Surachet Benjathummarak

Chikungunya virus was isolated in Thailand, 2010

Chikungunya fever (CHIKF) is an acute febrile illness caused by a mosquito-borne alphavirus, chikungunya virus (CHIKV). This disease re-emerged in Kenya in 2004, and spread to the countries in and around the Indian Ocean. The re-emerging epidemics rapidly spread to regions like India and Southeast Asia, and it was subsequently identified in Europe in 2007, probably as a result of importation of chikungunya cases. On the one hand, chikungunya is one of the neglected diseases and has only attracted strong attention during large outbreaks. In 2008–2009, there was a major outbreak of chikungunya fever in Thailand, resulting in the highest number of infections in any country in the region. However, no update of CHIKV circulating in Thailand has been published since 2009. In this study, we examined the viral growth kinetics and sequences of the structural genes derived from CHIKV clinical isolates obtained from the serum specimens of CHIKF-suspected patients in Central Thailand in 2010. We identified the CHIKV harboring two mutations E1-A226V and E2-I211T, indicating that the East, Central, and South African lineage of CHIKV was continuously circulating as an indigenous population in Thailand.

Phenotypic alterations in human saphenous vein culture induced by tumor necrosis factor-alpha and lipoproteins: a preliminary development of an initial atherosclerotic plaque model

BackgroundAtherosclerosis is a chronic progressive inflammatory disease of blood vessels particularly the arteries. The development of atherosclerotic plaques or atherogenesis is a complex process that is influenced by cardiovascular risk factors such as vascular inflammation and dyslipidemia. This study demonstrates the ability of tumor necrosis factor-alpha (TNF-α) and low density lipoproteins (LDL) to induce atherosclerotic plaque in human saphenous vein (HSV) organ culture.MethodsNormal HSV segments, from male patients who had coronary bypass graft, were cultured in DMEM containing 5% heat inactivated fetal bovine serum. TNF-α (5 ng/ml) was applied in combination with native LDL (nLDL) or oxidized LDL (oxLDL) at the dose of 50 μg/ml for 14 days. The phenotypic changes of the organ cultures characteristic of initial atherosclerotic plaques were evaluated. The effect of anti-atherogenic agent, 17-β estradiol (E2), was also determined.ResultsHistologic, histomorphometric, and immunohistochemical examinations revealed that HSV rings stimulated with TNF-α + nLDL or TNF-α + oxLDL can exhibit the essential morphological features of atherogenesis, including fibrous cap formation, cholesterol clefts, evident thickening of the intimal layer, increased proliferation of smooth muscle cells (SMC) and migration to the subendothelial layer, significant SMC foam cell formation, and increased expression of adhesion molecules in the vascular wall. Addition of E2 (50 nM) to the culture significantly modulated the critical changes. Consistently, mRNA profiling of the HSV model revealed that 50 of 84 genes of atherosclerosis were up-regulated.ConclusionsPhenotypic changes characteristic of the initial development of atherosclerotic plaques can be induced in HSV organ culture.

PPBP and DEFA1/DEFA3 genes in hyperlipidaemia as feasible synergistic inflammatory biomarkers for coronary heart disease

BackgroundCoronary heart disease (CHD) is an important complication of atherosclerosis. Biomarkers, which associate with CHD development, are potential to predict CHD risk. To determine whether genes showing altered expression in hyperlipidaemia (H) and coronary heart disease (CHD) patients compared with controls could be CHD risk biomarkers.MethodsControl, H, and CHD groups represented atherosclerosis to CHD development. Gene profiling was investigated in peripheral blood mononuclear cells using DNA microarrays. Eight selected genes expressed only in H and CHD groups were validated by real-time quantitative reverse transcription PCR and plasma protein determination.Resultsα-defensin (DEFA1/DEFA3), pro-platelet basic protein (PPBP), and beta and alpha2 hemoglobin mRNA expression was significantly increased in H and CHD groups compared with controls, but only plasma PPBP and α-defensin proteins were correspondingly increased.ConclusionPPBP and DEFA1/DEFA3 could be potential CHD biomarkers in Thai hyperlipidaemia patients.

Increased alpha-defensin expression is associated with risk of coronary heart disease: a feasible predictive inflammatory biomarker of coronary heart disease in hyperlipidemia patients

