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.

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.

Prevalence of soil-transmitted helminth infections and associated risk factors among elderly individuals living in rural areas of southern Thailand.

BACKGROUND: Soil-transmitted helminth (STH) infection is a neglected tropical disease affecting approximately 1.5 billion people worldwide. In past decades, most studies focused on STH infection in preschool-aged and school-aged children in different regions of Thailand. However, little is known about the prevalence and intensity of STH infection in the elderly population. Therefore, the aim of this study was to determine the current prevalence and intensity of STH infections and to identify associated risk factors among the elderly population. METHODS: A cross-sectional study was conducted from July to November 2019 to assess the prevalence of STH infections and associated risk factors among elderly populations living in five subdistricts of Thasala District, Nakhon Si Thammarat Province, Thailand. A total of 439 elderly individuals were selected using a random sampling technique. Each fresh stool sample was examined using the formalin ethyl acetate concentration technique (FECT), Kato-Katz thick smears and agar plate culture (APC). A structured questionnaire was used to obtain relevant information regarding associated risk factors for STH infection. RESULTS: The overall prevalence of STH infection was 15.7%. Hookworms (10.9%, 48/439) were the most prevalent STH species, followed by Strongyloides stercoralis (3.4%, 15/439) and Trichuris trichiura (2.1%, 9/439). Most elderly individuals infected with hookworms or T. trichiura had light-intensity infections. A higher prevalence of STH infection was observed among individuals aged older than 80 years (23.4%) than among those aged between 70 and 79 years (15.2%) and 60-69 years (14.5%). Males were 1.85-times more likely to present with STH infections than females. Not washing vegetables before eating increased the risk of STH infection by 3.19 times, while defecation in an open field increased the risk of STH infection by 2.65 times. CONCLUSIONS: The findings suggested that STH infection is prevalent, and that hookworms are the most common STH species among elderly populations in southern Thailand. Personal hygiene and deworming programs should be implemented among the elderly population to reduce the risk and prevent the spread of STH infections.

Effects of Gnathostoma spinigerum infective stage larva excretory-secretory products on NK cells in peripheral blood mononuclear cell culture: focused on expressions of IFN-γ and killer cell lectin-like receptors.

Human gnathostomiasis is mainly caused by third-stage larvae of Gnathostoma spinigerum (G. spinigerum L3). Excretory-secretory products (ES) released from infective helminthic larvae are associated with larval migration and host immunity modulation. Natural killer (NK) cells have important immune functions against helminth infection. Currently, the effects of ES from G. spinigerum L3 (G. spinigerum ES) on NK cell activity are unclear. This study investigated whether G. spinigerum ES affected human NK cells. Human normal peripheral blood mononuclear cell (PBMC) cultures were used to mimic immune cells within the circulation. PBMC were co-cultured with G. spinigerum ES (0.01-0.05 µg/ml) for 5 or 7 days. Levels of IFN-γ in cultured supernatants were measured by enzyme-linked immunosorbent assay. The expressions of mRNA encoding NK cell receptors, especially the C type killer cell lectin-like family (KLR; NKG2A, NKG2C, and NKG2D) and IFN-γ in ES induced PBMC were determined by quantitative reverse transcription-polymerase chain reaction (RT-qPCR). ES induced PBMC markedly decreased the levels of IFN-γ and increased the expressions of NKG2A and NKG2D on NK cells. In conclusion, low amounts of G. spinigerum ES modulated NK cells by downregulating the transcription of IFN-γ and upregulating the expressions of KLR (NKG2A and NKG2D receptors) during the 7-day observation period. These findings indicate more in-depth studies of NK cell function are required to better understand the mechanism involved in immune evasive strategies of human gnathostomiasis.

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.

Prevalence of parasitic contamination of raw vegetables in Nakhon Si Thammarat province, southern Thailand.

