Killing of ofloxacin-exposed pneumococci by human neutrophils.
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Blood Bactericidal Activity↗
Biomedical subjects
A Kharazmi
Publications and source records attributed to A Kharazmi.
Cellular and humoral immune responses in a population from the Baringo District, Kenya to Leishmania promastigote lipophosphoglycan.
In a cross-sectional house-to-house study in a leishmaniasis-endemic area in Kenya, the cellular and humoral immune response to Leishmania lipophosphoglycan (LPG) was determined. Clinical data, peripheral blood mononuclear cells, and plasma were obtained from 50 individuals over the age of eight years. Lymphoproliferation and interferon-gamma (IFN-gamma) production by these cells were examined. It was shown that cells from all six individuals in the population with a history of kala-azar responded to LPG in the lymphocyte proliferation assay, and four of these six responded in the IFN-gamma assay. In contrast, cells from 12 of 44 individuals from the study area with no history of kala-azar and none of the five Danish control samples responded to LPG. Antibodies against LPG were detected by enzyme-linked immunosorbent assay in 45 of 50 plasma samples. Our findings clearly show that mononuclear cells from kala-azar patients cured of infection were able to respond to the LPG preparation. The finding of a specific cellular immune response to LPG in 12 of 44 individuals with no history of kala-azar is consistent with previous epidemiologic studies, in which it has been shown that a proportion of L. donovani infections run a subclinical course. The high frequency of individuals with antibodies against LPG might indicate that a majority of the population had been exposed to the parasite.(ABSTRACT TRUNCATED AT 250 WORDS)
Adolescent↗
Some bacterial parameters influencing the neutrophil oxidative burst response to Pseudomonas aeruginosa biofilms.
Persistence of bacteria in spite of a normal host immune system and relevant antibiotic treatment is a key problem in many chronic infections, such as the bronchopulmonary P. aeruginosa infection in cystic fibrosis patients. The capability of bacteria to establish themselves in microcolonies or biofilms is an important protective mechanism of the microorganisms. We examined the human PMN oxidative burst response to P. aeruginosa in biofilm and in planktonic form. The PMN chemiluminescence response to P. aeruginosa in biofilms was reduced to 30.5-47.5% (p less than 0.04) and the superoxide response to 85.9% (p less than 0.02) of the response to equivalent numbers of planktonic bacteria. Mechanical disruption of the biofilms before the assays elicited a significantly increased response in the chemiluminescence experiments and to nonopsonized biofilms in the superoxide anion experiments. We conclude that biofilm bacteria, although able to stimulate the PMN, result in a reduced, suboptimal response leading to lack of efficient eradication of the bacteria in the chronic infection.
Cystic Fibrosis↗
Computer-assisted image analysis assay of human neutrophil chemotaxis in vitro.
We have developed a computer-based image analysis system to measure in-filter migration of human neutrophils in the Boyden chamber. This method is compared with the conventional manual counting techniques. Neutrophils from healthy individuals and from patients with reduced chemotactic activity were used. The cells migrating into the filter were counted automatically at depths 20 microns apart commencing from the upper surface on the filter. The major advantages of this method are reproducibility and the counting of many fields which provides better data for statistical analysis of the results. Another advantage of the assay is that it can be used to show the migration pattern of different populations of neutrophils from both healthy individuals and patients.
Chemotaxis, Leukocyte↗
Eosinophil protein X/eosinophil derived neurotoxin (EPX/EDN). Detection by enzyme-linked immunosorbent assay and purification from normal human urine.
