Sevoflurane Promotes Bactericidal Properties of Macrophages through Enhanced Inducible Nitric Oxide Synthase Expression in Male Mice.

BACKGROUND: Sevoflurane with its antiinflammatory properties has shown to decrease mortality in animal models of sepsis. However, the underlying mechanism of its beneficial effect in this inflammatory scenario remains poorly understood. Macrophages play an important role in the early stage of sepsis as they are tasked with eliminating invading microbes and also attracting other immune cells by the release of proinflammatory cytokines such as interleukin-1ß, interleukin-6, and tumor necrosis factor-α. Thus, the authors hypothesized that sevoflurane mitigates the proinflammatory response of macrophages, while maintaining their bactericidal properties. METHODS: Murine bone marrow-derived macrophages were stimulated in vitro with lipopolysaccharide in the presence and absence of 2% sevoflurane. Expression of cytokines and inducible NO synthase as well as uptake of fluorescently labeled Escherichia coli (E. coli) were measured. The in vivo endotoxemia model consisted of an intraperitoneal lipopolysaccharide injection after anesthesia with either ketamine and xylazine or 4% sevoflurane. Male mice (n = 6 per group) were observed for a total of 20 h. During the last 30 min fluorescently labeled E. coli were intraperitoneally injected. Peritoneal cells were extracted by peritoneal lavage and inducible NO synthase expression as well as E. coli uptake by peritoneal macrophages was determined using flow cytometry. RESULTS: In vitro, sevoflurane enhanced lipopolysaccharide-induced inducible NO synthase expression after 8 h by 466% and increased macrophage uptake of fluorescently labeled E. coli by 70% compared with vehicle-treated controls. Inhibiting inducible NO synthase expression pharmacologically abolished this increase in bacteria uptake. In vivo, inducible NO synthase expression was increased by 669% and phagocytosis of E. coli by 49% compared with the control group. CONCLUSIONS: Sevoflurane enhances phagocytosis of bacteria by lipopolysaccharide-challenged macrophages in vitro and in vivo via an inducible NO synthase-dependent mechanism. Thus, sevoflurane potentiates bactericidal and antiinflammatory host-defense mechanisms in endotoxemia.

Kingella kingae Surface Polysaccharides Promote Resistance to Human Serum and Virulence in a Juvenile Rat Model.

Kingella kingae is a Gram-negative coccobacillus that is increasingly being recognized as an important cause of invasive disease in young children. The pathogenesis of K. kingae disease begins with colonization of the oropharynx, followed by invasion of the bloodstream, survival in the intravascular space, and dissemination to distant sites. Recent studies have revealed that K. kingae produces a number of surface factors that may contribute to the pathogenic process, including a polysaccharide capsule and an exopolysaccharide. In this study, we observed that K. kingae was highly resistant to the bactericidal effects of human serum complement. Using mutant strains deficient in expression of capsule, exopolysaccharide, or both in assays with human serum, we found that elimination of both capsule and exopolysaccharide was required for efficient binding of IgG, IgM, C4b, and C3b to the bacterial surface and for complement-mediated killing. Abrogation of the classical complement pathway using EGTA-treated human serum restored survival to wild-type levels by the mutant lacking both capsule and exopolysaccharide, demonstrating that capsule and exopolysaccharide promote resistance to the classical complement pathway. Consistent with these results, loss of both capsule and exopolysaccharide eliminated invasive disease in juvenile rats with an intact complement system but not in rats lacking complement. Based on these observations, we conclude that the capsule and the exopolysaccharide have important redundant roles in promoting survival of K. kingae in human serum. Each of these surface factors is sufficient by itself to fully prevent serum opsonin deposition and complement-mediated killing of K. kingae, ultimately facilitating intravascular survival and promoting K. kingae invasive disease.

The fruRBA Operon Is Necessary for Group A Streptococcal Growth in Fructose and for Resistance to Neutrophil Killing during Growth in Whole Human Blood.