BACKGROUND: Soil-transmitted helminth (STH) infections are major public health problems in poor and developing countries that require fecal contamination of the environment for transmission. The consumption of raw vegetables without proper washing is one of the main routes of intestinal parasite acquisition. Therefore, this study was designed to detect the prevalence of intestinal parasitic contamination in commonly consumed raw vegetables sold in three central open-air markets in Nakhon Si Thammarat province, southern Thailand. METHODS: A total of 265 fresh vegetable samples consisting of peppermint, lettuce, coriander, leek, gotu kola, celery, Chinese cabbage, culantro, Thai basil, and Chinese morning glory were purchased from three central open-air markets in the Mueang, Thasala and Sichon districts from December 2016 to March 2017. Each sample was washed with physiological saline, shaken for 15 min, and then allowed to sediment. Finally, sedimentation was performed via the sedimentation concentration technique and examined using light microscopy for the detection of pathogenic parasites. RESULTS: The overall prevalence of parasitic contamination was 35.1% (93/265). The most predominant parasite was hookworms (42.9%), followed by Strongyloides stercoralis (10.6%), Trichuris trichiura (2.6%), Ascaris lumbricoides (2.6%), and Toxocara spp. (2.6%). The highest level of contamination was found in celery, with a prevalence rate of 63.3% (19/30), while the lowest contamination level was found in Chinese morning glory, with a prevalence rate of 2.0% (2/30). The prevalence of intestinal parasite contamination in Mueang district (51.5%) was significantly higher than that in Thasala district (17.9%) and Sichon district (30.6%) (P < 0.001). CONCLUSION: The results of the present study demonstrate that consumption of vegetables with parasite contamination in this area represents a potential route for the transmission of parasitic infection, particularly hookworm infection. Therefore, it is necessary for health authorities to educate consumers about the proper washing of vegetables prior to consumption. Preventive methods such as wearing gloves and washing hands after handling vegetables should also be advocated to sellers who are at risk of acquiring STH infections via skin penetration.

Prevalence of intestinal parasitic infections and associated risk factors for hookworm infections among primary schoolchildren in rural areas of Nakhon Si Thammarat, southern Thailand.

BACKGROUND: Soil-transmitted helminth (STH) infections are among the most common type of infections worldwide and are widely distributed in tropical areas. In rural areas of southern Thailand where most land is used for agriculture, children are at risk of acquiring parasites, especially STHs. Assessing the current situation regarding parasitic infection in these areas is a prerequisite for developing appropriate control measures. This study is aimed at determining the prevalence of intestinal parasitic infections, the intensity of STH infections and the associated risk factors among primary schoolchildren in Nopphitam District, Nakhon Si Thammarat Province, Thailand. METHODS: A cross-sectional study involving 299 schoolchildren between 7 and 12 years of age was conducted between January and March 2016. A questionnaire administered by direct interviews was used to collect sociodemographic information and data on associated risk factors. Stool samples were processed using direct wet smears, formalin-ethyl acetate sedimentation concentration, and the modified Kato-Katz technique. RESULTS: The overall prevalence of intestinal parasites among the 299 children was 16% (48 of 299), with 32 children infected with hookworms (10.7%), 10 with Blastocystis hominis (3.3%), seven with Giardia intestinalis (1.6%), one with Enterobius vermicularis (0.3%), and one with Trichuris trichiura (0.3%). The hookworm infection intensity, measured by the median eggs per gram (EPG) of stool, was 1200 EPG (Interquartile range (IQR): 360-3200). Most children had light-intensity hookworm infections, but two had heavy-intensity infections. When participants included in the sample were classified by age, children 10-12 years old demonstrated higher intestinal parasite prevalence than those aged 7-9 years (adjusted odds ratio (AOR) = 2.3, 95% CI: 1.1-4.9, P = 0.030). Inadequate handwashing before meals was statistically associated with hookworm infections (AOR = 2.3; 95% CI: 1.1-4.8, P = 0.037). CONCLUSIONS: This study highlights that hookworms are the most prevalent STH infection in the study area. Older age group (10-12 years) and inadequate handwashing before meals were statistically associated with hookworm infections. Accordingly, appropriate strategies and education on personal and environmental hygiene should be implemented. Moreover, the cost-effectiveness of mass drug administration in this area should be further investigated.