Eosinophil protein X/eosinophil derived neurotoxin (EPX/EDN) is one of the cationic proteins found in the granules of the human eosinophilic granulocytes. EPX was purified from extracts of granules isolated from blood buffy coat cells of healthy donors. Polyclonal anti-EPX antibodies were subsequently raised in rabbits. The anti-EPX-antibodies raised in rabbits showed no reactivity with other proteins in the granule extract. The sandwich ELISA utilized the biotin/avidin amplification system and measured EPX over the range of 60-2000 pg/ml. The intra- and interassay coefficients of variation were 6.5% and 8.2%, respectively, and the mean recoveries of 25 and 50 pg of purified EPX added to diluted serum samples were 106 +/- 16% (mean +/- SD; n = 12) and 112 +/- 14%, respectively. Using this assay we found high amounts of EPX in normal human urine (U-EPX). U-EPX was purified by a two step procedure involving affinity chromatography on heparin Sepharose and size exclusion chromatography on Sephadex G-50 superfine. Extracted EPX and U-EPX had ribonuclease activity and comigrated on agarose electrophoresis. They also showed immunological identity when evaluated with rabbit anti-EPX antibodies, but they differed slightly on SDS-PAGE probably due to differences in glycosylation. Our results support the findings that EPX/EDN is identical to a nonsecretory ribonuclease isolated from urine.
Blood Proteins↗
Detection of eosinophil cationic protein (ECP) by an enzyme-linked immunosorbent assay.
Eosinophil cationic protein (ECP) is a highly basic and potent cytotoxic single-chain zinc-containing protein present in the granules of the eosinophilic granulocytes. ECP appears to be involved in defence against parasites and in the tissue damage seen in subjects with allergic and inflammatory disease. To investigate ECP release from in vitro activated human eosinophils and to study the involvement of eosinophils in health and disease, we have developed a sensitive and specific enzyme immunoassay. ECP was purified from normal human peripheral blood eosinophils and polyclonal antibodies to ECP were subsequently raised in rabbits. The ELISA utilizes the biotin/avidin method and measures ECP within the range 15-1000 ng/l. The intra- and interassay coefficients of variation were 6% and 10%, respectively, and the recoveries of 12 and 25 pg of purified ECP added to diluted serum samples were 108 +/- 14.5% (mean +/- SD, n = 12) and 107 +/- 7.5%, respectively. The high sensitivity, reproducibility and specificity of this ELISA makes it suitable for the determination of minute amounts of ECP in in vitro systems as well as in various biological fluids.
Blood Proteins↗
Relationship between chemical composition and biological function of Pseudomonas aeruginosa lipopolysaccharide: effect on human neutrophil chemotaxis and oxidative burst.
There are conflicting data on the effect of bacterial lipopolysaccharides (LPS) on the function of human neutrophils. The present study was designed to examine the relationship between chemical composition and the modulatory effect of LPS on human neutrophil function. LPS was extracted from five strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients by the hot phenol-water method. Chemical characterization included neutral sugars, amino components, and fatty acids. Neutrophils isolated from peripheral blood of healthy individuals were preincubated with different concentrations of LPS. After preincubation, the chemotaxis and chemiluminescence of neutrophils to various stimuli were determined. It was shown that LPS from different strains did not exert the same degree of regulatory effect on neutrophil functions. LPS from strain 174-O:9 exerted the most pronounced effect on neutrophil function seen as inhibition of neutrophil chemotaxis toward the chemotactic peptide f-Met-Leu-Phe and zymosan-activated serum (ZAS) and priming of the cells for less than or equal to 8-fold enhancement of chemiluminescence response to f-Met-Leu-Phe. Conversely, LPS from strain 1118-O:3 had no effect on neutrophil chemotaxis and a slight effect on chemiluminescence. The major differences in chemical composition of the LPS from these two strains are in the rhamnose and heptose content of the O side chain and in the alanine content of the core region. These data indicate that chemical composition of the LPS molecule may play an important role in biological activity of LPS.
Chemotaxis, Leukocyte↗
Mechanisms involved in the evasion of the host defence by Pseudomonas aeruginosa.