Bacterial pathogens rely on the availability of nutrients for survival in the host environment. The phosphoenolpyruvate-phosphotransferase system (PTS) is a global regulatory network connecting sugar uptake with signal transduction. Since the fructose PTS has been shown to impact virulence in several streptococci, including the human pathogen Streptococcus pyogenes(the group A Streptococcus[GAS]), we characterized its role in carbon metabolism and pathogenesis in the M1T1 strain 5448. Growth in fructose as a sole carbon source resulted in 103 genes affected transcriptionally, where the frulocus (fruRBA) was the most induced. Reverse transcriptase PCR showed that fruRBA formed an operon which was repressed by FruR in the absence of fructose, in addition to being under carbon catabolic repression. Growth assays and carbon utilization profiles revealed that although the entire fruoperon was required for growth in fructose, FruA was the main transporter for fructose and also was involved in the utilization of three additional PTS sugars: cellobiose, mannitol, and N-acetyl-D-galactosamine. The inactivation of sloR, a fruA homolog that also was upregulated in the presence of fructose, failed to reveal a role as a secondary fructose transporter. Whereas the ability of both ΔfruR and ΔfruB mutants to survive in the presence of whole human blood or neutrophils was impaired, the phenotype was not reproduced in murine whole blood, and those mutants were not attenuated in a mouse intraperitoneal infection. Since the ΔfruA mutant exhibited no phenotype in the human or mouse assays, we propose that FruR and FruB are important for GAS survival in a human-specific environment.

Hypochlorous acid regulates neutrophil extracellular trap release in humans.

Neutrophil extracellular traps (NETs) comprise extracellular chromatin and granule protein complexes that immobilize and kill bacteria. NET release represents a recently discovered, novel anti-microbial strategy regulated non-exclusively by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generation of reactive oxygen intermediates (ROIs), particularly hydrogen peroxide. This study aimed to characterize the role of ROIs in the process of NET release and to identify the dominant ROI trigger. We employed various enzymes, inhibitors and ROIs to record their effect fluorometrically on in vitro NET release by human peripheral blood neutrophils. Treatment with exogenous superoxide dismutase (SOD) supported the established link between hydrogen peroxide and NET production. However, treatment with myeloperoxidase inhibitors and direct addition of hypochlorous acid (HOCl; generated in situ from sodium hypochlorite) established that HOCl was a necessary and sufficient ROI for NET release. This was confirmed by the ability of HOCl to stimulate NET release in chronic granulomatous disease (CGD) patient neutrophils which, due to the lack of a functional NADPH oxidase, also lack the capacity for NET release in response to classical stimuli. Moreover, the exogenous addition of taurine, abundantly present within the neutrophil cytosol, abrogated NET production stimulated by phorbol myristate acetate (PMA) and HOCl, providing a novel mode of cytoprotection by taurine against oxidative stress by taurine.

Corticosterone-immune interactions during captive stress in invading Australian cane toads (Rhinella marina).

Vertebrates cope with physiological challenges using two major mechanisms: the immune system and the hypothalamic pituitary-adrenal axis (e.g., the glucocorticoid stress response). Because the two systems are tightly integrated, we need simultaneous studies of both systems, in a range of species, to understand how vertebrates respond to novel challenges. To clarify how glucocorticoids modulate the amphibian immune system, we measured three immune parameters and plasma corticosterone (CORT), before and after inflicting a stressor (capture and captive confinement) on introduced cane toads (Rhinella marina) near their invasion front in Australia. Stress increased CORT levels, decreased complement lysis capacity, increased leukocyte oxidative burst, and did not change heterologous erythrocyte agglutination. The strength of the CORT response was positively correlated with leukocyte oxidative burst, and morphological features associated with invasiveness in cane toads (relative leg length) were correlated with stress responsiveness. No immune parameter that we measured was affected by a toad's infection by a parasitic nematode (Rhabdias pseudosphaerocephala), but the CORT response was muted in infected versus uninfected toads. These results illustrate the complex immune-stress interactions in wild populations of a non-traditional model vertebrate species, and describe immune adaptations of an important invasive species.

