Deactivation of macrophage oxidative burst in vitro by different strains of Histoplasma capsulatum

It was previously reported that Histoplasma capsulatum (Hc) yeast not only failed to stimulate a murine macrophage oxidative burst (OB), but they also blunted or abolished OB stimulation by a subsequent encounter with potent stimuli such as zymosan or phorbol 12-myristate 13-acetate (PMA). The present studies show that macrophage deactivation is proportional to the time of incubation and the dose of Hc yeast that induce the deactivated state. Hc yeast derived from a virulent strain (G217B) are more efficient inducers of macrophage deactivation than similar preparations derived from the avirulent Downs Hc strain. Yeast cells of two other pathogenic fungi, Candida albicans and Cryptococcus neoformans are shown to stimulate rather than deactivate a macrophage OB.     

Effect of remifentanil on respiratory burst of human neutrophilic granulocytes in vitro

Recovery chances for severely ill patients have been significantly improved by the progress of intensive care medicine. The success of any therapy, however, is still jeopardized by postoperative infections and septic complications. In the early stage of bacterial infections polymorphonuclear leukocytes (PMNL) play a decisive role. After PMNL activation, the production of oxygen radicals during the respiratory burst (RB) denature the phagocytosed micro-organisms. Remifentanil is a new opioid which has been safely administered to various patient groups and shows pharmacokinetic advantages in comparison to the already established opioids. As some intravenous anaesthetics can influence PMNL functions, we analysed, by flow cytometry, the in vitro influence of clinically relevant remifentanil concentrations on the respiratory burst. In our study remifentanil had no influence on the respiratory burst of human PMNL in vitro, regardless of the RB triggering agents chosen.     

Divergent effects of LPS on expression of IL-1 receptor family members in mononuclear phagocytes in vitro and in vivo

Three molecules, interleukin 1 (IL-1) receptor I (IL-1RI), IL-1 receptor II (IL-1RII or decoy) and IL-1 receptor accessory protein (IL-1R AcP or IL-1RIII), are involved in IL-1 binding and signal transduction. In addition, three homologous genes (T1/ST2, MyD88 and rsc786) have been identified. Expression of the signal transducing type I R and of the decoy type II R in human monocytes is regulated by pro- and anti-inflammatory signals. The present study was designed to evaluate comprehensively how a prototypic pro-inflammatory signal, bacterial lipopolysaccharide (LPS), affects expression of IL-1R family members in mononuclear phagocytes in vitro and in vivo. Resting human monocytes expressed high levels of IL-1RII, IL-1R AcP, MyD88 and rsc786, whereas low levels of IL-1RI and T1/ST2 were present. In vitro exposure to LPS augmented expression of IL-1RI, T1/ST2 and MyD88, whereas it inhibited that of IL-1RII and rsc786. Expression of IL-1R AcP in monocytes was less substantially affected by LPS. The expression of IL-1R family members was also studied in organs of mice given LPS. As expected on the basis of in vitro results, organs (e.g. spleen, lungs and peritoneal exudate cells) from LPS-treated mice showed increased levels of IL-1RI, T1/ST2 and MyD88. Intriguingly, while expression of IL-1RII was inhibited in peritoneal macrophages after LPS, in accordance with in vitro results, increased IL-1RII mRNA was observed in organs such as liver, lungs and spleen. This unexpected effect of LPS was drastically reduced in mice rendered neutropenic by 5-fluorouracil. Therefore, we conclude that the apparent induction of IL-1RII in certain organs of LPS-treated mice is due to recruitment of myeloid cells which express high levels of decoy RII. Therefore, members of IL-1R family are independently and divergently regulated in mononuclear phagocytes exposed to the prototypic pro-inflammatory signal LPS in vitro and in vivo.     

