel disease the main anti-inflammatory component of sulphasalazine is 5-ASA. Based on the same azo-splitting principle in the colon as sulphasalazine, the new drug olsalazine was developed. Olsalazine consists of two 5-ASA molecules joined by an azo bridge. Pharmacokinetic studies have shown that on oral administration there is little systemic absorption of olsalazine. Almost the whole dose passes into the colon, where the olsalazine is completely split into 5-ASA and subsequently excreted in faeces and urine. It can be concluded that from all toxicological and pharmacological aspects, olsalazine is a safe drug. Olsalazine will present the colonic mucosa with twice the amount of 5-ASA/unit compared with sulphasalazine (the present drug of choice) without concomitant delivery of sulphapyridine, which is believed to carry most of the drug's side-effects. Thus, olsalazine seems to be a suitable drug for long-term treatment of ulcerative colitis in man.
Aminosalicylic Acids↗
Rickets of prematurity: controversies in causation and prevention.
Metabolic bone disease in the growing premature infant is an important disorder owing to inadequate intake of calcium or phosphorus over an extended period of time. Prevention of serious bone disease is an important goal in the care of the VLBW infant. Attempts to reproduce intrauterine bone accretion rates in intrauterine life may be unnecessary and may result in potential complications.
Birth Weight↗
Expression of autoantigens in human colorectal carcinomas. Implications for a generic vaccine.
The antibody response of patients was used to characterize the autoantigens in human colorectal carcinoma. Twenty-seven primary and 13 metastatic carcinomas with paired normal tissues were extracted and transferred onto nitrocellulose membranes by the Western transfer technique. After the transfers were incubated with the serum of the patient from whom the tumor was derived, autoantigens were identified by indirect immunoperoxidase staining. All tumors contained at least one autoantigen. Six tumor-associated autoantigens, ranging in molecular weight from 26 to 58 kilodaltons (kD), were identified by antibodies in 25% or more of the sera. Eleven metastases expressed a 41-kD autoantigen that was present in only a third of the extracts of normal liver or lung. Thus, the number of dominant polypeptide autoantigens in colorectal carcinoma is restricted to six molecules. These autoantigens may be organ-associated antigens that are expressed by neoplastic cells. The 41-kD autoantigen may be a potential marker for metastases. A generic vaccine appears to be feasible for colorectal carcinoma since the number of dominant antigens is limited.
Antigens, Neoplasm↗
O-phenylenediamine oxidation by phorbol myristate acetate-stimulated human polymorphonuclear leukocytes: characterization of two distinct oxidative mechanisms.
O-Phenylenediamine (OPD) oxidation has been extensively utilized for the measurement of peroxidase-mediated catabolism of hydrogen peroxide. However, until now this system has not been evaluated for the measurement of hydrogen peroxide produced upon activation of the hexose monophosphate shunt (HMPS) in polymorphonuclear leukocytes (PMNs). OPD oxidation by phorbol myristate acetate (PMA)-stimulated PMNs was mediated by both hydrogen peroxide and superoxide produced by the activation of the HMPS. Furthermore, OPD oxidation by an oxidative mechanism independent of the HMPS was observed by the PMA stimulation of PMNs obtained from patients with chronic granulomatous disease (CGD). This HMPS-independent OPD oxidation was inhibited by superoxide dismutase or 1 mM potassium cyanide (KCN). Superoxide dismutase, catalase, or 1 mM potassium cyanide inhibited 50% OPD oxidation obtained with PMA-stimulated normal PMNs. PMA treatment of purified human myeloperoxidase (MPO) produced OPD oxidation which is inhibited by superoxide dismutase or 1 mM KCN. These data indicate that OPD oxidation observed with CGD PMNs is mediated by a PMA-induced oxidase activity of myeloperoxidase. OPD oxidation in the presence of 1 mM KCN is a method comparable in sensitivity with ferricytochrome c reduction for the evaluation of HMPS activity. Furthermore, the OPD assay can measure myeloperoxidase oxidase activity in PMA-stimulated PMNs.
Azides↗
Neutrophil activation in human inflammatory skin reactions.
To determine the functional activity of neutrophils emigrating to the sites of ongoing human allergic inflammatory reactions, we assessed the intensity of their intracellular oxidative metabolism by measuring the intracellular oxidation by H2O2 of the dye, 2',7'-dichlorofluorescein diacetate (DCFH-DA), to fluorescent 2',7'-dichlorofluorescein. The intensity of intracellular fluorescence was measured by flow cytometry. In 13 atopic subjects, two denuded skin blister bases were challenged for 5 hours with pollen antigen and two blister bases with buffer. Both the level of histamine and mean number of cells recovered at antigen sites were greater than at buffer sites. Oxidation of DCFH-DA by neutrophils recovered from both antigen and buffer sites was significantly greater than that of autologous peripheral blood neutrophils (PBNs), reflecting an increase in the spontaneous generation H2O2 of cells recovered from the sites of inflammation. DCFH-DA oxidation by cells recovered from sites of antigen incubation was significantly greater than cells from buffer site incubations. Neutrophils from both antigen and buffer sites could be stimulated further in vitro by phorbol myristate acetate to the same level of H2O2 generation as phorbol myristate acetate-stimulated PBNs. In five subjects studied, the noncellular component of chamber fluids collected at 1 and 5 hours stimulated PBNs to increase their oxidative metabolism by 42% to 62%; however, this increased level of intracellular H2O2 was still much less than the spontaneous H2O2 generation observed in cells recovered from sites of allergic inflammation. The increased oxidative metabolism of neutrophils in human allergic and inflammatory skin reactions has important pathophysiologic implications for the role of these cells in inflammatory responses.
Adult↗
Indirect enzyme-linked immunosorbent assay for measurement of human immunoglobulins E and G to purified cow's milk proteins: application in diagnosis of cow's milk allergy.
An indirect double-antibody enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of human immunoglobulin E (IgE) and IgG to the cow's milk proteins (CMP) alpha-casein, alpha-lactalbumin, and beta-lactoglobulin. Human serum albumin was used as the negative-antigen control. Rabbit anti-human IgE or IgG served as the primary antibody, and horseradish peroxidase-conjugated swine anti-rabbit immunoglobulin served as the secondary antibody. Positive control sera were obtained from patients with well-documented histories of cow's milk allergy, while negative control sera were obtained from cord bloods of healthy full-term infants and from normal adult volunteers without known milk allergy. Test sera were obtained from 41 children (ages, 3 months to 13 years; average age, 2.6 years) with suspected cow's milk allergy and clinical manifestations that included wheezing, rhinitis, atopic dermatitis, urticaria, or gastrointestinal disturbances. The patients were simultaneously evaluated by prick skin testing with scratch test antigen to whole CMP. Although only 13 (32%) of the 41 patients were positive by the prick skin test, 25 (61%) were positive by the IgE ELISA. Of the 25 IgE ELISA-positive patients, 20 were also positive by the IgG ELISA. There was concordance of positive results between skin testing and the IgE ELISA in only 9 patients (22%), and there was concordance of negative results in 12 patients (29%). Discordant results were observed in 20 patients (49%). These results indicate that the ELISA is more sensitive than prick skin testing in the identification of individuals with elevated levels of IgE to CMP.
Adult↗