Biomedical subjects

Venkatachalam Udhayakumar

Publications and source records attributed to Venkatachalam Udhayakumar.

At least 19 recordsLinked to original sources

PubMed2006

A comparative study of the genetic diversity of the 42kDa fragment of the merozoite surface protein 1 in Plasmodium falciparum and P. vivax.

We investigated the genetic diversity of the 42kDa fragment of the merozoite surface protein 1 (MSP-1) antigen in Plasmodium falciparum and P. vivax, as well as in non-human primate malarial parasites. This fragment undergoes a proteolytic cleavage generating two fragments of 19kDa (MSP-1(19)) and 33kDa (MSP-1(33)) that are critical in erythrocyte invasion. We found that overall the MSP-1(33) fragment exhibits greater genetic diversity than the MSP-1(19) regardless of the species. We have found evidence for positive natural selection only in the human malaria parasites by comparing the rate of non-synonymous versus synonymous substitutions. In addition, we found clear differences between the two major human malaria parasites. In the case of P. falciparum, positive natural selection is acting on the MSP-1(19) region while the MSP-1(33) is neutral or under purifying selection. The opposite pattern was observed in P. vivax. Our results suggest different roles of this antigen in the host-parasite immune interaction in each of the major human malarial parasites.

Amino Acid Motifs↗

PubMed2006

Pyrosequencing, a high-throughput method for detecting single nucleotide polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes of Plasmodium falciparum.

A pyrosequencing protocol was developed as a rapid and reliable method to identify the mutations of the dhfr and dhps genes of Plasmodium falciparum that are associated with antifolate resistance. The accuracy and specificity of this method were tested using six laboratory-cultured P. falciparum isolates harboring known single nucleotide polymorphisms (SNPs) in the genes dhfr (codons 50, 51, 59, 108, and 164) and dhps (codons 436, 437, 540, 581, and 613). The lowest threshold for detection of all the SNPs tested by pyrosequencing was the equivalent of two to four parasite genomes. Also, this method was highly specific for P. falciparum, as it did not amplify any DNA products from the other species of human malaria parasites. We also mixed wild-type and mutant-type parasite DNAs in various proportions to determine how pyrosequencing, restriction fragment length polymorphism (RFLP), and direct conventional sequencing (for dhfr) compared with each other in detecting different SNPs in the mixture. In general, pyrosequencing and RFLP showed comparable sensitivities in detecting most of the SNPs in dhfr except for the 164L mutation, which required at least twice the amount of DNA for pyroseqencing as for RFLP. For detecting SNPs in dhps, pyrosequencing was slightly more sensitive than RFLP and direct sequencing. Overall, pyrosequencing was faster and less expensive than either RFLP or direct sequencing. Thus, pyrosequencing is a practical alternative method that can be used in a high-throughput format for molecular surveillance of antimalarial-drug resistance.

Animals↗

PubMed2006

Antifolate resistance in Plasmodium falciparum: multiple origins and identification of novel dhfr alleles.

BACKGROUND: Sulfadoxine-pyrimethamine has been widely used as first-line therapy for uncomplicated malaria throughout sub-Saharan Africa. Recent studies conducted in Asia and Africa suggest the triple-mutant dhfr genotype (51I/59R/108N) may have been generated as a single event in Southeast Asia, with subsequent spread of the single lineage to the African continent, but this hypothesis needs further validation. METHODS: Direct sequencing of polymerase chain reaction (PCR) products, pyrosequencing, and cloning of PCR products were utilized to identify mutations in dhfr. To investigate the evolutionary history of dhfr alleles, we assayed microsatellite loci flanking dhfr along chromosome 4. RESULTS: A total of 15 of 479 samples from western Kenya showed the presence of I164L, in 5 different genotypes. We document C50R in 2 of our samples. Using microsatellite markers, we show 2 haplotypes for both the 51I/108N/164L and 51I/59R/108N/164L genotypes. Our results also show multiple lineages for the triple-mutant dhfr genotype in Africa. CONCLUSIONS: These findings highlight the importance of local characterization of alleles before molecular surveillance of drug-resistant alleles is considered in different endemic settings and populations.

