A comparative study of natural immune responses against Plasmodium vivax C-terminal merozoite surface protein-1 (PvMSP-1) and apical membrane antigen-1 (PvAMA-1) in two endemic settings

The mechanisms of cellular and humoral immune responses against P. vivax parasite remain poorly understood. Several malaria immunological studies have been conducted in endemic regions where both P. falciparum and P. vivax parasites co-exist. In this study, a comparative analysis of immunity to Plasmodium vivax antigens in different geography and incidence of Plasmodium spp. infection was performed. We characterised antibodies against two P. vivax antigens, PvMSP-1 and PvAMA-1, and the cross-reactivity between these antigens using plasma from acute malaria infected patients living in the central region of China and in the western border of Thailand. P. vivax endemicity is found in central China whereas both P. vivax and P. falciparum are endemic in Thailand. There was an increased level of anti-PvMSP-1/anti-PvAMA-1 in both populations. An elevated level of antibodies to total P. vivax proteins and low level of antibodies to total P. falciparum proteins was found in acute P. vivax infected Chinese, suggesting antibody cross-reactivity between the two species. P. vivax infected Thai patients had both anti-P. vivax and anti-P. falciparum antibodies as expected since both species are present in Thailand. More information on humoral and cell mediated immunity during acute P. vivax-infection in the area where only single P. vivax species existed is of great interest in the relation of building up anti-disease severity caused by P. falciparum. This knowledge will support vaccine development in the future

In vitro production of functional immune cells derived from human hematopoietic stem cells

Hematopoietic stem cells (HSC) from cord blood are potentially high sources for transplantation due to their low immunogenicity and the presence of the multipotent cells. These cells are capable of differentiating to produce various lineages of blood cells under specific conditions. We have enriched highly purified CD34+ cells from cord blood, determined in vitro growth of the cells in culture systems in the absence (condition A) or presence of GM-CSF and G-CSF (condition B), and determined the profile of immune cells during the period of cultivation by using flow cytometry. PhytohemagglutininA (PHA) was used as a mitogen to stimulate T lymphocytes derived from hematopoietic stem cells. GM-CSF and G-CSF prolonged the survival of the growing cells and also maintained expansion of cells in blastic stage. By day 12 of cultivation, when cell numbers peaked, various types of immune cells had appeared (CD14+ cells, CD40+HLA-DR+ cells, CD3+CD56+ cells, CD19+ cells, CD3+CD4+ cells, CD3+CD8+cells and CD3-CD56+). A significantly higher percentage of monocytes (p = 0.002) were observed under culture with GM-CSF, G-CSF when compared with culture without GM-CSF, G-CSF. In addition, T lymphocytes derived from HSC responded to 50 μg/ml of PHA. This is the first report showing the complete differentiation and proliferation of immune cells derived from CD34+ HSC under in vitro culture conditions. Lymphocytes, monocytes, dendritic cells and polymorph nuclear cells derived from HSC in vitro are unique, and thus may benefit various studies such as innate immunity and pathophysiology of immune disorders

The effect of mimicking febrile temperature and drug stress on malarial development

