Pattern of formylmethionyl-leucyl-phenylalanine-induced luminol- and lucigenin-dependent chemiluminescence in human neutrophils.

The stimulation of neutrophils by formylmethionyl-leucyl-phenylalanine results in a bimodal luminol-dependent chemiluminescence pattern. We observed, however, only a single peak chemiluminescence pattern in the response to the peptide when we used lucigenin as an amplifying substance. We suggest that lucigenin, the larger molecule, (510 daltons; luminol is 177 daltons) only exerts an extracellular effect.     

In vitro defects of phagocyte chemotaxis during pregnancy.

Pregnant women have an increased risk for some infections, particularly during the last trimester. Phagocytic emigration from the circulation into tissues is an important aspect of the initial immune response. Therefore, circulating phagocytes of 42 pregnant and 15 postpartum patients were studied in vitro for random and chemotactic (or directional) migration through membrane filters (Millipore Corp., Bedford, Mass.). Random migration of phagocytes from all 42 pregnant patients studied in each trimester was within normal limits. Chemotactic migration of 25 patients who were between 6 and 33 weeks of pregnancy was also similar to values obtained with control leukocytes (20 nonpregnant, normal females. However, phagocytes of 17 other women studied between week 34 of pregnancy and term showed marked depressions in chemotaxis (P less than 0.001 from control values). During labor and within 3 days of delivery, chemotactic migration increased to supranormal levels in 14 of 15 women studied. Sera from six pregnant patients with proven chemotactic defects did not reduce migration when incubated with normal phagocytes. These chemotactic defects appear to be intrinsic and may be important in predisposing to infections during late pregnancy.     

In vitro determination of anticryptosporidial activity of phytogenic extracts and compounds

Cryptosporidiosis caused by Cryptosporidium spp. is an important diarrhoeal disease observed in farm animals and humans, especially in young or immunocompromised individuals. A novel cell culture assay for testing extracts and pure compounds against Cryptosporidium parvum in 96-well microplate format was established and evaluated. It is based on previously described indirect fluorescent antibody techniques and was optimised for higher sample throughput. Rapid assessment of minimal inhibitory concentrations (MICs) was done by checking each well microscopically for the presence or absence of parasite stages. As a novelty, parasite development was quantified by enumeration of clusters of secondary infection (CSI), which typically appeared upon infection with a distinct parasite inoculum after a defined incubation time. Host cell (HCT-8) viability was measured by an integrated non-destructive water-soluble tetrazolium salt assay (WST-1), which facilitated discrimination of antiparasitic activity from possible cytotoxic effects of a test compound against the host cells. Host cell viability was regarded unimpaired when cultures had 75% or more viability when compared to control cultures without test substance. In this study, a maximum density of distinguishable CSI was obtained when cultures were infected with 2.5 × 10(3) oocysts and incubated for 48 h. The applicable inoculum has to be optimised for each batch of oocysts and before each experimental series. Parasite development was inhibited completely by monensin at 134 nM and silymarin at 50 mg/mL. These concentrations were non-toxic to the host cells and comparable to literature data. The percentages of parasite inhibition were determined for monensin and a 50% inhibitory concentration (IC(50)) of 36.6 nM (27.4-45.5) and a 90% inhibitory concentration of 65.9 nM (54.8-90.2) were calculated. The introduced assay is economic because relatively low parasite numbers may be used. If MICs are determined, evaluation is fast, as each well is viewed only briefly under the fluorescence microscope for presence or absence of CSI. Furthermore it is highly critical because only full parasite inhibition is assessed. Counting of CSI is more laborious and time-consuming, but it allows calculation of parasite inhibition rates and parameters like the half maximal inhibitory concentration (IC(50)). This assay shall be used to assess anticryptosporidial activities of various plant waste materials and by-products from the food and the pharmaceutical industries in the course of the EU project SAFEWASTES. Comparison with in vivo models should be performed to further corroborate the results. Automated evaluation by flow cytometry might facilitate higher sample throughput and reduce operator bias.     