Alleles↗

PubMed2006

Development, characterization and immunogenicity of a multi-stage, multi-valent Plasmodium falciparum vaccine antigen (FALVAC-1A) expressed in Escherichia coli.

A synthetic multistage, multi-epitope Plasmodium falciparum malaria antigen (FALVAC-1A) was designed and evaluated in silico, and then the gene was constructed and expressed in Escherichia coli. The FALVAC-1A protein was purified by inclusion body isolation, followed by affinity and ion exchange chromatography. Although FALVAC-1A was a synthetic antigen, it folded to a specific, but as yet incompletely defined, molecular conformation that was stable and comparable from lot to lot. When formulated with four different adjuvants, FALVAC-1A was highly immunogenic in rabbits, inducing not only ELISA reactivity to the cognate antigen and most of its component epitopes, but also in vitro activity against P. falciparum parasites as demonstrated by inhibition of sporozoite invasion, antibody dependent cellular inhibition and the immunofluorescence assay.

Amino Acid Sequence↗

PubMed2005

Modulation of immune responses during HIV-malaria co-infection in pregnancy.

Infection with either HIV or malaria during pregnancy often results in adverse outcomes for mother and child. Co-infection further increases the risks of these events, which include maternal anemia and babies with low birth weight. The immunological bases for the increased susceptibility of HIV-infected mothers to malaria and for the effect of co-infection on mother-to-child transmission of HIV are areas of major importance in public health. In this article, we review current data about humoral and cellular responses to HIV-placental-malaria co-infection and present an immunological hypothesis to explain the epidemiological findings.

Adult↗

PubMed2005

Placental malaria diminishes development of antibody responses to Plasmodium falciparum epitopes in infants residing in an area of western Kenya where P. falciparum is endemic.

To determine the effect of placental malaria (PM) infection on the development of antibody responses to malaria in infants, we measured immunoglobulin G levels to seven different Plasmodium falciparum epitopes by using plasma samples collected at monthly intervals from infants born to mothers with and without PM. Overall, PM was associated with diminished antibody levels to all of the epitopes tested, especially with infants aged >or=4 to 12 months, and the difference was statistically significant for four of the seven epitopes (P<0.0035). These findings suggest that PM can negatively influence the development of immune responses to malaria in infants.

Animals↗

PubMed2005

Immunohistological characterization of macrophage migration inhibitory factor expression in Plasmodium falciparum-infected placentas.

Previously, we have shown that macrophage migration inhibitory factor (MIF) was highly elevated in the placental intervillous blood (IVB) of Plasmodium falciparum-infected women. Here, we compared the expression of MIF in placental tissues obtained from P. falciparum-infected and -uninfected women. Immunoperoxidase staining showed a consistent pattern of MIF expression in syncytiotrophoblasts, extravillous trophoblasts, IVB mononuclear cells, and amniotic epithelial cells, irrespective of their malaria infection status. Cytotrophoblast, villous stroma, and Hofbauer cells showed focal staining. Only amniotic epithelial and IVB mononuclear cells from P. falciparum-infected placentas exhibited significantly higher level of MIF expression than uninfected placentas. Stimulation of syncytilized human trophoblast BeWo cells with P. falciparum-infected erythrocytes that were selected to bind these cells resulted in significant increases in MIF secretion, whereas control erythrocytes, lipopolysaccharides, and synthetic beta-hematin had minimal effect. These findings suggest that placental malaria modulates MIF expression in different placental compartments.

Animals↗

PubMed2005

Preliminary observations on the efficacy of a recombinant multistage Plasmodium falciparum vaccine in Aotus nancymai monkeys.

