ABSTRACT
BACKGROUND: About 10% of pre-school children has recurrent respiratory infections (RRI), which could be related to environmental and/or immunological factors. The aim of the present study has been to evaluate the impact of phagocytosis (FAG) and reactive oxygen intermediates (ROI) production deficiencies on pediatric RRI by the measurement of FAG and ROI activity of the polymorphonuclear neutrophils. METHODS: Serum immunoglobulins, IgG subclasses, lymphocytic subpopulations, FAG and ROI tests were measured in 90 children with RRI, in a moment of well-being and off all medications for at least 4 weeks. FAG and ROI tests were also measured in 19 healthy children. RESULTS: FAG (91.4 +/- 11.5%) and ROI (81.8 +/- 17.5%) of patients resulted in significantly decreased measurements compared to the control values (95.2 +/- 1.8% and 89.7 +/- 4.8%, respectively). No significant difference was manifest between the mean values of FAG and ROI tests among the patients when they were divided for age (above and below 6 years). A significant decreased likelihood of abnormal ROI (odds ratio, 0.3; 95% confidence interval, 0.07-0.97) was found in the patients with low IgA. CONCLUSIONS: The authors' results permit only to suppose an etiological role of FAG and ROI deficiencies of polymorphonuclear neutrophils in the genesis of pediatric RRI, irrespective of the age of the patients, and further studies are necessary for confirmation.
Subject(s)
Phagocytosis , Respiratory Tract Infections/physiopathology , Adolescent , Breast Feeding , Child , Child, Preschool , Female , Humans , Infant , Lymphocyte Subsets , Male , Odds Ratio , Reactive Oxygen Species/analysis , Recurrence , Respiratory Tract Infections/immunology , SmokingABSTRACT
Borrelia burgdorferi is the causative agent of Lyme disease. We investigated whether the in vitro co-cultivation of lymphocytes with spirochetes would induce apoptosis in human lymphocytes. Peripheral blood mononuclear cell were mixed with various ratio of cell/spirochetes (1:10, 1:20, 1:50, 1:100) and incubated in a humified atmosphere of 5% CO(2) at 37 degrees C. Apoptosis was determined at 0, 4, 24 h by Annexin V binding assay and propidium iodide staining, and by CD95 Apo-1 expression. Analysis was performed by multiparametric flow cytometry on CD3, CD4, CD8, CD19 subset of lymphocytes. The binding of Annexin V increased at 24 h in T lymphocytes infected by living spirochetes at ratio 1:50; similar results were obtained with inactivated or sonicated spirochetes and lipidated OspC. The rate of Annexin V binding and pattern of CD95 over-expression were different in CD3, CD4, CD8 and CD19 subset; interleukine-10 (IL-10) was measured in supernatants of cultures after treatment with Borrelia preparations and with OspA and OspC, lipidated or not. Our data suggest that spirochetes were able to induce apoptosis on lymphocytes; the phenomenon appears associated with number of spirochetes, incubation time and the release of IL-10 in co-cultures. Moreover apoptosis was probably Fas-mediated and the cells involved were prevalently CD4.
Subject(s)
Antigens, Bacterial , Apoptosis , Borrelia burgdorferi/growth & development , Borrelia burgdorferi/pathogenicity , Lipoproteins , Lymphocytes/microbiology , Lymphocytes/physiology , Annexin A5/metabolism , Antibodies, Monoclonal/metabolism , Antigens, Surface/metabolism , Bacterial Outer Membrane Proteins/metabolism , Bacterial Vaccines , Cell Culture Techniques , Cells, Cultured , Flow Cytometry , Humans , Interleukin-10/biosynthesis , Leukocytes, Mononuclear/microbiology , Leukocytes, Mononuclear/physiology , Lymphocyte Subsets/cytology , Lymphocyte Subsets/microbiology , Lymphocyte Subsets/physiology , Lymphocytes/cytology , Propidium/metabolism , fas Receptor/analysis , fas Receptor/immunologyABSTRACT
BACKGROUND: Phagosomal pH is thought to play an important role in the antimicrobial activity of polymorphonuclear leukocytes (PMNs). In this study, we set up a method for a rapid and accurate measurement of phagosomal pH in PMNs with the use of Candida albicans doubly labeled with a pH-insensitive and a pH-sensitive probe and flow cytometry. METHODS: Heat-killed, serum-opsonized C. albicans were doubly labeled with fluorescein, a pH-sensitive probe, and rhodamine, a pH-insensitive probe, and incubated with human PMNs. Flow cytometric readings of PMN-associated Candida were then taken, and the intraphagosomal pH was calculated on the basis of the ratio of fluorescein:rhodamine fluorescence by using a calibration curve obtained after equilibration of phagosomal pH with different external pH values after addition of digitonin. RESULTS: A rapid rise in phagosomal pH, which reached pH 7.8, was observed 2 min after initiation of phagocytosis and progressively declined to pH 6.9 after 15 min. Such a rise was not observed in PMNs with defective microbicidal activity (deficient in nicotinamide adenine dinucleotide phosphate oxidase), where phagosomal pH dropped to pH 6.6, 2 min after phagocytosis. The abnormal initial acidification in PMNs deficient in nicotinamide adenine dinucleotide phosphate oxidase was prevented by using lysosomotropic weak bases or the vacuolar-type H(+) pump inhibitor concanamycin A. CONCLUSIONS: Phagosomal pH of PMNs can be easily and accurately measured by dual fluorescence flow cytometry. The method can be applied to assess phagosomal pH in PMNs with defective microbicidal activity and to monitor the outcome of pharmacologic interventions aimed at correcting its abnormalities.
