miRNA-15/IL-10Rα axis promotes Kabasura Kudineer (Indian traditional Siddha formulation) induced immunomodulation by suppressing oxidative stress.

ETHNOPHARMACOLOGICAL RELEVANCE: Kabasura Kudineer (KK), the traditional Indian medicine of Siddha, effectively manages common respiratory symptoms such as flu, cold, and fever. However, there is no evidence of the immunomodulatory capacity of KK in the cultured Jurkat T-lymphocytes under the LPS insult studied. AIM OF THE STUDY: Assess the effect of the traditional Indian medicine of Siddha, Kabasura Kudineer (KK) on immunomodulation by suppressing oxidative damage in cultured Jurkat T cells in vitro. The miRNA activity on anti-inflammatory gene receptors and cellular nitric oxide levels also was studied. MATERIALS AND METHODS: Jurkat T cells were exposed to LPS treatment in the presence or absence of KK. Cell viability and nitric oxide (NO) were measured with MTT and Griess assay. Cellular antioxidant systems (glutathione and SOD) were determined using glutathione and SOD assay. Lipid peroxidation was measured using an MDA assay. MiRNA-15a-5p expression was performed using microRNA qPCR Assays. Both inflammatory and anti-inflammatory genes (IL-6, IL-1, IL-10, IL-13) were performed using a qPCR and ELISA assay. RESULTS: The data showed that reduced cell proliferation and exaggerated NO production was observed in LPS treated condition compared to the control condition. Further, LPS treatment increased lipid peroxidation and reduced antioxidant enzyme activities (SOD and glutathione) in cultured Jurkat T cells. However, treatment with KK or N-acetyl cysteine (NAC; antioxidant) treatment mitigates the above effect. Mechanistically, LPS-induced oxidative stress upregulated miR- 15-5p expression and suppressed IL-10 Receptor alpha (IL-10Rα) by binding to its 3'-UTR region. The deregulated expression of IL-10Rα expression leads to increased IL-6 and IL-1ß expression in LPS-induced Jurkat T cells; however, treatment with KK or NAC reversed the above effects. CONCLUSION: Collectively, our study revealed the previously undefined mechanistic role of Kabasura Kudineer (KK) that alleviates the LPS-induced oxidative damage associated with inflammation by inhibiting the miRNA-15-5p/IL-10Rα axis.

Nitric oxide promotes cell-matrix adhesion of endothelial progenitor cells under hypoxia condition via ITGA5 CpG promoter demethylation.

Hypoxia or low oxygen tension causes changes in the structure and functional phenotype of the endothelial progenitor cells (EPCs). EPCs are found to be involved in angiogenesis and vascular repair. However, EPC's role in cell-matrix adhesion under hypoxia conditions is not clearly established. Nitric oxide (NO) exerts a wide range of biological functions, especially in regulating the mobilization and vascular repair of EPCs. In contrast, the link between NO and its role in cell-matrix deadhesion under hypoxia is not studied yet. Here, we investigated the protective role of NO in hypoxia-induced cell-matrix deadhesion of EPCs through an epigenetic mechanism. The EPCs were exposed to 2% hypoxia in the presence or absence of 10 µM Spermine NONOate (NO donor). The result demonstrates that hypoxia exposure intensified mitochondrial oxidative damage and energy defects. Using miScript miRNA qPCR array-based screening, the study found miR-148 as a novel target of hypoxia-induced DNMT1 activation. Mechanistically, the study discovered that hypoxia suppressed miR-148 levels and stimulated EPCs cell-matrix deadhesion via increasing DNMT1 mediated Integrin alpha-5 (ITGA5) CpG promoter hypermethylation. Treatment with a mitochondria-targeted antioxidant, MitoTEMPO, or epigenetic DNMT inhibitor, 5'-azacitidine, or miR-148 overexpression in hypoxic EPCs culture, prevented the cell-matrix deadhesion compared to hypoxic EPCs. Further, treatment of spNO or transient expression of eNOS-GFP attenuated hypoxia-induced cell-matrix deadhesion via inhibition of ITGA5 CpG island promoter methylation. In conclusion, the study provides evidence that NO is essential for cell-matrix adhesion of EPCs by epigenetically mitigating ITGA5 CpG promoter hypermethylation under hypoxia conditions. This finding uncovers the previously undefined mechanism of NO-mediated diminution of hypoxia-induced cell-matrix deadhesion and dysfunction induced by low oxygen tension.