Pseudomonas aeruginosa, an extracellular opportunistic pathogen, utilizes two major mechanisms to evade the host defence system. One of these mechanisms is the production of a large number of extracellular products, such as proteases, toxins, and lipases. The two proteases, alkaline protease and elastase, inhibit the function of the cells of the immune system (phagocytes, NK cells, T cells), inactivate several cytokines (IL-1, IL-2, IFN-r, TNF), cleave immunoglobulins and inactivate complement. Inhibition of the local immune response by bacterial proteases provides an environment for the colonization and establishment of chronic infection. The other mechanism by which P. aeruginosa evades the host defence system is the biofilm mode of growth of the bacteria in chronic infections. The biofilm-grown bacteria induce a low phagocyte response, and provide a barrier for the bacteria against antibodies, complement, and the cells of the immune system. Protection from the host defence system combined with increased antibiotic resistance of the bacteria in the biofilm are the major reasons for the persistence of P. aeruginosa in chronic infections.
Animals↗
Extracellular lipase of Pseudomonas aeruginosa: biochemical characterization and effect on human neutrophil and monocyte function in vitro.
Lipase was isolated from P. aeruginosa by ultrafiltration of sterile-filtered culture supernatant. Gel filtration on Sepharose 4B yielded a broad peak corresponding to a molecular mass range of 100 to 1000 kDa. Electron microscopy of a negatively stained lipase preparation after Sepharose 4B chromatography revealed spherical particles with diameters ranging from 5 to 20 nm. Biochemical characterization and SDS polyacrylamide gel electrophoresis suggested that these particles consisted of protein and carbohydrate including lipopolysaccharide with the major enzyme activity being lipase. Various concentrations of this lipase preparation were preincubated with human peripheral blood neutrophils and monocytes. The chemotaxis and chemiluminescence of these cells were then determined. It was shown that lipase inhibited the monocyte chemotaxis and chemiluminescence, whereas it had no or very little effect on neutrophils. The inhibitory effect was concentration dependent and was abolished by heat treatment of the enzyme at 100 degrees C. Since monocytes are one of the important cells of the host defence system the inhibition of the function of these cells may contribute to the pathogenesis of infections caused by P. aeruginosa.
Cell Survival↗
Modulation of neutrophil and monocyte function by recombinant human granulocyte macrophage colony-stimulating factor in patients with lymphoma.
Granulocyte macrophage colony-stimulating factor (GM-CSF) has been shown to inhibit the chemotaxis and enhance the oxidative burst response of human neutrophils in vitro. The present study describes the effect of recombinant GM-CSF on the neutrophil and monocyte function in patients with lymphoma undergoing GM-CSF treatment. Patients with either Hodgkin's or non-Hodgkin's lymphoma were treated with various dosages (2-16 micrograms kg-1 body weight per day for 5 days) of rhGM-CSF by intravenous or subcutaneous route. Prior to and on day 5 of rhGM-CSF treatment, neutrophil and monocyte chemotaxis and chemiluminescence responses to f-Met-Leu-Phe, zymosan activated serum (ZAS) and opsonized zymosan (OZ) were determined. It was observed that chemotactic response of neutrophils to f-Met-Leu-Phe and ZAS was reduced, whereas the chemiluminescence response of both cell types to f-Met-Leu-Phe and zymosan was enhanced by up to 43-fold. rhGM-CSF treatment did not affect degranulation of the neutrophils as measured by release of vitamin B12 binding protein. Degree of modulation of neutrophil and monocyte function by rhGM-CSF was independent of rhGM-CSF dosages administered. These data suggest that phagocytic defence system may be enhanced by GM-CSF treatment and that this cytokine may be a useful therapeutic adjunct in compromised patients.
Cell Degranulation↗
Activation of human T lymphocytes by Leishmania lipophosphoglycan.