Differential analysis of bactericidal systems of blood serum with recombinant luminescent Escherichia coli and Bacillus subtilis strains.

Luminescence intensity of recombinant Escherichia coli and Bacillus subtilis strains with cloned luxCD(AB)E genes of the natural luminescent microorganism Photobacterium leiognathi was studied under the influence of 30 individual samples of human blood serum of different component composition. A relationship was found between the level of residual bioluminescence and degree of the bactericidal effect. Moreover, the inhibition of E. coli lux+ luminescence was shown to be related to activity of the complement-lysozyme system. The reaction of B. subtilis lux+ primarily depended on the presence of ß-lysin in the blood serum. These data provide an experimental substantiation of a new method of differential analysis of humoral factors of nonspecific innate immunity with recombinant luminescent bacteria.

Human disturbance alters endocrine and immune responses in the Galapagos marine iguana (Amblyrhynchus cristatus).

Anthropogenic disturbance is a relevant and widespread facilitator of environmental change and there is clear evidence that it impacts natural populations. While population-level responses to major anthropogenic changes have been well studied, individual physiological responses to mild disturbance can be equally critical to the long-term survival of a species, yet they remain largely unexamined. The current study investigated the impact of seemingly low-level anthropogenic disturbance (ecotourism) on stress responsiveness and specific fitness-related immune measures in different breeding stages of the marine iguana (Amblyrhynchus cristatus). Specifically, we found stress-induced elevations in plasma corticosterone among tourist-exposed populations relative to undisturbed populations. We also found changes in multiple immunological responses associated with stress-related effects of human disturbance, including bacterial killing ability, cutaneous wound healing, and hemolytic complement activity, and the responses varied according to reproductive state. By identifying health-related consequences of human disturbance, this study provides critical insight into the conservation of a well-known species that has a very distinct ecology. The study also broadens the foundation of knowledge needed to understand the global significance of various levels of human disturbance.

Influence of serum on interaction of Porphyromonas gingivalis ATCC 33277 and Aggregatibacter actinomycetemcomitans Y4 with an epithelial cell line.

BACKGROUND AND OBJECTIVE: The purpose of this study was to investigate the influence of serum on the interaction of periodontal pathogens with epithelial cells using an epithelial cell line (KB cells). This is important because serum is a key component of gingival crevicular fluid and may influence inflammatory responses in epithelial cells exposed to periodontal pathogens. MATERIAL AND METHODS: Porphyromonas gingivalis ATCC 33277 and Aggregatibacter actinomycetemcomitans Y4 were co-cultured with KB cells either with or without the addition of up to 10% human serum or 50 mg/mL human serum albumin. The numbers of free-floating, adherent and intracellular bacteria were determined up to 18 h after exposure of the epithelial cells to the pathogens. Additionally, the concentrations of interleukin (IL)-6 and IL-8 produced by the epithelial cells in response to exposure to the bacteria were determined. RESULTS: Serum and human serum albumin reduced the number of internalized A. actinomycetemcomitans Y4 organisms in the epithelial cells, increased the levels of IL-6 and IL-8 in the supernatants of infected cells (those with internalized A. actinomycetemcomitans) and influenced non-infected epithelial cells. Increased IL-6 and IL-8 concentrations were also detected in the supernatants of KB cells infected with P. gingivalis ATCC 33277. Interleukin-6 and IL-8 were detectable after addition of serum, probably as a result of inhibition of the activity of P. gingivalis cysteine proteinases by serum. CONCLUSION: Serum promotes the release of the cytokines IL-6 and IL-8 by epithelial cells. This mechanism is influenced by periodontal pathogens and may maintain clinical periodontal inflammation.