Oxidative autoaggression by phagocytes in human peritonitis

In 60 abdominal exudates from patients with the diagnosis of either acute or persistent (chronic) peritonitis, indicators of phagocyte-derived oxidative systems (myeloperoxidase, chemiluminescence) and proteases (elastase) were measured. These exudates reveal a picture of maximal inflammatory activation. Both types of exudates (30 each) showed a significant influx of inflammatory cells, with the mean leucocyte count being 73,000 microL and 32,000 microL-1 respectively. Local myeloperoxidase concentrations were approximately 1000-fold greater than that of normal plasma. Spontaneous and elicitable chemiluminescence--indicators of phagocyte respiratory burst activity--were dramatically increased. In addition, levels of extracellularly released elastase (from neutrophils) were found to be up to about 1000-fold that of normal plasma values. Although most of the elastase detected in the exudates was complexed with alpha 1-proteinase inhibitor (alpha 1 PI), enzymatically active elastase could be measured in approximately 60% of the samples being investigated. As there was an excess of immunoreactive alpha 1 PI in these exudates, the free elastase activity implies that much of the alpha 1 PI was inactive, presumably subjected to oxidative destruction. Moreover, a trypsin-inhibitory activity to antigen ratio below 1 (mean = 0.81) in 75% of the purulent exudates indicated also partial proteolytic degradation of alpha 1 PI. In contrast, 16 clear exudates (no bacteria, white cell count below 500 microL-1) taken from the non-infected peritoneal cavity of patients undergoing intra-abdominal surgery revealed a similar permeability increase of the peritoneum but did not show relevant oxidative and proteolytic activity or destruction of alpha 1 PI compared with purulent specimens. Thus, only the inflammatory process of peritonitis appears to result in an overwhelming local phagocytic activity that initiates and maintains protease inhibitor consumption and/or inactivation. The tremendous oxidative potential found in purulent exudates may cause destruction in a wide variety of defence systems.     

Eosinophil chemotaxis induced by several biologically active substances and the effects of apafant on it in vitro

Chemotaxis of guinea pig eosinophils induced by various stimuli in use of a modified Boyden chamber technique in vitro and the effect of a platelet-activating factor (PAF) antagonist, apafant (CAS 105219-56-5, WEB 2086 BS), on it were examined. The eosinophils were obtained by bronchoalveolar lavage from the animals treated by i.v. injection with Sephadex G-200 and purified by Percoll density gradient centrifugation. PAF significantly and potently induced the chemotaxis at a broad range of 10(-17) to 10(-7) mol/l, where no concentration-dependency was observed. Leukotriene B4 also induced the chemotaxis in a concentration-dependent manner at 10(-14) to 10(-12) mol/l and the enhanced migration was not declined until 10(-7) mol/l. Interleukin-5 (IL-5), IL-8 and regulated on activation normal T expressed and secreted (RANTES) only modestly enhanced the chemotaxis in some concentrations at 10(-13) to 10(-7) mol/l with or without significance and with no concentration-dependency while formyl-methionyl-leucyl-phenylalanine (FMLP), a known chemoattractant, increased the migration at 10(-7) to 10(-5) mol/l. Apafant at 10(-8) to 10(-6) mol/l strongly and concentration-dependently inhibited 10(-8) mol/l PAF-induced chemotaxis. However, the drug showed nominal or no influences on their chemotaxis stimulated by the other agonists, at the concentrations of which the enhanced migration was observed. From these results, it is concluded that IL-5, IL-8 and RANTES, different from PAF and LTB4, are not potent stimuli for the eosinophil chemotaxis and that apafant is a selective antagonist of PAF, which is expected to be therapeutically effective for PAF-associated diseases including bronchial asthma.     

Local changes in membrane potential intensify neutrophil oxidative burst

The aim of this paper is to study the effects of the pulsed electrical field/current alone or combined with ionomycin, fMLP and PMA, the chemical stimuli that operate through distinctly different activation pathways, on the time course of the oxidative burst response in human neutrophils. Neither the control groups nor the neutrophils treated with electrical field alone showed any increase in oxidative burst activity measured by the luminol-enhanced chemiluminescence technique. It was found that electrical treatment potentiates chemically induced activation with either of the chemical stimulators used. The integrated oxidative burst response--which represents a cumulative amount of oxygen metabolites produced during whole response--was 87% higher in neutrophils treated with a combination of ionomycin and an electric field than in solely ionomycin treated cells, while the peak level of the response was 114% higher. In neutrophils stimulated with fMLP the electrochemical treatment caused a 32% higher integral response as well as a 22% higher peak level compared to the neutrophils treated with chemical stimulant alone. The integrated oxidative burst response in the combined PMA and electric treatment was only 4.7% higher than in the cells treated with PMA alone, and no significant difference in the peak level was found. The results suggest that electric field treatment preferentially stimulates calcium-induced activation with ionomycin rather than calcium-dependent activation with fMLP or PMA.     

