brasiliensiswere analyzed, as well as the peptides P9, P13, P18, P19, P25 and P36 were selected for further studies. control. These data indicate a potential use of P2 as diagnostic tool in PCM. Its application for serological diagnosis of PCM may contribute to the development and standardization of simpler, faster and highly reproducible immunodiagnostic assessments at low cost. Keywords:gp75,Paracoccidioides brasiliensis, Paracoccidioidomycosis, Peptides, Phage display == Introduction == Paracoccidioides brasiliensisis the agent of paracoccidioidomycosis (PCM), a prevalent disease in Latin America. Despite the large number of cases frequently diagnosed, the exact epidemiology of PCM LJ570 is not clearly known since there is no compulsory notification of identified cases. Due to the lack of available data, PCM was recently added to the list of neglected diseases [1]. The infection probably begins by inhalation of fungal propagules that, once in the lungs, transform into the pathogenic yeast forms [2] with ability to disseminate through the lymphatic system and the bloodstream to any location in the hosts organism [3]. Two clinical forms of the PCM are recognized: acute form (AF), which is usually more rare and severe, with LJ570 involvement of the reticuloendothelial system; and chronic form (CF), which may develop in multiple ways, ranging from benign and localized (unifocal) to severe and disseminated (multifocal) disease, depending on the degree of depressive disorder of cellular immunity [4,5]. Regarding its diagnosis, serology has an important role in identifying the suspected cases of PCM. However, difficulties in the immunodiagnosis have been associated with cross-reactions with antibodies present in sera from patients with other mycosis or diseases, mainly due to antigen preparation used [6]. To improve serodiagnosis of PCM, great efforts have been made, such as using recombinant proteins [7,8] that in most cases, reduced cross-reaction [9]. Nevertheless, production LJ570 of these molecules still represents a high cost, being used only in research centers. Considering that for PCM diagnosis, only the epitopes able to induce specific humoral response may be sufficient in immunologic reactions, herein, we employed the phage display methodology to define peptides that mimic natural epitopes. It consists of a collection of millions of randomized peptides displayed on bacteriophage surfaces that can be used to identify ligands of molecules of interest [1012]. Peptides that mimic epitopes (mimotopes) have been identified by screening phage libraries with LJ570 monoclonal antibodies (mAb) in several models and those synthetic peptides have been used for diagnostic applications with promising results [1316]. In this study, a phage display approach was used for the first time to identify ligands mimicking antigenic epitopes fromP. brasiliensis. The protective monoclonal antibody 5E7C (anti-gp75) was used here to select binding peptides [17]. After selection and analysis procedures, one synthetic peptide selected (called P2) was significantly recognized by untreated PCM patients sera when compared with controls. These results open the way to new approaches using peptides for the diagnosis of PCM. == Materials and Methods == == Mice == Male BALB/c mice, 68 weeks old, were obtained from the animal facility, Federal University of So Paulo (UNIFESP), SP, Brazil. Animal handling and housing were performed in accordance with the NIH Guide for Care and Use of Laboratory LJ570 Animals. == Bacterial Strain and Growth Conditions == TheE. coliER2738 strain was obtained from New England Biolabs, Inc. (Beverly, Massachussets, USA) exhibiting rapid growth and suited for propagation of phages (M13). These bacteria have a mini-transposon that confers tetracycline resistance. It was produced in LuriaBertani agar made up of 20 g tetracycline/mL at 37C overnight with shaking. == Production ofP. brasiliensisExoantigen == All experiments were carried out withP. brasiliensisyeast cells from isolate Pb18 kindly Rabbit Polyclonal to KLF provided by Prof. Vera L. G. Calich.