The role of complement activity in the sensitivity of Salmonella O48 strains with sialic acid-containing lipopolysaccharides to the bactericidal action of normal bovine serum.

Sialic acids are important constituents of animal tissue glycoconjugates and are also present in the antigens of some bacterial strains. Capsular polysaccharides with sialic acid (NeuAc) have been extensively studied with regard to sensitivity to the bactericidal action of serum, whereas little is known in this regard about lipopolysaccharides (LPS) which contain NeuAc. Strains of Salmonella O48, able to infect animals and containing the same structures of LPS with NeuAc, were examined for their susceptibility to the bactericidal action of normal bovine serum (NBS). The strains showed varied sensitivity to the bactericidal action of NBS, which indicates that the expression of LPS containing NeuAc residues is not critical for the strains' resistance to the serum's activity. In this study the mechanisms of complement activation responsible for killing serum-sensitive Salmonella O48 rods by NBS were also established. Three such mechanisms were distinguished: activation of the classical/lectin pathways, important (decisive) in the bactericidal mechanism of complement activation, parallel activation of the classical/lectin and alternative pathways, and independent activation of the classical and lectin or the alternative pathway.

[Lactoferrin: a multifunctional protein]. / La lactoferrine : une protéine multifonctionnelle.

Lactoferrin (Lf) is an iron-binding glycoprotein of the transferrin family that is expressed and secreted by glandular cells and found in the secondary granules of neutrophils from which it is released in infected tissues and blood during the inflammatory process. Initially described as an iron-binding molecule with bacteriostatic properties, Lf is now known to be a multifunctional or multi-tasking protein. It is a major component of the innate immune system of mammals. Its protective effects range from direct anti-microbial activities against a large panel of microorganisms including bacteria, viruses, fungi, and parasites, to anti-inflammatory and anti-cancer activities. While iron chelation is central to some of the biological functions of Lf, other activities involve interactions of Lf with molecular and cellular components of both hosts and pathogens. Its powerful antimicrobial activities, immunomodulatory properties and prevention of septic shock, anti-carcinogenic functions and its growing importance in iron delivery and bone growth, combined with the data obtained either by in vivo studies or clinical trials, make this molecule and its derivatives very promising tools for health or nutritional applications.

Exposure of Avian Embryos to Cycling Incubation Temperatures Reduces Adult Bactericidal Ability.

In birds, the temperature at which eggs are incubated shapes many aspects of hatchling phenotype, but long-term effects are less studied. We studied the effect of incubation temperature and pattern on the subsequent development of innate immune function in Japanese quail (Coturnix japonica). We incubated quail eggs in one of three replicated treatments: control (37.5°C), low (36.0°C), and cyclical incubation. The cyclical treatment had the same average temperature as the low-temperature treatment (36.0°C) and an upper temperature that was the same as the control. When individuals were 5, 20, and 55 d of age (i.e., adults), we measured the ability of blood plasma to kill Escherichia coli. Throughout development there was a nonsignificant trend for immune function to be lower in the cycling treatment. In adulthood, however, individuals incubated at cycling temperatures had significantly lower immune function than control birds but did not differ from individuals incubated at constant low temperatures. Males and females responded similarly to the incubation treatment, but females developed a greater plasma bactericidal ability than males. We conclude that variation in innate immune function of adult birds is shaped by temperature fluctuations experienced during incubation.

Influence of lipid composition on membrane activity of antimicrobial phenylene ethynylene oligomers.

