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Biomedical subjects

R K Root

Publications and source records attributed to R K Root.

At least 37 records · Page 2Linked to original sources
PubMed1983

Role of myeloperoxidase in the respiratory burst of human neutrophils.

Myeloperoxidase (MPO), a heme enzyme present in the primary granules of polymorphonuclear leukocytes (PMNs), has been demonstrated to participate in the oxygen-dependent microbicidal activity of these cells. Evidence for the importance of MPO in this role comes in part from studies of normal PMNs treated with the heme enzyme inhibitor, sodium azide. MPO has also been suggested to regulate the respiratory activity of PMNs during phagocytosis. The role of MPO in PMN oxygen metabolism was examined by studying parameters of the respiratory burst of PMNs from a number of unrelated MPO-deficient subjects; in addition, the ability of heme enzyme inhibitors to duplicate the MPO-deficient state was studied by treating normal and MPO-deficient cells with these compounds. MPO-deficient PMNs were found to have a time-dependent hypermetabolic response as assessed by measurement of oxygen consumption, superoxide generation, hydrogen peroxide release, and hexose monophosphate shunt activity. Catabolic pathways for hydrogen peroxide were normal, suggesting the increased recovery of oxygen metabolites reflects increased production rather than decreased catabolism of H2O2. These observations support the concept that MPO may play an important role in terminating the respiratory burst of normal PMNs. The three heme enzyme inhibitors studied--sodium azide, potassium cyanide, and 3-aminotriazole--differed greatly in the degree to which they inhibited various enzymatic systems in the PMN. Nonetheless, as a group, they exerted qualitatively similar effects on oxygen metabolism of normal and of MPO-deficient PMNs. This indicates that many of the mechanisms by which heme enzyme inhibitors influence PMN metabolism are independent of the inhibition of MPO. Conclusions from studies using such treatment of PMNs should be interpreted with caution.

Catalase↗
PubMed1983

Inhibition of zymosan activation of human neutrophil oxidative metabolism by a mouse monoclonal antibody.

We have studied a neutrophil-specific murine monoclonal antibody, PMN7C3 (IgG3), which specifically alters PMN oxidative metabolism stimulated by serum-opsonized zymosan (STZ) or Candida albicans (STC). Polymorphonuclear cells (PMNs) exposed to PMN7C3 show a significant depression in O2- release (52.8% +/- 2.5% of control), H2O2 release (44.4% +/- 6.0% of control), and O2 consumption (73.9% +/- 2.6% of control) in response to STZ. O2 release in response to phorbol myristate acetate (PMA) was modestly reduced (78.4% +/- 3.7%) by PMN7C3 treatment, but not to the extent seen with STZ or STC. PMN7C3 did not affect O2 release by PMNs stimulated by zymosan opsonized with IgG or by S. aureus, A 23187, or FMLP. PMN7C3 was not cytotoxic, did not trigger oxidative metabolism when used as a stimulus, did not alter STZ-induced degranulation, and did not interfere with binding or uptake of STZ by PMNs. Exposure of PMNs to PMN7C3 decreased PMN rosette formation with erythrocytes coated with C3b (54% of control) or C3bi (63% of control), but had no affect on rosette formation with IgG-coated erythrocytes. PMN7C3 does not bind to monocytes and had no affect on rosette formation by this cell type. Binding of antibody PMN7C3 to the neutrophil surface inhibits the oxidative response to opsonized STZ or STC, possibly in part by altering the function or expression of C3b and C3bi receptors. Monoclonal antibodies such as PMN7C3 provide highly specific probes that may be used to define the molecular features of the stimulus-coupled response of PMN activation.

Antibodies, Monoclonal↗
PubMed1983

Enhancement of macrophage Fc-dependent phagocytosis by resident thymocytes: effect of a unique heat-stable lymphokine.