A vaccine trial was conducted to determine the efficacy of a multicomponent candidate vaccine, FALVAC-1, against Plasmodium falciparum in Aotus nancymai monkeys. After two immunizations, animals were challenged intravenously with parasites of the Vietnam Oak Knoll (FVO) strain of P. falciparum. The primary outcome was to determine the protective response of the monkeys to immunization with the FALVAC-1 antigen produced in baculovirus when combined with different adjuvants (alum, QS-21, ASO2a, CRL1005/oil, and CRL1005/saline) as compared with FALVAC-1 with FCA/FIA and antigen alone. When compared with the monkeys immunized with FALVAC-1 alone, FALVAC-1 with FCA/FIA reduced the mean parasite count (to Day 11), reduced the mean accumulated parasitemia (through Day 11), and extended the number of days to treatment. None of the other 5 antigen-adjuvant combinations were able to provide discernable levels of protection based on log(parasitemia) and log(cumulative parasitemia) to Day 11.

Adjuvants, Immunologic↗

PubMed2004

Assessing the effect of natural selection in malaria parasites.

There are few concepts that have been used across disciplines; one of them is natural selection. The impact that this process has on parasite genetic diversity is reviewed here by discussing examples on drug resistance and vaccine antigens. Emphasis is made on how mechanisms need to be addressed rather than associations, and how such investigations were out of reach of biomedical researchers only a decade ago.

Animals↗

PubMed2004

Hemozoin differentially regulates proinflammatory cytokine production in human immunodeficiency virus-seropositive and -seronegative women with placental malaria.

Pregnant women are at an increased risk for malarial infection. Plasmodium falciparum accumulates in the placenta and is associated with dysregulated immune function and poor birth outcomes. Malarial pigment (hemozoin) also accumulates in the placenta and may modulate local immune function. In this study, the impact of hemozoin on cytokine production by intervillous blood mononuclear cells from malaria-infected placentas was investigated. There was a dose-dependent, suppressive effect of hemozoin on production of gamma interferon (IFN-gamma), with less of an effect on tumor necrosis factor alpha (TNF-alpha) and interleukin-10, in human immunodeficiency virus-seronegative (HIV(-)) women. In contrast, IFN-gamma and TNF-alpha production tended to increase in HIV-seropositive women with increasing hemozoin levels. Production patterns of cytokines, especially IFN-gamma in HIV(-) women, followed different trends as a function of parasite density and hemozoin level. The findings suggest that the influences of hemozoin accumulation and high-density parasitemia on placental cytokine production are not equivalent and may involve different mechanisms, all of which may operate differently in the context of HIV infection. Cytokine production dysregulated by accumulation of hemozoin or high-density parasitemia may induce pathology and impair protective immunity in HIV-infected and -uninfected women.

Adolescent↗

PubMed2004

The burden of co-infection with human immunodeficiency virus type 1 and malaria in pregnant women in sub-saharan Africa.

In sub-Saharan Africa, human immunodeficiency virus (HIV) and malaria are among the leading causes of morbidity during pregnancy. We reviewed available information collected since the first report 15 years ago that HIV impaired the ability of pregnant women to control malaria parasitemia. Results from 11 studies showed that HIV-infected women experienced consistently more peripheral and placental malaria (summary relative risk = 1.58 and 1.66, respectively), higher parasite densities, and more febrile illnesses, severe anemia, and adverse birth outcomes than HIV-uninfected women, particularly in multigravidae. Thus, HIV alters the typical gravidity-specific pattern of malaria risk by shifting the burden from primarily primigravidae and secundigravidae to all pregnant women. The proportional increase of malaria during pregnancy attributable to HIV was estimated to be 5.5% and 18.8% for populations with HIV prevalences of 10% and 40%, respectively. Maternal malaria was associated with a two-fold higher HIV-1 viral concentrations. Three studies investigating whether placental malaria increased mother-to-child HIV-1 transmission showed conflicting results, possibly reflecting a complex balance between placental malarial immune responses and stimulation of HIV-1 viral replication. Further investigations of interactions between antiretroviral drugs, prophylaxis with cotrimoxazole, and antimalarial drugs in pregnant women are urgently needed. Although much has been learned in the past 15 years about the interaction between malaria and HIV-1 during pregnancy, many issues still require further information to improve our understanding. There is a clear need to strengthen the deployment of existing malaria and HIV prevention and intervention measures for pregnant women.