Expression of cleaved caspase-3 in renal tubular cells in Plasmodium falciparum malaria patients.

AIM: In Plasmodium falciparum malaria, the clinical manifestation of acute kidney injury (AKI) is commonly associated with acute tubular necrosis (ATN) in the kidney tissues. Renal tubular cells often exhibit various degrees of cloudy swelling, cell degeneration, and frank necrosis. To study individual cell death, this study evaluates the degree of renal tubular necrosis in association with apoptosis in malarial kidneys. METHODS: Kidney tissues from P. falciparum malaria with AKI (10 cases), and without AKI (10 cases) were evaluated for tubular pathology. Normal kidney tissues from 10 cases served as controls. Tubular necrosis was assessed quantitatively in kidney tissues infected with P. falciparum malaria, based on histopathological evaluation. In addition, the occurrence of apoptosis was investigated using cleaved caspase-3 marker. Correlation between tubular necrosis and apoptosis was analyzed. RESULTS: Tubular necrosis was found to be highest in P. falciparum malaria patients with AKI (36.44% ± 3.21), compared to non-AKI (15.88% ± 1.63) and control groups (2.58% ± 0.39) (all p < 0.001). In the AKI group, the distal tubules showed a significantly higher degree of tubular necrosis than the proximal tubules (p = 0.021) and collecting tubules (p = 0.033). Tubular necrosis was significantly correlated with the level of serum creatinine (r = 0.596, p = 0.006), and the occurrence of apoptosis (r = 0.681, p = 0.001). CONCLUSION: In malarial AKI, the process of apoptosis occurs in ATN.

Mast cell activation in the skin of Plasmodium falciparum malaria patients.

BACKGROUND: Mast cells (MCs) play an important role in the immune response and inflammatory processes. Generally, MCs can be stimulated to degranulate and release histamine upon binding to immunoglobulin E (IgE). In malaria, MCs have been linked to immunoglobulin (Ig) E-anti-malarial antibodies. This study investigated the response of MCs in the skin of patients with Plasmodium falciparum malaria. METHODS: Skin tissue samples were examined from ten uncomplicated and 20 complicated P. falciparum malaria cases. Normal skin tissues from 29 cases served as controls. Pre- and post-treatment tissues were included. Histopathological changes of the skin were evaluated using haematoxylin and eosin stain. MCs were investigated using toluidine blue staining. The percentage of MC degranulation was compared among groups and correlated with clinical data. RESULTS: MC degranulation was significantly higher in the complicated P. falciparum (43.72% ± 1.44) group than the uncomplicated P. falciparum (31.35% ± 3.29) (p <0.05) and control groups (18.38% ± 1.75), (p <0.0001). MC degranulation correlated significantly with the degree of parasitaemia (rs = 0.66, p <0.0001). Associated pathological features, including extravasation of red blood cells, perivascular oedema and leukocyte infiltration were significantly increased in the malaria groups compared with the control group (all p <0.001). CONCLUSIONS: MCs in the skin dermis are activated during malaria infection, and the degree of MC degranulation correlates with parasitaemia and disease severity.

A potential role for interleukin-33 and γ-epithelium sodium channel in the pathogenesis of human malaria associated lung injury.

