Chemokine-induced cutaneous inflammatory cell infiltration in a model of Hu-PBMC-SCID mice grafted with human skin.

Recently, certain chemokines and chemokine receptors have been preferentially associated with the selective recruitment in vitro of type 1 T cells, such as IP-10 and its receptor CXCR3, or type 2 T cells such as monocyte-derived chemokine (MDC) and eotaxin and their receptors CCR4 and CCR3. Very few models have provided confirmation of these findings in vivo. Taking advantage of the humanized SCID mouse model grafted with autologous human skin, the ability of the chemokines IP-10, MDC, eotaxin, and RANTES to stimulate cell recruitment was investigated. Intradermal IP-10 injection resulted in an influx of CD4+ T lymphocytes but also surprisingly in the recruitment of dendritic cells. MDC recruited mainly CD8+ T lymphocytes, and had little effect on eosinophils. As predicted, eotaxin was a potent inducer of eosinophil and basophil migration, also recruiting CD4+ T cells. RANTES, a ubiquitous chemokine associated with both type 1 and type 2 profiles, was able to recruit all cell types. CXCR3-positive cells were preferentially recruited by IP-10, whereas CCR3- and CCR4-positive cells were predominantly found after injection of eotaxin and MDC. Thus, in a human environment in vivo, some chemokines have the ability to recruit cells expressing chemokine receptors preferentially expressed on type 1 or type 2 cells. Further investigations revealed that MDC and eotaxin induced the recruitment of type 2, but not type 1, cytokine-producing cells. RANTES, on the other hand, induced the migration of both type 1 and type 2 cytokine-secreting cells, whereas IP-10 did not induce the recruitment of either subtype. These studies provide detailed information on the properties of MDC, eotaxin, IP-10, and RANTES as chemotactic molecules in skin in vivo. The use of the humanized SCID mouse model grafted with human skin is validated as a useful model for the evaluation of chemokine function in the inflammatory reaction, and suggests that therapeutic targeting of certain chemokines might be of interest in diseases associated preferentially with a type 1 or type 2 profile.

Demonstration of intracellular Staphylococcus aureus in bovine mastitis alveolar cells and macrophages isolated from naturally infected cow milk.

Numerous in vitro studies have demonstrated that Staphylococcus aureus may be internalized and survive in a bovine mammary epithelial cell line. We report here the presence of internalized and living S. aureus in alveolar cells and macrophages in milk samples of bovine mastitis. We used fluorochrome labeled monoclonal antibodies, specifically recognizing surface cell markers of bovine alveolar cells and macrophages, to isolate these two types of cells using fluorescence activated cell sorting. Extracellular bacteria and DNA were previously eliminated to exclude possible contamination. In order to detect intracellular bacterial DNA inside the isolated cells, we used PCR amplification of bacterial DNA and the PCR products were analyzed by Southern blot with a specific probe for Staphylococcus. The results showed the presence of Staphylococcus DNA inside the two isolated populations of cells, confirming that S. aureus could penetrate alveolar cells and macrophages. The demonstration of the presence of intracellular living S. aureus was determined by bacteriological culture of positive samples plated onto blood agar plates and by its further identification. Our results showed for the first time that living S. aureus and its DNA are present in both alveolar cells and macrophages in chronically infected cow milk.

Synergistic effect of diesel organic extracts and allergen Der p 1 on the release of chemokines by peripheral blood mononuclear cells from allergic subjects: involvement of the map kinase pathway.

