Inhibition of candidacidal activity of human neutrophil leukocytes by aminoglycoside antibiotics

We have tested the effect of five aminoglycoside antibiotics (gentamicin, sisomicin, tobramycin, ribostamycin, and amikacin) on the candidacidal activity of human neutrophils in vitro; all of them are inhibitory and can be grouped into three significantly different levels of toxicity. Gentamicin in the most toxic and sisomicin is the least toxic.     

Effect of human C-reactive protein on chemokine and chemotactic factor-induced neutrophil chemotaxis and signaling

C-reactive protein (CRP) is a unique serum pentraxin and the prototype acute phase reactant. CRP is a ligand for specific receptors on phagocytic leukocytes, and mediates activation reactions of monocytes/macrophages, but inhibits the respiratory burst of neutrophils (PMN). Since CRP selectively accumulates at inflammatory sites in which IL-8 is also produced, we tested the effects of CRP on the responsiveness of PMN to IL-8 and the bacterial chemotactic peptide, FMLP-phenylalanine (FMLPP). Purified human CRP inhibited the chemotactic response of PMN to IL-8 and FMLPP. A mouse IgM mAb that was generated against the leukocyte CRP receptor (CRP-R) also inhibited the chemotactic response. Incubation of purified CRP with activated PMN generated CRP-derived peptides that also inhibited chemotaxis. A synthetic CRP peptide (residues 27-38) that binds to the CRP-R had weak chemotactic activity, whereas two other CRP synthetic peptides (residues 174-185 and 191-205) inhibited chemotaxis of PMNs to both IL-8 and FMLPP. CRP did not alter receptor-specific binding of IL-8, but exerted its effect at the level of signaling. CRP augmented both IL-8- and FMLPP-induced mitogen-activated protein kinase (extracellular signal-regulated kinase-2) activity. CRP at acute phase levels increased both agonist-induced and noninduced phosphatidylinositol-3 kinase activity. The results suggest a role for CRP as a regulator of leukocyte infiltration at inflammatory sites.     

Alterations in peripheral blood leukocytes functions during enzootic bronchopneumonia of calves. Effect of treatment with antibiotics and immunomodulators

Foetal rat brain aggregation cultures were exposed to a single episode of anoxia and hypoglycaemia for 30 min. Lactate dehydrogenase specific activity was estimated in the culture medium after ischaemia as a marker of lost cell integrity. Release of lactate dehydrogenase was most prominent during the first 24 hr period after the ischaemic damage, then it gradually declined. Immediately after ischaemic exposure, the cultures were treated with different concentrations of L-deprenyl or tolcapone. Significantly lower amounts of lactate dehydrogenase leaked into the culture medium during the first 24 hr after the ischaemic episode in cultures treated with deprenyl or tolcapone (1-100 nM). These results suggest that deprenyl and tolcapone may reduce cell damage after ischaemia, at doses causing enzyme inhibition.     

Biochemical properties of human oral polymorphonuclear leukocytes

There is now some evidence that psychiatric disorders, such as major depression, schizophrenia and post-traumatic stress disorder are associated with significant alterations in the serum activity of peptidases, such as prolyl endopeptidase (PEP) and dipeptidyl peptidase IV (DPP IV). The aims of the present study were to examine the effects of psychological stress on serum PEP and DPP IV activity in humans. Thirty-eight university students had repeated measurements of serum PEP and DPP IV activity a few weeks before and after (baseline conditions) as well as the day before a difficult academic examination (stress condition). Subjects were divided into anxiety responders and nonresponders to stress according to their stress-induced increase in the Spielberger State Anxiety Inventory. Serum PEP activity was somewhat lowered by stress in female, but not male, students. Serum PEP activity was significantly higher in the two baseline conditions and during the stress condition in anxiety responders than in anxiety nonresponders. There were no significant effects of stress on serum DPP IV activity and no significant differences between anxiety responders and nonresponders. Serum PEP and DPP IV activity were significantly higher in men than in women. The results suggest that increased baseline serum PEP activity is related to stress-induced anxiety.     