BACKGROUND: The pathogenesis of pulmonary oedema (PE) in patients with severe malaria is still unclear. It has been hypothesized that lung injury depends, in addition to microvascular obstruction, on an increased pulmonary capillary pressure and altered alveolar-capillary membrane permeability, causing pulmonary fluid accumulation. METHODS: This study compared the histopathological features of lung injury in Southeast Asian patients (n = 43) who died from severe Plasmodium falciparum malaria, and correlated these with clinical history in groups with or without PE. To investigate the expression of mediators that may influence fluid accumulation in PE, immunohistochemistry and image analysis were performed on controls and sub-sets of patient with or without PE. RESULTS: The expression of leukocyte sub-set antigens, bronchial interleukin (IL)-33, γ-epithelium sodium channel (ENaC), aquaporin (AQP)-1 and -5, and control cytokeratin staining was quantified in the lung tissue of severe malaria patients. Bronchial IL-33 expression was significantly increased in severe malaria patients with PE. Malaria patients with shock showed significantly increased bronchial IL-33 compare to other clinical manifestations. Bronchial IL-33 levels were positively correlated with CD68+ monocyte and elastase + neutrophil, septal congestion and hyaline membrane formation. Moreover, the expression of both vascular smooth muscle cell (VSMC) and bronchial γ-ENaC significantly decreased in severe malaria patients with PE. Both VSMC and bronchial γ-ENaC were negatively correlated with the degree of parasitized erythrocyte sequestration, alveolar thickness, alveolar expansion score, septal congestion score, and malarial pigment score. In contrast AQP-1 and -5 and pan cytokeratin levels were similar between groups. CONCLUSIONS: The results suggest that IL-33 may play a role in lung injury during severe malaria and lead to PE. Both VSMC and bronchial γ-ENaC downregulation may explain pulmonary fluid disturbances and participate in PE pathogenesis in severe malaria patients.

Nuclear factor kappa B in urine sediment: a useful indicator to detect acute kidney injury in Plasmodium falciparum malaria.

BACKGROUND: Acute kidney injury (AKI) is one of the major complications of Plasmodium falciparum malaria, especially among non-immune adults. It has recently been revealed that activation of transcription factor nuclear factor kappa B (NF-κB) induces pro-inflammatory gene expression involved in the development of progressive renal inflammatory diseases. The aim of this study was to determine whether urinary sediment NF-κB p65 can act as a biomarker for AKI in patients with P. falciparum malaria. METHODS: Urinary sediments from malaria patients, including Plasmodium vivax malaria, uncomplicated P. falciparum malaria, complicated P. falciparum malaria without AKI (serum creatinine-Cr <3 mg/dl) and complicated P. falciparum malaria with AKI (Cr ≥3 mg/dl) were used to determine NF-κB p65 level by sandwich enzyme-linked immunosorbent assay (ELISA). Urinary sediments obtained from healthy controls were used as a normal baseline. Correlations between levels of urinary sediment NF-κB p65 and pertinent clinical data were analysed. RESULTS: Urinary sediment NF-κB p65 levels were significantly increased on the day of admission (day 0) and on day 7 post-treatment in complicated P. falciparum malaria patients with AKI, compared with those without AKI (p=0.001, p <0.001, respectively), P. vivax patients (all p <0.001) and healthy controls (all p <0.001). NF-κB p65 levels in urinary sediment cells showed a significant positive correlation with serum Cr (Day 0: rs=0.792; p <0.001, Day 7: rs=0.605; p <0.001) and blood urea nitrogen (BUN) (Day 0: rs=0.839; p <0.001, Day 7: rs=0.596; p <0.001). CONCLUSIONS: Urinary sediment NF-κB p65 level is a useful indicator for estimating renal tubular epithelial cell damage and subsequent development of AKI among patients with P. falciparum malaria.

Liver changes in severe Plasmodium falciparum malaria: histopathology, apoptosis and nuclear factor kappa B expression.