The organic compounds of diesel exhaust particles (DEP-PAHs) have been shown to favor immunoglobulin production and bronchial hyperresponsiveness and to affect cytokine and chemokine productions. To evaluate if diesel exhaust could act in synergy with a house dust mite allergen (Der p 1), peripheral blood mononuclear cells from allergic patients were exposed to DEP-PAHs, with or without purified Der p 1. DEP-PAHs and Der p 1 separately induced an increase in interleukin (IL)-8, regulated on activation, normal T cells expressed and secreted (RANTES), and tumor necrosis factor-alpha concentrations. Interestingly, a synergy between the two stimuli was also observed. In the case of monocyte chemotactic protein (MCP)-1, DEP-PAHs reduced the release, whereas Der p 1 enhanced it. A simultaneous exposure led to reduced production as compared with allergen exposure alone, but still represented an increase as compared with the control exposure. Mitogen-activated protein (MAP) kinase Erk1/2 antagonist mainly inhibited the release of MCP-1, whereas MAP kinase p38 antagonist mainly suppressed the release of IL-8 and RANTES. Messenger RNA expression correlated with protein measurements. Moreover, supernatants from cells exposed to both DEP-PAHs and Der p 1 had a significant chemotactic activity on neutrophils and eosinophils. These findings suggest that simultaneous exposure of allergic patients to DEPs and allergens could result in high local chemokine levels via MAP kinase pathways activation, increasing the likelihood of reaching a critical threshold leading to the initiation of respiratory allergic symptoms.

Demonstration of a fusion mechanism between a fluid bactericidal liposomal formulation and bacterial cells.

It was previously demonstrated that fluid liposomal-encapsulated tobramycin, named Fluidosomes, was successful in eradicating mucoid Pseudomonas aeruginosa in an animal model of chronic pulmonary infection, whereas free antibiotic did not reduce colony-forming unit (CFU) counts (C. Beaulac et al., Antimicrob. Agents Chemother. 40 (1996) 665-669; C. Beaulac et al., J. Antimicrob. Chemother. 41 (1998) 35-41). These liposomes were also shown to be bactericidal in in vitro tests against strong resistant P. aeruginosa 64 microg/ml). The time needed to reach the maximal fusion rate was about 5 h for the resistant strain comparatively to much shorter time for the sensitive strain. The specific characteristics of Fluidosomes could help overcome bacterial resistance related to permeability barrier and even enzymatic hydrolysis considering the importance of synergy in the whole process of antibiotic resistance.

A glycosyl hydrolase activity of mammalian reovirus sigma1 protein can contribute to viral infection through a mucus layer.

The mammalian reovirus sigma1 protein is responsible for viral attachment to host cells and hemagglutination properties of the virus. In the present study, sequence similarity between sigma1 and chicken-type lysozymes prompted us to investigate additional functions of the sigma1 protein. Expression in Pichia pastoris yeast cells showed that sigma1 can actually cleave lysozyme substrates, including complex sugars found in bacterial cell walls. Replacement by site-directed mutagenesis of acidic amino acid residues in sigma1 by their respective isosteric, uncharged, amino acid residues has allowed us to identify Glu36 and Asp54 as the catalytic pair involved in sigma1-mediated glycosidase activity. The enzyme appears inactive in virions but its activity is unmasked upon generation of infectious subviral particles (ISVPs) by partial proteolytic removal of the outer capsid proteins. Purified sigma1 protein and ISVPs can also hydrolyze mucins, heavily glycosylated glycoproteins that are a major component of the mucus layer overlaying the intestinal epithelium. Furthermore, reovirus infection of epithelial Madin Darby canine kidney cells was inhibited tenfold in cells expressing mucin at their apical surface, while this inhibition was overcome by ISVPs. Unmasking of sigma1 mucinolytic activity in the intestine, consecutive to proteolytic cleavage of virions to ISVPs, thus likely contributes to the known increase in infectivity of reovirus ISVPs compared to complete virions. This work presents the first evidence that some mammalian viruses have evolved mechanisms to facilitate their penetration through the protective barrier of the mucus layer in the intestinal tract.

Simultaneous flow cytometric measurement of Streptococcus suis phagocytosis by polymorphonuclear and mononuclear blood leukocytes.