Host defense peptides (HDPs), part of the innate immune system, selectively target the membranes of bacterial cells over that of host cells. As a result, their antimicrobial properties have been under intense study. Their selectivity strongly depends on the chemical and mostly structural properties of the lipids that make up different cell membranes. The ability to synthesize HDP mimics has recently been demonstrated. To better understand how these HDP mimics interact with bilayer membranes, three homologous antimicrobial oligomers (AMOs) 1-3 with an m-phenylene ethynylene backbone and alkyl amine side chains were studied. Among them, AMO 1 is nonactive, AMO 2 is specifically active, and AMO 3 is nonspecifically active against bacteria over human red blood cells, a standard model for mammalian cells. The interactions of these three AMOs with liposomes having different lipid compositions are characterized in detail using a fluorescent dye leakage assay. AMO 2 and AMO 3 caused more leakage than AMO 1 from bacteria membrane mimic liposomes composed of PE/PG lipids. The use of E. coli lipid vesicles gave the same results. Further changes of the lipid compositions revealed that AMO 2 has selectively higher affinity toward PE/PG and E. coli lipids than PC, PC/PG or PC/PS lipids, the major components of mammalian cell membranes. In contrast, AMO 3 is devoid of this lipid selectivity and interacts with all liposomes with equal ease; AMO 1 remains inactive. These observations suggest that lipid type and structure are more important in determining membrane selectivity than lipid headgroup charges for this series of HDP mimics.

Beta-lactam activity against penicillin-resistant Streptococcus pneumoniae strains exhibiting higher amoxicillin versus penicillin minimum inhibitory concentration values: an in vitro pharmacodynamic simulation.

BACKGROUND: Activity of simulated serum concentrations after oral therapy with 400 mg cefditoren pivoxil b.i.d., 500 mg cefuroxime axetil b.i.d. and 875/125 mg amoxicillin/clavulanic acid b.i.d. and t.i.d. regimens was explored over 24 h against Streptococcus pneumoniae. METHODS: Computerized pharmacodynamic simulations were performed against strains with penicillin/amoxicillin/cefuroxime/cefditoren minimum inhibitory concentrations (MICs, microg/ml) and serotypes: strain 1 (0.25/0.12/1/0.12; serotype 6A), strain 2 (2/4/ 2/0.25; serotype 6B), strain 3 (4/16/4/0.5; serotype 14), and strain 4 (4/16/8/1; serotype 14). RESULTS: Bactericidal activity (> or =3 log(10) reduction) at 12 and 24 h was obtained against all strains with cefditoren, against strains 1 and 2 with cefuroxime and amoxicillin/clavulanic acid t.i.d., but only against strain 1 with amoxicillin/clavulanic acid b.i.d.. Bactericidal activity at 24 h was related to T > MIC of >30% dosing interval, 1.7-2.0 log(10) reductions with T > MIC of 20-30%, and <1 log(10) reduction or regrowth with T > MIC of 0%. CONCLUSIONS: It is difficult to achieve pharmacodynamic coverage and bactericidal activity by physiological concentrations of oral beta-lactams against penicillin-resistant pneumococcal strains exhibiting higher amoxicillin versus penicillin MICs. Cefditoren may offer alternatives.

Distribution of secretory inhibitor of platelet microbicidal protein among urethral isolates with its correlation with prostatitis.

AIM: To report the detection in vitro of secretory inhibitor of platelet microbicidal protein (SIPMP) phenotypes of urethral isolates along with a comparison with isolates from patients with or without chronic bacterial prostatitis (CBP). METHODS: Urethral isolates of Staphylococcus spp. (n=4), diphtheroids (n=28), micrococci (n=15), streptococci (n=21), Enterobacteriaceae (n=9) and Enterococcus faecalis (n=19) from patients with or without CBP were tested. SIPMP production was tested by inhibition of platelet microbicidal protein (PMP) bioactivity against Bacillus subtilis and was expressed as percentage of inhibition of PMP bactericidal activity. RESULTS: A significantly higher proportion of CBP-strains (57.78% vs. 16.67%) reduced PMP-induced killing of Bacillus subtilis than non-CBP strains did (P<0.01). SIPMP levels of staphylococci and Enterococcus faecalis from the CBP group were significantly higher than those of the control group. CONCLUSION: These results suggest that SIPMP production is associated with the CBP source. Data from the present study might have significant implications for the understanding of the pathogenesis of CBP.