The effects of immunoglobulins on the convective permeability of human dentine in vitro

Immunoglobulin molecules are localized in the dentinal tubules of non-carious and carious teeth, but their possible role in caries invasion is not understood. This study sought to examine the effects of immunoglobulin molecules on dentine permeability using a fluid-filtration method. Crown segments cut from impacted human third molars were treated by filtration with 100 micrograms/ml IgG, 100 micrograms/ml IgA or 30 micrograms/ml IgM under a constant pressure. Flow rates were recorded and percent changes in flow rate analysed over time. Filtrates collected at various times were tested for changes in immunoglobulin concentrations by an enzyme-linked immunosorbent assay, and the percent retention of immunoglobulins to dentine was calculated. There was a decreasing non-linear exponential relation between the percent changes in flow rate and filtration time for all three immunoglobulins. The percentage of retained immunoglobulins was significantly related to the filtration time for all three classes of immunoglobulins. Immunoglobulin retention contributed to significant changes in flow rate with time. These in vitro results indicate the potential mechanism of immunoglobulins in decreasing tabular permeability.     

In vitro effects of diazepam on human ciliary function

This study demonstrated the in vitro effect of diazepam on human ciliary beat frequency (CBF) from fifteen normal subjects using brush cytology. Tube A contained culture medium, tube B the diluent that diazepam intravenous injectate was carried in and culture medium (controls). Tube C, D and E contained, 0.4 mg/l, 4.0 mg/l and 40.0 mg/l of diazepam in culture medium respectively. The mean effective diazepam concentration in plasma is 0.4 mg/l. CBF was measured photometrically. The most vigorous cilia were measured in 5 areas taking 10 readings on each sample, 30 minutes and 1 hour after mixing. Standard deviation (SD) and confidence limits were calculated along with significance testing (p < 0.05) using the paired t-test and ANOVA. The mean of the CBF of tubes A and B were 13.44 (SD 2.65) and 13.67 (SD 2.48). There was a reduction of the CBF with increasing concentrations of diazepam at 30 minutes, 11.32 (SD 2.14), 10.29 (SD 1.58) and 4.14 (SD 1.57) tubes C. D and E respectively. There was a significant lowering in CBF of 17% (p < 0.01) of diazepam at the mean effective plasma level (tube C) when compared against the controls. CBF decreased over time and at 1 hour was 10.57 (SD, 1.36), 9.02 (SD, 1.39) and 3.58 (SD, 1.31) tubes C, D and E respectively. A proposed mechanism of altered intracellular calcium flux via the action diazepam on GABA receptors is described.     

Comparison of several mouse and rat monoclonal antibodies against human fibrinogen

A rare case of desmoplastic melanoma arising from the maxillary gingiva of a 66-year-old woman is reported. This tumour metastasised to the submandibular lymph node 5 years after extirpation, and local recurrence was observed 2 years later. The gingival tumour showed the histopathological characteristics of desmoplastic melanoma and the metastasised tumour cells were immunohistochemically positive for S-100 protein, neuron specific enolase, HMB-45 highly specific for conventional melanoma, and Fontana-Masson staining. The gingival tumour, originally regarded as benign clinically, was actually a desmoplastic melanoma.     

In vivo effects of recombinant human granulocyte colony-stimulating factor on neutrophil oxidative functions in normal human volunteers

The effect of daily in vivo granulocyte colony-stimulating factor (G-CSF) treatment on neutrophil function was studied over a 14-day period using a luminescence system for differential measurement of oxidase and myeloperoxidase (MPO) dioxygenation activities in whole blood. Opsonin receptor-mediated phagocyte functions were also measured with this system. G-CSF produced a dose-dependent neutrophil leukocytosis and a proportional increase in oxidase activity per volume of blood. The oxidase activity per neutrophil remained relatively constant throughout the test period. However, both chemical- and opsonin-stimulated MPO oxygenation activities per neutrophil were greatly increased by treatment with maxima correlating temporally to initial G-CSF exposure during the early mitotic phase of neutrophil development. The possibility that peroxynitrite contributes to this maximum luminol-dependent activity was tested, but neither superoxide dismutase, a competitive inhibitor of peroxynitrite production, nor N-methyl-L-arginine, an inhibitor of nitric oxide synthase, exerted a significant inhibitory effect.     