A simple flow cytometric method was used to study simultaneously the phagocytosis of Streptococcus suis serotype 2 by polymorphonuclear and mononuclear blood leukocytes from swine and humans. Using this method with a bacteria-to-leukocytes ratio of 10:1 and after 60 min of incubation, 80.2 +/- 2.8% of swine granulocytes and 77.0 +/- 2.8% of swine monocytes were shown to contain FITC-labelled S. suis serotype 2 strain 735. Using the same strain, FITC-labelled bacteria were found in 95.5 +/- 3.2% of human granulocytes and in 92.8 +/- 3.6% of human monocytes. The phagocytosis rates of avirulent and virulent strains of S. suis were not significantly different.

Candida albicans serotype analysis by flow cytometry.

Candida albicans strains can be assigned to either of two major serogroups, A or B. Antigenic surface determinants present only in serotype A strains allow such a distinction, which has epidemiologic relevance. Reports have established that the relative distributions of the two serotypes can vary depending on the geographic origin of the isolates. A prevalence of susceptibility to an antifungal agent, flucytosine, was also observed with isolates of serotype A. More recently, it was suggested that the occurrence of serotype B isolates in various clinical forms of candidiasis is increasing. However, this latest finding remains controversial since serotyping results vary widely from one laboratory to another because of the lack of standardized methodologies. Difficulty in interpretation of results, which may lead to erroneous serotype identification, is the major setback associated with current methods. For this study, we thus devised a procedure that relies on flow cytometry and that may eliminate ambiguities in serotype determination. The validation of results was achieved with two types of serotype A-specific antisera, Iatron Factor 6 antiserum and an anti-C. albicans antiserum adsorbed on serotype B yeast cells. Agreement between results obtained with these two reagents was 100% with a wide array of Candida strains. These results confirmed the potential of the flow cytometric procedure as a reliable and reproducible method to establish the serotypes of C. albicans strains. Furthermore, some applications of this procedure to the epidemiological study of this human pathogen are presented.

Lack of involvement of nitric oxide in killing of Aspergillus fumigatus conidia by pulmonary alveolar macrophages.

Nitric oxide is an important antimicrobial mechanism of phagocytes from mice and rats, but in the case of human phagocytes, secretion is still controversial. We investigated whether nitric oxide is involved in the killing of Aspergillus fumigatus conidia by human or murine pulmonary alveolar macrophages. Stimulation of the macrophages with gamma interferon and Escherichia coli lipopolysaccharide had no effect on fungicidal activity against conidia in vitro, with or without the addition of tetrahydrobiopterin. Killing of conidia (means +/- standard deviations) by murine or human alveolar macrophages, before and after stimulation, was 44% +/- 13% and 49% +/- 12% (P = 0.34) and 24% +/- 5% and 29% +/- 10% (P = 0.20), respectively. Fungicidal activity was unaltered in the presence of the competitive inhibitor NG-monomethyl L-arginine, and nitrite was undetectable in cell supernatants. Peritoneal macrophages from B6C3F1 mice produced 18 mumol of nitrite per 10(6) cells in 18 h. In conclusion, nitric oxide does not appear to be involved in the fungicidal activity of murine or human alveolar macrophages against A. fumigatus conidia.

Acquired immunity in experimental murine aspergillosis is mediated by macrophages.