Measurement of respiratory burst products generated by professional phagocytes.

Activation of professional phagocytes, potent microbial killers of our innate immune system, is associated with an increase in cellular consumption of molecular oxygen (O2). The burst of 02 consumption is utilized by an NADPH-oxidase to generate highly-reactive oxygen species (ROS) starting with one and two electron reductions to generate superoxide anion (O2-) and hydrogen peroxide (H2O2), respectively. ROS are strongly bactericidal but may also cause tissue destruction and induce apoptosis in other immune competent cells of both the innate and the adaptive immune systems. Thus, the development of basic techniques to measure/quantify ROS generation/release by phagocytes during activation of the respiratory burst is of great importance, and a large number of techniques have been used for this purpose. Three of these techniques, chemiluminescence amplified by luminol/ isoluminol, the absorbance change following reduction of cytochrome c, and the fluorescence increase upon oxidation of p-hydroxyphenylacetate, are described in detail in this chapter. These techniques can be valuable tools in research spanning from basic phagocyte biology to more clinically-oriented research on innate immune mechanisms and inflammation.

Capture stress and the bactericidal competence of blood and plasma in five species of tropical birds.

In wild birds, relatively little is known about intra- or interspecific variation in immunological capabilities, and even less is known about the effects of stress on immune function. Immunological assays adaptable to field settings and suitable for a wide variety of taxa will prove most useful for addressing these issues. We describe a novel application of an in vitro technique that measures the intrinsic bacteria-killing abilities of blood. We assessed the capacities of whole blood and plasma from free-living individuals of five tropical bird species to kill a nonpathogenic strain of E. coli before and after the birds experienced an acute stress. Killing invasive bacteria is a fundamental immune function, and the bacteria-killing assay measures constitutive, innate immunity integrated across circulating cell and protein components. Killing ability varied significantly across species, with common ground doves exhibiting the lowest levels and blue-crowned motmots exhibiting the highest levels. Across species, plasma killed bacteria as effectively as whole blood, and higher concentrations of plasma killed significantly better. One hour of acute stress reduced killing ability by up to 40%. This assay is expected to be useful in evolutionary and ecological studies dealing with physiological and immunological differences in birds.

Normal human sera contain bactericidal IgG that binds to the oligosaccharide epitope expressed within lipooligosaccharides of Neisseria gonorrhoeae.

Although more than several investigators reported the presence of antibodies in normal human sera (NHS) that bind to lipooligosaccharide (LOS) of Neisseria gonorrhoeae, the specificities of those antibodies were not fully characterized. To identify anti-LOS antibodies in NHS, we used LOS from a serum-sensitive strain, JW31R, as an affinity ligand and purified IgG from NHS that bound to JW31R LOS. The affinity purified IgG (AP-IgG) binds to the oligosaccharide (OS) moiety of both the ligand LOS and its truncated form, 15253 LOS. Lipid A could be essential for maximum expression of the carbohydrate epitope that resides on 15253 OS. We also found that AP-IgG is capable of killing a serum-sensitive strain JW31R. The present work provided direct evidence that NHS contain bactericidal antibodies specific for a site close to the inner core OS expressed on gonococcal LOS. The present results not only show that anti-LOS antibodies specific for the inner core OS could play a major role in our defense against gram-negative bacteria. But also they demonstrated that such core OS or a nearby site could be utilized as possible targets for vaccine development against microbial infections.

Effects of granulocyte colony-stimulating factor on neutrophils and inflammatory cytokines in the early stage of severe acute pancreatitis in rats.