In vitro anticancer activity of fucoidan extracted from Sargassum cinereum against Caco-2 cells.

Fucoidan is a marine sulfated polysaccharide, which is extracted from brown seaweed that has a wide range of bioactivities including anti-cancer properties. However, the underlying mechanism of fucoidan on its anti-cancer and apoptotic activity against colon cancer cell line Caco-2 remains to be elucidated. Hence, the present study evaluated the cytotoxicity, apoptotic and anti-cancer activity of fucoidan extracted from brown seaweed Sargassum cinereum against Caco-2 cell line. Cytotoxicity, morphological examination of nuclei, mitochondrial membrane potential, flow cytometry, reactive oxygen species (ROS) formation and detection of apoptotic efficacy of fucoidan were assessed by different assay protocols. Fucoidan inhibited growth of Caco-2 cells in a dose-dependent manner. IC50 concentration of fucoidan was found to be 250 µg/ml. AO/EB, Hoechst and Annexin V/PI staining confirmed the apoptosis induced by fucoidan in Caco-2 cells. Fucoidan was also found to increase ROS production and augment mitochondrial membrane permeability. The findings of the study suggest that fucoidan exerts potent anti-cancer and apoptotic effect on Caco-2 cells by enhancing ROS production. Thus, fucoidan may be used as a promising therapeutic regimen against various cancer cell types.

Anopheles culicifacies sibling species B and E in Sri Lanka differ in longevity and in their susceptibility to malaria parasite infection and common insecticides.

Members of the Anopheles culicifacies Giles complex (Diptera: Culicidae) are well established as the predominant vectors of malaria in Sri Lanka. Until recently, only sibling species B was reported to be present in Sri Lanka, which was surprising as species B is a poor vector of malaria in India. This was clarified by the identification through Y-chromosome morphology that what was reported as B on the island is really a mixture of B and E. The fecundity, longevity and insecticide resistance of B and E are of relevance to malaria transmission and its control and are reported in this study. The mean egg production of these two sibling species did not differ significantly. The mean age of wild mosquitoes was assessed by the Polovodova technique of observing ovarian dilatations. More of species E than B had three or more dilatations, i.e. had reached an age at which sporozoites could have developed to maturity, although the difference between the species was of borderline significance. Following feeding on Plasmodium vivax or Plasmodium falciparum infected blood, some females of species E developed oocysts but none of species B did so. Both sibling species were found fully susceptible in laboratory tests to lambdacyhalothrin and deltamethrin, but resistant to DDT and partially resistant to malathion.

Antigenic relationships between adult and larval Anopheles tessellatus midgut glycoproteins and the midguts of other vector mosquitoes.

Glycoproteins expressed on the surface of midgut (MG) epithelium and the peritrophic matrix (PM) of vector mosquitoes (Diptera: Culicidae) are candidate molecules for interacting with pathogens. Antisera produced against Anopheles tessellatus Theobald female MG lectin-binding proteins (concanavalin A and wheat germ agglutinin) were used in Western blots to investigate MG/PM antigenic relationships between adult and larval An. tessellatus and with the MG glycoproteins of other vector mosquitoes: Anopheles culicifacies Giles, An. subpictus Grassi, An. varuna Iyengar, Aedes aegypti (L.) and Culex quinquefasciatus Say. Within An. tessellatus, strong antigenic cross-reactions were observed between adult and larval MG proteins, and between adult MG and PM proteins. Anopheles tessellatus adult MG antisera reacted with MG antigens from adult females of the other five mosquito species, with interspecific contrasts of relative molecular mass (Mr) of nearly all reacting antigens, except the strong 36 kDa band shared by An. tessellatus and Cx. quinquefasciatus. Cross-reactivity within female An. tessellatus may be due to the MG containing precursors to the PM glycoproteins and/or some common fully processed proteins, or perhaps carbohydrate epitopes that are shared between related or unrelated MG and PM glycoproteins. Cross-reactions between adult MG proteins from different mosquito species, mostly with differential Mr, reflect the presence of homologous proteins that may be relevant to specific vector competence.