BACKGROUND: Liver involvement in severe Plasmodium falciparum infection is commonly a significant cause of morbidity and mortality among humans. The clinical presentation of jaundice often reflects a certain degree of liver damage. This study investigated the liver pathology of severe P. falciparum malaria as well as the regulation and occurrence of apoptosis in cellular components of formalin-fixed, paraffin-embedded liver tissues. METHODS: The liver tissues used in the study came from patients who died from P. falciparum malaria with hyperbilirubinaemia (total bilirubin (TB)≥ 51.3 µmol/L or 3 mg/dl) (12 cases), P. falciparum malaria without hyperbilirubinaemia (TB<51.3 µmol/L) (10 cases); and patients who died due to accidents, whose liver histology was normal (the control group) (10 cases). The histopathology of the liver tissue was studied by routine histology method. Caspase-3 and nuclear factor kappa B (NF-κB) p65 expressions were determined using immunohistochemistry. RESULTS: The severity of liver histopathology, occurrence of apoptosis and NF-κB p65 activation in P. falciparum malaria were associated with higher TB level. Significant correlations were found between NF-κB p65 expression and apoptosis in Kupffer cells and lymphocytes in the portal tracts. CONCLUSIONS: Hyperplastic Kupffer cells and portal tract inflammation are two main features found in the liver tissues of severe P. falciparum malaria cases. In addition, NF-κB is associated with Kupffer cells and lymphocyte apoptosis in severe P. falciparum malaria.

Glomerular changes and alterations of zonula occludens-1 in the kidneys of Plasmodium falciparum malaria patients.

BACKGROUND: The process of cytoadhesion in Plasmodium falciparum malaria infection causes signaling processes that lead to structural and functional changes at the cellular level. Histopathological changes of acute kidney injury (AKI) in P. falciparum malaria often involve glomerular proliferation, thickening of the glomerular basement membrane, acute tubular necrosis, and interstitial inflammation. Focusing on the glomeruli, this study aimed to investigate glomerular and tight junction-associated protein- zonula occludens-1 (ZO-1) changes in P. falciparum malaria patients. METHODS: Kidney tissues were grouped into P. falciparum with AKI (Cr ≥ 265 µmol/L or 3 mg/dl), P. falciparum without AKI (Cr < 265 µmol/L), and normal kidney tissues (control group). Glomerular cells and the glomerular area were quantified and compared in three experimental groups. The tight junction was investigated immunohistochemically using tight junction-associated protein, ZO-1, protein marker. A further immunofluorescence study was performed in an endothelial cell (EC)-parasitized red blood cell (PRBC) co-culture system, to evaluate the tight junction protein. RESULTS: Glomerular cell proliferation was significant in P. falciparum with AKI (Cr ≥ 265 µmol/L). By contrast, the glomerular area decreased significantly. ZO-1 expression was significantly decreased in the AKI group compared with normal kidneys, and in kidney tissues without AKI (p < 0.05). This was further confirmed by the depletion in ZO-1 localization in ECs co-cultured with PRBCs. CONCLUSIONS: In P. falciparum malaria with AKI, the decrease in glomerular area, despite glomerular cell proliferation, could be due to the collapse of cellular structures secondary to damaged tight junction-associated protein, ZO-1.

A morphometric and histological study of placental malaria shows significant changes to villous architecture in both Plasmodium falciparum and Plasmodium vivax infection.

