Malaria Sera Research Articles

Immunofluorescence study of cytoskeleton in endothelial cells induced with malaria sera

BackgroundEndothelial 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.MethodsMorphology 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.ResultsThe 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.ConclusionsECs 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.

Linear and conformational determinants of visceral leishmaniasis diagnostic antigens rK28 and rK39

BackgroundRecombinant antigens rK39 (based on kinesin sequence) and rK28 (comprising kinesin and HASPB sequences) are a mainstay of serological diagnosis for visceral leishmaniasis (VL). However, their key epitopes and the significance of their structural conformation are not clearly defined, particularly in relation to reported cross-reactivity with sera from patients with malaria, schistosomiasis, and tuberculosis.MethodsTo assess the effect of conformation on antigenicity with Sudanese VL sera, antigens rK39 and rK28 were heat-denatured at 95 °C for 10 min and then assayed by enzyme-linked immunosorbent assay (ELISA). Amino acid sequences of rK39 and rK28 were submitted to NCBI BLASTp to assess homology with Plasmodium, Schistosoma, and Mycobacterium.ResultsHeat denaturation significantly diminished the antigenicity of rK39 compared to non-denatured antigen (P = 0.001), but not for rK28 (P = 0.275). In BLASTp searches, HASPB sequences from rK28 had similarities with sequences from Plasmodium, encompassing software-predicted B-cell epitopes.ConclusionsThe antigenicity of rK39 appears to be dependent on structural conformation, whereas that of rK28 depends on linear sequence. HASPB sequence homology with Plasmodium may be responsible for the reported cross-reactivity of rK28 with malaria sera. Further work is warranted to refine the specificity of these antigens.Graphical

Glycosylation of Trypanosoma cruzi TcI antigen reveals recognition by chagasic sera

Chagas disease is considered the most important parasitic disease in Latin America. The protozoan agent, Trypanosoma cruzi, comprises six genetic lineages, TcI-TcVI. Genotyping to link lineage(s) to severity of cardiomyopathy and gastrointestinal pathology is impeded by the sequestration and replication of T. cruzi in host tissues. We describe serology specific for TcI, the predominant lineage north of the Amazon, based on expression of recombinant trypomastigote small surface antigen (gTSSA-I) in the eukaryote Leishmania tarentolae, to allow realistic glycosylation and structure of the antigen. Sera from TcI-endemic regions recognised gTSSA-I (74/146; 50.7%), with no cross reaction with common components of gTSSA-II/V/VI recombinant antigen. Antigenicity was abolished by chemical (periodate) oxidation of gTSSA-I glycosylation but retained after heat-denaturation of conformation. Conversely, non-specific recognition of gTSSA-I by non-endemic malaria sera was abolished by heat-denaturation. TcI-specific serology facilitates investigation between lineage and diverse clinical presentations. Glycosylation cannot be ignored in the search for immunogenic antigens.

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.

Reactivity of sera from cases of Plasmodium vivax malaria towards three recombinant antigens based on the surface proteins of the parasite.

Sera collected in South Korea, from 61 cases of Plasmodium vivax malaria and, as controls, 40 healthy volunteers, were tested in ELISA for IgG or IgM reacting with any of three recombinant P. vivax proteins. The antigens used, representing the parasite's major merozoite surface protein (MSP), circumsporozoite surface protein (CSP) and Duffy-binding protein (DBP), had all been expressed in an Escherichia coli system and purified. The ELISA results were recorded as optical densities (OD). The highest ratio observed between the mean OD for a malaria serum and that for a control serum was that for IgG against MSP, although CSP gave a higher ratio than MSP or DBP in the IgM ELISA. In the ELISA for IgG, the OD for MSP were found to be correlated with those for DBP (r = 0.53; P < 0.5) but the OD for CSP were not correlated with those for MSP or DBP. As the most intense reactions observed were those between the IgG from the malaria sera and the recombinant MSP, the latter antigen may be useful in diagnostic tests and as a component of any vaccine used to protect against P. vivax malaria.

A Recombinant Protein from Schistosoma mansoni Useful for the Detection of S. mansoni and Schistosoma haematobium Antibodies

A recombinant Schistosoma mansoni protein has been identified as a useful antigen for the detection of S. mansoni and Schistosoma haematobium antibodies. The purified recombinant protein, Sm22.3, was assayed using an enzyme-linked immunosorbent assay format against a battery of 491 well defined sera, including S. mansoni, S. haematobium, and Schistosoma japonicum infection sera, normal human sera, sera from 9 other parasitic infections, and sera from 2 additional infections. The sensitivity for detecting S. mansoni and S. haematobium infections with this single recombinant protein is 80.1%. The specificity is 94.8%. However, 15 of the 16 cross-reactive sera are malaria infection sera, and we have data suggesting that these malaria sera are actually recognizing an epitope on the vector-derived 6Xhistidine tag of recombinant Sm22.3. If this is the case, then, the actual specificity of the assay is 99.6%.

Filariasis and malaria sera from Orissa (India) lack human immunodeficiency virus (HIV) antibody reactivity

Reports exist indicating a correlation between seropositivity for human T-lymphotrophic virus (HTLV) antibodies and certain parasitic infections in some parts of the world. In 274 filariasis and 119 malarla sera examined from Orissa, none was reactive in a test for antibodies to human immunodeficiency virus (HIV).

Changes in serum transferrin and iron concentrations in humans suffering from malaria with parasitaemia.

Sera from non-malarial (control) and Plasmodium falciparum-infected (malaria) subjects were assayed for transferrin and iron. The mean serum transferrin concentration in the malaria sera was found to be elevated by 44% above the control value. The mean serum iron was also raised in the malaria sera, but the increase (19%) was found to be well below that of transferrin. The mean saturation of serum transferrin with iron fell from 42 +/- 17% in the control group to 37 +/- 9% in the malaria group. It is suggested that the increase in transferrin concentration in the sera of malaria subjects probably resulted from an increase in the rate of transferrin synthesis, perhaps in response to the raised serum iron and/or the hypoxia which is known to be associated with malaria infection.