In vivo andin vitro HeNe laser effects on phagocyte functions

The goal of this work was to evaluate the effect of helium-neon (HeNe) laser irradiation on immunocompetent cells. We used the in vivo skin window method and in vitro granulocyte function tests. The study of cellular migration showed a marked decrease in vitro and in vivo in a dose-independent manner. Superoxide release was not modified by laser irradiation. The granulocyte's aggregation, when using PHA and PMA, presented a reduction that was statistically very significant, not as a subordinate dose. An increase of the release of ATP was demonstrated only at 4 joules and precedes granulocyte aggregation. When using Ca²⁺ ionophore A23187 as stimulus, laser irradiation at 1, 2 or 4J did not show any modification of granulocyte aggregation. The monoclonal antibody 60.1, which identifies a membrane antigen fundamental for aggregation and chemotaxis, is expressed in normal amounts on granulocyte membranes both before and after irradiation with a HeNe laser. In fact, laser irradiation preferentially attacks the area of the cellular centrosome that determines a modification of cellular morphology. The electron microscope and immunofluorescence study with a monoclonal antibody have pointed out a disorganization of the microtubles. The alteration of some of the granulocyte functions is correlated to the damage in the centrioles. The granulocyte mitocondrial system and surface membrane remain intact, and this explains the normal production and release of free radicals. Further experiments are necessary to evaluate the clinical application of lasers in various diseases with immunophagocytic pathogenesis.     

Measurement of phagocyte chemiluminescence in a microtitre plate format

Previously described assays of phagocyte chemiluminescence have required large numbers of cells and have not been able to follow responses from a large number of samples in single experiments. Recently, sensitive luminometers which employ a 96 well microtitre plate format have become available. We describe the application of this equipment to the measurement of phagocyte chemiluminescence using lucigenin to enhance the response and the estimation of the opsonic activity of serum. It was found that as few as 5 X 10(4) cells (polymorphonuclear leukocytes or monocytes) per well and a ratio of 10:1 zymosan particles to cells gave good results when opsonised with 10% whole serum. This method allows assays of opsonic activity to be performed in triplicate on large numbers of sera with a relatively small number of phagocytes and should aid the investigation of the role of opsonisation in infectious disease.     

In vitro activity of florphenicol

Florphenicol was active at a lower concentration than chloramphenicol against over half of 234 recent clinical bacterial isolates. The majority (98 %) of the isolates were inhibited by florphenicol at a concentration of 8 mg/l or less. Florphenicol was particularly effective against chloramphenicol resistant strains ofHaemophilus influenzae, Klebsiella aerogenes andBacteroides spp. Florphenicol was bacteristatic for salmonellae andEscherichia coli but bactericidal forHaemophilus influenzae. Florphenicol was slightly more active than chloramphenicol againstChlamydia trachomatis, Mycoplasma hominis andMycoplasma pneumoniae but less active againstUreaplasma urealyticum.     

Effect of neutrophil activating substances on intracellular generation of phagocyte chemiluminescence by means of luminol-bound microspheres

The intracellular chemiluminescence of granulocytes was measured by luminol-bound microspheres. After one minute of incubation, which was necessary for the granulocytes to phagocytize microspheres, intracellular chemiluminescence was generated. The intracellular chemiluminescence was enhanced by the introduction of granulocyte stimuli including concanavalin A, phorbol-myristate acetate, and formyl peptides. This incremental effect was the result of certain metabolic changes inside the cells. This newly discovered chemiluminescent reagent promises to be a useful tool for examining metabolic changes inside phagocytic cells.     

Dimethyl sulfoxide inhibits phagocyte influx into infected pleural spaces and phagocyte locomotion in vitro

Influx of polymorphonuclear leukocytes (PMNs) and monocytes (MNs) into pleural spaces was decreased in dimethyl sulfoxide (DMSO)-treated rabbits infected intrapleurally with Staphylococcus aureus. In addition, pleural fluids contained S. aureus longer and marked pleural thickening with fibrosis occurred in DMSO-treated rabbits. DMSO also inhibited stimulated locomotion of PMN and MN in vitro, suggesting that the aforementioned responses in vivo may have occurred because of DMSO-mediated inhibition of the locomotion of PMN and MN.     

In vitro inhibition of natural killer cell activity by doxycycline

The effect of four tetracyclines, tetracycline, rolitetracycline, oxytetracycline and doxycycline, on human natural killer (NK) cell-mediated cytotoxicity was examined in vitro. Doxycycline was the only tetracycline which inhibited the NK cell activity. At concentrations of 10 micrograms/ml the drug caused approximately 50% inhibition of NK cell mediated cytotoxicity against K562 target. The inhibition was not a result of a toxic effect of the drug on NK cells. These results support the previous findings that doxycycline shows immunosuppressive properties.     