Anopheles culicifacies Y-chromosome dimorphism indicates sibling species (B and E) with different malaria vector potential in Sri Lanka.

In Sri Lanka, malaria is transmitted mainly by Anopheles culicifacies Giles sensu lato (Diptera: Culicidae). In India, this nominal taxon comprises sibling species A, B, C, D and E, distinguished by their chromosome morphology. Species B (identified by polytene chromosome sequence Xab, 2g1 + h1) is not such an efficient vector of malaria as other members of the An. culicifacies complex in India. All specimens of An. culicifacies s.l. examined from Sri Lanka possess Xab, 2g1 + h1 polytenes, previously interpreted as species B, despite their important vector status. Recently, species E was described from Rameshwaram Island (Tamil Nadu, India) between Sri Lanka and the Indian mainland, where both species B and E are sympatric. Species B and E share polytene sequence Xab, 2g1 + h1 but differ by the mitotic Y-chromosome being acrocentric in species B, submetacentric in species E, the latter implicated as vector of vivax malaria. From May 1999 to January 2000, we surveyed Y-chromosomes of male progeny from An. culicifacies Xab, 2g1 + h1 females collected from cattle bait in diverse malarious districts of Sri Lanka: Badulla, Monaragala, Puttalam and Trincomalee. Karyotypes of readable quality were obtained from 42/83 families examined, with overall proportions 24% acrocentric and 76% submetacentric Y-chromosome carriers, both types being sympatric in at least 3/4 localities sampled. By analogy with the situation on Rameshwaram Island, we interpret these observations to demonstrate widespread presence of two members of the An. culicifacies complex in Sri Lanka, their karyotypes being compatible with species B and E, the latter predominant and having greater vector potential.

Antibodies to a merozoite surface protein promote multiple invasion of red blood cells by malaria parasites.

The 40-50 kDa merozoite surface antigen (MSA2) is a candidate molecule for use in a malaria vaccine. The gene for MSA2 from the 3D7 isolate of Plasmodium falciparum was amplified by polymerase chain reaction and cloned into the bacterial expression vector pGEX-3X to obtain a fusion protein of MSA2 with Schistosoma japonicum glutathione S-transferase. The recombinant fusion protein was used to immunize rabbits. After four injections, the sera had Western blotting and immunofluorescence titres of 10(-6). Immune sera, and immunoglobulin (Ig)G, F(ab)'2, F(ab) prepared from the immune sera, were assessed for their effects on the growth of 3D7 parasites in vitro by microscopy and a [3H]-hypoxanthine incorporation assay. The antibodies did not significantly inhibit red blood cell invasion and parasite growth when added to cultures as 10% v/v serum or as immunoglobulin preparations at concentrations up to 200 microg ml(-1). However, in the presence of IgG or F(ab)'2, but not F(ab), antibodies to MSA2, the proportions of red blood cells invaded by more than one merozoite increased significantly. Multiple invasion is attributed to merozoites cross-linked by bivalent antibodies, attaching to and subsequently invading the same red cell. These observations have a bearing on the evasion of host immune responses by the parasite and the use of full-length recombinant MSA2 protein in a malaria vaccine.

Mammalian cell expression of malaria merozoite surface proteins and experimental DNA and RNA immunisation.