This study describes Leishmania antigen-induced activation of lymphocytes isolated from Kenyan donors, previously treated for visceral leishmaniasis, and from Danish and Kenyan controls. Peripheral blood mononuclear cells (PBMC) from cured Kala-Azar patients proliferated and produced Interferon-gamma in vitro in response to lipophosphoglycan (LPG) isolated from Leishmania major. The proliferative response was mainly due to activation of CD2-positive T cells. PBMC from controls did not respond to LPG, but to sonicates prepared from both L. major and L. donovani promastigotes. The surface glycoprotein GP 63 failed to activate PBMC from any of the donors tested. These results show that the individuals cured from visceral leishmaniasis had expanded T-cell clones recognizing LPG, conceivably as a result of Leishmania infection. The LPG preparation was without detectable protein contamination. Thus, the results suggest that human T lymphocytes can respond to glycolipid antigens.
Animals↗
Induction of beta-lactamase production in Pseudomonas aeruginosa biofilm.
Imipenem induced high levels of beta-lactamase production in Pseudomonas aeruginosa biofilms. Piperacillin also induced beta-lactamase production in these biofilms but to a lesser degree. The combination of beta-lactamase production with other protective properties of the biofilm mode of growth could be a major reason for the persistence of this sessile bacterium in chronic infections.
Culture Media↗
Inhibition of human monocyte chemotaxis and chemiluminescence by Pseudomonas aeruginosa elastase.
The in vitro effect of Pseudomonas aeruginosa elastase on human monocyte function was examined. Mononuclear cells isolated from the peripheral blood of healthy individuals were incubated with various concentrations of elastase, and the chemotactic activity and chemiluminescence response of these cells were measured after washing the cells free of the enzyme. Elastase at concentrations of 0.1 micrograms/ml and higher inhibited the chemotactic activity of monocytes towards F-Met-Leu-Phe and zymosan-activated serum. The inhibition of chemiluminescence response was observed at as low as 0.125 micrograms/ml elastase. Monocytes/macrophages are known to be involved in the inflammation observed in the lungs of patients infected by P. aeruginosa. The inhibition of monocyte function by elastase may serve as an escape mechanism for this bacterium from these cells.
Bacterial Proteins↗
Phagocyte function after splenic autotransplantation.
This study was designed to examine the role of splenectomy and autotransplantation with regard to the leukocyte/differential cell counts and the function of peripheral blood phagocytes. Eleven groups of 40 Wistar male rats in each group either underwent total splenectomies or sham operations. The splenectomized groups underwent autotransplantations with 10% through 90% of the weight of the intact spleen. The leukocyte count and the oxidative burst response of the blood leukocytes were measured in each group. It was shown that a total splenectomy did not alter the leukocyte/differential cell counts. Furthermore, the blood picture remained basically unchanged after an autotransplantation with 10% through 90% of the weight of the intact spleen. The phagocyte oxidative burst response was measured by chemiluminescence. The chemiluminescence response of these cells was reduced after a total splenectomy. The phagocyte oxidative burst response returned to normal levels following an autotransplantation. There was no correlation between the amount of autotransplanted spleen and the degree of the oxidative burst response. These findings indicated that a splenectomy results in a diminished phagocyte oxidative burst response and that a spleen autotransplantation returns this function to normal levels.
Animals↗
Indomethacin in vitro and in vivo abolishes post-exercise suppression of natural killer cell activity in peripheral blood.
The present study was designed to explain the mechanism of the post-exercise down-regulation of human natural killer (NK) cell activity recently described by us. Fifteen young, healthy volunteers underwent 60 min of bicycle exercise at 75% of maximal oxygen uptake (VO2max). Six of the volunteers were exercised twice with an interval of at least one month. At the second exercise test they received oral indomethacin. Blood samples were collected before and during the last minutes of exercise as well as 2 h and 24 h after work. The NK cell activity (lysis fixed number of mononuclear cells) increased during bicycle exercise, dropped to a minimum 2 h later and returned to pre-exercise levels within 24 h. During bicycle exercise the percentage of NK cells (CD16+ cells) of mononuclear cells increased significantly but returned to normal within 2 h after exercise. Two hours after exercise, however, increased monocyte cell count and neutrophils were found. The in vitro release of prostaglandin E2 from mononuclear cells was increased. Furthermore, the neutrophil chemiluminescence response was also increased in the 2 h post-exercise period; this response is associated with prostaglandin E2 production by neutrophils. Indomethacin, whether administered in vivo or in vitro, fully restored the suppressed post-exercise NK cell activity. Finally, the NK cell activity of monocyte depleted mononuclear cells did not decrease below basal levels after exercise. These findings strongly indicate that prostaglandins released from monocytes and neutrophils are involved in the post-exercise down-regulation of NK cells.
