CANDIDA ALBICANS ALLERGENIC EXTRACT 1 1000 INDUCES TOLLLIKE

CANDIDA ALBICANS ALLERGENIC EXTRACT (1: 1000) INDUCES TOLL-LIKE RECEPTOR EXPRESSION IN HUMAN NEUTROPHILS & MONOCYTES & INCREASES PRODUCTION OF INTERFERON-ALPHA IN PERIPHERAL BLOOD CELLS SILVIA BELLOCCHIO, Ph. D, LUIGINA ROMANI, MD, Ph. D, MICROBIOLOGY SECTION, DEPARTMENT OF EXPERIMENTAL MEDICINE & BIOCHEMICAL SCIENCE UNIVERSITY OF PERUGIA, ITALY ROBERT J. SIGNORE, DO, DERMATOLOGY PRIVATE PRACTICE, TINLEY PARK, ILLINOIS INTRODUCTION Candida albicans allergenic extract 1: 1000 (CAE) has been used for intralesional injection immunotherapy for verruca vulgaris and plantar warts as an off-label usage. However, its mechanism of action is not completely understood. CAE is FDA approved for the diagnosis and treatment of patients with immediate hypersensitivity allergy to C. albicans (Figure 1). Dendritic cells, macrophages, & granulocytes provide a first line of defense against infectious microbes. Toll-like receptors (TLRs) expressed on these cells recognize molecular structures present in the pathogens. Upon binding of a pathogen ligand, TLRs trigger a cascade of signaling pathways which ultimately activates NFkappa. B. In mammals, this leads to induction of cytokine genes & establishment of innate immunity. TLR 7, TLR 8, and TLR 9 induce antiviral responses by producing cytokines such as interferon-alpha (IFN-α). We present evidence that CAE induces TLR expression in human neutrophils and monocytes. We also found that CAE increases IFN-α production by human peripheral blood cells (PBCs). Figure 2 Figure 1 MATERIALS AND METHODS Reagents and Cell Culture The cell culture medium used was RPMI 1640 with 10% FCS. All reagents contained <0. 125 endotoxin units/ml of endotoxin as checked by Limulus amebocyte lysate assay. Candida albicans allergenic extract (1: 1000) (CAE) was obtained from Greer Laboratories, Inc. , Le. Noir, North Carolina. Circulating human monocytes (MONOs), polymorphonuclear cells (PMNs), & peripheral blood cells (PBCs) were separated from blood of normal donors (>97% pure as determined by Giemsa morphology) by Percoll (Pharmacia Fine Chemicals, Uppsala, Sweden) gradient centrifugation as described (Muzio M, 1994) Cytokine Assays For cytokine determination, purified human PBCs were resuspended in RPMI medium with no serum but with polymixin B, to avoid nonspecific activation by serum components & endotoxin, & were exposed to 100 μl Candida allergenic extract (dilution 1: 100) for 24 hours. Levels of cytokines in culture supernatants were determined by Kit ELISA (R&D Systems, Inc. , Space Import-Export Srl, Milan, Italy). The detection limits (pg/ml) of the assays were >0 for h. TNF-α , >0 for h. IFN-α. Reverse Transcriptase – Polymerase Chain Reaction (RT-PCR) Total RNA was extracted from purified peripheral human PMNs & MONOs using the TRIZOL reagent according to manufacturer (Invitrogen Srl Life Technologies, Milano, Italy) after the exposure to CAE (50 or 100 μl of a 1: 100 dilution of the stock). Reverse transcription (RT) was done as described (Bellocchio S, 2004). PCR amplification was performed with Ampli Taq Gold polymerase (Applied Biosystems, Foster City, CA, USA). The forward & reverse PCR primers used for human TLRs & HPRT were as described (Bellocchio S, 2004). Semiquantitative PCR used the “primer dropping” method in which HPRT was coamplified as an internal control in all reactions as described. The data shown in Figure 2 are representative of three experiments. RESULTS CAE induced expression of TLR 2 & TLR 9 in PMNs and TLR 10 in MONOs and increased expression of TLR 8 in PMNs & MONOs (Figure 2). Following stimulation of PBCs with CAE, production of IFN-α was increased fourfold whereas tumor necrosis factor-alpha (TNF-α) production was only slightly reduced, *p<0. 05 (Figure 3). COMMENTS We are not aware of any previous reports demonstrating that CAE induces expression of TLRs in human neutrophils & MONOs. Nor are we aware of any previous reports showing that CAE increases production of IFN-α by human PBCs. Our preliminary study shows that Candida albicans allergenic extract stimulates innate immunity. MONOs are the precursors of macrophages, which are present in the infiltrate of regressing warts. Macrophages make numerous cytokines. This study is an important first step in beginning to understand the mechanisms behind Candida immunotherapy of human papilloma virus induced skin disease, such as verruca vulgaris & plantar warts. Figure 3 REFERENCES Signore R. Candida albicans Intralesional Injection Immunotherapy of Warts. Cutis 2002; 70: 185 -192. 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Expression and Function of Toll-Like Receptors 2 and 4 in Human Keratinocytes. Int Immunol. 2003; 15 (6): 721 -730. Abreu M, Arditi M. Innate Immunity and Toll-Like Receptors: Clinical Implications of Basic Science Research. J Pediatrics. 2004; 144: 421 -429. Abbas A, Lichtman A. Basic Immunology (2 nd Ed). Philadelphia, Saunders. 2004. Heil F, Hemmi H, Hochrein H, et al. Species-Specific Recognition of Single. Stranded RNA via Toll-Like Receptor 7 and 8. Science Magazine. 2004; 303: 1526 -1529. The authors have no financial conflicts of interest regarding the products described The authors received no outside financial support for this study The use of Candida albicans allergenic extract (1: 1000) for the immunotherapy of warts represents an “off-label” usage