The gene for a 45 kDa merozoite surface protein (MSA-2) of the human malaria parasite Plasmodium falciparum was PCR amplified and cloned into eukaryotic expression vectors VR1012 and pcDNA3 to yield plasmids P1 and P2, respectively. The coding sequences for two N-terminal fragments of the 185 kDa merozoite surface protein (MSA-1) gene were similarly PCR amplified and cloned into vectors VR1020 and VR1012 to yield plasmids P3 and P4, respectively. The MSA-1 signal peptide sequence, present in P4, was replaced with the human tissue plasminogen activator signal sequence in P3. The four plasmids expressed the cloned genes under the control of the cytomegalovirus promoter and carried 3' bovine growth hormone termination/poly A signals. P1, P3 and P4 also contained the cytomegalovirus intron A enhancer sequence. MSA-1 expression was more readily detected than MSA-2 in Cos cells transfected with P3/P4 and P1/P2 respectively. The MSA-2 gene was also cloned into the phagemid pBluescript IISK+ with and without a 3' poly A tail composed of 35 A residues. MSA-2 was synthesised in HeLa cells infected with a recombinant vaccinia virus carrying T7 RNA polymerase when MSA-2 recombinant pBluescript was transfected into the cells. Inoculation with P1 intramuscularly or intradermally and with P2 intradermally into rabbits led to the production of antibodies to MSA-2 detectable by immunofluorescence and Western blotting. Antibodies were also produced against MSA-1 after intramuscular/intradermal inoculation with P3 and P4. Inoculation of rabbits with MSA-2 mRNA yielded better antibody titres when a poly A tail was present. Antibody levels were maintained for > 9 weeks after the final immunisation. However the immune sera failed to inhibit in vitro parasite growth.

Model multiple antigenic and homopolymeric peptides from non-repetitive sequences of malaria merozoite proteins elicit biologically irrelevant antibodies.

Three model peptides containing B-epitopes from conserved, non-repetitive regions of the merozoite surface antigens, MSA2 and MSA1, and the erythrocyte binding protein EBP of Plasmodium falciparum were synthesised. The peptides incorporated GPG spacers and C residues at the N and C termini, and were polymerised by oxidation to form cystine bridges. Multiple copies of essentially the same peptide sequences were also synthesised on a branching lysyl matrix to form a tetrameric multiple antigen peptide. Rabbits were immunised with the polymerised and multiple antigen peptides, in alum followed by Freund's adjuvant, and the antibody responses examined by IFA and ELISA. Reproducible antibody responses were obtained against the MSA1 and EBP but not MSA2 peptides. IgG antibody levels detected by ELISA after three injections of antigen in alum, increased significantly after further immunisation in Freund's adjuvant. IgG levels were largely maintained for at least 23 weeks after the final immunisation. IgM antibodies, generally detectable only after immunisation in Freund's adjuvant, were absent 23 weeks later. Antibody titres against the native protein on fixed parasites, assayed by IFA, were three to five orders of magnitude lower than the corresponding ELISA titres against the peptides. Antibody-dependent inhibition of P. falciparum growth in vitro could not be demonstrated with the immune rabbit sera. The MSA1 and EBP peptides elicited cross-reactive antibodies. The results suggest that the selected non-repetitive sequences are conformationally constrained in the native proteins and only a small proportion of the anti-peptide antibodies bind to the native proteins. The significance of the findings for the development of peptide vaccines and the use of peptides in immunoassays is discussed.

Mosquito midgut glycoproteins and recognition sites for malaria parasites.

Midgut glycoproteins of the malaria vector Anopheles tessellatus were partially characterised by gel electrophoresis and lectin binding. Specific binding to wheat germ agglutinin (WGA) and Concanavalin A (Con A) indicated the presence of N-linked core oligosaccharides in many proteins. Rabbit antibodies were produced against wheat germ agglutinin binding proteins (WGABP). These antibodies also recognised distinct proteins in the peritrophic membrane which is secreted into the midgut to enclose a bloodmeal. Rabbit anti-WGABP antibodies ingested in a bloodmeal containing infective gametocytes of the human malaria parasites Plasmodium falciparum and P. vivax tended to reduce infectivity of the parasites to vector mosquitoes. Chitotriose added to a bloodmeal also inhibited parasite development in the mosquito. The results are consistent with a hypothesis that N-acetyl glucosamine residues in mosquito midgut glycoproteins and/or midgut chitin and proteoglycan function as recognition sites for malaria parasites.

Interactions of human malaria parasites, Plasmodium vivax and P.falciparum, with the midgut of Anopheles mosquitoes.

