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***< 0 We generated a heterodimeric Fc fusion protein, GBP1+6-Fc, by separately fusing GBP1 and GBP6 to oppositely polarized Fc fragments that can only bind to each other and not homodimerically (Fig 2C)

2008;82:6631. one serotype does not provide protection to contamination with the other serotypes meaning secondary or sequential infections are common (2, 3). Severe complications of dengue haemorrhagic fever (DHF) are more likely during secondary versus primary infections (2, 3). In 1977 Halstead suggested ADE to explain severe DENV infections (4). ADE has been widely analyzed and results from the high sequence divergence between DENV such that antibody to the first infection may not be of sufficient avidity to neutralise a secondary contamination (5). The partial Cetirizine cross reactivity may cause a degree of opsonisation that promotes computer virus uptake into Fc bearing cells such as monocytes/macrophages, a major site of DENV replication was cultured in Leibovitz L-15 medium supplemented with 10% heat-inactivated foetal bovine serum (FBS), 0.26% tryptose phosphate broth (TPB), 100 units/ml penicillin, 100 g/ml streptomycin and 2 mM L-Glutamine at 28C. For endotoxin-free conditions, cells were produced in the absence of TPB. Vero, a cell collection derived from the kidney of African green monkeys and U937, a human monocytic cell collection, were produced in MEM and RPMI1640, respectively. The media were supplemented with 10% FBS, 100 models/ml penicillin, 100 g/ml streptomycin and 2 mM L-Glutamine in a 37C humidified 5% CO2 incubator. Monocyte-derived dendritic cells (DC) were prepared as previously explained (20). LoVo cells were cultured in Nutrient combination (Ham) F12 medium made up of 20% FBS. Conjugated antibodies against human or mouse Ig (DAKO) Cetirizine were used. Pooled convalescent dengue hyperimmune human serum (PCS) (hemagglutination titre 1/25600), pooled non-dengue immune serum (PND) (hemaggutination inhibition titre and anti-dengue Ab ELISA unfavorable) and mouse anti-DENV envelope, 4G2, were kindly provided by AFRIMS, Thailand. NS1-F3, 2G6 and 1H10 are anti-NS1 and anti-prM mAb, respectively (21, 22). Computer virus stock DENV serotype 1 (Hawaii), serotype 2 (16681), serotype 3 (H87) and serotype 4 (H241) were propagated in C6/36 cells and computer virus supernatant was collected and stored at ?80C. The DENV stock propagated from C6/36 Cetirizine and MDDC’s were free from endotoxin and mycoplasma detected by Limulus amebocyte lysate assay (Whittaker M.A.) and PCR using the mycoplasma detection set (TAKARA BIO INC), respectively. For poorly infectious DENV, C6/36 cells were infected with DENV2. Four days after infection, culture medium was replaced by new L-15 made up of 1.5% FBS and 0.26% TPB with 10 or 20 mM NH4Cl for 2 hrs and the medium was then replaced again. At 24 hrs after the medium made up of NH4Cl was added, computer virus particles were harvested and precipitated by 10% PEG 8000. Fully immature computer virus was produced on LoVo cells as previously explained (13). Briefly, computer virus was produced by infecting LoVo cells with DENV2 strain 16681 at MOI of 10 and computer virus was harvested at 2 days. Focus forming assay The titres of computer virus were determined by a focus forming assay on Vero cells and expressed as focus-forming models (FFU) per ml. Briefly, computer virus was serially diluted and incubated with Vero cells for 2 hrs at 37C. The monolayers were then overlaid with 1.5% carboxymethylcellulose and incubated at 37C for 3 Cetirizine days. Virus foci were stained with anti-E antibody (4G2) followed by peroxidase-conjugated anti-mouse Ig CBL and visualized by the addition of DAB substrate. Generation of dengue-specific human monoclonal Abs DENV-specific human mAb’s were generated as previously explained (8). Briefly, IgG+ memory B cells were positively selected from PBMC.