The effects of anti-rheumatoid drugs on the in vitro activity of human serum hyaluronidase

Fourteen antirheumatoid drugs were tested for their effects on the in vitro hyaluronidase activity of normal human serum. Four drugs produced significant changes in enzyme activity. Different results were obtained with ovine testicular hyaluronidase when diluted with either saline or inactivated human serum. No increase in serum hyaluronidase activity was found in patients with rheumatoid arthritis. There was no evidence for the existence of tissue specific isoenzymes of hyaluronidase in the serum of either normal subjects or patients with rheumatoid arthritis.     

Comparison of the in vitro activity of several cephalosporin antibiotics against gram-negative and gram-positive bacteria resistant to cephaloridine

The in vitro activity of each of two oral [cefatrizine (BL-S640), cephalexin] and three parenteral (cefamandole, cefazolin, cephapirin) cephalosporin antibiotics was compared with that of cephalothin against 168 clinical isolates of gram-negative and gram-positive bacteria selected as resistant to 20 mug of cephaloridine per ml on the basis of agar dilution susceptibility test data. Each of the five other cephalosporins inhibited a greater percentage of gram-negative bacillary isolates than did cephalothin or cephaloridine, with minimal inhibitory concentration values ranging 2- to 50-fold lower. Significant differences between minimal inhibitory concentrations of the compounds tested were also observed in tests against strains of Streptococcus faecalis and of methicillin-resistant Staphylococcus aureus. Potential advantages of including more than a single cephalosporin antibiotic in the panel of antibiotics used for routine susceptibility testing, suggested by these observations, are discussed.     

Selective protection against conidia by mononuclear and against mycelia by polymorphonuclear phagocytes in resistance to Aspergillus. Observations on these two lines of defense in vivo and in vitro with human and mouse phagocytes

By comparing natural immunity to Aspergillus fumigatus (AF) in vivo with the action of human or mouse phagocytes against AF in vitro, we delineated two sequential lines of defense against AF. The first line of defense was formed by macrophages and directed against spores. Macrophages prevented germination and killed spores in vitro and rapidly eradicated conidia in vivo, even in neutropenic and athymic mice. The second was the neutrophilic granulocyte (PMN), which protected against the hyphal form of AF. Human and mouse PMN killed mycelia in vitro. Normal, but not neutropenic mice, stopped hyphal growth, and eradicated mycelia. Either line of defense acting alone protected mice from high challenge doses. Natural immunity collapsed only when both the reticuloendothelial system and PMN were impaired. These findings are in keeping with the clinical observation that high doses of cortisone and neutropenia are the main risk factors for invasive aspergillosis. Cortisone inhibited the conidiacidal activity of mouse macrophages in vivo and of human or mouse mononuclear phagocytes in vitro. Cortisone damaged this first line of defense directly and not through the influence of T lymphocytes or other systems modifying macrophage function as shown in athymic mice and in vitro. In addition, daily high doses of cortisone in mice reduced the mobilization of PMN so that the second line of defense was also impaired. Thus, cortisone can break down natural resistance on its own. Myelosuppression rendered mice susceptible only when the first line of defense was overpowered by high challenge doses, by activated spores that cannot be killed by macrophages, or by cortisone suppression of the conidiacidal activity of macrophages. The host, thus, can call upon two independent phagocytic cell lines that form graded defense systems against aspergillus. These lines of defense function in the absence of a specific immune response, which seems superfluous in the control and elimination of this fungus.     

