Recent advances on T-cell exhaustion in malaria infection.

T-cell exhaustion reportedly leads to dysfunctional immune responses of antigen-specific T cells. Investigations have revealed that T cells expand into functionally defective phenotypes with poor recall/memory abilities to parasitic antigens. The exploitation of co-inhibitory pathways represent a highly viable area of translational research that has very well been utilized against certain cancerous conditions. Malaria, at times, evolve into a sustained chronic state where T cells express several co-inhibitory molecules (negative immune checkpoints) facilitating parasite escape and sub-optimal protective responses. Experimental evidence suggests that blockade of co-inhibitory molecules on T cells in malaria could result in the sustenance of protective responses together with dramatic parasite clearance. The role of several co-inhibitory molecules in malaria infection largely remain unclear, and here we discussed the potential applicability of co-inhibitory molecules in the management of malaria with a view to harness protective host responses against chronic disease and associated consequences.

Adulticidal activity of essential oil of Lantana camara leaves against mosquitoes.

BACKGROUND & OBJECTIVES: Development of insect resistance to synthetic pesticides, high operational cost and environmental pollution have created the need for developing alternative approaches to control vector-borne diseases. In the present study we have investigated the insecticidal activity of essential oil isolated from the leaves of Lantana camara against mosquito vectors. METHODS: Essential oil was isolated from the leaves of L. camara using hydro-distillation method. Bioassay test was carried out by WHO method for determination of adulticidal activity against mosquitoes. Different compounds were identified by gas chromatography-mass spectrometry analysis. RESULTS: LD(50) values of the oil were 0.06, 0.05, 0.05, 0.05 and 0.06 mg/cm(2) while LD(90) values were 0.10, 0.10, 0.09, 0.09 and 0.10 mg/cm(2) against Ae. aegypti, Cx. quinquefasciatus, An. culicifacies, An. fluvialitis and An. stephensi respectively. KDT(50) of the oil were 20, 18, 15, 12, and 14 min and KDT(90) values were 35, 28 25, 18, 23 min against Ae. aegypti, Cx. quinquefasciatus, An. culicifacies, An. fluviatilis and An. stephensi, respectively on 0.208 mg/cm(2) impregnated paper. Studies on persistence of essential oil of L. camara on impregnated paper revealed that it has more adulticidal activity for longer period at low storage temperature. Gas chromatographic-mass spectrometric analysis of essential oil showed 45 peaks. Caryophyllene (16.37%), eucalyptol (10.75%), alpha-humelene (8.22%) and germacrene (7.41%) were present in major amounts and contributed 42.75 per cent of the total constituents. INTERPRETATION & CONCLUSIONS: Essential oil from the leaves of L. camara possesses adulticidal activity against different mosquito species that could be utilized for development of oil-based insecticide as supplementary to synthetic insecticides.

Genetic characterization and evolutionary inference of TNF-α through computational analysis

TNF-α is an important human cytokine that imparts dualism in malaria pathogenicity. At high dosages, TNF-α is believed to provoke pathogenicity in cerebral malaria; while at lower dosages TNF-α is protective against severe human malaria. In order to understand the human TNF-α gene and to ascertain evolutionary aspects of its dualistic nature for malaria pathogenicity, we characterized this gene in detail in six different mammalian taxa. The avian taxon, Gallus gallus was included in our study, as TNF-α is not present in birds; therefore, a tandemly placed duplicate of TNF-α (LT-α or TNF-β) was included. A comparative study was made of nucleotide length variations, intron and exon sizes and number variations, differential compositions of coding to non-coding bases, etc., to look for similarities/dissimilarities in the TNF-α gene across all seven taxa. A phylogenetic analysis revealed the pattern found in other genes, as humans, chimpanzees and rhesus monkeys were placed in a single clade, and rats and mice in another; the chicken was in a clearly separate branch. We further focused on these three taxa and aligned the amino acid sequences; there were small differences between humans and chimpanzees; both were more different from the rhesus monkey. Further, comparison of coding and non-coding nucleotide length variations and coding to non-coding nucleotide ratio between TNF-α and TNF-β among these three mammalian taxa provided a first-hand indication of the role of the TNF-α gene, but not of TNF-β in the dualistic nature of TNF-α in malaria pathogenicity.

Plasma IP-10, apoptotic and angiogenic factors associated with fatal cerebral malaria in India.

