David M. Lupan

Liposomal entrapment of the neutrophil-derived peptide indolicidin endows it with in vivo antifungal activity

Indolicidin, a cationic tridecapeptide amide isolated from the granules of bovine neutrophils, has been found to possess potent antimicrobial activity in vitro but its nonselective toxicity could restrict its therapeutic utility. We found that the concentration at which indolicidin disrupts washed human red blood cell membranes coincided with the concentration at which indolicidin self associates. Because of a preponderance of hydrophobic residues, we believed that indolicidin would partition into liposomes which would restrict its exchange with biological tissues and consequently reduce its toxicity. Fluorescence spectroscopy of indolicidin added to 100 nm liposomes comprised of POPC, POPC/cholesterol (60:40 mol%), DPPC, or DPPC/cholesterol (60:40) revealed a large blue-shift and an increase in intensity of the emission profile indicating insertion into the bilayer. Of the lipids tested, POPC exhibited the highest degree of indolicidin binding as determined by fluorescence and encapsulation efficiency. By sequestering indolicidin within the lipid bilayer of 100 nm POPC liposomes we significantly reduced its toxicity to CHO/K1 cells. Likewise, the systemic toxicity of liposomal indolicidin in Balb/c mice was decreased dramatically relative to aqueous solutions; the maximum dose at which no deaths occurred was 0.4 mg/kg for free indolicidin versus 40 mg/kg for indolicidin-POPC. Because of this decrease in toxicity, we were able to administer liposomally encapsulated material at significantly higher concentrations than unencapsulated aqueous material and achieve efficacy in treating animals systemically infected with Aspergillus fumigatus. Liposomal but not free indolicidin was found to be effective in obtaining cures. This report is the first description of the in vivo therapeutic activity of a neutrophil-derived antimicrobial peptide and suggests that liposomal treatment modalities will provide effective strategies for endowing this class of compounds with pharmacological utility.

Siderophore production by the pathogenic yeast, Candida albicans.

Biochemical assays were used to determine that some strains of Candida albicans were capable of simultaneous secretion of both the hydroxamate and phenolate-type siderophores when grown in a deferrated medium at 37 degrees C. All isolants of C. albicans released hydroxamate-type siderophores into the culture medium; whereas, approximately 40% of the strains simultaneously secreted phenolate-type siderophores. The presence of phenolate and hydroxamate-type siderophores in the culture medium was further confirmed by assaying the culture media with type specific siderophore-dependent bacterial auxotrophs. This is the first report showing production of both classes of siderophores by a pathogenic yeast.

Utilization of siderophores by Candida albicans.

Candida albicans secretes both hydroxamate and phenolate-type siderophores when grown under iron-restricted conditions. The inhibition of candidal growth by iron limitation was reversed by the addition of supplemental hydroxamate on phenolate siderophores. Both siderophores produced equal stimulation of growth suggesting that C. albicans could utilize both siderophores with equal efficiency. Addition of heterologous siderophores from both bacteria and fungi also supported growth of the yeast in a deferrated medium. These results suggest that C. albicans has an iron-uptake mechanism which enables it to obtain iron by utilizing candidal and non-candidal siderophores.

Effect of temperature on siderophore production by Candidaalbicans

The purpose of this study was to examine the effect of elevated temperature on growth and siderophore production by Candida albicans. The results showed that an increase in incubation temperature from 37 degrees C to 41 degrees C produced a marked decrease in both the rate and quantity of siderophore production. Elevated temperature was unable to suppress growth of C. albicans in either a control culture medium or a deferrated culture medium. A significant suppression of growth compared to the controls was observed in the deferrated media at both 37 degrees C and 41 degrees C. However with time, the growth of cells in the deferrated media showed partial recovery which was followed by an increase in siderophore production. Thus, elevation of temperature to suppress growth and siderophore production by C. albicans appears to be an ineffective host defense mechanism.

Serological diagnosis of petriellidiosis (allescheriosis). I. Isolation and characterization of soluble antigens from Allescheria boydii and Monosporium apiospermum.

Soluble antigens in culture filtrates of three strains of Petriellidium boydii and three strains of Monosporium apiospermum were examined. Antigens were separated from concentrated crude filtrates by anion-exchange chromatography. A single major peak (Antigen 1), constituting a significant proportion of the total recoverable carbohydrate, was the only product isolated from each of four chromatographed filtrates. Depending on the fungus strain, Antigen 1 consisted of 90–96% carbohydrate, 3–4% protein, and 2–4% nucleic acid. Antigen 1 was found to consist of a population of molecules with a heterogeneous molecular size when assayed by gel filtration chromatography; however, isolated fractions of Antigen 1 proved to be immunologically identical when examined by Ouchterlony immunodiffusion. In addition, Antigen 1 from each strain was immunologically identical to similar preparations of Antigen 1 from the other five fungus strains. Chromatography of culture filtrates from two strains of M. apiospermum revealed a second peak (Antigen 2), which was found to consist of 70% carbohydrate, 16% protein, and 4% nucleic acid. Although Antigen 2 contained four times as much protein as Antigen 1, the two preparations were immunologically identical by immunodiffusion tests. Ion-exchange chromatography proved to be a useful procedure for isolating antigens of P. boydii and M. apiospermum from culture filtrates.

Serological diagnosis of petriellidiosis (allescheriosis). II. Indirect (passive) hemagglutination assay for antibody to polysaccharide antigens of Pertriellidium (Allescheria) boydii and Monosporium apiospermum.

The indirect (passive) hemagglutination assay (IHA) was utilized for detection of humoral antibody resulting from an experimental or a natural petriellidiosis. Polysaccharide antigens from three strains ofP. boydii and three strains ofM. apiospermum were isolated from culture filtrates of these fungi by anion-exchange chromatography and were coupled to sheep erythrocytes using chromium chloride. Rabbits were inoculated by the subcutaneous route with viable spores of these fungi and were bled periodically to obtain serum samples. IHA assay of serial specimens revealed varying levels of humoral antibody. Antibody stimulation by the subcutaneous route of inoculation appeared advantageous inasmuch as high IHA titers (1∶1024 to 1∶4096) could be achieved without endangering the life of the animal or exposing investigators to draining lesions. When serum specimens from six human beings with culturally proven petriellidiosis were tested by the IHA method, five were shown to have antibody levels of 1 ∶ 16 to 1 ∶ 512. IHA assay of serum samples obtained from normal human beings showed measurable levels (1 ∶ 4 to 1 ∶ 32) of antibody in 29 of 30 specimens. A serum pool composed of the higher titered specimens from the normal group was adsorbed with killed whole spores ofM. apiospermum, and the attached serum components were eluted by altering the pH. The immune globulin that had adsorbed to the spores was found, by immunoelectrophoresis, to be IgG. An indirect hemagglutination inhibition assay, developed from the IHA assay, provided a means for determining whether heterologous fungus antigens were capable of cross-reacting with petriellidium antibody. Soluble antigens from several other genera of fungi failed to react with petriellidium antibody in the IHA assay, suggesting that the reaction of petriellidium antibody with homologous antigen was genus specific.

Elastase activity of fungi with anamorphs similar to Coccidioides immitis.

The elastin digestion assay was examined to determine if it would facilitate the identification of Coccidioides immitis when non-pathogenic fungi resembling C. immitis are encountered. Fungal isolants tested have anamorphs that closely resemble the macroscopic or microscopic morphology of C. immitis. Elastin hydrolysis was measured by elastin-agar plate assays. Approximately 80% of the isolants hydrolyzed elastin; thus, the elastin digestion assay as a differential test appears to have little value.