In vitro augmentation of natural killing activity by OK-432

OK-432, a streptococcal preparation, augmented the natural killing (NK) activity of peripheral blood lymphocytes of normal donors and cancer patients against both NK sensitive and resistant human target cells in vitro. The enhancement of NK activity was evident after 4 h pretreatment and maximum by 16-24 h. The manifestation of OK-432 induced augmentation required active cell metabolism, RNA and protein synthesis but no DNA synthesis of lymphocytes. The supernatants produced by OK-432 stimulated lymphocyte cultures had no enhancing substance nor interferon. Anti-interferon antibodies did not inhibit boosting activity of OK-432. Large granular lymphocytes were involved in both spontaneous and OK-432 induced cytotoxic activity. The proportion of lymphocytes conjugating to target cells was not changed by OK-432. These results suggest that OK-432 augments cytotoxic activity of large granular lymphocytes having ability to recognize target cells independent of interferon induction.     

In vitro osteogenetic activity of pearl

In vivo and in vitro studies have shown that shell nacre and hydroxyapatite (HA) are promising bioactive materials for bone repair. In this work, the osteogenetic activity of pearl is evaluated by soaking it in simulated body fluid (SBF) and cell culture, taking shell nacre and HA as control materials at the same time. After soaking in SBF, HA particles were rapidly formed on the surface of pearl, the dissolution of CaCO3 and the binding between organic components and Ca2+ ions in pearl provide favorable conditions for the HA precipitation, and the whole process follows a dissolution-binding-precipitation mechanism. Calcium surplus, not conventional calcium deficiency, is found in HA crystal structure; it implies that type B-HA is formed on pearl surface in this study. HRTEM observation shows that HA is poorly crystallized with so many dislocations and shuttle-like amorphous areas. Cell culture reveals that pearl could stimulate osteoblast proliferation, which proceeded more quickly and smoothly than that on shell nacre and HA, and abundant extracellular matrix occupied the whole pearl surface by 5 days. It is concluded that pearl is a superior osteoinductive material with high osteogenetic activity.     

In vitro antifungal activity of mercurobutol

Mercurobutol, in its commercial form, exhibits fungistatic and fungicidal activities against both yeasts: Candida albicans, C. tropicalis, Torulopsis glabrata, Pityrosporum spp., and filamentous fungi: dermatophytes, Aspergillus fumigatus, mainly saprophytic or potentially pathogenic fungi of the cutaneo-mucous flora. Minimal inhibitory concentrations on this antiseptic indicate a high susceptibility of the species tested, that could justify the enlargement of the indication of the drug to the complementary treatment of most cutaneous mycosis.     

In vitro immunomodulatory activity of Bo-yang-hwan-o-tang

Bo-yang-hwan-o-tang (BHT), an herbal decoction has been mainly used for improvement of blood flow in oriental medicine. Its in vivo immunomodulation was recently demonstrated but the effective mechanisms have not been described. This study was carried out to evaluate in vitro immunomodulatory activity of BHT. Water extract of BHT significantly promoted in vitro proliferative responses of mouse spleen cells (SPC) and also further enhanced the proliferation of SPC stimulated with anti-CD3 antibody. Unexpectedly, addition of BHT extract did not affect proliferation of both resting and CD3-activated T cells, whereas it showed a strong mitogenic activity on B cells. Flow cytometric analysis of CFSE-stained SPC showed that BHT-mediated enhancement of CD3-activated SPC proliferation is due to T cell, but not B cell, division. Mixed culture experiment combining T and mitomycin C-treated B cells demonstrated that BHT-mediated enhancement of CD3-activated T cell proliferation was dependent on the presence of B cells. However, B cell-derived factors were not involved in BHT effect on T cell proliferation. In the presence of B cells, BHT treatment resulted in a great enhancement in IL-2 production of CD3-activated T cells, and BHT effect on T cell proliferation was completely abrogated by addition of exogenous IL-2, indicating that IL-2 plays a critical role in BHT-mediated enhancement of CD3-activated T cell proliferation. Taken together, our data revealed that BHT possesses a potent B cell mitogenic activity and also can enhance activated T cell response through B cell regulation.     