Present understanding of the development of sexual stages of the human malaria parasites Plasmodium vivax and P.falciparum in the Anopheles vector is reviewed, with particular reference to the role of the mosquito midgut in establishing an infection. The sexual stages of the parasite, the gametocytes, are formed in human erythrocytes. The changes in temperature and pH encountered by the gametocyte induce gametogenesis in the lumen of the midgut. Macromolecules derived from mosquito tissue and second messenger pathways regulate events leading to fertilization. In An.tessellatus the movement of the ookinete from the lumen to the midgut epithelium is linked to the release of trypsin in the midgut and the peritrophic matrix is not a firm barrier to this movement. The passage of the P.vivax ookinete through the peritrophic matrix may take place before the latter is fully formed. The late ookinete development in P.falciparum requires chitinase to facilitate penetration of the peritrophic matrix. Recognition sites for the ookinetes are present on the midgut epithelial cells. N-acetyl glucosamine residues in the oligosaccharide side chains of An.tessellatus midgut glycoproteins and peritrophic matrix proteoglycan may function as recognition sites for P.vivax and P.falciparum ookinetes. It is possible that ookinetes penetrating epithelial cells produce stress in the vector. Mosquito molecules may be involved in oocyst development in the basal lamina, and encapsulation of the parasite occurs in vectors that are refractory to the parasite. Detailed knowledge of vector-parasite interactions, particularly in the midgut and the identification of critical mosquito molecules offers prospects for manipulating the vector for the control of malaria.

Different effects of modulation of mosquito (Diptera:Culicidae) trypsin activity on the infectivity of two human malaria (Hemosporidia:Plasmodidae) parasites.

Trypsin production in the malaria vector Anopheles tessellatus Theobald peaks between 12 and 21 h after a blood meal. The presence of leupeptin or soybean trypsin inhibitor in a blood meal delayed the onset of maximal trypsin activity. Trypsin inhibitors in an infective blood meal increased the infectivity of Plasmodium vivax Grassi and decreased infectivity of P. falciparum Welch to An tessellatus. The opposite effects of trypsin inhibitors on infectivity of the 2 malaria parasites were attributed to differences in the biology of the parasites within the midgut of the vector, particularly the time of ookinete formation and the requirement for activation of a chitinase.

Antimidgut antibodies inhibit peritrophic membrane formation in the posterior midgut of Anopheles tessellatus (Diptera: Culicidae).

The effect on midgut structure of feeding rabbit antibodies in a blood meal was investigated in Anopheles tessellatus Theobald. No detectable ultrastructural changes in midgut cells were observed. However, antimidgut antibodies inhibited formation of the peritrophic membrane in the posterior midgut. The results indicate that a peritrophic membrane in the posterior midgut is not essential for survival of the mosquito after a blood meal or for establishing a Plasmodium vivax Grassi infection.

Antibodies to Anopheles midgut reduce vector competence for Plasmodium vivax malaria.

Anopheles tessellatus mosquitoes ingested Plasmodium vivax gametocytes in human erythrocytes suspended in rabbit sera with and without anti-mosquito midgut antibodies. When the mosquito bloodmeal contained anti-midgut antibodies, fewer oocysts of P.vivax developed on the mosquito midgut and the proportion of mosquitoes becoming infected was significantly reduced. Complement inactivated serum also reduced the infection rate and load. A second bloodmeal containing anti-midgut antibodies, given 48 or 72 h later, did not enhance the transmission-blocking effect. IgG purified from anti-midgut sera was shown to mediate the transmission-blocking effect.

Dynamics of natural antibody responses to malaria parasite surface proteins in the intermediate rainfall zone of Sri Lanka.

Antibodies against repetitive epitopes on Plasmodium falciparum and P. vivax circumsporozoite (CS) proteins and epitopes on the 45 kDa and 185-200 kDa P. falciparum merozoite surface proteins were measured by radioimmunoassay in a two year longitudinal study in Nikawehera village located in the intermediate rainfall zone of Sri Lanka. The prevalence and concentrations of specific antibodies were in many, but not all instances, greater in adults than in children who were aged 7-15 yr at the beginning of the study. The concentrations and prevalence of antibodies were associated with malaria transmission levels previously determined from entomological and hospital admission data in the area. Antibody responses to epitopes on different P. falciparum antigens, two different epitopes within the 185-200 kDa merozoite surface protein and between the P. falciparum and P. vivax CS repeats were significantly correlated. Antibody concentrations against a conserved epitope in the 185-200 kDa protein were significantly higher in P. falciparum infected individuals than in non-parasitaemic subjects. Antibody concentration and prevalences in Nikawehera were lower than at Weheragala, a site located 70 km away in the dry zone of Sri Lanka. It is postulated that lower levels of immunity in the population in areas such as Nikawehera, that are adjacent to more highly malaria endemic areas, may promote epidemics when conditions favour transmission.