A number of studies have substantiated the pivotal role of innate defense mechanisms in protection against invasive aspergillosis. However, experiments demonstrating increased resistance to lethal intravenous (i.v.) infection with Aspergillus fumigatus conidia in cortisone-treated or untreated mice preinfected with a sublethal dose of conidia and protection of turkeys inoculated subcutaneously with a killed A. fumigatus germling vaccine against subsequent aerosol challenge led us to speculate that acquired immunity may also contribute to host defense against Aspergillus infection. Five-week-old male BALB/c mice were inoculated i.v. with 1.0 x 10(4) viable conidia or saline and challenged i.v. with 1.0 x 10(6) conidia after 7, 15, or 21 days. No protection against challenge was found after 7 days. However, significant and reproducible protection was observed after 15 and 21 days. Mortality was reduced from 90% in control mice to 53% in preinfected mice 40 days after challenge (P = 0.0002). Increased survival was correlated with decreased content of chitin in lungs, liver, and kidneys 4 and 7 days after challenge (P < 0.05). Mice were again inoculated with 1.0 x 10(4) conidia or saline, and after 21 days, 1.0 x 10(8) or 2.0 x 10(8) splenocytes were transferred to naive syngeneic recipients; 2.0 x 10(8) immune splenocytes conferred significant protection (P = 0.0001) against i.v. challenge with 1.0 x 10(6) conidia, and mortality decreased from 83 to 48% 40 days after challenge. Transfer of immune serum offered no protection despite the presence of antibody against a hyphal homogenate of A. fumigatus, which was absent in the sera of control mice. Protection by immune splenocytes was maintained after selective depletion of T cells but was abolished after removal of plastic-adherent splenocytes. Adherent cells were characterized as macrophages by using morphological criteria, nonspecific esterase, and MAC-1 monoclonal antibody. Production of hydrogen peroxide by peritoneal and splenic macrophages from preinfected mice was the same as and lower than, respectively, that from uninfected controls. However, phagocytosis of conidia by peritoneal or splenic macrophages from mice preinfected i.v. or intratracheally was significantly increased after 2 and 3 h of coculture compared with that from uninfected animals, whereas in vitro killing of conidia by splenic macrophages was unaltered. Peritoneal or splenic macrophages from control or preinfected mice failed to kill hyphae in vitro. Killing of hyphae by polymorphonuclear leukocytes was not significantly different between mice preinfected i.v. and uninfected controls. Taken together, the results indicate that acquired immunity mediated by activated macrophages can be demonstrated in experimental murine aspergillosis.(ABSTRACT TRUNCATED AT 400 WORDS)

Production and characterization of monoclonal antibodies to cell wall antigens of Aspergillus fumigatus.

Two murine monoclonal antibodies (MAbs) against Aspergillus fumigatus were produced and characterized. Splenocytes from cell wall-immunized BALB/c mice were fused with SP2/0 myeloma cells. The hybridomas were screened with a cold alkali (CA) extract of mycelium containing protein, mannose, and galactose, and two MAbs of the immunoglobulin M class were purified from ascites fluid. MAbs 1 and 40 were characterized by double immunodiffusion against CA antigen, indirect enzyme immunoassay with mannans of Candida albicans serotypes A or B or Candida tropicalis, indirect immunofluorescence with C. albicans- or A. fumigatus-infected tissues, indirect immunofluorescence with smears of other pathogenic fungi, Western blotting (immunoblotting) with the lectin concanavalin A or BS-1 from the seeds of Bandeirea simplicifolia, and immunoelectron microscopy. MAb 1 did not cross-react with Candida mannan and recognized a periodate-sensitive, pronase- and heat-resistant epitope in CA antigen and three mannose- and galactose-containing components (80, 62, and 49 kilodaltons) of a mycelial homogenate. Immunoelectron microscopy demonstrated binding of MAb 1 to the inner cell wall and intracellular membranes of hyphae and conidia of A. fumigatus. Circulating antigen was detected in experimental invasive aspergillosis by inhibition enzyme immunoassay with MAb 1 and CA antigen. MAb 40 was a nonprecipitating antibody cross-reactive with Candida species, and competition for an epitope located diffusely in the cell wall of A. fumigatus hyphae was demonstrated by incubating MAb 40 with mannan of C. albicans serotype A. These results suggest that MAb 1 recognizes immunodominant oligogalactoside side chains of A. fumigatus galactomannan, while MAb 40 binds to mannopyranosyl side chains common to A. fumigatus galactomannan and C. albicans mannan.