Peter G. Sohnle

Zinc-Reversible Antimicrobial Activity of Recombinant Calprotectin (Migration Inhibitory Factor—Related Proteins 8 and 14)

Recombinant calprotectin, consisting of 2 individual peptide chains also called migration inhibitory factor-related protein (MRP)-8 and MRP14, was tested for antimicrobial activity in a Candida albicans growth inhibition assay. Both chains contain HEXXH zinc-binding sites and might be expected to manifest zinc-reversible, antimicrobial activity similar to that of native calprotectin. When tested alone, neither MRP8 nor MRP14 showed activity in the Candida growth assay. A synthetic 20-amino acid peptide containing the HEXXH sequence of MRP14, along with a nearby HHH sequence, was also inactive in this assay. However, equimolar concentrations of MRP8 and MRP14 demonstrated a potent growth inhibitory effect that was reversible by 30 microM zinc. Truncated MRP14 (missing the C-terminal GHHHKPGLGEGTP tail) used in combination with MRP8 demonstrated zinc-reversible activity that was somewhat less than that with complete MRP14. These results suggest that intact calprotectin, consisting of a heterodimer of MRP8 and MRP14, is necessary to form a zinc-binding site capable of inhibiting microbial growth.

Generation of free radical intermediates from foreign compounds by neutrophil-derived oxidants.

A large number of foreign compounds, including many drugs, industrial pollutants, and environmental chemicals, can be oxidized under appropriate conditions to potentially toxic free radical intermediates. We evaluated the ability of the oxidants produced by the neutrophil myeloperoxidase system to generate free radical intermediates from several such compounds. Sodium hypochlorite or hypochlorous acid produced by human peripheral blood neutrophils and trapped in the form of taurine chloramine were both found to be capable of producing free radicals from chlorpromazine, aminopyrine, and phenylhydrazine. These radical intermediates were demonstrated by visible light spectroscopy and by direct electron spin resonance (for the chlorpromazine and aminopyrine radicals) or by spin-trapping (for the phenyl radical generated from phenylhydrazine). Stable oxidants produced by the neutrophils (i.e., those present in the supernatants of stimulated neutrophils in the absence of added taurine) also were found to be capable of generating free radical intermediates. The production of the oxidants and the ability of neutrophil supernatants to generate these radicals were almost completely eliminated by sodium azide, a myeloperoxidase inhibitor. We suggest that the oxidation by neutrophils of certain chemical compounds to potentially damaging electrophilic free radical forms may represent a new metabolic pathway for these substances and could be important in the processes of drug toxicity and chemical carcinogenesis.

Histidine-Based Zinc-Binding Sequences and the Antimicrobial Activity of Calprotectin

Calprotectin is a protein in neutrophil cytoplasm and abscess fluids that appears to inhibit microbial growth through competition for zinc. This study was undertaken to identify specific sites that might be responsible for the proteins zinc-binding antimicrobial activity. A review of published calprotectin amino acid sequences revealed the HEXXH motif of thermolysin-type metalloproteases and an HHH polyhistidine sequence near the C-terminus of the proteins heavy chain. Reagent polyhistidine had antimicrobial activity against Candida albicans similar to that of calprotectin. Also, one type of HEXXH-containing thermolysin was inactive in the C. albicans assay, whereas a protein tagged with six C-terminal histidines did have calprotectin-like zinc-reversible antimicrobial activity. The activity of polyhistidine, as well as that of calprotectin itself, was reversed by addition of zinc or treatment with the histidine-modifying compound diethylpyrocarbonate. These results suggest that calprotectins antimicrobial activity may be related to certain histidine-based zinc-binding sequences.