Adult↗
Functional and phenotypic changes in human lymphocytes after coincubation with Leishmania donovani in vitro.
In this paper we describe functional and phenotypic changes in T cells after in vitro coincubation of peripheral blood mononuclear cells (PBMC) and Leishmania donovani parasites at different parasite/peripheral blood mononuclear cell ratios. The phytohemagglutinin (PHA)-induced lymphoproliferative response was reduced by the coincubation, and at the maximal parasite/peripheral blood mononuclear cell ratio used (7.5:1), the average response was less than 40% of the response in the absence of parasites. The cause of the reduction in lymphoproliferation is not clear, but it requires live parasites. Interleukin-1 production was unaffected, the levels of soluble interleukin-2 receptor in supernatants were not changed by the coincubation, and the addition of exogenous interleukin-2 failed to revert the suppressive effect of the parasites. In addition to the reduction in lymphocyte proliferation, phenotypic lymphocyte changes were observed. Cell surface expression of the CD3 antigen, which is part of the CD3-T-cell receptor complex, was significantly reduced with increasing parasite/peripheral blood mononuclear cell ratios; the reduction was general in the sense that the parasites caused a shift in the fluorescent intensities of anti-CD3 labeled cells toward lower values, without affecting the distribution pattern. In contrast, the parasites altered the CD25 (interleukin-2 receptor) expression on PHA-stimulated cells from a homogenous CD25-positive population to two populations, one small and without CD25 expression and the other, larger population with only a slight reduction in size and CD25 expression. In addition to the changes in expression of surface antigens, a general reduction in the size of PHA-stimulated lymphocytes after coincubation with the parasites was observed. The data presented thus suggest that the inhibition of the proliferative response to PHA by live L. donovani in vitro is associated with early processes in lymphocyte activation. Further studies on the inhibitory phenomena described may be of potential significance in the investigation of the suppressive mechanisms in human visceral leishmaniasis.
Animals↗
Pseudomonas aeruginosa alginate in cystic fibrosis sputum and the inflammatory response.
Alginate, a viscous polysaccharide from mucoid Pseudomonas aeruginosa, may interfere with the host defenses in patients with cystic fibrosis and chronic P. aeruginosa lung infection. The alginate concentration in the sol phase of expectorated sputum was quantitated by a biochemical method and a newly developed enzyme-linked immunosorbent assay. There was a high degree of correlation between the methods, and the concentration of alginate ranged from 4 to 101 micrograms/ml with a median of 35.5 micrograms/ml when measured by enzyme-linked immunosorbent assay. Alginate could not be detected in the bronchial secretions from patients without P. aeruginosa infection. In vitro investigation of alginate did not show any activation of the alternative pathway of complement, as determined by a hemolytic kinetic assay and by testing for neutrophil chemotaxis. At a high concentration, P. aeruginosa alginate caused a slight activation of the classical pathway of complement. Alginate did not cause neutrophil chemotaxis by itself but was able to reduce the neutrophil chemotactic response to N-formylmethionylleucylphenylalanine and for zymosan-activated serum. P. aeruginosa and seaweed alginates were able to prime neutrophils for increased N-formylmethionylleucylphenylalanine-induced neutrophil oxidative burst, as determined by chemiluminescence. Because of its ability to prevent attraction of neutrophils to the site of infection, lack of complement activation, and ability to enhance neutrophil oxidative burst, alginate from P. aeruginosa may contribute to the persistence and pathogenesis of chronic P. aeruginosa infection in cystic fibrosis.
Alginates↗