Lymphocytes, activated by lectins or specific antigens, have been shown to enhance macrophage phagocytosis through the elaboration of a heat-labile soluble factor(s). Recent evidence from our laboratory revealed that resident (nonactivated) murine thymocytes and splenic lymphocytes increase peritoneal macrophage glucose metabolism through the elaboration of a heat-stable soluble factor(s). Therefore, we investigated the effect of resident lymphocyte subpopulations on macrophage Fc-dependent phagocytosis. Thioglycollate-elicited and resident peritoneal macrophages from BALB/c mice were cultured in serum-free media with syngeneic resident thymocytes or splenic T lymphocytes. Macrophage Fc-dependent phagocytosis was assayed by measuring the ingestion of 51CrSHEA. After 4 days in vitro, resident thymocytes produced a mean 160 (+/- 31) and 136% (+/- 22) increase in Fc-dependent phagocytosis by thioglycollate-elicited (thio-macrophages) and resident peritoneal macrophages, respectively. Splenic T lymphocytes increased thio-macrophage phagocytosis by 112% (+/- 41) under similar conditions. Macrophage Fc-dependent phagocytosis was increased after 24 hr of co-culture by supernatant derived from resident thymocytes and could be further enhanced by supernatant from Con A-activated thymocytes. Supernatant from guinea pig embryo fibroblasts did not increase macrophage phagocytosis. The soluble factor(s) was produced by resident thymocytes after 24 hr of preculture. This factor was active despite heating at 100 degrees C for 30 min whereas the effect of Con A-activated thymocyte supernatant was heat-labile. The stimulatory effect of resident thymocyte supernatant was not observed when the macrophages and supernatant were cultured in 2% FCS. In contrast to the factor(s) produced by resident thymocytes, the factor(s) in FCS that increased phagocytosis was heat-labile. These data suggest thymocytes and splenic T lymphocytes promote macrophage Fc-dependent phagocytosis in the absence of antigenic or lectin stimulation. This previously unrecognized effect of resident thymocytes is due to a unique heat-stable soluble factor(s) that is concealed in the presence of serum.

Animals↗
PubMed1982

Genetic disorders of leukocyte function: what they tell us about normal antimicrobial mechanisms of human phagocytic cells.

Analysis of three inherited defects of granulocyte function (Chediak-Higashi Syndrome, CHS; Chronic Granulomatous Disease, CGD; Myeloperoxidase Deficiency, MPO) has highlighted critical events for the antimicrobial function of these cells and placed others in perspective. Prompt phagosomal fusion may be more important for digestion of organisms rather than killing as indicated by the mild bactericidal defects in the CHS. The formation of O2- and H2O2 during the phagocytic respiratory burst is central for the broad antimicrobial activity of granulocytes. MPO, on the other hand, while perhaps normally participating in granulocyte antimicrobial action, appears to be essential only for the effective killing of eukaryotic organisms such as certain fungal strains. While the non-oxidative killing mechanism of neutrophils have stimulated much recent interest and were the first to be defined no specific inherited defects have been discovered which are clinically important. Genetic disorders of macrophage effector function remain to be clearly defined as do those of eosinophils. The lessons learned from the study of the granulocyte defects discussed have provided both the technology and approach to the analysis of the antimicrobial and cytocidal mechanisms of these important phagocytic cells.

Blood Bactericidal Activity↗
PubMed1982

Neutrophil response and function during acute cytomegalovirus infection in guinea pigs.

The mobilization and functional characteristics of polymorphonuclear leukocytes (PMNs) at the site of an inflammatory stimulus were studied during acute cytomegalovirus infection in guinea pigs. Weanling Hartley strain guinea pigs were inoculated subcutaneously with approximately 10(6) 50% tissue culture infective doses of virulent salivary gland-passaged guinea pig cytomegalovirus. The virus was uniformly present in bone marrow, buffy coat, and casein-elicited peritoneal exudate cells 5 to 7 days after the inoculation. The mean numbers of circulating PMNs in the animals were 2,862/microliters in uninfected controls and 880/microliters in infected animals. The total peritoneal PMNs recovered 14 h after casein injection were 491 X 10(6) and 237 X 10(6) in control and infected animals, respectively. The number of 50% tissue culture infective doses of guinea pig cytomegalovirus per 10(6) purified peritoneal PMNs was 10(2.17). Neutrophil O2 consumption was similar in infected and control animals in response to either stimulation by a neutrophil activator, phorbol myristate acetate, or phagocytosis of Staphylococcus aureus. However, the maximal rate of H2O2 release and the percent killing of S. aureus by peritoneal exudate cells from infected animals were significantly reduced compared with uninfected controls during acute infection. Granulocytopenia, a decreased mobilization of PMNs to the site of the inflammatory stimulus, a diminished cellular release of H2O2 in response to a bacterial stimulus, and a modest reduction in bacterial killing were demonstrated during experimental acute cytomegalovirus infection. Such reductions in granulocyte number and function at inflammatory sites during this type of infection could alter antimicrobial defenses.