INFLUENCE OF BACTERIAL ADHERENCE TO INTRAVASCULAR CATHETERS ON IN-VITRO ANTIBIOTIC SUSCEPTIBILITY

Slime-producing and non-slime-producing strains of coagulase-negative staphylococci (CNS) were evaluated for nafcillin susceptibility in the presence and absence of polyvinylchloride (PVC) catheters. Semiquantitative roll cultures of catheters with adherent organisms after exposure to predicted bactericidal concentrations of nafcillin were carried out to assess survival of these organisms. Slime-producing and non-slime-producing CNS had similar minimum inhibitory (MIC) and bactericidal (MBC) concentrations in the absence of catheters and similar MIC in the presence of catheters. However, the mean MBC of slime-producing CNS, and to a lesser extent of non-slime-producing strains was higher in the presence than in the absence of catheters. Slime-producing CNS were recovered from PVC catheters after overnight incubation in cidal concentrations (greater than 4.0 micrograms/ml) of nafcillin (average 350 colony-forming units per 1 cm). Thus nafcillin-sensitive CNS strains, particularly those producing slime, are able to survive exposure to cidal concentrations of the drug when adherent to PVC catheters.

Marijuana smoking and fungal sensitization

The possible role of marijuana (MJ) in inducing sensitization to Aspergillus organisms was studied in 28 MJ smokers by evaluating their clinical status and immune responses to microorganisms isolated from MJ. The spectrum of illnesses included one patient with systemic aspergillosis and seven patients with a history of bronchospasm after the smoking of MJ. Twenty-one smokers were asymptomatic. Fungi were identified in 13 of 14 MJ samples and included Aspergillus fumigatus, A. flavus, A. niger, Mucor, Penicillium, and thermophilic actinomycetes. Precipitins to Aspergillus antigens were found in 13 of 23 smokers and in one of 10 controls, while significant blastogenesis to Aspergillus was demonstrated in only three of 23 MJ smokers. When samples were smoked into an Andersen air sampler, A. fumigatus passed easily through contaminated MJ cigarettes. Thus the use of MJ assumes the risks of both fungal exposure and infection, as well as the possible induction of a variety of immunologic lung disorders.

In vitro quantitative adherence of bacteria to intravascular catheters.

Adapting standard techniques, a simple in vitro system was devised to compare quantitative bacterial adherence to iv catheters of different compositions. Upon brief immersion of catheters in suspensions of Staphylococcus aureus, coagulase-negative staphylococci, and Escherichia coli, organisms adhered to catheter surfaces. After overnight growth in broth, organisms remained adherent and formed colonies, as shown by light and scanning electron microscopy. In addition, quantitative adherence using a blood agar roll technique, expressed as bacteria per square centimeter of catheter surface area per 10(6) colonies per milliliter inoculum, was calculated. Adherence was greater on polyvinylchloride (PVC) catheters (geometric mean 342) than on Teflon catheters (geometric mean 49.6) for coagulase-negative staphylococci (P less than 0.005). Also, the number of coagulase-negative staphylococci adherent to PVC catheters was significantly greater than for E. coli (geometric mean 70.6) at analogous inocula (P less than 0.02). Differences in bacterial adherence to the surface of iv devices may be important in the pathogenesis of catheter-associated infections. This in vitro method could prove useful in testing bacterial adherence properties of newly developed catheter materials, and allow development of catheters less prone to be associated with bacterial adherence and catheter-related infections.

Effect of Bone Biopsy in Guiding Antimicrobial Therapy for Osteomyelitis Complicating Open Wounds

BackgroundOsteomyelitis associated with infected overlying wounds represents a difficult diagnostic and therapeutic problem; bone biopsies can be done during debridement of the overlying wounds, but it is unclear how often the results of these bone cultures actually affect subsequent antibiotic decisions. The present study was undertaken to evaluate the usefulness of bone biopsies in guiding antibiotic therapy for this type of osteomyelitis. MethodsCulture results of 44 bone biopsies taken during surgical debridement in 41 patients over the period from June 1994 to August 1998 were compared with those from the overlying wounds to determine whether the data affected the subsequent choice of antibiotics. The study design was that of a retrospective chart review in which the standard operative and microbiological procedures in place at the Milwaukee Veterans Affairs Medical Center were used. ResultsSixty-one wound and 55 bone isolates were obtained during this study. Thirty-one isolates were found in bone, but not the overlying wound; diphtheroids were the most common organism obtained in this fashion. Correlation between wound and bone isolates was generally poor. Antibiotics were subsequently changed in 20 of the 44 cases after results of the bone biopsy became known, with the bone isolates already being covered in 10 cases and the bone biopsy results ignored in 14 cases. ConclusionBecause bone biopsy results seem to aid in tailoring antibiotic therapy in almost half the cases when bone is sampled during wound debridement surgery, this technique may be very helpful in certain cases and should be regularly undertaken when these procedures are carried out.