Antibody and clinical responses in volunteers to immunization with malaria peptide-diptheria toxoid conjugates.

Twenty residue peptides from the 185-200-kD and 45-kD merozoite surface antigens of the malaria parasite Plasmodium falciparum were covalently linked to diphtheria toxoid as a carrier and used to immunize human volunteers with aluminium hydroxide as an adjuvant. Significant antibody levels were elicited by two boosting injections. The antibodies reacted with acetone-methanol fixed merozoite membranes in an immunofluorescence assay, but no inhibition of merozoite reinvasion could be detected in in vitro cultures containing the antibodies. Antibody levels against the immunizing peptides declined markedly within 77 days after the third injection. No hypersensitivity was observed against the peptides. However, the volunteers developed hypersensitivity against diphteria toxoid, and in particular a pronounced type III (Arthus) hypersensitivity after three injections with the toxoid. This effect might appear to limit the use of peptide-diphtheria toxoid conjugates for human immunization. Several biochemical, haematological and immunological tests done on the volunteers showed no other adverse effects from the immunizations.

Population dynamics of anthropophilic mosquitoes during the northeast monsoon season in the malaria epidemic zone of Sri Lanka.

Mosquito-borne diseases are a major health problem in Sri Lanka. Human biting mosquitoes were collected during the night (18.00-06.00 hours) at Nikawehera village, in the malaria endemic intermediate rainfall zone of the country. Collections were made at monthly intervals in the period October 1991 to April 1992, which included the main rainy season due to the northeast monsoon (October-January). Thirteen Anopheles, eleven Culex, three Aedes, three Mansonia and one Armigeres species were identified, including known vectors of malaria, Bancroftian filariasis, Japanese encephalitis and dengue fever. Mosquito human-biting rates were highest in December. The main malaria vector Anopheles culicifacies showed peak biting between 18.00 and 23.00 hours whereas the predominant culicines Culex fuscocephala and Cx quinquefasciatus preferred to bite after midnight. In 1991-92 the prevalence of some species of anophelines at Nikawehera differed markedly from that observed in 1990-91 and the possible reasons are discussed.

Antibodies to epitopes on merozoite and sporozoite surface antigens as serologic markers of malaria transmission: studies at a site in the dry zone of Sri Lanka.

Antibodies against repetitive epitopes on Plasmodium falciparum and P. vivax circumsporozoite (CS) proteins and epitopes on the 45-kD and 185-200-kD P. falciparum merozoite surface antigens were measured by radioimmunoassay in Weheragala, a malaria-endemic site in the dry zone of Sri Lanka. Antibodies were measured in sera collected in February at the end of the main malaria transmission season and three months later in May during the low transmission period. Ninety-seven percent of the sample population had antibodies to the P. falciparum CS repeat in February and a significant proportion possessed antibodies directed against all epitopes tested. Concentrations and prevalence of antibodies to the CS repeats decreased with time after the end of malaria transmission in adults and children. Similar temporal changes were observed with antibodies to the epitopes on merozoite surface antigens. Children 7-15 years of age had lower antibody concentrations against most epitopes than adults. Antibody concentrations to two different epitopes within the same merozoite surface antigen showed significant association as did antibody levels against the P. falciparum CS repeat and the predominant P. vivax CS repeat. However, antibody concentrations did not correlate with the presence of blood-stage malaria infections.

Fecundity of Anopheles tessellatus reduced by the ingestion of murine anti-mosquito antibodies.

High titres of antibodies to antigens derived from head/thorax, midgut or abdomen of Anopheles tessellatus were produced in inbred mice. These antibodies, when ingested in a bloodmeal, reduced the fecundity of An. tessellatus by up to 29% in different experiments. It is postulated that antibodies directed against antigens shared between the head/thorax, abdomen and midgut tissues are involved in the reduction of fecundity.