Glucose-modified low density lipoprotein enhances human monocyte chemotaxis

In response to stimulation with immobilized anti-CD3 antibody, splenocytes from C57BL/6 and BALB/c mice principally produced INF-gamma and IL-4, respectively. However, both splenocytes equally proliferated in response to ConA. We compared the changes after inoculation with BCG (1 mg/mouse) in their capacity to produce IL-4 or IFN-gamma in response to anti-CD3 antibody and to proliferate in response to ConA. Splenocytes from C57BL/6 and BALB/c mice, that had been inoculated with BCG 4 weeks before, produced IFN-gamma with diminished IL-4 production in response to anti-CD3 antibody. Furthermore these splenocytes became anergic to ConA stimulation and died due to cell apoptosis in stead of proliferation. However, we observed the strain difference at 12 weeks after BCG-infection. BCG-primed C57BL/6 splenocytes, that continuously produced IFN-gamma in response to anti-CD3 antibody, failed to proliferate in response to ConA. In contrast, BCG-primed BALB/c splenocytes, that increased IL-4 production but decreased IFN-gamma production when stimulated with anti-CD3 antibody, could proliferate well in response to ConA. Since the splenocytes of BALB/c mice became ConA responsive along with their shifting from Th1 dominant immune response at 4 weeks to Th2 dominant immune response at 12 weeks after BCG-inoculation, IL-4 was assumed to play a crucial role in activation of anergic T cells. Therefore, we stimulated splenocytes from both strains of mice infected with BCG 4 weeks before with ConA in the presence or absence of IL-4. Splenocytes from BCG-infected BALB/c mice showed marked proliferation, while those from BCG-infected C57BL/6 mice failed. We found that IL-4 protected against ConA-induced cell apoptosis in BALB/c splenocytes but not C57BL/6 splenocytes.     

Effect of plasma, steroids, or steroid products on the adhesion of human opsonized thrombocytes to human leukocytes

An in vitro system was designed in which human thrombocytes were opsonized with rabbit antihuman platelet antibody and incubated with human leukocytes in EDTA-plasma in order to measure opsonized platelet-leukocyte adhesion. Antiplatelet antibody opsonization enhanced platelet-leukocyte adhesion 2.6-fold over control nonopsonized platelets in 50 out of 56 experiments. A plasma factor was found necessary for the support of opsonized platelet-leukocyte adhesion. This factor was EDTA-stable, heat labile, and labile to freezing and thawing. Hydrocortisone at 10-2 M was extremely effective in preventing opsonized platelet-leukocyte adhesion in 11 out of 14 experiments (2 S.D. below test-control value). Plasmas from 23 different patients ingesting 20 to 120 mg. of prednisone per day, were also tested. Seventeen out of 23 (74 per cent) did not support opsonized platelet-leukocyte adhesion at in vivo steroid concentrations of 10-6 M. Dilutions of 6 of these plasma samples with pooled normal plasma from 8- to 16-fold were still incapable of supporting opsonized platelet-leukocyte adhesion, whereas higher dilutions were capable. "Nonsteroid" plasma from 9 out of 17 (53 per cent) hospitalized "sick" patients were also incapable of supporting opsonized platelet-leukocyte adhesion. "Steroid plasma" from 5 out of 6 patients with severe thrombocytopenic purpura refractory to steroid treatment, did support leukocyte adhesion of opsonized platelets. It is concluded that a plasma factor is required for opsonized platelet-leukocyte adhesion. A steroid metabolite or byproduct factor is capable of inhibiting the adhesion of antibody-coated human platelets to leukocytes at an in vivo "steroid" concentration of 10-7 M. This factor does not appear to be present in most patients refractory to steroid treatment for autoimmune thrombocytopenic purpura.     

Effects of erythromycin on chemoattractant-activated human polymorphonuclear leukocytes

1. Erythromycin (2-100 micrograms ml-1) produced a concentration-related inhibition of superoxide generation and elastase release induced by in vitro exposure of human polymorphonuclear leukocytes (PMNs) to the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (FMLP; 30 nM). 2. By contrast, erythromycin (100 micrograms ml-1) did not alter the leukotriene B4 production elicited by FMLP (30 nM; in the presence of thimerosal 20 microM) or the intracellular calcium changes promoted by FMLP (30 nM; in the absence or presence of thimerosal 20 microM). 3. These results indicate that by reducing chemoattractant-triggered release of oxidative and proteolytic mediators from human PMNs, erythromycin may have clinically useful antiinflammatory effects.     

