Recognition of fungal RNA by TLR7 has a nonredundant role in host defense against experimental candidiasis.

Despite convincing evidence for involvement of members of the Toll-like receptor (TLR) family in fungal recognition, little is known of the functional role of individual TLRs in antifungal defenses. We found here that TLR7 was partially required for the induction of IL-12 (IL-12p70) by Candida albicans or Saccharomyces cerevisiae. Moreover, the IL-12p70 response was completely abrogated in cells from 3d mice, which are unable to mobilize TLRs to endosomal compartments, as well as in cells from mice lacking either the TLR adaptor MyD88 or the IRF1 transcription factor. Notably, purified fungal RNA recapitulated IL-12p70 induction by whole yeast. Although RNA could also induce moderate TLR7-dependent IL-23 and tumor necrosis factor-alpha (TNF-α) secretion, TLR7 and other endosomal TLRs were redundant for IL-23 or TNF-α induction by whole fungi. Importantly, mice lacking TLR7 or IRF1 were hypersusceptible to systemic C. albicans infection. Our data suggest that IRF1 is downstream of a novel, nonredundant fungal recognition pathway that has RNA as a major target and requires phagosomal recruitment of intracellular TLRs. This pathway differs from those involved in IL-23 or TNF-α responses, which we show here to be independent from translocation of intracellular TLRs, phagocytosis, or phagosomal acidification.

Recognition of yeast nucleic acids triggers a host-protective type I interferon response.

Although type I interferons (IFN-α/ß) have been traditionally associated with antiviral responses, their importance in host defense against bacterial pathogens is being increasingly appreciated. Little is known, however, about the occurrence and functional role of IFN-α/ß production in response to pathogenic yeasts. Here, we found that conventional DCs, but not macrophages nor plasmacytoid DCs, mounted IFN-ß responses after in vitro stimulation with Candida spp. or Saccharomyces cerevisiae. These responses absolutely required MyD88, a Toll-like receptor (TLR) adaptor molecule, and were partially dependent on TLR9 and TLR7. Moreover, Candida DNA, as well as RNA, could recapitulate the IFN-ß response. After intravenous challenge with Candida albicans, most mice lacking the IFN-α/ß receptor died from their inability to control fungal growth, whereas all WT controls survived. These data suggest that recognition of yeast nucleic acids by TLR7 and TLR9 triggers a host-protective IFN-α/ß response.

[Interferon inductor cupping of postvaccinal complications after variolation].

Efficacy of arbidol and ridostin in cupping postvaccinal complications due to variolation was studied by the clinico-virological, hematological and biochemical indices and it was shown that arbidol was efficient in cupping development of dermal complications, lowered the severity of the postvaccinal reaction and stimulated the cellular and humoral immune response. Ridostin, a high molecular interferon inductor, was highly efficient in cupping all the forms of the postvaccinal complications, including the neurological and cutaneous ones.

[Allergenic and immunotropic properties of yeast RNA-tilorone hydrochloride molecular complex].

Immunotropic properties of the interferon-inducing molecular complex (MC) yeast RNA--tilorone hydrochloride have been under study. MC was experimentally studied in vivo to establish its influence on the amount of antibody-forming cells and the level of antibody formation. The influence of MC on the oxygen-generating activity of spleen macrophages was established in the HCT test. MC in a dose of 1.25 mg/kg was shown to considerably activate immunocompetent cells, thus producing pronounced influence on humoral immunity. In addition, the study showed the dose dependence of the influence of MC on individual elements of the immune system as well as differences in the dynamics of immunomodulation caused by the use of high and low doses of MC. The data thus obtained made it possible to regard MC as a promising immunomodulator.

Monoclonal antibody against DNA adducts with osmium structural probes.

Osmium tetroxide complexes with nitrogen ligands (Os,L) have been widely used as probes of the DNA structure. A monoclonal antibody OsBP7H8 against DNA adducts with Os,L was produced in mice. OsBP7H8 does not bind to proteins or total yeast RNA modified with Os,2,2'-bipyridine (bipy) nor to the unmodified nucleic acids and proteins. The antibody recognizes DNA modified with Os,bipy (DNA-Os,bipy) or with OsO4,1,10-phenanthroline (DNA-Os,phen) but it does not cross-react with oxidized DNA and with DNA adducts of osmium tetroxide complexes with other ligands (such as pyridine, TEMED and bathophenanthroline disulfonic acid). The affinity of OsBP7H8 to DNA-Os,phen is about five-fold higher as compared to DNA-Os,bipy. The antibody can be thus applied either for recognition of single-stranded and distorted regions in DNA (after DNA modification with Os,bipy) or for detection of both single-stranded and double-stranded DNAs (after DNA modification with Os,phen). A new simplified procedure for the dot-blot analysis is proposed, not requiring the purification of DNA-osmium adduct prior to its application to the membrane.

Nucleotide actions on humoral immune responses.