BACKGROUND: Malaria in pregnancy remains a major health problem. Placental malaria infection may cause pathophysiological changes in pregnancy and result in morphological changes to placental villi. Quantitative histomorphological image analysis of placental biopsies was performed to compare placental villous architecture between active or treated placental malaria cases and controls. METHODS: A total of 67 placentas were studied from three clinical groups: control patients who did not have malaria (n = 27), active (n = 14) and treated (n=26) malaria cases, including both Plasmodium falciparum and Plasmodium vivax infections. Image analysis of histological placental sections was performed using ImageJ software to measure the number and size (area) of terminal villi, perimeter measurement per villus and total perimeter per unit area, and number of capillaries per villus (vascularity). Histological features of placental malaria were scored and these results were correlated with malaria status and clinical outcomes. RESULTS: Villous size correlated with vascularity (p <0.0001) but was inversely correlated with observed villi per unit area, (p = 0.0001). Significantly greater villous area and vascularity was observed in UK controls. Indices of histological malaria infection were significantly greater in active versus treated malaria cases. Active placental malaria cases showed significantly smaller villous area (p <0.0084), vascularity (p <0.0139) and perimeter (p <0.0006) than treated malaria cases or controls, but significantly more villi per unit area (p <0.0001). Villous size in treated malaria cases was significantly larger than active placental malaria cases (p <0.001) and similar to controls. There was a significant relationship between villous number and anaemia at the time of infection (p <0.0034), but not placental weight, birth weight or gestational age at delivery. No differences were found between histology or villous morphology comparing infections with P. falciparum or P. vivax. CONCLUSIONS: These results imply that villous size, perimeter and vascularity are acutely decreased during active placental malaria, decreasing the surface area available for gas exchange per villus. However the increased number of villi per unit area offsets this change and persists after treatment. Histopathological and villous architectural changes may be reversed by early detection and appropriate anti-malarial treatment.

Cellular-mediated immune responses in the liver tissue of patients with severe Plasmodium falciparum malaria.

The immune responses against Plasmodiumfalciparum malaria infections are complex and poorly understood. No published studies have yet reported the lymphocyte subsets involved in the human liver tissue of P. falciparum malaria patients. To understand the cellular-mediated immune responses in the liver during malaria infection, we determined the numbers of the various lymphocyte subsets in tissue samples obtained at autopsy from patients who died with P. falciparum malaria infection. All the liver tissue specimens had been stored at the Department of Tropical Pathology, Faculty of Tropical Medicine, Mahidol University, Thailand. On the basis of total bilirubin (TB) levels prior to death, patients were divided into 2 groups: those with hyperbilirubinemia [total bilirubin (TB) > or =51.3 micromol/l) (n = 9)] and those without hyperbilirubinemia (TB < 51.3 micromol/l) (n = 12). Normal liver specimens (n = 10) were used as controls. An immunohistochemistry method was used to analyze the types and numbers of lymphocytes (T and B lymphocytes), and Kupffer cells, using specific antibodies against CD3+, CD4+, CD8+, CD20+, and CD68+. Our findings reveal the numbers of T lymphocytes (CD3+ T-cells) and their subsets (CD4+ and CD8+ T-cells) were significantly greater in the portal tracts and sinusoids of liver tissue obtained from P. falciparum malaria cases with hyperbilirubinemia than those without hyperbilirubinemia or controls. CD8+ T-cells were the major lymphocyte subset in the liver tissue of patients with severe falciparum malaria. A significant positive correlation was seen between the numbers of CD4+ and CD8+ T-cells and the liver enzyme levels among P. falciparum malaria patients. The number of CD68+ cells (Kupffer cells) was significantly greater in the liver sinusoids of P. falciparum malaria cases with hyperbilirubinemia than those without hyperbilirubinemia. These findings suggest T-cells, especially CD8+ T-cells and Kupffer cells are an important part of the cellular immune response in the liver tissue of P. falciparum infected patients.

Detection of Gnathostoma spinigerum larva in the brain with complete follow-up after surgical treatment of human neurognathostomiasis.