Africa South of the Sahara↗

PubMed2003

In vivo acquisition of hemozoin by placental blood mononuclear cells suppresses PGE2, TNF-alpha, and IL-10.

In areas of high malaria endemicity, women have increased susceptibility to malaria during pregnancy characterized by placental parasitemia. Our previous studies in children with malaria demonstrate that suppression of leukocyte-derived prostaglandin-E(2) (PGE(2)) is associated with enhanced pathogenesis. To examine the role of PGE(2) as an immunoregulatory molecule in placental malaria, PGE(2) was determined in cultured intervillous blood mononuclear cells (IVBMCs) from aparasitemic and parasitemic women. PGE(2) was significantly lower in parasitemic women at all gravidities. Women with a positive antenatal peripheral parasitemia who were negative for placental malaria (PM) at term produced the highest PGE(2) levels. Suppression of PGE(2) was associated with increasing amounts of hemozoin (malarial pigment) acquired during the natural infection. PGE(2) regulatory cytokines, tumor necrosis factor (TNF)-alpha and interleukin (IL)-10, were non-significantly increased in IVBMC containing an intermediate amount of hemozoin and significantly suppressed in IVBMC with high levels of hemozoin. Results presented here show that in vivo acquisition of high levels of hemozoin by IVBMC leads to decreased synthesis of PGE(2), IL-10, and TNF-alpha.

Cells, Cultured↗

PubMed2003

Gonadotropin-releasing hormone alters the T helper cytokine balance in the pregnant rat.

The interactions between immune-endocrine and reproductive systems are heightened during pregnancy as an adaptive mechanism, and are regulated by a complex array of hormones and cytokines that control the survival of a semiallogeneic conceptus. GnRH can exert direct effects on the immune system via its receptor (GnRH-R) on lymphoid cells. In the present study, we employed in vitro, ex vivo, and in vivo approaches to investigate the role of GnRH in the modulation of T helper cytokines in pregnant rats undergoing termination of pregnancy. Day 8 pregnant rats were infused with a GnRH agonist (GnRH-Ag) for 24 h using an osmotic minipump. Sham control rats were infused with the vehicle, saline. Lymphocytes were isolated from sham and treated rats and polyclonally stimulated with immobilized anti-CD3 antibody. The levels of the signature T helper 1 (Th-1) cytokines (interferon-gamma [IFN-gamma] and interleukin-2 [IL-2]) and Th-2 cytokines (IL-4 and IL-10) were measured in culture supernatants. Using immunoflourescence confocal microscopy, we demonstrated for the first time the spatial localization of GnRH-R protein on the surface of lymphocytes. We observed a marked increase in IFN-gamma and inhibition of IL-4 production from lymphocytes of pregnant rats treated in vitro with different doses of GnRH-Ag. Further, the responsiveness of lymphocytes to produce IFN-gamma was markedly increased in cells cultured ex vivo from GnRH-Ag infused rats, whereas the capacity of lymphocytes to produce IL-4 was significantly inhibited. In addition, GnRH-Ag infusion in pregnant rats induced a shift toward Th-1 cytokines in the serum. We did not observe any significant difference in IL-2 and IL-10 production in response to GnRH-Ag. Our results suggest an additional function for GnRH as a Th-1 inducer and Th-2 inhibitor. GnRH can thus skew the cytokine balance to predominantly Th-1 type in pregnancy, leading to the termination of pregnancy in rats.