Previous studies indicate the importance of dietary nucleotides in maintaining optimal cellular immunity. Our studies using murine spleen cells showed that polynucleotides significantly increase in vitro antibody production in response to T-cell-dependent antigen. They seem to exert actions on T-helper cells at antigen presentation, perhaps during cognitive cell-cell interactions. They do not augment the actions of cloned, antigen-specific, activated T-helper cells, nor do they increase antibody production in response to T-cell-independent antigen or polyclonal B-cell activation. Polynucleotides increase in vitro human immunoglobulin production in response to T-cell-dependent stimuli and antigen. Humoral immune responses to T-cell-dependent antigen were depressed in mice fed a nucleotide-free diet, but were restored by a mononucleotide-nucleoside mixture. Responses to T-cell-independent antigen remained intact in these mice. The mononucleotide-nucleoside mixture had no effect on in vitro antibody production and did not further increase humoral immune responses in mice fed regular lab chow. These results suggest that the in vivo actions of polynucleotides on humoral immune responses may reflect local immune responses, perhaps at the site of inflammation. Mononucleotides and nucleosides may be incorporated into the tissue nucleotide pool fairly rapidly in a state of relative nucleotide deficiency and help restore T-cell-dependent humoral immune responses. Our findings may further support the importance of dietary nucleotides.

[Study of immunoregulating properties of an interferon inducer in animals with various reactions to the antigen]. / Issledovanie immunoreguliatornykh svoistv induktora interferona u oppozitnoreagiruiushchikh na antigen zhivotnykh.

Influence of dsRNA isolated from killer yeast of S. cerevisiae on humoral and cellular immune responses in mice CBA/CaY and C57Bl/6Y with opposing reaction to the antigen was studied. It was shown that after administration of the yeast dsRNA preparation to the animals simultaneously with the antigen there was an increase in the number of the antibody forming cells in the spleen and the titer of hemolytic antibodies in blood serum of the animals with high and low reactions to the antigen. After sensitization with different doses of sheep red blood cells (10(7) and 10(8)) the preparation had immunomodulating action on development of DTH in mice CBA/CaY. The effect of the dsRNA preparation on the immunity system depended on the preparation dose, antigen loading and animal genotype and was the most marked in mice CBA/CaY with interferon levels in blood serum 2-3 times higher than those in mice C57Bl/6Y.

Role of protein processing, intracellular trafficking and endocytosis in production of and immunity to yeast killer toxin.

Yeast strains harboring M1-dsRNA and its packaging virus ScV-L secrete a disulfide-linked, heterodimeric toxin which kills sensitive yeast cells by disrupting plasma membrane function. The mature toxin is derived from a precursor (preprotoxin) which undergoes post-translational processing steps during export via the established yeast secretory pathway. Cleavage by both the KEX1 and KEX2 endopeptidases is required for expression of killing activity. The same 1.0 kb open reading frame on M1-dsRNA directs the expression of immunity to toxin. Differentially processed derivatives of protoxin, as well as protoxin itself, have been proposed to serve as mediators of immunity. To understand the mechanisms by which the killing and immunity phenotypes can be derived from a common precursor, we have: 1) studied cellular processes implicated in expression of the phenotypes; and 2) developed a system to produce mutants defective in immunity, killing, or both. In the first approach, the role played by both endocytosis and vesicular traffiking in expression of killing and immunity was examined. Strains defective in endocytosis (end1, end2) or vacuolar protein localization (vpl3, vpl6) were transformed with a plasmid encoding killer toxin under control of the pho5 promoter. When induced by phosphate starvation, both end mutants and all vpl mutants expressed killing activity. Immunity to exogenous toxin, however, was significantly decreased in strains carrying both vpl mutant alleles and in one of the endocytosis mutants (end1]. This suicidal phenotype (rex for resistance expression) has been described previously in M1-containing strains as a leaky phenocopy.(ABSTRACT TRUNCATED AT 250 WORDS)

[Killer systems of Saccharomyces cerevisiae yeasts]. / Killernye sistemy drozhzhei Saccharomyces cerevisiae.

The killer systems of Saccharomyces cerevisiae are a peculiar group of cytoplasmic symbionts of primitive eukaryotes. The genetic material of these symbionts is double-stranded RNA. Their basic properties are linearity of genome, its fragmentation, resulting in two separately replicating major and minor segments, and the ability to control the synthesis of secretory proteins--mycocins which can kill the taxonomically related strains. Secretion of mycocins also confers immunity to their action. The strains containing killer symbionts are toxigenic and resistant to their own toxins, while those with no killer double-stranded RNA are sensitive to mycocins. The killer systems of Saccharomyces cerevisiae possess some properties relevant to viruses and evidently are evolved during the evolution of infectious viruses. Occurrence of such systems in monocellular eucaryotic organisms is an example of genome complication in the course of putting together the virus-like components. The peculiarities of replication and expression of killer systems and their utilization for the construction of vector molecules are discussed.

[RNA as a modulator of secondary immunological deficiency and dysnucleotidosis]. / RNK kak moduliator vtorichnoi immunologicheskoi nedostatochnosti i disnukleotidoza.

Secondary immunological deficiency was identified in patients with chronic pneumonia, rheumatism, peptic ulcer or chronic alcoholism. The routine therapy resulted in aggravation of the immunity defects in the patients with pulmonary affections and chronic alcoholism. The combined therapy with the use of sodium nucleinate promoted elimination of the immunological deficiency, whereas in the patients with peptic ulcer, elimination of the increased content of 17-HKS in the urine and dysnucleotidose.