<p>Abstract</p> <p>Background</p> <p>Malaria remains one of the most important tropical diseases of human with 1–2 million deaths annually especially caused by <it>P. falciparum</it>. During malarial life cycle, they exposed to many environmentally stresses including wide temperature fluctuation and pharmacological active molecules. These trigger malarial evolutionarily adaptive responses. The effect of febrile temperature on malarial growth, development and drug susceptibility by mimicking patient in treatment failure before and after drug uptake was examined.</p> <p>Methods</p> <p>Sensitivities of <it>P. falciparum </it>to antimalarial drug (chloroquine, mefloquine, quinine and artesunate) were investigated based on the incorporation of [³H] hypoxanthine into parasite nucleic acids or radioisotopic technique. The number of parasites was examined under microscope following Giemsa staining and the parasite development at the end of each phase was counted and comparison of parasite number was made. The proteome was separated, blotted and hybridized with anti-Hsp70s primary antibody. The hybridized proteins were separately digested with trypsin and identified by MALDI-TOF peptide mass fingerprint.</p> <p>Results</p> <p>The results show that febrile temperature is capable of markedly inhibiting the growth of field isolate <it>P. falciparum </it>but not to K1 and 3D7 standard strains. K1 and 3D7 grown under heat shock developed greater and the reinfection rate was increased up to 2-folds when compared to that of non-heat shock group. The IC₅₀value of K1 toward chloroquine, mefloquine and quinine under heat shock was higher than that of K1 under non-heat shock which is opposite to that of 3D7. Heat shock caused death in field isolated parasite. It was also found that the febrile temperature coped with chloroquine uptake had no effect to the development, drug sensitivity and the parasite number of K1 strain. In the opposite way, heat shock and chloroquine shows extremely effect toward 3D7 and field isolate PF91 as shown by higher number of dead parasites compared to that of control group. After culture under high temperature with artesunate, the total parasite number of all strains including K1, 3D7 and PF91 was extremely decreased and the parasite was not found at the end. Additionally, the expression of <it>pf</it>Hsp70s was found in all strains and conditions as shown in 120 kDa hybridized band. However, the proteome extracted from K1 grown under heat shock with chloroquine, anti-<it>pf</it>Hsp70 interacted with additional three bands identified by MALDI-TOF as elongation factor-1α (83 kDa), pf<it>Hsp</it>86 (60 kDa) and phosphoethanolamine <it>N</it>-methyltransferase (43 kDa).</p> <p>Conclusion</p> <p>In conclusion, febrile temperature was capable of markedly inhibiting the growth of field isolate <it>P. falciparum </it>while the development, reinfection rate and drug (chloroquine, mefloquine and quinine) resistant level of standard strain K1 was enhanced. However, the febrile temperature coped with chloroquine had no effect to the development, drug sensitivity and the parasite number of K1 strain. In the opposite way, heat shock and chloroquine showed extremely effect toward 3D7 and field isolate PF91 as shown by some died parasites. Heat shock protein 70 (<it>pf</it>HSP70) of strain K1 under heat shock with chloroquine might involved in many pathways in order to sustain the parasite.</p

Increased fluidity and oxidation of malarial lipoproteins: relation with severity and induction of endothelial expression of adhesion molecules

INTRODUCTION: Oxidative stress has been demonstrated in malaria. The potential oxidative modification of lipoproteins derived from malaria patients was studied. These oxidized lipids may have role in pathogenesis of malaria. METHOD: The plasma lipid profile and existence of oxidized forms of very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL) were investigated in malaria (17 mild and 24 severe patients) and 37 control subjects. Thiobarbituric acid reactive substances (TBARs), conjugated dienes, tryptophan fluorescence and fluidity of lipoproteins were determined as markers of oxidation. The biological effect of malarial lipoproteins was assessed by the expression of adhesion molecules on endothelial cells. RESULTS: Malarial lipoproteins had decreased cholesterol (except in VLDL) and phospholipid. The triglyceride levels were unchanged. The cholesterol/phospholipid ratio of LDL was decreased in malaria, but increased in VLDL and HDL. TBARs and conjugate dienes were increased in malarial lipoproteins, while the tryptophan fluorescence was decreased. The fluidity of lipoproteins was increased in malaria. These indicated the presence of oxidized lipoproteins in malaria by which the degree of oxidation was correlated with severity. Of three lipoproteins from malarial patients, LDL displayed the most pronounced oxidative modification. In addition, oxidized LDL from malaria patients increased endothelial expression of adhesion molecules. CONCLUSION: In malaria, the lipoproteins are oxidatively modified, and the degree of oxidation is related with severity. Oxidized LDL from malarial patients increases the endothelial expression of adhesion molecules. These suggest the role of oxidized lipoproteins, especially LDL, on the pathogenesis of disease

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

BACKGROUND: Malaria 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. METHODS: The 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. RESULTS: At 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 (r(s) = −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. CONCLUSIONS: This 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

Modulating effects of plasma containing anti-malarial antibodies on in vitro anti-malarial drug susceptibility in Plasmodium falciparum

