Manoranjan Ranjit

Association of TNF level with production of circulating cellular microparticles during clinical manifestation of human cerebral malaria

Microparticles (MPs) resulting from vesiculation of different cell types in Plasmodium falciparum infection correlate with the level of proinflammatory cytokine TNF that may thereby determine the disease severity. Using TruCount tube based flow cytometric method for the exact quantification of MP and enzyme linked immunosorbent assay for the measurement of TNF, we conducted a hospital based case control study on P. falciparum malaria patients to scrutinize and infer the link between the two. In 52 cerebral malaria (CM), 21 multi-organ-dysfunction (MOD), 12 non cerebral severe malaria (NCSM) and 43 uncomplicated malaria patients, the MP level was found to be significantly elevated in febrile malaria patients compared to healthy controls and a striking decrease in MP level was observed with the clearance of the P. falciparum infection in the patients upon follow-up. The lowering of the parasite density with the level of plasma TNF and the positive correlation of the cytokine with the cell derived MPs and negative correlation with the respective cell count in human malaria patients suggests that TNF may be a key stimulant to the cells resulting in the release of MPs in malaria infection.

The CCTTT pentanucleotide microsatellite in iNOS promoter influences the clinical outcome in P. falciparum infection

To assess the hypothesis that nitric oxide (NO) is critical in the pathogenesis of cerebral malaria, we analyzed those single nucleotide polymorphisms (SNPs) and microsatellite (MS) of the promoter region of inducible nitric oxide synthase (iNOS) gene which are known to enhance the NO production in vivo. A total of 428 (204 severe, 224 mild) adult patients living in the eastern part of India were analyzed. The single nucleotide substitutions −954G→C was found to be very rare, and −1173C→T was absent in this population. But interestingly, longer forms of MS were found to be significantly associated with severe malaria (ORu2009=u20092.89, 95% CIu2009=u20091.955–4.295, Pu2009<u20090.0001), and the linear regression analysis revealed that the risk of severe malaria significantly increases as the summed repeat number in an individual increase (ORu2009=u20091.16, Pu2009=u20090.0013). Further, the median plasma level of nitrate/nitrite (NOx) was observed to be high in mild patients compared to severe patients, and the level of parasitemia was significantly low among mild patients than severe ones. These findings suggest that the CCTTT repeats in iNOS may play a key role in the pathogenesis of severe malaria.

Gene polymorphisms in angiotensin I converting enzyme (ACE I/D) and angiotensin II converting enzyme (ACE2 C -> T) protect against cerebral malaria in Indian adults.

To explore the hypothesis that angiotensin II may play a role in the susceptibility to cerebral malaria (CM), we performed a genetic association study of malaria patients in Orissa, India analyzing three SNPs (ACE2 C-->T, iNOS C-->T, eNOS Glu-->Asp) and two I/D polymorphisms (ACE I/D and IL-4 B1/B2). Our results showed that the D allele of ACE I/D polymorphism, responsible for increased Ang II production had a significant association with mild malaria and the ACE2 C-->T substitution had gender specific effect of possibly reduced expression of ACE2 in presence of T allele in women leading to increased level of Ang II and hence protection against CM. Combined genotype analysis of eNOS Glu-->Asp substitution responsible for increased NO production in Plasmodium falciparum infected individuals and ACE I/D polymorphism also showed stronger association of (Glu-Asp+Asp-Asp/ID+DD) genotypes with mild malaria (P<0.0001). Whether by its antiplasmodial activity and/or by some unknown mechanisms, Ang II protects from susceptibility to cerebral malaria remains to be investigated. These genetic findings may contribute to the understanding of malaria pathogenesis.

Association of angiotensin-converting enzyme and angiotensin-converting enzyme-2 gene polymorphisms with essential hypertension in the population of Odisha, India

Abstract Background: Hypertension is a serious health issue worldwide and essential hypertension, which includes 90–95% of the cases, is influenced by both genetic and environmental factors. Identification of these factors may help in control of this disease. The Insertion/Deletion (I/D) polymorphism in Angiotensin-Converting Enzyme (ACE) gene and rs2106809 (Cu2009>u2009T) polymorphism in Angiotensin-Converting Enzyme 2 (ACE2) gene have been reported to be associated with essential hypertension in different populations. Aim: To investigate the association of ACE I/D and ACE2 rs2106809 polymorphisms with essential hypertension in the population of Odisha, an eastern Indian state. Subjects and methods: A total of 246 hypertensives (159 males and 87 females) and 274 normotensives (158 males and 116 females) were enrolled in the study. Detailed anthropometric data, tobacco, alcohol and food habits were recorded and 2u2009ml of venous blood was collected for biochemical and genetic analysis. Results: The DD genotype of ACE and TT genotype of ACE2 were significantly high among female hypertensives, while T allele of ACE2 was linked to male hypertensives. In the male population, alcohol was also identified as a potential risk factor. Conclusion: Among females, ACE I/D and ACE2 rs2106809 polymorphisms, while among males, ACE2 rs2106809 polymorphism and alcohol consumption are associated with essential hypertension in the study population.