BACKGROUND: Plasmodium falciparum in a subset of patients can lead to cerebral malaria (CM), a major contributor to malaria-associated mortality. Despite treatment, CM mortality can be as high as 30%, while 10% of survivors of the disease may experience short- and long-term neurological complications. The pathogenesis of CM is mediated by alterations in cytokine and chemokine homeostasis, inflammation as well as vascular injury and repair processes although their roles are not fully understood. The hypothesis for this study is that CM-induced changes in inflammatory, apoptotic and angiogenic factors mediate severity of CM and that their identification will enable development of new prognostic markers and adjunctive therapies for preventing CM mortalities. METHODS: Plasma samples (133) were obtained from healthy controls (HC, 25), mild malaria (MM, 48), cerebral malaria survivors (CMS, 48), and cerebral malaria non-survivors (CMNS, 12) at admission to the hospital in Jabalpur, India. Plasma levels of 30 biomarkers ((IL-1beta, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, Eotaxin, FGF basic protein, G-CSF, GM-CSF, IFN-gamma, IP-10, MCP-1 (MCAF), MIP-1alpha, MIP-1beta, RANTES, TNF-alpha, Fas-ligand (Fas-L), soluble Fas (sFas), soluble TNF receptor 1 (sTNF-R1) and soluble TNF receptor 2 (sTNFR-2), PDGF bb and VEGF)) were simultaneously measured in an initial subset of ten samples from each group. Only those biomarkers which showed significant differences in the pilot analysis were chosen for testing on all remaining samples. The results were then compared between the four groups to determine their role in CM severity. RESULTS: IP-10, sTNF-R2 and sFas were independently associated with increased risk of CM associated mortality. CMNS patients had a significantly lower level of the neuroprotective factor VEGF when compared to other groups (P < 0.0045). The ratios of VEGF to IP-10, sTNF-R2, and sFas distinguished CM survivors from non survivors (P < 0.0001). CONCLUSION: The results suggest that plasma levels of IP-10, sTNF-R2 and sFas may be potential biomarkers of CM severity and mortality. VEGF was found to be protective against CM associated mortality and may be considered for adjunctive therapy to improve the treatment outcome in CM patients.

Genetic characterization and evolutionary inference of TNF-alpha through computational analysis.

TNF-alpha is an important human cytokine that imparts dualism in malaria pathogenicity. At high dosages, TNF-alpha is believed to provoke pathogenicity in cerebral malaria; while at lower dosages TNF-alpha is protective against severe human malaria. In order to understand the human TNF-alpha gene and to ascertain evolutionary aspects of its dualistic nature for malaria pathogenicity, we characterized this gene in detail in six different mammalian taxa. The avian taxon, Gallus gallus was included in our study, as TNF-alpha is not present in birds; therefore, a tandemly placed duplicate of TNF-alpha (LT-alpha or TNF-beta) was included. A comparative study was made of nucleotide length variations, intron and exon sizes and number variations, differential compositions of coding to non-coding bases, etc., to look for similarities/dissimilarities in the TNF-alpha gene across all seven taxa. A phylogenetic analysis revealed the pattern found in other genes, as humans, chimpanzees and rhesus monkeys were placed in a single clade, and rats and mice in another; the chicken was in a clearly separate branch. We further focused on these three taxa and aligned the amino acid sequences; there were small differences between humans and chimpanzees; both were more different from the rhesus monkey. Further, comparison of coding and non-coding nucleotide length variations and coding to non-coding nucleotide ratio between TNF-alpha and TNF-beta among these three mammalian taxa provided a first-hand indication of the role of the TNF-alpha gene, but not of TNF-beta in the dualistic nature of TNF-alpha in malaria pathogenicity.

Alleles -308A and -1031C in the TNF-alpha gene promoter do not increase the risk but associated with circulating levels of TNF-alpha and clinical features of vivax malaria in Indian patients.