Sterically stabilized phospholipids attenuate human neutrophils chemotaxis in vitro

The purpose of this study was to determine whether sterically stabilized liposomes (SSL) and poly(ethylene glycol)-distearoylphosphatidylethanolamine (PEG-DSPE) attenuate polymorphonuclear neutrophils (PMNs) chemotaxis in vitro and, if so, whether incorporation of vasoactive intestinal peptide (VIP), a pleiotropic neuropeptide, on the surface of SSL amplifies SSL-induced responses. Using a modified blind-well chamber chemotaxis assay, we found that N-formyl-methionyl-leucyl-phenylalanine (FMLP; 0.1 microM) and zymosan opsonized with purified human complement (2 x 10(9) yeast wall particles/ml) elicit significant human PMNs chemotaxis (95+/-9 and 103+/-3 cells/high power field; p<0.05). These effects are significantly attenuated by SSL and PEG-DSPE (p<0.05). By contrast, aqueous VIP and VIP on SSL have no significant effects on FMLP- and zymosan-induced responses. We conclude that certain sterically stabilized liposomes and phospholipids attenuate human PMNs chemotaxis in vitro and that VIP does not modulate this response.     

Phagocytosis of human blood leukocytes: a simple micromethod

A simple micromethod for testing human blood leukocyte phagocytosis employing synthetic hydrophilic particles based on 2-hydroxyethylmethacrylate is described. The normal level of phagocytosing leukocytes in healthy children was 20.1 +/- 2.7%, and in healthy adult donors 34.3 +/- 6.1%. The method was found suitable for routine testing in both clinical and laboratory practice.     

In vitro infection of primary and retrovirus-infected human leukocytes by human foamy virus

The infectivity of human foamy virus (HFV) was examined in primary and cultured human leukocytes. Cell-free infectious viral stocks of HFV were prepared from the human kidney cell line 293 transfected with an infectious molecular clone of HFV. HFV productively infects a variety of human myeloid and lymphoid cell lines. In addition, primary cell cultures enriched for human CD4+, monocytes and brain-derived microglial cells, were readily infected by HFV. Interestingly, while infected primary CD4+ lymphocytes and microglial cells showed marked cytopathology characteristic of foamy virus, HFV-infected monocyte-derived macrophages failed to show any cytopathology. In addition, marked cytotoxicity due to HFV infection was seen in both human T-cell leukemia virus type 1- and human immunodeficiency virus type 1-infected T-cell lines and in human immunodeficiency virus type 1-infected monocytoid cell lines. Thus, HFV infection produces differential cytopathology in a wide host range of primary human leukocytes and hematopoietic cell lines.     

Further characterization of NADPH oxidase activity of human polymorphonuclear leukocytes

Mn2+ was shown to catalyze a nonenzymatic oxidation of NADPH in the presence of superoxide anion by means of an isotopic assay for measurement of the oxidation of NADPH to NADP+. Human polymorphonuclear leukocyte granule NADPH oxidase activity was evaluated in the absence of Mn2+ and was found to be higher in granules from phagocytizing cells than in granules from resting cells. The drug phorbol myristate acetate, which affects the oxidative metabolism of the neutrophil like phagocytosis, was found to activate granule NADPH oxidase activity. Superoxide dismutase was shown to inhibit NADPH oxidase activity both in the presence and absence of added Mn2+. The NADPH oxidase reaction in the absence of Mn2+ was optimal at pH 5.5, and was more linear with increasing time and protein concentration than in the presence of Mn2+. No activity was measurable in granules isolated from resting cells until the level of NADPH added was above 0.25 mM. Activity was present in granules isolated from cells challenged with opsonized zymosan, even at 0.05 mM NADPH, and was higher than the activity found in granule fractions from resting cells at all levels of NADPH tested. The addition of as little as 0.1 muM NADH to the reaction mixture was found to inhibit granular NADPH oxidase activity, indicating a possible regulatory role for NADH. These results suggest that NADPH oxidase may be the enzyme that initiates the metabolic events accompanying phagocytosis.     

Staurosporine stimulates phospholipase D activation in human polymorphonuclear leukocytes

Oropharyngeal pressure during swallowing was studied in a total of 40 healthy adult males and females in two age groups (21-27 yr and 62-75 yr). Effects of bolus volume, bolus viscosity, age, and gender were analyzed, and dry and bolus swallows were compared. The duration of the intrabolus pressure, reflecting the pressure exerted by the tongue on the bolus and preceding the generation of the pharyngeal pressure, was significantly affected by bolus volume. The duration of oropharyngeal pressure was affected by age, gender, and bolus type (bolus vs. dry swallow). Peak oropharyngeal pressure was not affected by any of the test factors, although there was a tendency for older subjects to have higher pressures than young subjects.     