Animals↗
PubMed1981

Fungal infection in chronic granulomatous disease. The importance of the phagocyte in defense against fungi.

Among 245 cases of chronic granulomatous disease which were evaluated, fungal infection occurred in 20.4 percent. Fungi encountered include Aspergillus, Torulopsis and Candida. In 18 percent of the patients with fungal infection, the disease was limited to soft tissue or bone; all did well. Most of the patients had fungal pneumonia and/or widely disseminated disease; diagnosis was usually confirmed by open lung biopsy. Patients with pneumonia or disseminated disease who received no therapy succumbed to infection, whereas more than half the patients who received antifungal therapy were cured. Modalities of treatment included antifungal chemotherapy, surgical removal of infected tissue and granulocyte transfusion. Although several patients showed dramatic improvement during granulocyte transfusions given in combination with antifungal chemotherapy, the improvement achieved was not statistically significant when compared with that achieved with chemotherapy alone. These results emphasized the importance of phagocytic cells in defense against fungi and the need for further evaluation of granulocyte transfusion therapy in compromised hosts in whom fungal infections develop.

Adolescent↗
PubMed1981

Interactions between antibiotics and human neutrophils in the killing of staphylococci.

Normal and antibiotic-pretreated staphylococci were incubated with human neutrophils to determine the interactions between cells and antimicrobials in the killing of the organisms. Staphylococcus aureus 502A pretreated during log-phase growth with subinhibitory ((1/4) minimum inhibiting concentration) (MIC) concentrations of penicillin G were more susceptible to killing by normal neutrophils than untreated bacteria (intracellular survival 0.17+/-0.04 vs. 1.5+/-0.38%, mean+/-SEM, respectively, at 35 min in 14 experiments; P < 0.01 by t test). Furthermore, this enhanced susceptibility to killing was observed even when phagosome formation was inhibited by cytochalasin B (65.6+/-4.6% pencillintreated vs. 30.5+/-4.5% untreated killed at 30 min in 14 experiments, P < 0.001). Pretreatment of S. aureus with vancomycin similarly enhanced susceptibility to killing by cytochalasin B-treated polymorphonuclear leukocytes (PMN), whereas pretreatment with gentamicin did not. The enchancement of killing by pretreatment with cell wall-active antibiotics was present in a dose-response fashion to 1/16th the MIC. It required specific antimicrobial activity; i.e., penicillin activity was inhibited by penicillinase or by incubation with bacteria at 4 degrees C. It also required active cellular metabolism and intact neutrophils. For antibiotic-pretreated bacteria to be killed by normal and cytochalasin B-treated cells, phagocytosis or binding to the cells was essential via a serum opsonindependent mechanism. In experiments with the cytochalasin B-treated cells, all bound penicillin-treated bacteria were killed vs. only a fraction (70%) of the bound untreated bacteria. Penicillin in 10 times the MIC had no direct effects on PMN phagocytic, metabolic, or microbicidal functions against a nonsusceptible organism, Candida albicans. The results indicate a cooperative effect between cell wall-active antibiotics at low concentrations and human PMN in the killing of staphylococci. The model establishes conditions for the study of the mechanisms involved in the cooperation of these bactericidal systems.

Cytochalasin B↗
PubMed1981

Myeloperoxidase deficiency: prevalence and clinical significance.

Leukocyte differential counting by flow cytochemistry has shown 28 subjects with partial or complete neutrophil myeloperoxidase (MPO) deficiency in a population of about 60 000 patients screened at a general hospital. Partial (13 patients) or complete (13 patients) MPO deficiency was confirmed by examination of cytochemical stains in 26, biochemical measurement of total enzymatic activity in eight, and flow cytometry in six patients. None had apparent hematologic disorders. Only four patients had infections; of these, two had major systemic infections (one, candidiasis; one, bacteremia). In assays of leukocyte function only minor defects in killing of Staphylococcus aureus by MPO-deficient cells were noted whereas killing of Candida albicans was much more impaired. Family studies in eight patients have shown various degrees of partial or complete MPO deficiency in first-degree relatives of six. The findings indicate that the incidence of MPO deficiency is much higher than previously suspected. Although MPO appears to be necessary for killing of Candida species by neutrophils, the importance of its role in normal antibacterial defense must be re-evaluated.