Abortifacient Agents↗

PubMed2003

Relationship between plasma Interleukin-12 (IL-12) and IL-18 levels and severe malarial anemia in an area of holoendemicity in western Kenya.

In this study, we investigated whether levels of interleukin-12 (IL-12) and IL-18 in plasma are associated with severe malarial anemia outcomes in an area of holoendemicity in western Kenya. We compared plasma IL-12 and IL-18 levels in six groups of children grouped into the categories aparasitemic, asymptomatic, mild malaria, high-density uncomplicated malaria (UC), moderate malarial anemia (MMA), or severe malarial anemia (SMA). IL-12 levels were significantly reduced in children with SMA (P < 0.05) but not in other groups compared to children in the aparasitemic control group. IL-18, a cytokine known to be critical for the induction of gamma interferon along with IL-12, was produced more frequently (70%) in children with UC (P = 0.06) than in children in the aparasitemic control group (32%). However, in the SMA group the IL-18 response rate declined to 30%, which was similar to that in the aparasitemic control group, which showed a 32% response rate. This finding suggests that the IL-18 response may be impaired in children with SMA. In summary, the results from this study support the hypothesis that impairment of IL-12 and/or IL-18 response may contribute to the development of severe malarial anemia in areas of holoendemicity for malaria.

Blackwater Fever↗

PubMed2003

Levels of macrophage inflammatory protein 1 alpha (MIP-1 alpha) and MIP-1 beta in intervillous blood plasma samples from women with placental malaria and human immunodeficiency virus infection.

Macrophage inflammatory protein-1 alpha (MIP-1 alpha) and MIP-1 beta play an important role in modulating immune responses. To understand their importance in immunity to placental malaria (PM) and in human immunodeficiency virus (HIV)-PM coinfection, we investigated levels of these chemokines in the placental intervillous blood plasma (IVB plasma) and cord blood plasma of HIV-negative PM-negative, HIV-negative PM-positive, HIV-positive PM-negative, and HIV-positive PM-positive women. Compared to HIV-negative PM-negative women, the MIP-1 beta concentration in IVB plasma was significantly elevated in HIV-negative PM-positive women and HIV-positive PM-positive women, but it was unaltered in HIV-positive PM-negative women. Also, PM-infected women, irrespective of their HIV status, had significantly higher levels of MIP-1 beta than HIV-positive PM-negative women. The MIP-1 alpha level was not altered in association with either infection. The IVB plasma levels of MIP-1 alpha and MIP-1 beta positively correlated with the cord blood plasma levels of these chemokines. As with IVB plasma, only cord plasma from PM-infected mothers had significantly elevated levels of MIP-1 beta compared to PM-negative mothers, irrespective of their HIV infection status. MIP-1 beta and MIP-1 alpha levels in PM-positive women were positively associated with parasite density and malaria pigment levels. Regardless of HIV serostatus, the IVB MIP-1 beta level was significantly lower in women with PM-associated anemia. In summary, an elevated level of MIP-1 beta was associated with PM. HIV infection did not significantly alter these two chemokine levels in IVB plasma.

Adult↗

PubMed2003

Plasma interleukin-12 in malaria-tolerant papua new guineans: inverse correlation with Plasmodium falciparum parasitemia and peripheral blood mononuclear cell nitric oxide synthase activity.

