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.

Apoptotic changes and aquaporin-1 expression in the choroid plexus of cerebral malaria patients.

BACKGROUND: Cerebral malaria (CM) is associated with sequestration of parasitized red blood cells (PRBCs) in the capillaries. Often, the association of CM with cerebral oedema is related with high mortality rate. Morphological changes of the choroid plexus (CP) and caspase-3 expression in CM have not been reported. In addition, limited knowledge is known regarding the role of aquaporin (AQP)-1 in CM. The present study evaluated changes in the CP, explored apoptotic changes and AQP-1 expression in CP epithelial cells (CPECs) in fatal CM patients. METHODS: CP from fatal Plasmodium falciparum malaria patients (5 non-CM [NCM], 16 CM) were retrieved and prepared for histopathological evaluation. Caspase-3 and AQP-1 expressions in CPECs were investigated by immunohistochemistry. RESULTS: Histologically, apoptotic changes in CPECs were significantly observed in the CM group compared with the NCM and normal control (NC) groups (p < 0.05). These changes included cytoplasmic and nuclear condensation/shrinkage of CPECs and detachment of CPECs from the basement membrane. The apoptotic changes were positively correlated with caspase-3 expression in the nuclei of CPECs. In addition, AQP-1 expression in CPECs was significantly decreased in the CM group compared with the NCM and NC groups (all p < 0.001). A negative correlation (rs = - 0.450, p = 0.024) was documented between caspase-3 expression in the nuclei of CPECs and AQP-1. CONCLUSIONS: Apoptotic changes and altered AQP-1 expression may contribute to CPEC dysfunction and subsequently reduce cerebrospinal fluid production, affecting the water homeostasis in the brains of patients with CM.

Protective Effect of an Anti-HMGB-1 Neutralizing Antibody on Hemozoin-Induced Alveolar Epithelial Cell in a Model of Malaria Associated ALI/ARDS.

BACKGROUND: We aimed to determine whether neutralizing high mobility group box-1 (HMGB-1) prevents the release of HMGB-1 and proinflammatory cytokines on hemozoin (Hz)-induced alveolar epithelial cell in a model of malaria associated ALI/ARDS. METHODS: This study was conducted in the Department of Tropical Pathology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand in 2020. Human pulmonary alveolar epithelial cells (HPAEpiCs) were exposed to medium alone or 20 µM Hz for 24 h and incubated with different concentrations (1, 5, and 10 µg/ml) of anti-HMGB-1 monoclonal antibody (mAb) for various times (0, 4, 12, 24, and 48 h). The levels of HMGB-1, TNF-α and IFN-γ in the supernatants were measured by ELISA. The mRNA expression of RAGE, TLR-2 and TLR-4 were analyzed by real-time PCR. RESULTS: The HPAEpiCs treated with 10 µg/ml anti-HMGB-1 mAb showed a significant reduction in HMGB-1 release into the supernatant compared with those treated with 1 and 5 µg/ml anti-HMGB-1 mAb. The levels of TNF-α and IFN-γ were significantly decreased in the supernatant of HPAEpiCs treated with 1, 5, and 10 µg/ml anti-HMGB-1 mAb for 4, 12, 24, and 48 h compared with those stimulated with Hz alone. The mRNA expression levels of RAGE, TLR-2, and TLR-4 were significantly decreased after 24 h of anti-HMGB-1 antibody treatment at all concentrations. CONCLUSION: An anti-HMGB-1 antibody could be an effective agent for inhibiting the release of HMGB-1, TNF-α and IFN-γ. Furthermore, a neutralizing anti-HMGB-1 antibody could be applicable for the treatment of malaria-associated ALI/ARDS.

M1 macrophage features in severe Plasmodium falciparum malaria patients with pulmonary oedema.

BACKGROUND: Pulmonary oedema (PE) is a serious complication of Plasmodium falciparum malaria which can lead to acute lung injury in severe cases. Lung macrophages are activated during malaria infection due to a complex host-immune response. The molecular basis for macrophage polarization is still unclear but understanding the predominant subtypes could lead to new therapeutic strategies where the diseases present with lung involvement. The present study was designed to study the polarization of lung macrophages, as M1 or M2 macrophages, in the lungs of severe P. falciparum malaria patients, with and without evidence of PE. METHODS: Lung tissue samples, taken from patients who died from severe P. falciparum malaria, were categorized into severe malaria with PE and without PE (non-PE). Expression of surface markers (CD68+, all macrophages; CD40+, M1 macrophage; and CD163+, M2 macrophage) on activated lung macrophages was used to quantify M1/M2 macrophage subtypes. RESULTS: Lung injury was demonstrated in malaria patients with PE. The expression of CD40 (M1 macrophage) was prominent in the group of severe P. falciparum malaria patients with PE (63.44 ± 1.98%), compared to non-PE group (53.22 ± 3.85%, p < 0.05), whereas there was no difference observed for CD163 (M2 macrophage) between PE and non-PE groups. CONCLUSIONS: The study demonstrates M1 polarization in lung tissues from severe P. falciparum malaria infections with PE. Understanding the nature of macrophage characterization in malaria infection may provide new insights into therapeutic approaches that could be deployed to reduce lung damage in severe P. falciparum malaria.

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.

Release of endothelial activation markers in lungs of patients with malaria-associated acute respiratory distress syndrome.