Adolescent↗
PubMed1981

The oxidative metabolism of thioglycollate-elicited mouse peritoneal macrophages: the relationship between oxygen, superoxide and hydrogen peroxide and the effect of monolayer formation.

The oxygen and glucose metabolism of peritoneal macrophages harvested from untreated mice (resident cells) and mice given an i.p. injection of thioglycollate broth (thioglycollate cells) were examined. Thioglycollate cells consumed approximately 3 times as much O2 at rest and during phagocytosis as resident cells, but oxygen reduction products (superoxide and hydrogen peroxide) could be recovered in only minimal amounts despite triggering by phagocytosis or exposure to PMA. Indirect evidence for the formation of oxygen reduction products such as O2- by thioglycollate cells was obtained by observation of the major pathways for glucose oxidation and NBT dye reduction. When thioglycollate cells were allowed to adhere to a glass surface O2- and H2O2 were easily recovered in the extracellular medium with a 20-fold increase above cells in suspension exposed to PMA. This study suggests that thioglycollate-elicited macrophages have a vigorous oxidative metabolism but that recovery, and perhaps utilization, of O2 reduction products formed will depend on the conditions of incubation. These events may be significant both for the study of parameters of macrophage "activation" in vitro as well as the function of these cells in vivo.

Animals↗
PubMed1980

Enterococcal meningitis: combined vancomycin and rifampin therapy.

Intrathecal vancomycin and oral rifampin have been used together to successfully treat a patient with enterococcal meningitis who was allergic to penicillin and who had failed a course of treatment with chloramphenicol. This therapy was tolerated very well and represents an alternate mode of therapy which should be considered in penicillin allergic patients with enterococcal meningitis.

Administration, Oral↗
PubMed1979

Neutrophil function and host resistance.

The part played by the phagocytic cells against invading pathogens has been known since the work of Metchnikoff nearly a century ago. This review deals primarily with the role of the neutrophilic polymorphonuclear leukocyte in host defense against microbial infections. The overall function of these cells in protection from infection is dependent on a number of steps. First, an adequate number of functionally mature neutrophils have to be produced and released into the circulation by the bone marrow. Cells must circulate normally and be capable of adhering to capillary and venule walls overlying inflammatory sites. The next step involves the exit of phagocytes from the blood stream through the capillary wall and emigration into the tissues to establish contact with the invading pathogens. This process is accomplished by the locomotive characteristics of these cells and chemotaxis. Most organisms must then be phagocytized to be killed. Two discrete phases are involved in phagocytosis; the "recognition" and attachment phase followed by the ingestion phase. After phagocytosis a series of coordinated morphologic and biochemical events are set into motion which leads to eventual death and lysis of the ingested microbes. A variety of antimicrobial mechanisms are involved in this final step and indicate that these cells have an appreciable reserve capacity if one mechanism is impaired. Recent evidence which clarifies mechanisms involved in all these stages is discussed.

Animals↗
PubMed1979

The role of antibody and complement in phagocytosis by rabbit alveolar macrophages.

The relative importance of antibody and complement in the phagocytosis of Staphylococcus aureus and Pseudomonas aeruginosa, two common bacterial pathogens, by alveolar macrophages from rabbits was studied. Normal rabbit serum was a satisfactory opsonin for the phagocytosis of S. aureus but not for P. aeruginosa. Normal rabbit serum opsonized S. aureus by both the classic and the alternative complement pathways; loss of both pathways destroyed opsonic activity. The presence of complement was not required for maximal phagocytosis when 10% staphylococcal immune serum was used. However, an intact alternative complement pathway enhanced phagocytosis when the concentration of staphlyococcal immune serum was lowered to 0.3%. Similarly, 10% pseudomonas immune serum opsonized P. aeruginosa without complement. When the concentration of pseudomonas immune serum was lowered to 1%, either the classic or the alternative complement pathway could significantly enhance phagocytosis of P. aeruginosa. Similar results were obtained with alveolar macrophages activated with bacille Calmette-Guérin. These studies demonstrate the importance of complement in enhancing phagocytosis by alveolar macrophages of bacterial pathogens when antibody concentration is the limiting factor.

Animals↗