Data are presented as medians with 95% confidence intervals

Data are presented as medians with 95% confidence intervals. Effects of trypsin, heparin and EGTA Proteolysis of IRBC surface proteins by high concentrations of trypsin ( 10 ug/mL) completely inhibited adherence. of the antigenically variant membrane protein (PfEMP1) to the GW843682X placental glycosaminoglycan chondroitin sulphate A, and to secondary receptors such as hyaluronic acid and immunoglobulins [1]C[5]. is generally regarded as a more GW843682X benign parasite than accounts for approximately half of all malaria outside Africa [7]. Like has also exerted a considerable selective pressure on human development although pathological processes are less well understood. causes rosetting (adherence to uninfected erythrocytes) [8] but until recently it has not been considered to cytoadhere [9]. infections in pregnancy also cause abortions [10] and reduce birthweight which increases the risk of neonatal death, even though mechanism underlying early fetal loss or the intrauterine growth retardation is usually unclear [11]. We have investigated the adherence of (N?=?33) to the placental glycosaminoglycans chondroitin sulphate A (CSA) and hyaluronic acid (HA), the putative receptors for placental adherence [10], [11]. Materials and Methods Ethics statement This study was a part of clinical studies which have been approved by the Ethics Committee, Faculty of Tropical Medicine, Mahidol University. All participants gave fully informed consent to providing a 5 mL blood sample. Written informed consent was provided by study participants. Parasites Synchronous new isolates ( 80% ring stage) of were obtained from non-pregnant adult patients with acute vivax malaria admitted to the Hospital for Tropical diseases, Bangkok and joined into clinical studies. Malaria parasite species were confirmed by PCR [12]. All blood samples were recorded using a code identifier, and the subsequent experiments were conducted and the results were interpreted blinded to the patient data. Blood samples were taken into heparinized tubes and cultured as explained previously [13]. Briefly, blood samples were centrifuged at 500 g at 4C then plasma was discarded. White blood cells were removed by GW843682X a CF-11 column or Plasmodipur? filter. After 24 hours of cultivation, trophozoite-infected reddish GW843682X cells were utilized for further experiments. The parasite density of clinical isolates with less than 0.5% parasitaemia was augmented by concentration using a 66% Percoll? gradient [14] or a magnetic separation column [15]. The synchronous trophozoite-IRBCs were enriched to 80C90% parasitaemia and the concentrate then adjusted to 1% parasitaemia at 1% haematocrit in PV-MCM media for the static adherence assay [16], [17], and to 2% Haematocrit in 1% albumax for the laminar sheer circulation adherence assay. Highly synchronized ring stage parasites ( 0.5% parasitaemia) were utilized for assessing the relationship between adherence and stage of parasite development. The A4 clone, selected for adhesion to CSA (kindly provided by Dr David Roberts) was used as the control. Rosette formation (the adherence of two or more uninfected reddish cells to the infected cells) was counted for 100 IRBCs as explained previously [18]. Static adherence assay Adherence of parasitized erythrocytes to umbilical vein endothelial cells was assessed GW843682X as explained previously [19]. The reagents evaluated as potential receptors in the adherence assay were: purified CD36 and ICAM-1 (kindly provided by Arnab Pain), Thrombospondin (TSP; provided by Rachanee Udomsangpetch), CSA (from bovine trachea @amgiS cat no C8529, or CSA covalently linked to phosphatidylethanolamine, kindly provided by Stephen Rogerson), Rabbit polyclonal to PIWIL2 CSC (chondroitin sulphate C), de-6-O-sulphated CSA [20], dextran sulphate with molecular excess weight of 500 kD (Sigma), and HA (from bovine vitreous humor, Sigma @ cat no H7630)..