Background Schistosomiasis is an important helminth infection of humans. Betulinic acid

Background Schistosomiasis is an important helminth infection of humans. Betulinic acid The former yielded eight differentially-expressed host proteins in the serum at different disease stages including transferrin and alpha 1- antitrypsin. The latter suggested the presence of a surprising number Betulinic acid of parasite-origin proteins in the serum during both the acute (n = 200) and chronic (n = 105) stages. The Orbitrap platform also identified many differentially-expressed host-origin serum proteins during the acute and chronic stages (296 and 220 respectively). The presence of one of the schistosome proteins, glutathione S transferase (GST: 25 KDa), was confirmed by Western Blot. This study provides proof-of-principle for an approach that can yield a large number of novel candidate biomarkers for Schistosoma infection. Introduction Schistosomiasis is a public health problem of global importance [1]. For both surveillance and the optimal treatment of patients, rapid and sensitive diagnostic tests are needed that can detect infection soon after exposure and when parasite burden is low. Although the current gold-standard test for is microscopic detection of the eggs in stool, eggs first appear only 6C8 weeks after infection. This method also has poor sensitivity when few parasites are present and during the chronic stage of infection when the passage of eggs is typically low [2]. Other tools used to diagnose and monitor schistosomiasis include the detection of circulating antigens or antibodies and ultrasound to assess liver fibrosis and hepatosplenomegaly [3], [4], [5]. Polymerase Betulinic acid chain reaction (PCR) has been used to detect DNA in human fecal samples [6], [7]. All of these tests have important limitations related to their complexity, expense, sensitivity and/or cross-reactivity with other helminth infections. Most cannot discriminate between active and past infections [8], [9]. Therefore, there is a need for new schistosomiasis diagnostic options. Mass spectrometry (MS) has the potential to modernize diagnostics through the discovery of specific biomarkers or proteomic profiles associated with infection or disease stage. In addition to possible diagnostic advances, the application of MS techniques to serum samples from the well-described schistosome-infected mouse model also has the potential to provide novel insights into parasite biology. Although the choice of proteomic platform and the optimal timing of sampling were unknown at the launch of these studies, we hoped to identify candidate biomarkers at different time-points after infection, representing the different pathological stages of the disease: ie: EARLY prior to egg production (~3 weeks post-infection), ACUTE: ~6 weeks post-infection when eggs are Betulinic acid being starting to be deposited in the liver, and CHRONIC: ~12 weeks post-infection when there is a well-defined granulomatous reaction in the liver. Because several different MS platforms are available, each with its particular strengths and weaknesses, we opted to explore three complimentary approaches. We used high throughput surface-enhance, laser-desorption and ionization, time-of-flight mass spectrometry (SELDI-TOF MS) to compare uninfected and infected sera as a proof-of-principal exercise. We subsequently used sample fractionation and differential gel electrophoresis prior to analysis on two more precise MS platforms; specifically matrix-assisted, laser-desorption and ionization (MALDI-TOF MS) and Velos Orbitrap MS. By using multiple proteomic platforms in parallel, we demonstrated that serum protein profiles differ extensively between infected and uninfected mice, offering a rich source of potential biomarkers. Materials and Methods Mouse infection and serum collection Twenty-six female CD1, six-week old mice were purchased from The Charles River (St. Constant, Qubec). All animal experiments were approved by the Facility Animal Care Committee of McGill University and followed the guidelines of the Canadian Council on Animal Care. Mice (5/group) were infected intraperitoneally (IP) with 50, 100, 150 or 200 cercariae (snails were obtained from the Biomedical Research Institute; Bethesda, MD). Control animals (n = 6) were IP injected with PBS. Mice were maintained in ventilated cages and monitored once per week. Blood samples were collected by saphenous bleeding before infection, Rabbit polyclonal to IL9 at three weeks and six weeks post-infection. Mice were sacrificed by CO2 narcosis and blood was collected by direct cardiac puncture at 12 weeks post-infection. All sera were kept at ?80C until analyzed. Fractionation method We used sample fractionation prior to SELDI and MALDI analysis. Serum samples at different time points were fractionated as previously described [10], [11]. Briefly, ProteinChip serum fractionation kit (Bio-Rad) was used to fractionate the samples into six pH fractions.