Urai Chaisri

A quantitative ultrastructural study of renal pathology in fatal Plasmodium falciparum malaria

Objective  To use electron microscopy to examine the role of parasitized red blood cell (PRBC) sequestration in the pathogenesis of acute renal failure in severe falciparum malaria.

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

BackgroundCerebral 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.MethodsThe 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.ResultsNF-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.ConclusionsThis 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.

Electron‐microscopic examination of Rickettsia tsutsugamushi‐infected human liver

SummaryA 33 year‐old Thai woman was diagnosed with scrub typhus infection according to clinical symptoms, eschar lesions compatible with the disease, and specific antibody to Rickettsia tsutsugamushi detected by indirect immunoperoxidase. Percutaneous transhepatic needle biopsies were taken before and 7 days after treatment with tetracycline to study the pathology of the liver. The liver tissue was evaluated by light microscopy, using H & E and Pinkertons stains, and by transmission electron microscopy (TEM). Before treatment it showed reactive hepatitis. Rickettsia organisms within the hepatocytes and sinusoids detected by Pinkertons stain appeared as tiny bright‐red organisms. By TEM, the rod‐shaped double‐membrane Rickettsiae appeared intact in the cytoplasm of Kupffers cells and hepatocytes. After tetracycline treatment, moderate levels of acidophilic and ballooning liver cells were observed. The degree of cytoplasmic organelle damage varied, including fatty metamorphosis, depletion of glycogen granules, loss of the mitochondrial cristae, dilatation of endoplasmic reticulum and cytoplasmic vacuolation. Rickettsia organisms cannot be visualized by Pinkertons stain but were detected by TEM, in markedly vacuolated hepatocytes, in congested sinusoids and in Kupffers cells. Intranuclear Rickettsia were discovered in the endothelial nucleus, showing various degrees of injury. Some were mildly degenerated, while others exhibited clumping of nucleoprotein at the cytoplasm periphery and large vacuolation centrally. Many indented organisms were found, and binary fission during Rickettsiae multiplication was always affected. Electron‐microscopic examination of hepatic injury associated with scrub typhus is rare. This is the first ultrastructural localization of Rickettsiae in the infected human liver.

Identification of amino acids required for receptor binding and toxicity of the Bacillus sphaericus binary toxin.

Bacillus sphaericus produces a mosquito-larvicidal binary toxin composed of BinB and BinA subunits. BinA is important for toxicity, whereas BinB acts as a specific receptor-binding component. To study the functional significance of two regions that are only present in BinB, four block mutations and two single mutations were initially introduced: (111)YLD(113)-->(111)AAA(113), (115)NNH(117)-->(115)AAA(117), (143)GEQ(145)-->(143)AAA(145), (147)FQFY(150)-->(147)AAAA(150), N114A and F146A. Only the replacements at (147)FQFY(150) resulted in a total loss of toxicity to Culex quinquefasciatus larvae. Further single alanine substitutions in this region, F147A, Q148A, F149A and Y150A, were introduced to identify residues playing a critical role in mosquito-larvicidal activity. Larvicidal activity assays revealed that only F149A and Y150A mutants exhibited a total loss of toxicity. The in vitro interaction assays demonstrated that all BinB mutants are able to interact with BinA. Immunohistochemistry analysis revealed that only the Y150A mutant was unable to bind to the larval midgut, suggesting an important role of this residue in receptor binding of the BinB subunit. Conservative aromatic substitutions at F149 and Y150 resulted in full recovery of larvicidal activity, indicating that the aromaticity of F149 and Y150 is a key determinant of larvicidal activity, possibly playing a key role in the membrane interaction and receptor binding.

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

BackgroundMalaria 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.MethodsA 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.ResultsVillous 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.ConclusionsThese 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.

Activation of nuclear factor kappa B in peripheral blood mononuclear cells from malaria patients.

BackgroundMalaria parasites and their products can activate a specific immune response by stimulating cytokine production in the host’s immune cells. Transcription nuclear factor kappa B (NF-κB) is an important regulator for the control of many pro-inflammatory genes, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF). The activation and expression of NF-κB p65 in peripheral blood mononuclear cells (PBMCs) of malaria patients were investigated and correlated with the levels of IL-10 and TNF to study the nature of NF-κB p65 and its linkage to inflammatory cytokines.MethodsThe sample group comprised 33 patients admitted with malaria caused by Plasmodium vivax (n = 11), uncomplicated Plasmodium falciparum (n = 11), and complicated Plasmodium falciparum (n = 11). Peripheral blood was collected at admission and on day 7 for PBMC isolation. Healthy subjects were used as a control group. The expressions of NF-κB p65 in the PBMCs from malaria patients and the plasma levels of IL-10 and TNF were measured by using enzyme-linked immunosorbent assay (ELISA). The immunofluorescence technique was used to determine NF-κB nuclear translocation.ResultsAt admission, patients with P. vivax and uncomplicated P. falciparum had significantly elevated phospho-NF-κB p65 levels in the PBMCs compared with those of healthy controls. However, patients with complicated P. falciparum malaria had decreased levels of phospho-NF-κB p65. On day 7 post-treatment, significantly increased phospho-NF-κB p65 was found in the PBMCs of patients with complicated P. falciparum, compared with healthy controls. The plasma level of IL-10 was elevated in day 0 in patients with complicated P. falciparum malaria and was found to be negatively correlated with phospho-NF-κB p65 level (rs = −0.630, p = 0.038). However, there was no correlation between phospho-NF-κB p65 expression and TNF level in patients with complicated P. falciparum malaria.ConclusionsThis is the first report demonstrating alterations in NF-κB p65 activity in the PBMCs of malaria patients. The altered lower features of NF-κB p65 in the PBMCs of patients with complicated P. falciparum at admission could be due to a suppressive effect of high IL-10 associated with complicated P. falciparum malaria.