Human gnathostomiasis is a food-borne zoonotic helminthic infection widely reported in Latin America, Asia, and Southeast Asia. Consuming raw, or under-cooked fresh-water fish is the leading cause of this helminthic infection, which is clinically characterized by signs of inflammation, itching sensation, or irritation with migratory swelling. Neurological symptoms resulting from neurognathostomiasis vary, and there is scant information due to the rareness of patient brain samples. This study aimed to demonstrate the first evidence of human neurognathostomiasis by the detection of Gnathostoma spinigerum larva in patient's brain during craniotomy, supported by histopathological, immunological and proteomic evidence. Clinical symptoms were obtained from medical history and physical examination with laboratory investigations, including magnetic resonance imaging (MRI), left temporal craniotomy, histopathology of brain tissue, and Western blot analysis, were performed to elucidate the causative pathogens for diagnosis. In addition, the host-parasite interaction of the parasite invading the patient's brain was characterized through proteomics. Histopathology revealed worms with the characteristic cuticular spines of G. spinigerum which were detected and identified. These histopathological findings were consistent with a positive Western blot showing a 24-kDa reactive-band for gnathostomiasis. Proteomic analysis revealed the presence of G. spinigerum serpin and serine protease in the patient's serum. Moreover, the leucine-rich alpha-2-glycoprotein was indicated as a systemic biomarker of early brain injury related to invasion by G. spinigerum. Therefore, our study provides the initial evidence of human neurognathostomiasis due to G. spinigerum larval invasion along with successful craniotomy and proven larval detection including complete follow-up, and the disease prognosis after surgical treatment.

Nuclear factor kappa B modulates apoptosis in the brain endothelial cells and intravascular leukocytes of fatal cerebral malaria.

BACKGROUND: Cerebral malaria (CM) caused by Plasmodium falciparum is known to be associated with the sequestration of parasitized red blood cells (PRBCs) in the microvasculature and the release of soluble cytokines. In addition, the involvement of signaling molecules has gained wide interest in the pathogenesis of CM. An important signaling factor, nuclear factor kappa B (NF-κB) is known to regulate apoptosis. This work aimed to study the expression of NF-κB p65 and its correlation with apoptosis in the brain of fatal CM. METHODS: The expression of NF-κB p65 and cleaved caspase-3 in the brain of fatal P. falciparum malaria cases was investigated by immunohistochemistry. Histopathological features were analysed together with the correlations of NF-κB p65 and cleaved caspase-3 expression. RESULTS: NF-κB p65 activation and cleaved caspase-3 expression were significantly increased in the neurons, glial cells, vascular endothelial cells (ECs) and intravascular leukocytes of the brain in fatal CM, compared with the control brain (p < 0.001) and non-cerebral malaria (NCM) (p = 0.034). The percentage of neurons that expressed nuclear NF-κB p65 showed a positive correlation with the total score of histopathological changes (rs = 0.678; p = 0.045). Significant positive correlations were established between vascular ECs NF-κB index and ECs apoptotic index (rs = 0.717; p = 0.030) and between intravascular leukocytes NF-κB index and leukocytes apoptotic index (rs = 0.696; p = 0.037) in fatal CM. CONCLUSIONS: This study documented that NF-κB p65 is one of the signaling factors that modulates apoptosis in the brain ECs and intravascular leukocytes of fatal CM.

Antimalarial efficacy and toxicological assessment of medicinal plant ingredients of Prabchompoothaweep remedy as a candidate for antimalarial drug development.

