Deep Candida infections. Serology for laboratory diagnosis and antifungal activity of lactoferrin derived fragments

Sammanfattning: Deep Candida infections (DCI) in patients who are immunocompromised or in intensive care units present both diagnostic and therapeutic problems. Serological tests for diagnosis of invasive fungal infections are used with varying results. Moreover, long term therapies with antifungal agents have led to the emergence of resistant Candida strains. Our aim was to investigate cell wall components of Candida albicans in order to find antigenic markers of DCI, and to explore the anti-fungal activity of peptide fragments of the innate defense factor lactoferrin (LF).The relative ability of antibodies to specifically recognize C. albicans cell wall components, such as native cell wall fragments (CW), phosphopeptidomannan (PPM), and b(1-6)(1-3)glucan was evaluated in ELISA by using serum from C. albicans-immunized rabbits and serum from patients with DCI. It was found that PPM is the dominant antigen in the cell wall of C. albicans. Moreover, analysis of DCI patients for IgG subclass antibodies against the above antigens showed that IgG2 anti- PPM and IgG1 anti-CW antibodies were highly discriminatory for recognition of DCI when compared with two control groups consisting of women with suspected superficial Candida infection of the nipples and healthy blood donors. Determination of circulating Candida cell wall material in the very same patients and control groups revealed that b(1-6)(1-3)glucan, as determined by a commercially available biochemical assay based on a coagulation cascade in the cell lysate of hemocytes from the Limulus crab, was detectable in high concentrations in the serum of all patients, but not in any of the controls. Polyclonal and monoclonal (Mab) antibodies against b(1-3) glucans were produced. One novel Mab recognized not only an epitope in the free form of b(1-6)(1-3)glucan, but uniquely also on the cell surface of C. albicans, C. krusei, C. glabrata, C. parapsilosis and Cryptococcus neoformans as revealed by immunofluorescence. Antibodies with high specificity against b(1-3) glucans might become useful tools in the serodignosis of DCI, but also for investigations regarding the localization of b(1-3) glucans in the cell wall of Candida.Lactoferrin (LF)-derived synthetic fragments (12-, 23-, and 25 amino acid long peptide sequences) homologous to the antimicrobial, surface-exposed a-helix and b-sheet region from the N terminal end of the human LF exerted fungicidal activity against C. albicans, C. krusei, C. kefyr, C. parapsilosis and Cryptococcus neoformans analysed by a microplate assay. C. glabrata was, however, resistant. The killing of yeast cells by the peptides was accompanied by cytoplasmic and mitochondrial permeabilization as analysed by propidium iodide staining and rhodamine 123-loaded mitochondria in yeast cells. By peroral treatment with each of the two longer LF peptides (23 and 25 a.a.) of mice with gastric colonization of C. albicans the yeast cells were significantly reduced. In conclusion, two IgG subclass antibodies and circulating b(1-3)glucan seem to be promising early specific markers in the laboratory diagnosis of DCI. Complementary studies on patients followed prospectively will provide definite information to their future usefulness. The novel Mab and polyclonal antibodies to b(1-3)glucans might become useful tools in the serodiagnosis of DCI by improving the precision of the glucan determination. Colonization by Candida species increases the risk of DCI in immunocompromised patients, thus antifungal peptide fragments derived from antimicrobial proteins of the host such as lactoferrin might be of prophylactic and therapeutic use.

  Denna avhandling är EVENTUELLT nedladdningsbar som PDF. Kolla denna länk för att se om den går att ladda ner.