The biological significance of TNF promoter polymorphism and infectious disease association prompted us to investigate whether TNF-alpha -308 G/A and -1031 T/C promoter polymorphisms are associated with Plasmodium vivax infection, cellular TNF-alpha level and possibly with clinical symptoms by employing PCR-RFLP methods. An overall significant elevation of serum TNF-alpha, IL-6 content (p=0.0002, p=0.002, respectively), whereas highly significant depletion of IL-10 content (p=0.0001) was observed in vivax patients. In addition, TNF-alpha concentration in patients with and without fever were found to be significant (p=0.0001, p=0.0004, respectively). The genotypic distribution for -308 G/A and -1031 T/C positions were found non significant, but it was clinically potent to observe statistically significant distribution of genotypes (p=0.032) in patients with and without fever. Furthermore, the TNF-alpha level in TNF1 and TNF2 genotype for -308 position was significantly higher (p=0.010, p=0.006 respectively). In case of -1031 position TNF-alpha level was significant in ancestral (TT) genotype (p=0.0007) in patients compared to healthy subjects and significantly higher in rare (CC) genotype (p=0.021) as compared to ancestral genotype. In addition, the two polymorphisms 308G/A and -1031T/C were in highly significant LD (D'=0.7992, r(2)=0.6005, p=0.0001) in the patients as well as it is interesting to report that the distribution of novel 308A: 1031C alleles associated haplotypes are nearly the same in patients (0.2610) and in healthy subjects (0.2636). In view of present observation of promoter polymorphism with TNF-alpha level and other clinical parameters of vivax infection, we suggest that evaluation of TNF level and its polymorphisms in the promoter region may be considered to be reliable molecular and immunological markers, possess promising rational for diagnostic potential and immunotherapeutic interventions in clinical vivax malaria. Genetic variation in the promoter region is of biological significance and may play important roles in host defense mechanisms against vivax infection by enhancing cell-mediated immunity and stimulating the protective immunological cascade.

A simple, artificial-membrane feeding method for the radio-isotope labelling of Aedes aegypti polypeptides in vivo.

An artificial feeding unit has been designed and constructed to feed laboratory-bred Aedes aegypti with a radio-isotope (35S), so that the mosquitoes' polypeptides can be labelled in vivo. In the unit, a piece of Parafilm M barrier film is stretched over the bottom, outer surface of a polystyrene Petri dish, to create a small gap in which the mixture of blood and radio-isotope is placed. Warm water is placed in the dish, to keep the blood at about 37 degrees C. When such units were placed on net-covered rearing cages, almost all (80%-90%) of the female mosquitoes in the cages took bloodmeals from them. When checked by polyacrylamide-gel electrophoresis and autoradiography 1 h after feeding had begun, the blood-fed mosquitoes were found to have radio-labelled polypeptides. The unit is simple, easy to handle, disposable and can be used to offer small blood samples (>or=50 microl) to Ae. aegypti and, presumably, other mosquito species.

Short Communication: detection of dengue virus antigens in desiccated mosquitoes: an improved tool for surveillance.

We developed a surveillance tool to monitor dengue virus (DENV) infection in vector mosquitoes using a dengue antigen-capture enzyme immunoassay (EIA) on desiccated specimens stored at room temperature (RT). We tested the effect of storage on the stability of DEN1, DEN2, DEN3 and DEN4 antigens. Although desiccated infected Aedes aegypti mosquitoes stored between 31 and 34 degrees C (RT) showed greater reactivity in the EIA than those stored at -80 degrees C, a significant difference between the two was observed only with DEN2. Storage between 31 and 34 degrees C for up to 4 weeks (the longest period tested) did not affect the reactivity in the EIA, indicating the stability of DENV antigens.

Identification of telomerase activity in gametocytes of Plasmodium falciparum.

Telomerase, a specialized cellular reverse transcriptase, compensates the chromosome shortening during the replication of most eukaryotic cells and contributes to cellular immortalization in cell culture (in vitro) and cancerous cell (in vivo). In the present study, the telomerase activity in the gametocytes of Plasmodium falciparum was investigated. Here, we report for the first time, the presence of telomerase activity in the gametocytes of P. falciparum using P. falciparum telomere repeat amplification protocol (Pf-TRAP) assay and Southern blot hybridization. Telomerase inhibitors such as 7-deaza-dGTP and AZT-TP, when used with the cytoplasmic extract of gametocytes in the Pf-TRAP assay, efficiently inhibit the product, which confirms the presence of telomerase in the gametocytes. The presence of telomerase activity in the laboratory adapted local (eastern India) isolates of P. falciparum indicates that telomerase might be the major player in chromosomal end protection during replication. The finding suggests that telomerase can be a potent target for the transmission blocking vaccine and drugs for combating malaria caused by P. falciparum.