Laminin and fibronectin promote the chemotaxis of human malignant plasma cell lines

We examined chemotaxis of human plasma cells (PCs) in response to extracellular matrix proteins (ECMs) in the human PC cell lines FR4ds and OPM-1ds. The FR4ds cells expressed beta 1+, beta 3-, alpha 2-, alpha 3-, alpha 4+, alpha 5+, alpha 6+, and alpha v+ integrins, whereas the OPM-1ds cells expressed beta 1+, beta 3-, alpha 2-, alpha 3+, alpha 4+, alpha 5-, alpha 6+, and alpha v+. Fibronectin (FN) and laminin (LN) promoted the chemotaxis of the PCs. An inhibitory assay with anti-integrin monoclonal antibodies (MoAbs) showed that anti-alpha 4 MoAb partially inhibited the chemotaxis of FR4ds and completely inhibited the chemotaxis of OPM-1ds. Anti-alpha 5 MoAb alone had no effect on either of these two lines. Nevertheless, anti-alpha 5 MoAb completely inhibited chemotaxis when it was added with anti-alpha 4 in FR4ds, demonstrating a novel complementary role of VLA-5 toward VLA-4 in the chemotaxis induced by FN. LN facilitated chemotaxis both in OPM-1ds expressing alpha 3 and alpha 6 integrins and in FR4ds expressing alpha 6 integrin alone. Anti-alpha 6 MoAb completely inhibited FR4ds chemotaxis, whereas anti-alpha 3 and -alpha 6 MoAb had synergistic inhibitory effects on the chemotaxis of OPM-1ds. These results indicated that the distribution of PCs in human tissue are determined by at least two factors: the concentration of the ECM proteins FN and LN and the expression of integrins.     

Effect of bacteria on chemotaxis of peripheral blood lymphocytes from nonatopic asthmatics

The chemotactic response of peripheral blood lymphocytes from nonatopic asthmatics and healthy subjects in the gradients of various bacterial strains obtained from the airways of the asthmatic patients was investigated. The dominant autologous strains were found to be effective chemoattractants for lymphocytes form the asthmatics. However, none of the bacterial strains investigated in this study induced increased motility of lymphocytes from healthy subjects. These findings might explain the mechanisms of the accumulation of lymphocytes in the airways of nonatopic asthmatics.     

Mobilizing lipocortin 1 in adherent human leukocytes downregulates their transmigration

Polymorphonuclear leukocyte (PMN) migration into sites of inflammation is fundamental to the host defense response. Activation of endothelial cells and PMNs increases the expression or activation of adhesion molecules, culminating in rolling and subsequent adherence of these cells to the vascular wall. Further activation of adherent PMNs, possibly by endothelial cell ligands, leads, within a few minutes, to extravasation itself. This process is not clearly understood, but adhesion molecules or related proteins, as well as endogenous chemokines, may play an important role. The anti-inflammatory glucocorticoids delay extravasation, which implies that an inhibitory regulatory system exists. Resting PMNs contain abundant cytoplasmic lipocortin 1 (LC1, also called annexin I)', and the activity profile of this protein suggests that it could reduce PMN responsiveness. To investigate this we have assessed neutrophil transmigration both in vivo and in vitro and examined the content and subcellular distribution of LC1 in PMNs by fluorescence-activated cell-sorting (FACS) analysis, western blotting and confocal microscopy. We report that LC1 is mobilized and externalized following PMN adhesion to endothelial monolayers in vitro or to venular endothelium in vivo and that the end point of this process is a negative regulation of PMN transendothelial passage.     

Effect of antibiotics on interferon production in mice

Neomycin, chloramphenicol, gentamycin, rifampicin and oxytetracycline were tested for their influence on virus induced interferon (IF) production in mice. Repeated intraperitoneal (i.p.) injections of antibiotics were given to produce significant serum concentrations during the time of IF production, induced by i.p. injections of Newcastle disease virus (NDV). IF titres in antibiotic-treated mice determined 6 hours after induction were compared to titres in control mice given NDV only. The IF production was not significantly modified in most of the antibiotic treated mice. Only the highest dose of chloramphenicol (2500 mug/mouse) appeared to cause a reduction in IF production (p less than 0.10). Addition of antibiotics in vitro did not alter the antiviral titres of IF.     