Intranasal, Liposome-Adjuvanted Cockroach Allergy Vaccines Made of Refined Major Allergen and Whole-Body Extract of Periplaneta americana

Background: Cockroach (CR) allergens frequently cause severe asthma in CR-sensitized subjects. Allergen-specific immunotherapy causes a shift of allergic Th2 responses towards Th1 and/or regulatory T cell (Treg) responses which reduce airway inflammation and prevent disease progression. Data are relatively limited on immunotherapy via CR allergy vaccine. Methods: The therapeutic efficacy of an intranasal liposome-adjuvant vaccine made of a refined Periplaneta americana arginine kinase (AK) was compared to the liposome-entrapped P. americana crude extract (CRE) vaccine. Adult BALB/c mice were rendered allergic to CRE. Three allergic mouse groups were immunized intranasally on alternate days with 8 doses of liposome-entrapped CRE (L-CRE), liposome-entrapped AK and placebo, respectively. One week later, all mice received a nebulized CRE provocation. Evaluation of vaccine efficacy was performed 1 day after provocation. Results: Liposome-entrapped native AK attenuated airway inflammation after the CRE provocation and caused a shift of allergic Th2 to Th1 and Treg responses. The L-CRE also induced a shift from the Th2 to the Th1 response but did not induce a Treg response and could not attenuate the airway inflammation upon allergen reexposure. Conclusions: Intranasal liposome-adjuvant CR allergy vaccine containing native AK (Per a 9) is better than L-CRE in attenuating allergic airway inflammation. The findings of this study not only document a more comprehensive and beneficial immune response induced by the refined allergen vaccine but also raise the point that the shift from the Th2 to the Th1 response alone might not correlate with improved airway histopathology, clinical outcome and quality of life.

Humanized-monoclonal antibody against heterologous Leptospira infection

Patients with leptospirosis are commonly treated with antibiotics. Jarisch-Herxheimer reaction caused by toxic bacterial substances massively released as a result of the antibiotic mediated-bacterial lysis occurs in some patients which may aggravate the existing severe clinical manifestations. In this study, a humanized-murine single-chain monoclonal antibody (HuScFv) was produced and tested as an alternative of antibiotics for treatment of leptospirosis. Complementary DNA was prepared from total RNA of a murine hybridoma clone secreting monoclonal antibody (MAb) specific to LipL32 of pathogenic Leptospira spp. The MAb had therapeutic efficacy in Leptospira challenged hamsters. The VH and VL coding sequences were amplified using the cDNA as a template. The sequences were linked to form a single-chain variable murine DNA fragment (muscFv). CDR sequences of the muscFv were grafted onto the best matching human VH and VL immunoglobulin frameworks. After cloning of the humanized murine DNA sequences (huscFv) into a phagemid vector and the vector was introduced into competent Escherichia coli, the HuScFv was produced. On the same weight basis, the HuScFv possessed equal neutralizing activities to the murine ScFv counterpart against heterologous Leptospira-mediated hemolysis in vitro and rescued hamsters from a heterologous Leptospira lethal challenge. The HuScFv antibody has high therapeutic potential as an alternative to antibiotics for human leptospirosis, especially for drug hypersensitive patients.

Ultrastructural and Real-time Microscopic Changes in P. falciparum-infected Red Blood Cells Following Treatment with Antimalarial Drugs

Ultrastructural changes to P. falciparum-infected red blood cells were examined in vitro after treatment with antimalarial drugs. Artesunate had the most rapid parasitocidal effect. All three drugs caused structural changes within the parasite, including dilatation of the parasitophorus vacuole membrane, depletion of ribosomes, mitochondrial swelling, and decreased formation of hemozoin crystals. The structure of surface knobs and Maurer’s clefts were similar to controls but reduced in number. Only depletion of free ribosomes correlated with antimalarial drug exposure. Drug treatment decreased movement of hemozoin granules within parasites on real-time microscopy, before recognizable morphological changes of parasite death.

Human transbodies to VP40 inhibit cellular egress of Ebola virus-like particles.

A direct acting anti-Ebola agent is needed. VP40, a conserved protein across Ebolavirus (EBOV) species has several pivotal roles in the virus life cycle. Inhibition of VP40 functions would lessen the virion integrity and interfere with the viral assembly, budding, and spread. In this study, cell penetrable human scFvs (HuscFvs) that bound to EBOV VP40 were produced by phage display technology. Gene sequences coding for VP40-bound-HuscFvs were subcloned from phagemids into protein expression plasmids downstream to a gene of cell penetrating peptide, i.e., nonaarginine (R9). By electron microscopy, transbodies from three clones effectively inhibited egress of the Ebola virus-like particles from human hepatic cells transduced with pseudo-typed-Lentivirus particles carrying EBOV VP40 and GP genes. Computerized simulation indicated that the effective HuscFvs bound to multiple basic residues in the cationic patch of VP40 C-terminal domain which are important in membrane-binding for viral matrix assembly and virus budding. The transbodies bound also to VP40 N-terminal domain and L domain peptide encompassed the PTAPPEY (WW binding) motif, suggesting that they might confer VP40 function inhibition through additional mechanism(s). The generated transbodies are worthwhile tested with authentic EBOV before developing to direct acting anti-Ebola agent for preclinical and clinical trials.