Cell-Mediated Immunity to Pityrosporum Orbiculare in Tinea Versicolor

Pityrosporum orbiculare, the presumed etiologic agent of tinea versicolor, was cultured in vitro and antigenic extracts prepared from the cultured organisms. Studies with lymphocytes from human cord blood and peripheral blood of guinea pigs demonstrated that such extracts were not mitogenic. Further studies in guinea pigs indicated that the animals could be sensitized by the injection of P. orbiculare extract in Freunds complete adjuvant and that this extract could elicit lymphocyte transformation and delayed skin test responses in sensitized animals. A group of 12 tinea versicolor patients and 15 normal subjects were studied in vitro for cell-mediated immunity to P. orbiculare extract. The majority of the subjects tested in both groups demonstrated positive lymphocyte transformation responses to this extract, as well as to standard mitogens and common microbial antigens. However, lymphocytes from tinea versicolor patients produced significantly less leukocyte migration inhibitory factor activity when stimulated by Candida albicans and P. orbiculare extracts than did lymphocytes from normal subjects. This was also true if only subjects with positive lymphocyte transformation responses to these antigens were considered. Leukocyte migration inhibitory factor responses to streptokinase/streptodornase were not significantly different between the two groups. Therefore, it appears that although both normal subjects and tinea versicolor patients demonstrate prior sensitization to antigens of P. orbiculare, the effector function of lymphocytes from most tinea versicolor patients appears to be impaired in that they produce subnormal amounts of the mediator leukocyte migration inhibitory factor when stimulated with antigenic extracts of this organism.

NEUTROPHIL DEATH AS A DEFENCE MECHANISM AGAINST CANDIDA ALBICANS INFECTIONS

In studies of experimental Candida albicans infections, growth of invading organisms sometimes ceased before the organisms reached the neutrophil infiltrates. Lysates of human neutrophils inhibited the directed growth of candida pseudohyphae in agarose gel and suppressed the proliferation of candida yeast in broth cultures, but did not kill the organisms or prevent their germination. The growth-inhibitory material released from disrupted neutrophils had an estimated molecular weight of 30 kD and differed from most previously described neutrophil antimicrobial factors in that it was present in cell sap rather than granules, and did not appear in the supernatant after stimulation of the cells. Neutrophil death and dissolution may represent an alternative host defence mechanism against invasive C albicans infection.

Deposition of complement components in the cutaneous lesions of chronic mucocutaneous candidiasis

Abstract By immunofluorescent techniques, deposits of C3 and properdin were found along the basement membranes in the cutaneous lesions of three of six patients with chronic mucocutaneous candidiasis; candida antigen was found with C3 and properdin in the lesion of one. Immunoglobulin and C4 were not found in any specimen. Soluble Candida albicans components and intact organisms were found to be capable of activating the alternative complement pathway. The findings suggest that the intense inflammatory infiltrates in the lesions of chronic mucocutaneous candidiasis occur as a consequence of complement activation. It is proposed that soluble products are released from the infecting organisms in the epidermis, diffuse to the upper dermis, and directly activate complement there by the alternative pathway, resulting in the release of complement-derived chemotactic factors. Activation of complement in this manner and the resulting inflammatory infiltrates do not rid the hosts of the infecting organisms as the cutaneous infections persist for months if untreated. However, the inflammatory infiltrate may prevent the organisms from invading more deeply into the skin.