BackgroundAtherosclerosis is a multifactorial disorder of the heart vessels that develops over decades, coupling inflammatory mechanisms and elevated total cholesterol levels under the influence of genetic and environmental factors. Without effective intervention, atherosclerosis consequently causes coronary heart disease (CHD), which leads to increased risk of sudden death.Polymorphonuclear neutrophils play a pivotal role in inflammation and atherogenesis. Human neutrophil peptides (HNPs) or alpha (α)-defensins are cysteine-rich cation polypeptides that are produced and released from activated polymorphonuclear neutrophil granules during septic inflammation and acute coronary vascular disorders. HNPs cause endothelial cell dysfunction during early atherogenesis. In this cross-sectional study, control, hyperlipidemia and CHD groups were representative as atherosclerosis development and CHD complications. We aimed to assess the correlation between α-defensin expression and the development of CHD, and whether it was a useful predictive indicator for CHD risk.MethodsFirst, DNA microarray analysis was performed on peripheral blood mononuclear cells (PBMCs) from Thai control, hyperlipidemia and CHD male patients (n = 7). Gene expression profiling revealed eight up-regulated genes common between hyperlipidemia and CHD patients, but not controls. We sought to verify and compare α-defensin expression among the groups using: 1) real-time quantitative RT-PCR (qRT-PCR) to determine α-defensin mRNA expression (n = 10), and 2) enzyme-linked immunosorbent assay to determine plasma HNP 1–3 levels (n = 17). Statistically significant differences and correlations between groups were determined by the Mann–Whitney U test or the Kruskal–Wallis test, and the Rho-Spearman correlation, respectively.ResultsWe found that α-defensin mRNA expression increased (mean 2-fold change) in the hyperlipidemia (p = 0.043) and CHD patients (p = 0.05) compared with the controls. CHD development moderately correlated with α-defensin mRNA expression (r = 0.429, p = 0.023) and with plasma HNP 1–3 levels (r = 0.486, p = 0.000).ConclusionsIncreased α-defensin expression is a potential inflammatory marker that may predict the risk of CHD development in Thai hyperlipidemia patients.

Antibody germline characterization of cross-neutralizing human IgGs against 4 serotypes of dengue virus

Abstract Dengue virus (DENV), a re-emerging virus, constitutes the largest vector-borne disease virus, with 50–100 million cases reported every year. Although DENV infection induces lifelong immunity against viruses of the same serotypes, the subsequent infection with the heterologous serotypes can cause more severe form of the disease, such as Dengue Haemorrhagic Fever (DHF) or Dengue Shock Syndrome (DSS). However, there is neither approved vaccine nor specific drugs available to treat this disease. In this study, previously developed 19 human monoclonal antibodies (HuMAbs) showing strong to moderate cross neutralizing activity were selected. Most of them (13/19) were targeted to domain II of envelop glycoprotein. To understand and clarify the recognition properties, the maturation mechanisms comprising Variable/Diversity/Joining (VDJ) recombination, Variable Heavy (VH)/Variable Light (VL) chain pairing, variability at junctional site, and somatic hypermutation (SHM) of those antibodies were studied and compared with their predecessor germline sequences. IMGT/V-QUEST database was applied to analyze the isolated VH and VL sequences. To confirm the correction of isolated VH/VL, 3 HuMAbs (1A10H7, 1B3B9, 1G7C2) was transiently expressed in HEK293T cell. All three clones of the expressed recombinant IgG (rIgG) showed the same binding and neutralizing activity as same as those from hybridomas. The data obtained in this study will elucidate the properties of those HuMAbs for further genetic modification, and its binding epitopes.

The role of the BAFF/APRIL system in the T cell-independent specific response to blood stage Plasmodium falciparum hemozoin

Background The B cell activating factor (BAFF) and a proliferation‐inducing ligand (APRIL) are tumor necrosis factor family members that regulate B cell maturation, proliferation, survival and function. We have previously shown that blood‐stage Plasmodium falciparum hemozoin (HZ) can act as a T‐independent antigen (TI Ag) that induces the production of specific IgG to soluble crude P. falciparum Ag through the BAFF pathway. However, we have not yet clarified whether HZ need APRIL signaling in the TI response. Here, we aimed to clarify whether both BAFF and APRIL signaling pathways play roles in HZ induction of specific antibody production without T‐cell help. Methods Normal monocytes alone or co‐cultured with naïve B cells were stimulated by HZ (10 &mgr;M) in vitro. Naïve B cell cultures, with HZ alone or with exogenous recombinant BAFF (rBAFF) and recombinant APRIL (rAPRIL) plus recombinant IL‐4 (rIL‐4) for 6 and 10 days were used as controls to investigate activation of B cells. At various times, the levels of sBAFF, sAPRIL, and HZ‐specific IgG in the culture supernatants were assessed by enzyme‐linked immunosorbent assay. The BAFF and APRIL expression levels on the HZ‐stimulated monocytes and their specific receptors on activated B cells, including the BAFF receptor (BAFF‐R), the transmembrane activator and calcium‐modulator and cyclophilin ligand interactor (TACI) and the B cell maturation antigen (BCMA), were determined by flow cytometry. mRNA expression levels for the receptors were validated using Real‐Time quantitative PCR. Results HZ‐activated monocytes released sBAFF and sAPRIL during the 72 h stimulation period. Increased mRNA encoding of their cognate receptors, BAFF‐R, TACI, and BCMA, and increased HZ‐specific IgG levels were also observed in HZ induction within the monocyte and B cell co‐culture. The experiments under control conditions revealed that HZ alone could induce B cell culture to produce a small amount of the specific IgG compared with those in medium alone or rBAFF + rAPRIL + rIL‐4. Conclusion Taken together, we suggest that in the TI response HZ stimulates monocyte and B cell co‐culture to produce specific IgG through BAFF, APRIL and other independent complimentary signaling pathways. HighlightsP. falciparum hemozoin (HZ) can induce the T cell‐independent immune response.Human monocyte/naïve B cell co‐cultures or naïve B cells stimulated by HZ.Increased sBAFF, sAPRIL and cognate receptors levels in the co‐culture.HZ‐specific IgG production observed under conditions with/without BAFF/APRIL system.Without T cells, HZ induce specific IgG through BAFF/APRIL and other pathways. Graphical abstract P. falciparum HZ induce T independent specific antibody production via BAFF/APRIL and other independent signaling pathways. Figure. No caption available.