<p>Abstract</p> <p>Background</p> <p>The efficacy of anti-malarial drugs is determined by the level of parasite susceptibility, anti-malarial drug bioavailability and pharmacokinetics, and host factors including immunity. Host immunity improves the <it>in vivo </it>therapeutic efficacy of anti-malarial drugs, but the mechanism and magnitude of this effect has not been characterized. This study characterized the effects of 'immune' plasma to <it>Plasmodium falciparum</it>on the <it>in vitro </it>susceptibility of <it>P. falciparum </it>to anti-malarial drugs.</p> <p>Methods</p> <p>Titres of antibodies against blood stage antigens (mainly the ring-infected erythrocyte surface antigen [RESA]) were measured in plasma samples obtained from Thai patients with acute falciparum malaria. 'Immune' plasma was selected and its effects on <it>in vitro </it>parasite growth and multiplication of the Thai <it>P. falciparum </it>laboratory strain TM267 were assessed by light microscopy. The <it>in vitro </it>susceptibility to quinine and artesunate was then determined in the presence and absence of 'immune' plasma using the ³H-hypoxanthine uptake inhibition method. Drug susceptibility was expressed as the concentrations causing 50% and 90% inhibition (IC₅₀and IC₉₀), of ³H-hypoxanthine uptake.</p> <p>Results</p> <p>Incubation with 'immune' plasma reduced parasite maturation and decreased parasite multiplication in a dose dependent manner. ³H-hypoxanthine incorporation after incubation with 'immune' plasma was decreased significantly compared to controls (median [range]; 181.5 [0 to 3,269] cpm versus 1,222.5 [388 to 5,932] cpm) (<it>p</it>= 0.001). As a result 'immune' plasma reduced apparent susceptibility to quinine substantially; median (range) IC₅₀6.4 (0.5 to 23.8) ng/ml versus 221.5 (174.4 to 250.4) ng/ml (<it>p </it>= 0.02), and also had a borderline effect on artesunate susceptibility; IC₅₀0.2 (0.02 to 0.3) ng/ml versus 0.8 (0.2 to 2.3) ng/ml (<it>p </it>= 0.08). Effects were greatest at low concentrations, changing the shape of the concentration-effect relationship. IC₉₀values were not significantly affected; median (range) IC₉₀448.0 (65 to > 500) ng/ml versus 368.8 (261 to 501) ng/ml for quinine (<it>p </it>> 0.05) and 17.0 (0.1 to 29.5) ng/ml versus 7.6 (2.3 to 19.5) ng/ml for artesunate (<it>p </it>= 0.4).</p> <p>Conclusions</p> <p>'Immune' plasma containing anti-malarial antibodies inhibits parasite development and multiplication and increases apparent <it>in vitro </it>anti-malarial drug susceptibility of <it>P. falciparum</it>. The IC₉₀was much less affected than the IC₅₀measurement.</p

RECRUDESCENCE IN ARTESUNATE-TREATED PATIENTS WITH FALCIPARUM MALARIA IS DEPENDENT ON PARASITE BURDEN NOT ON PARASITE FACTORS

Artemisinin derivatives are first-line antimalarial drugs in Thailand. No firm evidence of clinically relevant artemisinin resistance exists. When used as monotherapy, artesunate has been associated with a high treatment failure (recrudescence) rate, which could be due to low-level artemisinin resistance. To understand the causes of recrudescence, we retrospectively studied a cohort of 104 malaria patients treated with artesunate monotherapy, 32 of whom recrudesced. There was no difference in in vitro artesunate sensitivities between 6 nonrecrudescent isolates and 16 paired admission and recrudescent isolates. Paired admission and recrudescent isolates from 10 patients were genotyped; only 3 had pfmdr1 mutations. Patients with admission parasitemias >10,000 per µl had a 9-fold higher likelihood of recrudescence (adjusted odds ratio) compared with patients with lower parasitemias. This study suggests (1) recrudescence after treatment with artesunate is not the result of inherent parasite resistance, and (2) admission parasitemia may be useful in choosing therapeutic options