Interleukin-12 (IL-12) has been inversely associated with disease severity in human and murine malaria, and a polymorphism in the IL-12 p40 subunit gene (IL12B) has been associated with susceptibility to human cerebral malaria and reduced nitric oxide (NO) production. To better define the relationships between IL-12, NO, malaria parasitemia, and IL12B polymorphisms during malarial tolerance, plasma IL-12 levels and peripheral blood mononuclear cell NO synthase (NOS) activity were measured in asymptomatic Papua New Guineans exposed to intense malaria transmission. The IL-12 level was strongly inversely correlated with the density of Plasmodium falciparum parasitemia (rho = -0.45; P < 0.001) and was predicted to decrease by 19% (95% confidence interval [CI], 10 to 27%) for each twofold increase in P. falciparum parasitemia. This is consistent with a suppressive effect of parasitemia on IL-12 production, an effect previously shown in vitro and in rodent models of disease. The IL-12 level was inversely correlated with NOS activity (r = -0.22; P = 0.007), with each twofold increase in NOS activity being predictive of a 25% (95% CI, 7 to 38%) decrease in plasma IL-12 levels. This probably reflects additional down-regulation of IL-12 by the high basal NO production and monocyte NOS expression found in the malaria-tolerant state. Neither the IL-12 level nor NOS activity was associated with either of two IL12B polymorphisms, reflecting the diversity of genetic control over immune responses in different populations.

Adolescent↗

PubMed2002

A new NOS2 promoter polymorphism associated with increased nitric oxide production and protection from severe malaria in Tanzanian and Kenyan children.

BACKGROUND: Nitric oxide (NO) is a mediator of immunity to malaria, and genetic polymorphisms in the promoter of the inducible NO synthase gene (NOS2) could modulate production of NO. We postulated that NOS2 promoter polymorphisms would affect resistance to severe malaria. METHODS: We assessed genomic DNA from healthy children and from those diagnosed with malaria from Tanzania (n=47 and n=138, respectively) and Kenya (n=1106) for polymorphisms by single-stranded conformational polymorphism (SSCP) analysis and sequencing. We also measured in-vivo NO production in Tanzanian children. FINDINGS: We identified a novel single nucleotide polymorphism, -1173 C-->T, in the NOS2 promoter that was significantly associated with protection from symptomatic malaria (odds ratio 0.12, 95% CI 0.03-0.48, p=0.0006) in 179 Tanzanian children, and significantly associated with protection from severe malarial anaemia (adjusted relative risk 0.25, 95% CI 0.09-0.66, p=0.0005) in 1106 Kenyan children studied over 5 years. The risk of parasitaemia was not significantly different in wild-type or -1173 C-->T individuals. -1173 C-->T protection in Tanzanians was independent of the previously recognised NOS2-954 G-->C polymorphism. The (CCTTT)(n) NOS2 polymorphism (Tanzania and Kenya) was not associated with severe malaria outcomes. -1173 C-->T was associated with increased fasting urine and plasma NO metabolite concentrations in Tanzanian children, suggesting that the polymorphism was functional in vivo. Interpretation The NOS2 promoter -1173 C-->T single nucleotide polymorphism is associated with protection against cerebral malaria and severe malarial anaemia. Increased NO production in individuals with the -1173 C-->T polymorphism lends support to a protective role for NO against these syndromes. Targeted interventions to increase NO delivery or production could provide novel preventive and therapeutic strategies against these major causes of mortality in African children.

Anemia↗

PubMed2002

Increased efficacy of sulfadoxine-pyrimethamine in the treatment of uncomplicated falciparum malaria among children with sickle cell trait in Western Kenya.

The role of the sickle cell hemoglobin type as a determinant of treatment outcome with sulfadoxine-pyrimethamine was retrospectively studied in young children with uncomplicated falciparum malaria who lived in an area with intense perennial malaria transmission. Between 1993 and 1997, 2795 treatments involving 813 children were monitored. Sickle cell trait (HbAS) was present in 17.7% of the children. Two-and-a-half percent of the children experienced early clinical treatment failure by day 2-3, and 17.3% of the children were parasitemic on day 7. Treatments in HbAS children were less likely than those in HbAA children to result in persistence of parasitemia by day 3 (relative risk [RR], 0.66; 95% confidence interval [CI], 0.47-0.93; P=.02) or in parasitologic treatment failure on day 7 (RR, 0.51; 95% CI, 0.36-0.71; P<.0001). These results suggest that the HbAS phenotype should be included among factors that determine sulfadoxine-pyrimethamine treatment outcome.

Animals↗