Human single-chain variable fragment antibody expressed in E. coli with optimal in vitro cross-neutralizing and no enhancing activity

Single chain fragment variable (scFv) is a small molecule antibody comprising of only the variable region of heavy and light chain responsible for antigen binding. For dengue disease, the Fc region of antibody molecule was reported to be involved with dengue complication caused by Antibody-dependent enhancement (ADE). We attempted to produce small molecule scFv human monoclonal antibody (HuMAb), which lacking the Fc portion to eliminate the ADE effect of the IgG. This scFv antibody was produced in Escherichia coli. The biologically active form of scFv antibody was successfully generated. 23-1C2D2-scFv showed neutralizing activity similar to the IgG obtained from parental hybridoma, but lacked enhancing activity in all studied concentrations. This antibody was targeted to the 101WXN103 motif of dengue envelop protein domain II, studied by western blot analysis with truncated E protein and random peptide phage display. This scFv is verified as a candidate for further development as therapeutic candidate for DENV infection.

Generation and characterization of cross neutralizing human monoclonal antibody against 4 serotypes of dengue virus without enhancing activity

Background Dengue disease is a leading cause of illness and death in the tropics and subtropics. Most severe cases occur among patients secondarily infected with a different dengue virus (DENV) serotype compared with that from the first infection, resulting in antibody-dependent enhancement activity (ADE). Our previous study generated the neutralizing human monoclonal antibody, D23-1B3B9 (B3B9), targeting the first domain II of E protein, which showed strong neutralizing activity (NT) against all four DENV serotypes. However, at sub-neutralizing concentrations, it showed ADE activity in vitro. Methods In this study, we constructed a new expression plasmid using the existing IgG heavy chain plasmid as a template for Fc modification at position N297Q by site-directed mutagenesis. The resulting plasmid was then co-transfected with a light chain plasmid to produce full recombinant IgG (rIgG) in mammalian cells (N297Q-B3B9). This rIgG was characterized for neutralizing and enhancing activity by using different FcγR bearing cells. To produce sufficient quantities of B3B9 rIgG for further characterization, CHO-K1 cells stably secreting N297Q-B3B9 rIgG were then established. Results The generated N297Q-B3B9 rIgG which targets the conserved N-terminal fusion loop of DENV envelope protein showed the same cross-neutralizing activity to all four DENV serotypes as those of wild type rIgG. In both FcγRI- and RII-bearing THP-1 cells and FcγRII-bearing K562 cells, N297Q-B3B9 rIgG lacked ADE activity against all DENV serotypes at sub-neutralizing concentrations. Fortunately, the N297Q-B3B9 rIgG secreted from stable cells showed the same patterns of NT and ADE activities as those of the N297Q-B3B9 rIgG obtained from transient expression against DENV2. Thus, the CHO-K1 stably expressing N297Q-B3B9 HuMAb can be developed as high producer stable cells and used to produce sufficient amounts of antibody for further characterization as a promising dengue therapeutic candidate. Discussion Human monoclonal antibody, targeted to fusion loop of envelope domainII (EDII), was generated and showed cross-neutralizing activity to 4 serotypes of DENV, but did not cause any viral enhancement activity in vitro. This HuMAb could be further developed as therapeutic candidates.

Genomic studies of envelope gene sequences from mosquito and human samples from Bangkok, Thailand

Dengue virus (DENV) is an RNA virus showing a high degree of genetic variation as a consequence of its proofreading inability. This variation plays an important role in virus evolution and pathogenesis. Although levels of within-host genetic variation are similar following equilibrium, variation among different hosts is frequently different. To identify dengue quasispecies present among two hosts, we collected patient samples from six acute DENV cases and two pools of Aedes aegypti mosquitoes and analyzed the genetic variation of regions of the viral envelope gene. Among human and mosquito samples, we found three major clusters originating from two subpopulations. Although several shared lineages were observed in the two hosts, only one lineage showing evidence of neutral selection was observed among two hosts. Taken together, our data provide evidence for the existence of a DENV quasispecies, with less genetic variation observed in mosquitoes than humans and with circulating lineages found in both host types.