Molecular monitoring of antimalarial drug resistance among Plasmodium falciparum field isolates from Odisha, India

In the absence of definite marker for artemisinin (ART) resistance, molecular monitoring of its partner drug sulfadoxine pyrimethamine (SP) in artemisinin based combination therapy (ACTs) together with chloroquine (CQ) for which ART is negatively correlated, may predict the effectiveness of ACT. We analyzed 201 Plasmodium falciparum field isolates for drug resistance markers for CQ (pfcrt and pfmdr1), pyrimethamine (pfdhfr) and sulfadoxine (pfdhps). Our study reveals high prevalence and non-random association of resistant mutants (K76T and N86Y) of CQ markers (pfcrt and pfmdr1). The predominance of highly resistant pfdhfr genotypes for SP with intragenic and intergenic pair-wise linkage disequilibrium between single nucleotide polymorphisms of resistant mutants of pfdhfr (C59R and S108N) and pfdhps (S436A, A437G, K540E) warn on further inclusion of SP in ACT. These findings suggest the replacement of SP in ACT with alternative partner drug for better efficacy.

Promoter Polymorphisms in the ATP Binding Cassette Transporter Gene Influence Production of Cell-Derived Microparticles and Are Highly Associated with Susceptibility to Severe Malaria in Humans

ABSTRACT Microparticle (MP) efflux is known to be mediated by the ABCA1 protein, and the plasma level of these cell-derived MPs is elevated considerably during human malarial infection. Therefore, two polymorphisms at positions −477 and −320 in the promoter of the ABCA1 gene were genotyped and tested for association with the plasma MP level in four groups of malaria patients segregated according to the clinical severity, i.e., cerebral malaria (CM), multiorgan dysfunction (MOD), noncerebral severe malaria, and uncomplicated malaria (UM). The TruCount tube-based flow cytometric method was used for the exact quantification of different cell-derived MPs in patients. Polymorphisms in the ABCA1 gene promoter were analyzed by use of the PCR/two-primer-pair method, followed by restriction fragment length polymorphism, in 428 malaria patients. The level of circulating plasma MPs was significantly higher in febrile patients with Plasmodium falciparum infection, especially in CM patients compared to healthy individuals. The homozygous wild-type −477 and −320 genotype was observed to be significantly higher in patients with severe malaria. These patients also showed marked increases in the plasma MP numbers compared to UM patients. We report here for the first time an association of ABCA1 promoter polymorphisms with susceptibility to severe malaria, especially to CM and MOD, indicating the protective effect of the mutant variant of the polymorphism. We hypothesize that the −477T and −320G polymorphisms affect the downregulation of MP efflux and may be a predictor of organ complication during P. falciparum malarial infections.

Aldosterone synthase C-344T, angiotensin II type 1 receptor A1166C and 11-β hydroxysteroid dehydrogenase G534A gene polymorphisms and essential hypertension in the population of Odisha, India

Essential hypertension which accounts 90–95% of the total hypertension cases is affected by both genetic and environmental factors. This study was undertaken to investigate the association of aldosterone synthase C-344T, angiotensin II type I receptor A1166C and 11- β hydroxysteroid dehydrogenase type 2 G534A polymorphisms with essential hypertension in the population of Odisha, India. A total of 246 hypertensive subjects (males, 159; females, 87) and 274 normal healthy individuals (males, 158; females, 116) were enrolled in this study based on the inclusion and exclusion criteria. Analysis of genetic and biochemical data revealed that in this population the CT and TT genotypes of aldosterone synthase C-344T polymorphism, frequency of alcohol consumption and aldosterone levels were significantly high among the total as well as male hypertensives, while the AC and CC genotypes of angiotensin II type I receptor A1166C polymorphism were significantly high among the total as well as female hypertensives. High density lipoprotein levels were higher in male hypertensives.

