Immunofluorescence study of cytoskeleton in endothelial cells induced with malaria sera.

BACKGROUND: Endothelial cells (ECs) play a major role in malaria pathogenesis, as a point of direct contact of parasitized red blood cells to the blood vessel wall. The study of cytoskeleton structures of ECs, whose main functions are to maintain shape and provide strength to the EC membrane is important in determining the severe sequelae of Plasmodium falciparum malaria. The work investigated the cytoskeletal changes (microfilaments-actin, microtubules-tubulin and intermediate filaments-vimentin) in ECs induced by malaria sera (Plasmodium vivax, uncomplicated P. falciparum and complicated P. falciparum), in relation to the levels of pro-inflammatory cytokines. METHODS: Morphology and fluorescence intensity of EC cytoskeleton stimulated with malaria sera were evaluated using immunofluorescence technique. Levels of tumour necrosis factor (TNF) and interferon (IFN)-gamma (γ) were determined using enzyme-linked immunosorbent assay (ELISA). Control experimental groups included ECs incubated with media alone and non-malaria patient sera. Experimental groups consisted of ECs incubated with malaria sera from P. vivax, uncomplicated P. falciparum and complicated P. falciparum. Morphological scores of cytoskeletal alterations and fluorescence intensity were compared across each experiment group, and correlated with TNF and IFN-γ. RESULTS: The four morphological changes of cytoskeleton included (1) shrinkage of cytoskeleton and ECs with cortical condensation, (2) appearance of eccentric nuclei, (3) presence of "spiking pattern" of cytoskeleton and EC membrane, and (4) fragmentation and discontinuity of cytoskeleton and ECs. Significant damages were noted in actin filaments compared to tubulin and vimentin filaments in ECs stimulated with sera from complicated P. falciparum malaria. Morphological damages to cytoskeleton was positively correlated with fluorescence intensity and the levels of TNF and IFN-γ. CONCLUSIONS: ECs stimulated with sera from complicated P. falciparum malaria showed cytoskeletal alterations and increased in fluorescence intensity, which was associated with high levels of TNF and IFN-γ. Cytoskeletal changes of ECs incubated with complicated P. falciparum malaria sera can lead to EC junctional alteration and permeability changes, which is mediated through apoptotic pathway. The findings can serve as a basis to explore measures to strengthen EC cytoskeleton and alleviate severe malaria complications such as pulmonary oedema and cerebral malaria. In addition, immunofluorescence intensity of cytoskeleton could be investigated as potential prognostic indicator for malaria severity.

The activation of BAFF/APRIL system in spleen and lymph nodes of Plasmodium falciparum infected patients.

Previous studies have reported activation of the B cell-activating factor (BAFF)/a proliferation-inducing ligand (APRIL) system in T independent immunity against malaria infection. Plasmodium falciparum (P. falciparum) infected animal model is not feasible. Therefore, little is known about the occurrence of BAFF/APRIL system and changes in falciparum lymphoid tissues. This study aimed to investigate the expression of BAFF/APRIL system components in lymphoid tissues from P. falciparum infected patients. Spleen and lymph node samples from 14 patients were collected at autopsy. Normal spleens and bacterially infected tonsils served as controls. The protein and/or mRNA expression of BAFF/APRIL and their cognate receptors, BAFF-R, TACI and BCMA, were determined by immunohistochemistry and RT-qPCR, respectively. The spleens of the patients exhibited significantly higher BAFF-R protein expression than normal spleens. Although without appropriate control, BCMA protein was markedly observed only in the lymph nodes. BAFF and BCMA mRNA levels were also significantly elevated in the spleen tissues of the patients compared with normal spleens. The overall BAFF-R protein levels in the lymphoid tissues of the patients correlated positively with parasitaemia. These findings are the first to confirm that BAFF/APRIL system activation in lymphoid tissues and is positively correlated with the parasitaemia levels in falciparum malaria.

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

BACKGROUND: Atherosclerosis 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. METHODS: First, 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. RESULTS: We 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). CONCLUSIONS: Increased α-defensin expression is a potential inflammatory marker that may predict the risk of CHD development in Thai hyperlipidemia patients.