Abstract
Except immunoglobulins and components of complement system, the important part of congenital humoral immunity are antimicrobial (poly) peptides (AMP). Main antimicrobial mechanism of AMP action is the destruction of cytoplasmic membrane. In host organism number of AMP act simultaneously, therefore method for the measuring of overall AMP activity would be helpful. The method was recently applied and modified for estimation of AMP activity in serum: aliquots of low-molecular fraction of serum added to test-culture of Candida albicans, and the part of killed cells, i.e. total activity of AMP, calculated by spectroscopy. Aim of present study is the estimation of AMP activity in mice sera after immunization with cells of clinical yeasts compare to the immunoglobulins G. Yeasts strains-Candida albicans, Cryptococcus neoformans, Rhodotorula mucilaginosa, Trichosporon cutaneum, Geotrichum candidum, and Saccharomyces cerevisiae-were cultivated in liquid defined media up to the end of exponential growth phase. Obtained yeasts cells used for immunization of mice, but yeast antigens-for the estimation of IgG-antibodies levels in mice sera by ELISA method. AMP activity were measured as described above. Immunization of mice with yeasts cells was accompanied not only with increase of specific IgG levels, but with elevation of total activity of AMP: for example specific IgG in sera of mice immunized by C. albicans cells was 16-fold higher than in non-immunized (control), and AMP activity-1,5 fold higher than in control. Correlation between specific humoral response at the immunoglobulins level and humoral response at the AMP-activity level (Pirson`s coefficient r=0,735; p ≤ 0,05) as a result of immunization of mice by fungal cells was demonstrated for the first time. The method may be used for study of humoral immunity associated with action of AMP in various animal and human models.
Highlights
The clinically important yeasts are progressively increased group of fungi due to following reasons-appearance of new identification methods and elevation of number of immunosuppressed individuals
We reported about cultivation of 7 specia of these genera and isolation of specific yeast antigens which are appropriate for the immunodiagnostics of yeast mycoses by the estimation of specific immunoglobulins level [2]
Significance of differences between levels of immunoglobulin G (IgG)-antibodies to yeast antigens in homologous serum and this indexes in non-homologous sera were calculated by Mann-Whitney U test (p ≤ 0,05)
Summary
The clinically important yeasts are progressively increased group of fungi due to following reasons-appearance of new identification methods and elevation of number of immunosuppressed individuals. Methods of quantitative estimation of individual AMP are known [8,9], but number of AMP act simultaneously, an adequate method for the measuring of overall AMP activity would be helpful Such method was recently applied for estimation of AMP activity of vaginal fluid [10,11], based on the action of aliquots of the substrate on test-culture of Candida albicans and calculation of killed cells percent. Later we have modified the method with adapt it for the serum by use of two techniquesremoval of high-molecular (≥ 100 kDa) protein fraction (i.e. removal of complement system proteins) and estimation the result by spectroscopy [12,13] Use this approach we measured the total AMP activity in sera of patients with bronchopulmonary candidiasis and showed that the activity was elevated (like the specific serum immunoglobulins) in patients compare to control group [13]. The aim of present study is the estimation of AMP activity levels in mice before and after
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