Accumulation of bisphosphonates in human artery and their effects on human and rat arterial function in vitro

Of 91 limb-salvage procedures using prosthetic reconstructions because of primary or metastatic bone and soft-tissue tumors 26 revisions were performed in 16 patients. Revision was due to polyethylene wear (9 cases), aseptic loosening (8 cases), recurrent hip dislocation (3 cases), prosthetic stem fracture (2 cases), infection (2 cases), leg length discrepancy (1 case), and traumatic dislocation of a saddle prosthesis (1 case). The follow-up period for tumor control varied from 1.5 to 22 years with a median of 13.5 years. The follow-up period after the last revision operation varied from 0.5 to 12 years with a median of 3 years. At the last follow-up, the functional results had deteriorated compared with after the primary operation in 5 patients and had improved in 2 patients. In the remaining patients, the results did not change.     

Temperature effects on Legionella pneumophila killing by and multiplication in phagocytes of guinea pigs

We examined the effects of temperature on the interaction between Legionella pneumophila and phagocytes of guinea pigs. The body temperatures of guinea pigs infected with a sublethal dose (1.2 x 10(4) CFU) or a lethal dose (1.0 x 10(5) CFU) of L. pneumophila elevated from 38.4 +/- 0.15 C to 40.2 +/- 0.42 C or 40.3 +/- 0.62 C, respectively. The intracellular bacterial killing by and bacterial proliferation in the phagocytes were examined at 33, 37, 40, and 42 C, using in vitro culture systems of peritoneal macrophages or polymorphonuclear leukocytes (PMN) of guinea pigs. In all the macrophages incubated at different temperatures, significant intracellular bacterial killings were observed at 4 hr after in vitro phagocytosis. After 24 hr of incubation, there was about a 100-fold increase of CFU and the number reached a maximum after 48 hr of incubation in the macrophages incubated at 42 C as well as 37 and 40 C, suggesting that macrophages support the intracellular bacterial growth in hyperthermia. In the PMN, L. pneumophila CFU 4 hr or 12 hr after the infection were significantly lower at 42 C than those at 37 C (P < 0.05), indicating that the bactericidal capacity of PMN was enhanced at 42 C compared to 37 C. However, in all the PMN incubated at different temperatures, there were about 10-fold increases of CFU 24 hr after the infection, suggesting that PMN as well as macrophages support intracellular bacterial growth in hyperthermia. The extracellular bacterial growth was examined at 33, 37, 40, and 42 C in buffered yeast extract (BYE) broth or RPMI 1640 medium containing 50% guinea pig serum as a permissive or non-permissive liquid medium for the bacterial growth, respectively. Inhibition of bacterial growth in BYE broth at 42 C, and a decrease of CFU in RPMI 1640 medium containing 50% guinea pig serum at 42 C were observed. In conclusion, hyperthermia may be beneficial by restricting extracellular bacterial survival, but it exerts no beneficial effect on the restriction of intracellular bacterial growth in phagocytes, though PMN showed enhanced initial killing at 42 C. These results suggest that fever, or hyperthermia itself, may not largely contribute as a nonspecific host defense early in the course of legionellosis.     

Flow-cytometric evaluation of oxidative burst in phagocytic cells of children with cystic fibrosis

To assess the value of fine-needle aspiration (FNA) cytology for the diagnosis of amyloid, we retrospectively studied all FNA cases diagnosed as having amyloid during a 6-yr period (1990-1996). FNA was performed on both superficial and deep locations. A total of 6 cases containing amyloid was studied, including primary medullary thyroid carcinoma, metastatic medullary thyroid carcinoma to a vertebrae, multiple myeloma, squamous-cell carcinoma of the lung metastatic to a hilar lymph node, primary pulmonary amyloid, and amyloid tumor in a vertebral body in a patient with primary systemic amyloidosis. Despite the location or disease association, the cytologic appearance of amyloid in all cases was similar. On Diff-Quik stain, amyloid appeared as amorphous, irregular, waxy basophilic to metachromatic clumps of material. Papanicolaou stain revealed cyanophilic to organophilic clumps of material with occasional prominent fissures. In all 6 cases, amyloid was confirmed by Congo red stain and in 3 cases by a thioflavin T stain. In 4 of the 6 cases (67%), amyloid was associated with an underlying malignancy. In 3 cases malignant cells were admixed with the amyloid, and in another case malignancy was present at a distant site. We conclude that FNA biopsy is a helpful initial procedure for the evaluation of patients with amyloid deposits. The clinical implications of amyloid found in any particular body site include both benign and malignant conditions. The presence of an associated neoplasm must be especially considered in the differential diagnosis of amyloid deposits.     