Genetic diversity of PfEMP1-DBL 1-α and its association with severe malaria in a hyperendemic state of India

Objective: To find out the extent of dully-binding-like (DBL) a gene diversity and the rosetting potential of the parasite population in association with severe malaria. Methods: Genotyping of DBL a domain was done by PCR using three sets of primers (FR, F1R2 and F2R2) and the rosetting frequency was assessed by parasite culture followed by ethidium, bromide staining and visualization under a fluorescent microscope. Results: The significant association of high parasite density with severe malaria and the positive correlation between rosetting frequency and parasite density in vivo (P=0.613, P＜0.0001) were observed. Moreover, the parasite strains having multiple fragments of F2R2 region and b variant of FR region of DBL 1-α showed increased rosetting frequency and supported the strain specific association of disease severity. Conclusions: The findings suggest that rosetting mediated higher parasitemia might have contributed to the development of severe disease. As the rosetting domain of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), the DBL a hinds to multiple host receptors; the significant association of multiple fragments of F2R2 region with severe malaria suggests several receptor-ligand interactions as the possible mechanisms of pathogenesis. Alternatively, the high percentage distribution of smaller fragments with mild malaria suggests the lack of adequate rosetting epitopes that might have contributed to low rosetting frequency in mild malaria.

A new allele (eNOS4e) in the intron 4 (VNTR) of eNOS gene in malaria infected individuals of the population of Orissa (an eastern Indian state)

To the Editor Nitric oxide (NO) has been proposed to play a crucial role in malaria pathogenesis. It is produced during enzymatic conversion of L-arginine to L-citrulline by three members of the nitric oxide synthase (NOS) family of proteins, inducible (iNOS), endothelial (eNOS) and neural (nNOS). However the mechanism of its action during the disease process is still controversial. On one hand, it has been proposed that NO is produced in excess and kills the Plasmodium parasites with the unfortunate side effects of mediating pathogenesis through oxidative damage or aberrant signaling in the brain and contributing to anemia. On the other hand, the finding that hypoargininaemia and low levels of plasma nitrates and nitrites (NOx), the stable degradation products of NO correlate with the development of severe Plasmodium falciparum malaria suggests that level of NO production might be low during malaria (reviewed in Ref. [1]). Whereas other reports showed no correlation with disease progression [2]. Moreover, NO was found to down regulate the expression of ICAM-1 and VCAM-1 on endothelial cells leading to a hypothesis that NO plays a protective role by decreasing the adherence of infected erythrocytes [3]. The eNOS derived NO has an important role of vasodilation, inhibition of platelet aggregation and endothelial cell activation [1]. But no data are available about eNOS variants and their association with malaria outcome. Several allelic variants of eNOS gene have been identified such as a T/C substitution in the 50-flanking region near promoter at 786, a 27-base pair (bp) variable number of tandem repeat (VNTR) in intron 4, and a G/T substitution at position 894 in exon 7 and their association with human diseases has been studied. The VNTR polymorphism located in intron 4 of eNOS (eNOS 4b/a polymorphism) has been reported to be significantly associated with plasma NOx concentration, affects the transcription efficiency in a haplotype specific fashion in linkage disequilibrium with the T-786C polymorphism in the promoter region [4]. Thus attempt was taken to look for the association of eNOS (VNTR) intron 4 polymorphism with malaria in Orissa, an eastern Indian state, hyperendemic for malaria with perennial transmission. A total of 249 patients with mild malaria and 195 patients with cerebral malaria admitted to SCB medical College and Hospital, Cuttack were analysed. The genomic DNA was extracted from the peripheral blood leucocytes by phenol–chloroform extraction method and genotyping of eNOS intron 4 VNTR polymorphism was done by PCR using primers 50 AGG CCC TAT GGT AGT GCC

Antimalarial activity of Artemisia nilagirica against Plasmodium falciparum

Malaria is one of the most prevalent vector borne infectious disease and a serious global health problem in the world. Treatment for malaria is commonly inadequate due to the lack of quality assured limited number of effective drugs, underline how important it is to discover new antimalarial plants from number of natural sources. In the present study, the efficacy of antimalarial activity was studied by taking six various (n-hexane, chloroform, petroleum ether, ethanol, methanol and aqueous) organic leaf extracts of Artemisia nilagirica (Clarke) Pamp. against malarial parasite Plasmodium falciparum. Promising antiplasmodial activity was found in all tested extracts; however, maximum 50% inhibitory concentration (IC50) values were noticed after 32xa0h of incubation, which is 5.76xa0±xa00.82, 7.09xa0±xa01.09, 9.88xa0±xa01.13, 10.24xa0±xa01.52, 11.37xa0±xa01.77 and 50.15xa0±xa06.16xa0µg/ml in methanol, chloroform, n-hexane, petroleum ether, ethanol and aqueous extracts, respectively. In conclusion, A. nilagirica leaf extract possesses antiplasmodial activity which may be used as a potent plant-based antimalarial drug in the future by investigating the hidden phytochemical/(s).