Quantitation of endopeptidase 24.11 and endopeptidase 24.15 in human blood leukocytes

In order to know the involvement of multiple gene alterations in the pathogenesis of human lung cancer, we examined the genes of K-, H-ras (codons 12, 13, 61), p53(exons 5-9) and the retinoblastoma susceptibility gene (RB)(exons 20-22) using the polymerase chain reaction/single-strand conformation polymorphism method in 32 human lung cancer cell lines (5 squamous-cell carcinomas, 10 adenocarcinomas, 3 large-cell carcinomas, 14 small-cell carcinomas). In 18 non-small-cell lung cancer lines, gene alterations were found in 4 for K-ras (22%), none for H-ras (0%), 4 for p53 (22%) and none for the RB (0%) gene. In 14 small-cell lung cancer (SCLC) lines, no gene alterations were found in K-ras (0%), or H-ras (0%), but 6 were found for p53 (43%) and 3 for the RB (21%) gene. Coincident abnormalities of K-ras and p53, or K-ras and RB genes were not found in any cell lines, and those of the p53 and RB genes were found in only 2 SCLC lines. No association was observed between these three gene alterations and N-myc amplification. Although the above three genes may be involved to some extent in the pathogenesis of lung cancer, more factors are required for its development.     

Stress-associated modulation of proto-oncogene expression in human peripheral blood leukocytes.

Changes in the cellular immune response associated with psychological stress were studied by using an academic stress model with medical students. The authors examined the expression of 2 proto-oncogenes, c-myc and c-myb, in peripheral blood leukocytes (PBLs) obtained from medical students at the time of examinations and at a baseline period approximately 1 mo prior to the examinations. The level of messenger ribonucleic acid (mRNA) expression of both proto-oncogenes was significantly lower in PBLs obtained during examinations than in those from the baseline period. In addition, a significant decrease in the level of mRNA to the glucocorticoid receptor and gamma interferon was also found in the same preparations. The decrease in mRNA content of c-myc, c-myb, the glucocorticoid receptor, and gamma interferon in PBLs obtained from Ss during examinations is consistent with data from previous studies using the same model that have demonstrated a down-regulation of T-lymphocyte activation and proliferation response to mitogens. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Receptor-mediated activation of multiple serine/threonine kinases in human leukocytes

Activation of the microbicidal response of phagocytes requires cytosolic ATP and is associated with extensive protein phosphorylation, suggesting the involvement of protein kinases in the signal transduction cascade. An in vitro renaturation assay was used to identify the protein kinase(s) activated by chemoattractants in human blood neutrophils. Four distinct kinases were activated by the chemotactic peptide formyl-methionyl-leucyl-phenyl-alanine with molecular masses of 72, 65, 49, and 41 kDa (designated PK72, PK65, PK49, and PK41, respectively). PK72 and PK65 were activated very rapidly (5-15 s), yet transiently. By comparison, PK49 and PK41 responded in a slower, more sustained manner. Treatment of extracts of activated cells with alkaline phosphatase reverted the stimulation of the kinases, suggesting that phosphorylation is the post-translational modification that underlies activation of the kinases. Stimulation of PK72 and PK65 by chemoattractant was independent of calcium and protein kinase C. In contrast, elevation of cytosolic free calcium levels was sufficient and appeared to be necessary for full activation of PK49 and PK41. While phorbol esters can mimic the effects of formyl-methionyl-leucyl-phenylalanine on PK49 and PK41, inhibition of protein kinase C by staurosporine did not prevent the receptor-mediated activation of these kinases. PK41 most likely corresponds to the Erk-1 isoform of mitogen-activated protein (MAP) kinase. Accordingly, PK41 effectively phosphorylated myelin basic protein, known to be a good substrate for Erk-1. The electrophoretic mobility of PK49 is similar to that of MAP kinase-kinase (MAP/Erk kinase). However, immunoprecipitation experiments indicated that PK49 is not MAP/Erk kinase. The identity of this and other kinases remains to be defined, but possible candidates are discussed. In addition to autophosphorylating, PK72, PK65, and PK41 were shown to effectively phosphorylate exogenous substrates. These kinases may therefore play a role in signal transduction during stimulation by chemoattractants.