Influence of the age and sex on respiratory burst of human monocytes

The usefulness of peripheral human lymphocytes as a bioindicator for ionizing radiation effect was tested in a survey of Malaysian workers in two industries producing technologically enhanced naturally occurring radioactive material (TENORM). Workers in amang processing plants who have been with the plant for an average of 12.9 years and who were exposed to radioactive dust showed significantly higher frequencies of chromosomal aberration compared to control and even ilmenite-processing workers. Such frequency was not significantly different between workers in ilmenite-processing plant and control. The differences in duration of employment, occupational hygiene, together with the difference in the percentage of 'old' and 'new' aberrations among the groups sampled were used to explain the high chromosomal aberration frequency among amang workers. The presence of significantly high chromosome damage (dicentrics and fragments) in workers who were chronically exposed to doses below 50 mSv per year or 20 mSv per year averaged over 5 years (ICRP, 1991) provided additional experimental data on the dose-effect relationship at these low-dose ranges. The results confirm the usefulness of using human lymphocytes as a bioindicator for chronic exposure to ionizing radiation and in cases where physical radiation detectors are not available.     

Decreased CD11b expression, phagocytosis, and oxidative burst in urban particulate pollution-exposed human monocytes and alveolar macrophages

Elevated levels of air pollution particulates < or = 10 microm in diameter (PM10) have been associated with an increase in mortality and morbidity due to pulmonary complications, including pneumonia. Impairment of inflammatory and host defense functions of the alveolar macrophage (AM) may be a precipitating factor. The present study was undertaken to determine whether human AM and blood derived monocytes (MO) modulate the expression of receptors important for phagocytosis of opsonized microbes (CD11b, CD11c), gram-negative bacteria (CD14), extracellular matrix interaction (CD29), and immune responses (CD11a, CD54, HLA-DR) when exposed to particulates obtained from urban air (UAP). Furthermore, phagocytosis of and oxidant generation by opsonized yeast were investigated in particle-exposed cells. AM and MO exposed to UAP for 18 h expressed significantly lower levels of CD11b and CD29. CD14 expression was markedly decreased in MO but not in AM, and CD11c was reduced in AM but not in MO. CD11a, CD54, and HLA-DR were unaltered in both phagocyte populations. Decreased receptor expression was not dependent on particle load in the cells. Phagocytosis of Saccharomyces cerevisiae and the chemiluminescence response were also significantly inhibited by UAP. Time-course studies revealed that decreased oxidant generation was evident already at 3 h postexposure, while significant effects on phagocytosis and CD11b expression were found at 18 h. These data indicate that exposure to particulate pollution is likely to impair host defense functions of AM and MO, which are important in elimination of a variety of pathogens in the lung.     

Toluene-induced oxidative stress in several brain regions and other organs

The in vivo dose-response relationship between toluene and reactive oxygen species (ROS) formation in rat brain, liver, kidney, and lung, and the time-course of these effects has been characterized. The rate of oxygen radical formation was measured using the probe 2',7'-dichlorofluorescin diacetate. In vivo exposure to various doses of toluene (0.5, 1.0, and 1.5 g/kg ip) elicited a dose-dependent elevation of ROS generation within crude mitochondrial fractions obtained from rat lung and kidney, and within crude synaptosomal fractions from cerebellum. ROS formation in crude mitochondrial fractions from liver, and crude synaptosomal fractions from striatum and hippocampus, reached a maximum value at relatively low doses of toluene. Of the brain regions, the hippocampus had the highest induced levels of ROS. In vivo exposure to a single dose of toluene (1.5 g/kg ip), revealed that toluene-induced ROS reached a peak within 2 h, which correlated directly with measured toluene blood levels. This elevated oxidative activity was maintained throughout the next 24 h, even though blood values of toluene decreased to negligible amounts. These results demonstrate that exposure to toluene results in broad systemic elevation in the normal rate of oxygen radical generation, with such effects persisting in the tissues despite a rapid decline in toluene blood levels. Acute exposure to toluene may lead to extended ROS-related changes, and this may account for some of the clinical observations made in chronic toluene abusers.