Quentin N. Myrvik

Adhesive colonization of biomaterials and antibiotic resistance

This study addresses the problem of antibiotic resistance in adhesive, biomaterial-centred infections. It is suggested that this anionic, extracapsular, polysaccharide slime produced by bacteria protects them from antibiotics and sequesters critical ions from the surface of biomaterials. Biofilm-enclosed bacteria on the surface of stainless steel substrata in a test chamber were challenged with incremental levels of tobramycin. In this setting, the minimum inhibitory concentration and minimum bactericidal level of tobramycin for Staphylococcus epidermis were well above normal.

Oral Administration of Chitin Down-Regulates Serum IgE Levels and Lung Eosinophilia in the Allergic Mouse

Previous studies showed that local macrophages phagocytose nonantigenic chitin particles (1–10 μm polymers of N-acetyl-d-glucosamine) through mannose receptors and produce IL-12, IL-18, and TNF-α. These cytokines lead to the production of IFN-γ by NK cells. To determine whether chitin could down-regulate Th2 responses, chitin was given orally (8 mg/day for 3 days before and 13 days during ragweed allergen immunization) in BALB/c and C57BL/6 mice. These ragweed-immunized mice were given ragweed intratracheally on day 11. Three days after the challenge, the immunized mice with saline (controls) showed increases in serum IgE levels and lung eosinophil numbers. The chitin treatment resulted in decreases of these events in both strains. To dissect the inhibitory mechanisms of Th2 responses, spleen cells (4 × 106 cells/ml) isolated from the ragweed-immunized mice (controls) were cultured in the presence of ragweed and/or chitin for 3 days (recall responses). Ragweed alone stimulated the production of IL-4 (0.6 ng/ml), IL-5 (20 U/ml), and IL-10 (3.2 ng/ml), but not IFN-γ. Ragweed/chitin stimulation resulted in significant decreases of IL-4, IL-5, and IL-10 levels and the production of IFN-γ (48 U/ml). Moreover, spleen cells isolated from the chitin-treated mice showed ragweed-stimulated IFN-γ production (15 U/ml) and significantly lower levels of the Th2 cytokines, suggesting that the immune responses were redirected toward a Th1 response. Collectively, these results indicate that chitin-induced innate immune responses down-regulate Th2-facilitated IgE production and lung eosinophilia in the allergic mouse.

Antibiotic resistance of biomaterial-adherent coagulase-negative and coagulase-positive staphylococci.

Whether or not bacterial populations are massively enclosed in slime, it appears that antibiotic resistance, when compared to suspension organisms, is related to surface adhesion and to the specific material of the substratum. These findings are of significance in the understanding and treatment of biomaterial-localized infections.

Surgical biomaterials and differential colonization by Staphylococcus epidermidis

The data presented in this communication demonstrate preferential colonization of certain biomaterials by Staphylococcus epidermidis. Using a laminar flow biomaterial colonization chamber and surgical-grade biomaterials (stainless steel, aluminium ceramic, methyl methacrylate and high-density polyethylene), the pattern of colonization was quantitated using plate count techniques and electron microscopy. Under comparable conditions, methyl methacrylate was colonized by S. epidermidis in greater numbers than the other biomaterials. Increased bacterial colonization and slime production on methyl methacrylate was time-dependent and 15 times higher than on stainless steel and aluminium and four times higher than on high-density polyethylene. The data reveal that certain biomaterials may promote infection by favouring colonization by potential pathogens. This variable should be explored extensively in an in vivo setting because of its implication in clinical infections.

Th1 Adjuvant N-Acetyl-d-Glucosamine Polymer Up-Regulates Th1 Immunity but Down-Regulates Th2 Immunity against a Mycobacterial Protein (MPB-59) in Interleukin-10-Knockout and Wild-Type Mice

ABSTRACT Treatment of mice with heat-killed (HK) Mycobacterium bovis BCG or 1- to 10-μm chitin particles (nonantigenic N-acetyl-d-glucosamine polymers) is known to induce innate immune responses, including gamma interferon (IFN-γ) production, which plays a Th1 adjuvant role. However, HK BCG further induces prostaglandin E2-releasing spleen macrophages (Mφ) (PGE2-Mφ), which potentially inhibit Th1 adjuvant activities. We found that chitin particles did not induce PGE2-Mφ formation. To further assess whether chitin has Th1 adjuvant effects, interleukin-10 (IL-10)-knockout (KO) mice and their wild-type (WT, C57BL/6) controls were immunized with a 30-kDa MPB-59 mycobacterial protein mixed with chitin. Immunization with MPB-59 alone induced Th2 responses, characterized by increases in total serum immunoglobulin E (IgE) and specific serum IgG1 levels and spleen Th2 cells producing IL-4, IL-5, and IL-10. No IFN-γ-producing spleen Th1 cells, specific serum IgG2a, or delayed-type hypersentivity (DTH) footpad reactions were detected. On the other hand, chitin–MPB-59 immunization significantly increased spleen Th1 responses, DTH reaction, and serum IgG2a levels along with decreases of Th2 responses. The magnitude of these Th1 adjuvant effects was greater in IL-10-KO mice than in WT mice. In contrast, immunization with HK BCG–MPB-59 showed little or no Th1 adjuvant effect. These data indicate that chitin has a unique Th1 adjuvant effect on the development of Th1 immunity against a mycobacterial antigen. IL-10 down-regulates the adjuvant effect of chitin.