BACKGROUND: Malaria-associated acute respiratory distress syndrome (MA-ARDS) is an understudied complication of malaria and is characterized by pulmonary inflammation and disruption of the alveolar-capillary membrane. Its pathogenesis remains poorly understood. Since endothelial activation plays an important role in other malarial complications, the expression of two endothelial activation markers, von Willebrand factor (VWF) and angiopoietin-2 (ANG-2), was investigated in the lungs of patients with MA-ARDS. METHODS: Post-mortem lung sections of Plasmodium falciparum-infected patients without alveolar oedema (NA), P. falciparum-infected patients with alveolar oedema (MA-ARDS), and uninfected people who died accidentally with no pathological changes to the lungs (CON) were immunohistochemically stained for VWF and ANG-2, and were evaluated with semi-quantitative analysis. RESULTS: Alveolar oedematous VWF and ANG-2 and intravascular VWF staining were significantly increased in patients with MA-ARDS versus infected and uninfected control groups. The levels of VWF in the alveolar septa and endothelial lining of large blood vessels of patients with MA-ARDS was significantly decreased compared to controls. ANG-2 expression was increased in the alveolar septa of malaria patients without alveolar oedema versus control patients, while ANG-2+ leukocytes were increased in the alveoli in both infected patient groups. CONCLUSIONS: This study documents a high level of VWF and ANG-2, two endothelial activation markers in the oedematous alveoli of post-mortem lung sections of Thai patients with MA-ARDS. Decreased detection of VWF in the endothelial lining of blood vessels, in parallel with an increased presence of intravascular VWF staining suggests marked endothelial activation and Weibel-Palade body release in the lungs of patients with MA-ARDS.

FTY720 restores endothelial cell permeability induced by malaria sera.

Increased endothelial cell (EC) permeability in severe Plasmodium falciparum malaria contributes to major complications of severe malaria. This study explored EC permeability in malaria, and evaluated the potential use of FTY720 to restore EC permeability. ECs were incubated with sera from malaria patients (P. vivax, uncomplicated and complicated P. falciparum malaria). Cellular permeability was investigated using a fluorescein isothiocyanate (FITC)-dextran permeability assay. FTY720, an analogue of sphingosine-1-phosphate (S1P), was tested for its potential action in maintaining EC integrity. ECs incubated with sera from malaria patients with complicated P. falciparum showed higher fluorescein leakage compared with ECs incubated with sera from P. vivax (p < 0.001) and uncomplicated P. falciparum (p < 0.001). ECs pretreated with FTY720 before incubation with malaria sera had significantly decreased fluorescein leakage compared with no FTY720 treatment. In addition, FTY720 treatment significantly reduced fluorescein leakage for both uncomplicated (at 45 min) (p = 0.015), and complicated P. falciparum malaria (15 min) (p = 0.043). The permeability increase induced by complicated P. falciparum sera was significantly reversed and prevented by FTY720 in vitro. FTY720 may have clinical applications to protect against endothelial barrier dysfunction in severe P. falciparum malaria.

Exploring pancreatic pathology in Plasmodium falciparum malaria patients.

Hypoglycaemia is an important complication of Plasmodium falciparum malaria infection, which can be lethal if not treated. A decrease in blood sugar (BS) level has been correlated with disease severity, parasitaemia and the use of certain antimalarial drugs. This study explored the relationship between pancreatic pathology, including the expressions of insulin and glucagon in the islets of Langerhans, and the BS levels in P. falciparum malaria patients. Pancreatic tissues from malaria patients were divided into three groups, namely those with BS < 40 mg/dl, BS = 40-120 mg/dl, and BS > 120 mg/dl. In P. falciparum malaria, pancreatic tissues showed numerous parasitised red blood cells (PRBCs) in the capillaries, oedema, acinar necrosis and the presence of inflammatory cells. The islet size and the expression of insulin were significantly increased in P. falciparum malaria patients with hypoglycaemia. In addition, insulin expression was positively correlated with islet size and negatively correlated with BS levels. This pioneer study documents an increase in insulin expression and an increase in islet size in hypoglycaemic patients with P. falciparum malaria. This could contribute to the pathogenesis of hypoglycaemia and provides evidence for the potential need to effectively manage the hypoglycaemia seen in malaria infection.

Increased synapsin I expression in cerebral malaria.

Synapsin I is a neuronal phosphoprotein contained in the synaptic vesicles of mammalian central and peripheral nervous systems. It regulates both neurotransmitter release and synaptic formation. Variations in synapsin I expression in the brain have been reported to cause brain malfunction. In severe malaria, neurological complications, such as convulsion, delirium and coma, suggest abnormalities in the release of neurotransmitters. This study evaluated synapsin I expression in cerebral malaria (CM). An immunohistochemical method was used to study the semi-quantitative and qualitative expression of synapsin I in the brain of CM patients (10 cases) who died with Plasmodium falciparum, compared with non-cerebral malaria (NCM) (4 cases), and control brain tissues (5). Synapsin I was expressed in the gray matter of the cerebral cortex and the molecular layer of the cerebellum, as a diffusely dense precipitate pattern in the neuropil, with no immunoreactivity in the neurons, neuronal dendrites, glial cells, endothelial cells, and Purkinje cells. The findings were similarly demonstrated in CM, NCM, and control brain tissues. However, in the granular layer of the cerebellum, a significant increase in synapsin I expression was observed in the granule cells, and the glomerular synaptic complex, from the CM group, compared with the NCM, and control brain tissues (all P < 0.05). Parasitemia showed a positive correlation with synapsin I expression in the granule cells (on admission: Spearman's ρ = 0.600, P = 0.023) (before death: Spearman's ρ = 0.678, P = 0.008), and glomerular synaptic complex (before death: Spearman's ρ = 0.571, P = 0.033). It was hypothesized that CM causes pre-synaptic excitation and eventually activation of synapsin I, leading to increased neurotransmitter release. Synapsin I inhibitor should be investigated further as a target for a therapeutic intervention to alleviate neurological symptoms in severe malaria.