BACKGROUND: The high mortality rate of severe acute pancreatitis (SAP) is closely associated with secondary infections of pancreatic and peripancreatic tissues. It was reported that granulocyte colony-stimulating factor (G-CSF) increased the number of leukocytes and enhanced their functions. However, an inflammatory response may be enhanced by an increased number of leukocytes. Our purpose was to study the roles of G-CSF in peritoneal-exudate neutrophils and inflammatory cytokines in the early stage of experimental SAP. METHODS: SAP was induced by injecting 0.2 ml of 3% taurocholate acid into the biliopancreatic duct in male Wistar rats. G-CSF (90 microg/kg body weight) or saline was administered 1 h before the SAP induction. The number of neutrophils and their phagocytic and bactericidal activities were evaluated, and the concentrations of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-1beta in plasma and ascitic fluid were measured 1 h and 3 h after the SAP induction. RESULTS: The number of peritoneal-exudate neutrophils (PENs) at 3 h was increased by G-CSF administration (81 +/- 50 x 10(5) cells/total exudate), as compared with that shown with saline administration (28 +/- 13 x 10(5) cells/total exudate; P < 0.05). The numbers of phagocytic and bactericidal neutrophils were also elevated by G-CSF administration. G-CSF administration did not increase the concentrations of TNF-alpha, IL-6, and IL-1beta in the plasma and ascitic fluid. CONCLUSIONS: G-CSF increases the numbers of neutrophils and enhances their functions against bacteria, but it does not enhance intraabdominal and systemic inflammatory responses in the early stage of SAP.

Differentiated U937 cells exhibit increased bactericidal activity upon LPS activation and discriminate between virulent and avirulent Listeria and Brucella species.

In the study of interactions between facultative intracellular pathogens and macrophages, monocytic cell lines have the advantages of showing defined states of activation and lacking genetic variation among donors, thus yielding reproducible results. Nonpathogenic Escherichia coli K12 were killed at similar rates in the U937 cell line differentiated into macrophage-like cells by phorbol myristate acetate (PMA) or by the combination of retinoic acid (RA) and vitamin D3 (VD). Complete elimination was reached only when cells were activated by lipopolysaccharide for 30 min prior to infection, and it was further enhanced when bacteria were opsonized by specific immunoglobulin G. Both types of differentiation led to intracellular multiplication of virulent Listeria monocytogenes and to elimination of the animal pathogen Listeria ivanovii. For both strains, conditions for intracellular survival were more favorable in PMA-differentiated U937. During infection, RA/VD-differentiated U937 could discriminate between the human pathogen Brucella suis S1, which strongly multiplied, and the animal pathogen Brucella canis, which survived without multiplication. U937 cells differentiated by RA and VD therefore represent a basic model in bacteria-human macrophage interactions.

Identification of alternative pathway serum complement activity in the blood of the American alligator (Alligator mississippiensis).

Incubation of different dilutions of alligator serum with sheep red blood cells (SRBCs) that had not been sensitized with antibodies resulted in concentration-dependent hemolytic activity. This hemolytic activity was not affected by the presence of ammonium hydroxide and methylamine, known inactivators of the classical complement cascade. However, the hemolytic activities were inhibited by EDTA and salicylaldoxime, indicating that the alternate pathway is primarily responsible for these activities. Immunofixation of electrophoretically-resolved alligator serum proteins with antihuman C3 polyclonal antibodies resulted in detection of a protein antigenically similar to human C3 in alligator serum. SDS-PAGE, followed by Western blot analysis, revealed the presence of two alligator serum proteins with nearly identical molecular weights as human C3alpha and C3beta. SRBC hemolysis and antibacterial activity by alligator serum was significantly reduced in the presence of antihuman C3 antibodies. The hemolytic effect of alligator serum was shown to occur rapidly, with significant activity within 5 min and maximal activity occurring at 15 min. SRBC hemolysis was also temperature-dependent, with reduced activity below 15 degrees C and above 30 degrees C. These data suggest that the antibiotic properties of alligator serum are partially due to the presence of a complement-facilitated humoral immune response analogous to that described in mammalian systems.