Nitrogen Dioxide Effects on Alveolar Macrophages

Rabbits were exposed to 5, 15, 25, or 50 ppm of nitrogen dioxide for three hours. Their alveolar cells were harvested and tested for phagocytic activity, rate of oxygen uptake, and hexosephosphate shunt activity. Other groups of animals were given parainfluenza 3 virus intrafracheally prior to NO2 exposure in order to measure the effect of this gas on virus-induced resistance to rabbitpox virus. Virus-induced resistance and phagocytic activity were suppressed by 15 ppm of NO2. In contrast, 50 ppm stimulated oxygen uptake and hexosephosphate shunt activity. Suppression of phagocytic activity and virus-induced resistance were observed to be the most sensitive indicators of NO2 alterations of alveolar cells.

Nitrogen dioxide inhibition of viral-induced resistance in alveolar monocytes.

Rabbit alveolar monocytes harvested from rabbits injected intratracheally with a 5 × 105 tissue culture infective dose (TCID00) of para-influenza-3 virus are resistant to an in vitro challenge with rabbit pox virus. However, if the rabbits are exposed to 25 ppm nitrogen dioxide (NO2) for three hours immediately after the para-influenza-3 virus inoculation or at 0, 3, 6, 12, or 24 hours before the inoculation of virus, the previously observed resistance does not develop. This refractory state lasts at least 96 hours in that the alveolar macrophages from animals exposed to NO2 are unable to produce interferon when inoculated with para-influenza-3 virus in vitro. Exposure to NO2 also appears to increase the adsorption rate of para-influenza-3 virus in the lungs of rabbits, but it does not inactivate or enhance the infectivity of the virus employed.

Differential uptake of neutral red by macrophages from three species of estuarine fish

Macrophage endocytosis is part of the immune response to foreign material and includes the processes of phagocytosis, ingestion of particulate material, and pinocytosis, the uptake of liquid droplets. This work reports the development of a quantitative method to measure in vitro uptake of neutral red by fish macrophages, based on the spectrophotometric measurement of ingested neutral red dye. We have used this method to monitor the rates of uptake in three species of fish: spot, Leiostomus xanthurus; hogchoker, Trinectes maculatus; and summer flounder, Paralichthys dentatus. For the species tested, there appeared to be an intrinsic pattern of uptake which was highest in flounder, lowest in hogchoker and intermediate in spot. The excellent reproducibility of this assay should make it useful for detecting toxicological effects in the environment.

The Effects of Asbestos on Macrophages

The exact role of the alveolar macrophage in the pathogenesis of asbestosis is not known. Most studies of the effect of asbestos on macrophages have been concerned with the in vitro biochemical or cytotoxic properties of the dust and have made use of peritoneal macrophages. In general, chrysotile had a toxic effect on the macrophages, whereas amphibole varieties did not. Most forms of absetos, however, are actively fibrogenic in man and animals, and there is no clear correlation between in vitro cytotoxicity of various forms of asbestos and their fibrogenicity. Recent experiments in which animals are exposed to asbestos in vivo provide evidence of alteration of macrophage activity, as demonstrated by changes in surface morphology and IgG receptor sites, as well as released of various secretory products. Deposition of complement components found on the surface of alveolar marcophages from animals exposed to asbestos could be a manifestation of a humoral immune response directed against an altered cell. The capacity of macrophages to participate in inflammation, tissue repair, and immunity suggests an immunopathogenic concept for the development of asbestosis.

Splenic PGE2-releasing macrophages regulate Th1 and Th2 immune responses in mice treated with heat-killed BCG

Hosts infected with low doses of mycobacteria develop T helper cell type 1 (Th1) immunity, but at relatively higher doses, a switch to Th2 immunity occurs. Prostaglandin E2 (PGE2) is a proposed mediator of the Th1‐to‐Th2 shift of immune responses, and mycobacterial products induce PGE2‐releasing macrophages (PGE2‐MØ) in the mouse spleen in a dose‐dependent manner. Splenic PGE2‐M Ø from Balb/c mice, given 0.01 or 1 mg heat‐killed (HK) Mycobacterium bovis bacillus Calmette‐Guerin (BCG) intraperitoneally (i.p.), were characterized by the ex vivo release of PGE2 (>10 ng/106 cells), cytokine production, and expression of PGG/H synthase (PGHS)‐1, PGHS‐2, cytosolic PGE synthase (PGES), and microsomal PGES‐1. At Day 14 after the treatment, mice treated with 1 mg, but not 0.01 mg, BCG had increased levels of PGHS‐2+ PGE2‐MØ, total serum immunoglobulin E (IgE), and serum IgG1 antibodies (Th2 responses) against heat shock protein 65 and purified protein derivative. Cultures of spleen cells isolated from these mice expressed interleukin (IL)‐4 and IL‐10 in recall responses. Treatment of mice receiving 1 mg BCG with NS‐398 (a PGHS‐2 inhibitor, 10 mg/kg i.p., daily) resulted in enhanced interferon‐γ (IFN‐γ) production with reduced IL‐4 and IL‐10 production in recall responses. This treatment also resulted in decreased total serum IgE levels. Treatment of C57Bl/6 mice with HK‐BCG (0.5 mg dose) also induced a mixture of Th1 and Th2 responses, although IFN‐γ production was markedly increased, and IL‐4 was decreased compared with Balb/c mice. Thus, our results indicate that by 14 days following treatment of mice with high doses of HK‐BCG, splenic PGE2‐MØ formation is associated with a PGHS‐2‐dependent shift from Th1‐to‐Th2 immune responses.