Donald E. Campbell

Persistent decreases in blood plasmacytoid dendritic cell number and function despite effective highly active antiretroviral therapy and increased blood myeloid dendritic cells in HIV-infected individuals.

Dendritic cells (DC) have an instrumental role in the activation and function of both innate and adaptive immune responses. In humans, at least two distinct DC subsets have been characterized based on phenotypic markers: the myeloid DC (MDC) and the plasmacytoid DC (PDC). Both subsets are critical producers of cytokines (IL-12 for MDC and type I/II IFNs for PDC) and are functionally different. We show in this study that HIV+ individuals have a significant decrease in the number of the Lin−HLA-DR+CD123+ and BDCA-2+ PDC compared with uninfected donors (p = 0.0001). HIV+ individuals also have a sustained impairment in viral-induced IFN-α production (p < 0.0001). The decrease of the PDC subsets did not correlate with CD4 count or viral load and was not reversed in subjects under virally suppressive treatment, suggesting an irreversible change after infection. By contrast, the absolute number and median frequency of MDC in HIV-infected individuals were similar to those observed in uninfected controls, while a significant decrease was present in subjects with >5000 HIV-1 copies/ml. The inverse association with viral load of the MDC number, but not of IFN-α secretion or the number of PDC, suggests a role for MDC in viral control. Our data suggest that DC subsets are differentially reconstituted during the immune recovery associated with antiviral therapy. The persistent impairment of certain DC subsets may result in a sustained defect in DC-mediated innate immune functions despite an effective treatment regimen.

Purification of human monocytes on gelatin-coated surfaces.

Human peripheral blood monocytes secrete a cell membrane-associated glycoprotein, cold insoluble globulin (fibronectin). Since fibronectin binds to gelatin-coated surface, we developed a simple technique for separation of human peripheral blood monocytes from whole mononuclear cell preparation. These preparations are characterized by a high monocyte purity (more than 90%), low platelet contamination and excellent viability.

Cytofluorographic analysis of effects of interferons on expression of human cytomegalovirus proteins

The appearance of cytomegalovirus (CMV) proteins in infected fibroblasts was determined by flow cytometry. The sequential production of immediate early (IE), early (E), and late (L) proteins reacting with respective monoclonal antibodies (mAbs) E13, 58/5, and 24/4 was determined in fibroblasts infected with the AD-169 strain of CMV. The percentage of cells expressing CMV proteins and the intensity of fluorescence within cells were determined from day 1 to day 7 post-infection. The effect of interferons (IFNs) alpha, beta, gamma on expression of CMV proteins was analyzed using flow cytometry. IFNs inhibited E and L protein production at days 3 and 6 post-infection in a dose-dependent manner. This inhibitory effect on protein expression was associated with a reduction in release of infectious CMV into culture media. The method described here for detection of CMV proteins using flow cytometry may be useful for basic studies of gene expression and for diagnostic purposes.

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.

Cytokines Released by Human Peripheral Blood Mononuclear Cells Inhibit the Production of Early and Late Cytomegalovirus Proteins

Cytomegalovirus‐infected human fibroblasts are susceptible to lysis by natural killer cells and cytotoxic T cells. The purpose of this study was to determine whether non‐lytic mechanisms might also contribute to the control of cytomegalovirus infection. The appearance of cytomegalovirus proteins in infected fibroblasts was determined by flow cytometry. Infected fibroblasts incubated with peripheral blood mononuclear cells for 3 days expressed less early and late proteins than fibroblasts incubated without peripheral blood mononuclear cells. Supernatants generated by the cocultivation of peripheral blood mononuclear cells with cytomegalovirus‐infected fibroblasts inhibited the production of cytomegalovirus early and late proteins. The soluble factors in supernatants which contributed to the inhibitory effect were identified as interferons α, β and γ, and tumor necrosis factors α and β. The ability of supernatants to inhibit the production of cytomegalovirus early protein was mimicked by combinations of corresponding recombinant cytokines. The inhibition of cytomegalovirus protein production by cytokines produced by peripheral blood mononuclear cells may contribute to early containment of cytomegalovirus infection.

Cervical and vaginal cytokine determinations in pregnant women: methodologic issues.

Investigations of the role cytokines play in preterm birth are complicated by a number of methodologic issues that arise as to the most feasible and efficient methods to measure cytokines. The purpose of this article is to review methodologic issues surrounding the measurement of vaginal and cervical cytokines, specifically IL-1β, IL-6, and TNF-α, in pregnant women experiencing preterm labor. Specifically, two quantification methods, weight and protein assay, and cytokine specimens from two different sites, vaginal and cervical, were compared. There were no significant correlations between cytokine levels using the protein versus weight quantification method. The weight method had more negative values and thus the protein quantification method was more accurate. There were high correlations between cervical and vaginal IL-1β levels and IL-6 levels, but cervical and vaginal TNF-α levels were not correlated.

Flow Cytometric Analysis of Oxidase Activity of Neutrophils from Chronic Granulomatous Disease Patients

Phagocyte cells, neutrophils and macrophages constitute the main defense mechanisms of the human body against invading microorganisms. The bactericidal activity of polymorphonuclear leukocytes (PMNs) involves the activation of a cell membrane associated NADPH oxidase enzyme“. Following activation of the NADPH oxidase enzyme, a cascade of oxidation/reduction reactions occurs, with the generation of several oxygen radicals. Superoxide (0 2 − ) hydrogen peroxide (H202), hydroxyl radical (HO) singlet oxygen (0) are the main oxygen by-products of the respiratory burst of PMNs2 and have an important role in killing of bacteria. The enzyme NADPH oxidase has a major function in the generation of O 2 − radical while other enzymes including superoxide dismutase3, myeloperoxidase4, catalase and glutathione peroxidase5 serve a regulatory activity in the respiratory burst. NADPH oxidase differs from other cellular oxidase enzymes (i.e., mitochondrial), since it is a cyanide insensitive enzyme6. The activation of the NADPH oxidase enzyme with different soluble stimulants as phorbol esters7, formyl-methionyl-leucyl-phenylalanine8 and γ-hexachlorocyclohexane9 results in the generation of active oxygen radicals and mimics the biochemical events which occur during phagocytosis in vivo.