In vitro antibacterial activity of sucralfate

The effect of sucralfate (12.5 mg/ml) on the growth ofEscherichia coli (ATCC 25922),Enterococcus faecalis (ATCC 29212) and two isolates ofPseudomonas aeruginosa (ATCC 27853 and a multi-resistant clinical isolate) was studied in vitro at pH values of 3.0, 4.5, 6.0 and 7.4. A bacteriostatic effect of sucralfate was demonstrated forPseudomonas aeruginosa at a pH of 6.0 and 7.4 and forEscherichia coli andEnterococcus faecalis at a pH of 6.0. The bacteriostatic effect was most pronounced at high pH values. Sucralfate had no bactericidal effect on the bacteria tested at the concentration used.     

Glucocorticoid and ACTH regulation of rat peritoneal phagocyte chemiluminescence and nitric oxide production in culture

In order to study the adrenocortical regulation of monocyte/macrophage functions further, leucocytes from the rat peritoneum were incubated in vitro with glucocorticoid concentrations up to 10 μmol L^?1 and with adrenocorticotropic hormone (ACTH) up to 100 μg mL^?1. The monocyte/macrophage production of reactive oxygen molecules was measured by luminol amplified chemiluminescence, and the production of nitric oxide (NO) was measured as nitrite (NO₂^?). Dexamethasone in vitro in nanomolar concentrations inhibited monocyte/macrophage chemiluminescence and also nitric oxide production; the potency was dexamethasone > methylprodnisolone > prednisolone. ACTH enhanced both activated chemiluminescence and endotoxin-induced nitric oxide production, but only at concentrations about 20–100 μg mL^?1, and there was no significant effect of physiological concentrations. In summary, the results of the present study further confirm and substantiate that glucocorticoids in low pharmacological concentrations have a general inhibitory effect on monocyte/macrophage production of reactive oxygen molecules through the specific glucocorticoid receptors, while the stimulatory effect of ACTH is only observed by very high, non-physiological concentrations. Furthermore, since low concentrations of glucocorticoids inhibited the production of these reactive oxygen molecules in vitro, indirect mechanisms involving hormones and other elements outside the immune system are not essential for the effect of glucocorticoids on monocytes/macrophages.     

A turbidimetric assay in an ELISA reader for the determination of mononuclear phagocyte procoagulant activity

A method is described for the simultaneous determination of plasma recalcification time in 60-96 replicates. It is based on turbidimetry in a thermostatic (37 degrees C) ELISA reader photometer. Dose-response curves with thromboplastin showed a double-logarithmic correlation between recalcification time and thromboplastin concentration. Various ways of determining the coagulation time by turbidimetry were compared. Samples of mononuclear cells or monocyte-derived macrophages showed that procoagulant activity (PCA), expressed in thromboplastin units, was directly proportional to the cell number. The method may be of value for the determination of mononuclear phagocyte PCA and for the other applications of plasma recalcification time.     

青霉源抗菌肽类霉肽素的体外抑菌活性研究

目的 研究抗菌肽类霉肽素(AF)的体外抑菌活性.方法 通过抑菌圈法和二倍稀释法检测AF的抗菌谱和对金黄色葡萄球菌的MIC;通过检测在AF中传代菌的敏感性确定细菌是否容易对AF产生抗药菌;将AF和细菌在不同温度和pH环境作用后检测抑菌活性,明确AF发挥活性的最适温度和pH值.结果 AF对大部分供试细菌和抗药菌有效,对金黄色葡萄球菌的MIC为0.8 mg/ml,金黄色葡萄球菌在AF中传代200代后敏感性不变.在14℃到30℃之间AF的抑菌活性随温度升高而降低,在pH 3到pH 10之间AF的活性随pH值升高而降低.结论 AF是一种对抗性菌有效的广谱抗菌肽,而且不容易诱导细菌产生抗药性.     

青霉源抗菌肽类霉肽素的体外抑菌活性研究

目的研究抗菌肽类霉肽素（AF）的体外抑菌活性。方法通过抑菌圈法和二倍稀释法检测AF的抗菌谱和对金黄色葡萄球菌的MIC;通过检测在AF中传代菌的敏感性确定细菌是否容易对AF产生抗药菌;将AF和细菌在不同温度和pH环境作用后检测抑菌活性,明确AF发挥活性的最适温度和pH值。结果AF对大部分供试细菌和抗药菌有效,对金黄色葡萄球菌的MIC为0．8mg／ml,金黄色葡萄球菌在AF中传代200代后敏感性不变。在14℃到30℃之间AF的抑菌活性随温度升高而降低,在pH3到pH10之间AF的活性随pH值升高而降低。结论AF是一种对抗性菌有效的广谱抗菌肽,而且不容易诱导细菌产生抗药性。