BACKGROUND: Drug resistance exists in almost all antimalarial drugs currently in use, leading to an urgent need to identify new antimalarial drugs. Medicinal plant use is an alternative approach to antimalarial chemotherapy. This study aimed to explore potent medicinal plants from Prabchompoothaweep remedy for antimalarial drug development. METHODS: Forty-eight crude extracts from Prabchompoothaweep remedy and its 23 plants ingredients were investigated in vitro for antimalarial properties using Plasmodium lactate dehydrogenase (pLDH) enzyme against Plasmodium falciparum K1 strain and toxicity effects were evaluated in Vero cells. The plant with promising antimalarial activity was further investigated using gas chromatography-mass spectrometry (GC-MS) to identify phytochemicals. Antimalarial activity in mice was evaluated using a four-day suppressive test against Plasmodium berghei ANKA at dose of 200, 400, and 600 mg/kg body weight, and acute toxicity was analyzed. RESULTS: Of the 48 crude extracts, 13 (27.08%) showed high antimalarial activity against the K1 strain of P. falciparum (IC50 <  10 µg/ml) and 9 extracts (18.75%) were moderately active (IC50 = 11-50 µg/ml). Additionally, the ethanolic extract of Prabchompoothaweep remedy showed moderate antimalarial activity against the K1 strain of P. falciparum (IC50 = 14.13 µg/ml). Based on in vitro antimalarial and toxicity results, antimalarial activity of the aqueous fruit extract of Terminalia arjuna (IC50 = 4.05 µg/ml and CC50 = 219.6 µg/ml) was further studied in mice. GC-MS analysis of T. arjuna extract identified 22 compounds. The most abundant compounds were pyrogallol, gallic acid, shikimic acid, oleamide, 5-hydroxymethylfurfural, 1,1-diethoxy-ethane, quinic acid, and furfural. Analysis of the four-day suppressive test indicated that T. arjuna extract at dose of 200, 400, and 600 mg/kg body weight significantly suppressed the Plasmodium parasites by 28.33, 45.77, and 67.95%, respectively. In the acute toxicity study, T. arjuna extract was non-toxic at 2000 mg/kg body weight. CONCLUSIONS: The aqueous fruit extract of T. arjuna exerts antimalarial activity against Plasmodium parasites found in humans (P. falciparum K1) and mice (P. berghei ANKA). Acute toxicity studies showed that T. arjuna extract did not show any lethality or adverse effects up to a dose of 2000 mg/kg.

Efficacy of artesunate combined with Atractylodes lancea or Prabchompoothaweep remedy extracts as adjunctive therapy for the treatment of cerebral malaria.

BACKGROUND: Cerebral malaria is one of the most serious complications of Plasmodium infection and causes behavioral changes. However, current antimalarial drugs have shown poor outcomes. Therefore, new antimalarials with neuroprotective effects are urgently needed. This study aimed to evaluate the effects of selected extracts as monotherapy or adjunctive therapy with artesunate on antimalarial, anti-inflammatory, antioxidant, and neuroprotective properties in experimental cerebral malaria (ECM). METHODS: ECM was induced in male C57BL/6 mice by infection with Plasmodium berghei ANKA (PbA). Ethanolic extracts of Atractylodes lancea (a dose of 400 mg/kg) and Prabchompoothaweep remedy (a dose of 600 mg/kg) were evaluated as monotherapy and adjunctive therapy combined with artesunate at the onset of signs of cerebral malaria and continued for 7 consecutive days. Parasitemia, clinical scores, and body weight were recorded throughout the study. At day 13 post-infection, mouse brains were dissected and processed for the study of the inflammatory response, oxidative stress, blood-brain barrier (BBB) integrity, histopathological changes, and neurocognitive impairments. RESULTS: Ethanolic extracts of A. lancea and Prabchompoothaweep remedy alone improved cerebral malaria outcome in ECM, whereas artesunate combined with extracts of A. lancea or Prabchompoothaweep remedy significantly improved the outcome of artesunate and crude extracts alone. Using real-time PCR, PbA-infected mice that had received the combination treatment showed significantly reduced gene expression of inflammatory cytokines (TNF-α, IL-1ß, IL-6, and IL-10), chemokines (CXCL4 and CXCL10), and adhesion molecules (ICAM-1, VCAM1, and CD36). The PbA-infected mice that received the combination treatment showed a significantly decreased malondialdehyde level compared to the untreated group. Similarly, the Evans blue dye assay revealed significantly less dye extravasation in the brains of infected mice administered the combination treatment, indicating improved BBB integrity. Combination treatment improved survival and reduced pathology in the PbA-infected group. Additionally, combination treatment resulted in a significantly reduced level of cognitive impairment, which was analyzed using a novel object recognition test. CONCLUSIONS: This study demonstrated that artesunate combined with A. lancea or Prabchompoothaweep remedy extracts as adjunctive therapy reduced mortality, neuroinflammation, oxidative stress, BBB integrity protection, and neurocognitive impairment in the ECM.