In pregnant women, Plasmodium falciparum-infected erythrocytes expressing the VAR2CSA antigen bind to chondroitin sulfate A in the placenta causing placental malaria. The binding site of VAR2CSA is present in the ID1-ID2a region. This study sought to determine if pregnant Cameroonian women naturally acquire antibodies to ID1-ID2a and if antibodies to ID1-ID2a correlate with absence of placental malaria at delivery. Antibody levels to full-length VAR2CSA and ID1-ID2a were measured in plasma samples from 745 pregnant Cameroonian women, 144 Cameroonian men, and 66 US subjects. IgM levels and IgG avidity to ID1-ID2a were also determined. As expected, antibodies to ID1-ID2a were absent in US controls. Although pregnant Cameroonian women developed increasing levels of antibodies to full-length VAR2CSA during pregnancy, no increase in either IgM or IgG to ID1-ID2a was observed. Surprisingly, no differences in antibody levels to ID1-ID2a were detected between Cameroonian men and pregnant women. For example, in rural settings only 8-9% of males had antibodies to full-length VAR2CSA, but 90-96% had antibodies to ID1-ID2a. In addition, no significant difference in the avidity of IgG to ID1-ID2a was found between pregnant women and Cameroonian men, and no correlation between antibody levels at delivery and absence of placental malaria was found. Thus, the response to ID1-ID2a was not pregnancy specific, but predominantly against cross-reactivity epitopes, which may have been induced by other PfEMP1 antigens, malarial antigens, or microbes. Currently, ID1-ID2a is a leading vaccine candidate, since it binds to the CSA with the same affinity as the full-length molecule and elicits binding-inhibitory antibodies in animals. Further studies are needed to determine if the presence of naturally acquired cross-reactive antibodies in women living in malaria endemic countries will alter the response to ID1-ID2a following vaccination with ID1-ID2a.
Placental infections with Plasmodium falciparum are associated with fetal growth restriction resulting in low birth weight (LBW). The mechanisms that mediate these effects have yet to be completely described; however, they are likely to involve inflammatory processes and dysregulation of angiogenesis. Soluble endoglin (sEng), a soluble receptor of transforming growth factor (TGF)-? previously associated with preeclampsia in pregnant women and with severe malaria in children, regulates the immune system and influences angiogenesis. We hypothesized that sEng may play a role in development of LBW associated with placental malaria (PM). Plasma levels of sEng were measured in women (i) followed prospectively throughout pregnancy in Cameroon (n = 52), and (ii) in a case-control study at delivery in Malawi (n = 479). The relationships between sEng levels and gravidity, peripheral and placental parasitemia, gestational age, and adverse outcomes of PM including maternal anemia and LBW were determined. In the longitudinal cohort from Cameroon, 28 of 52 women (54%) experienced at least one malaria infection during pregnancy. In Malawi we enrolled two aparasitemic gravidity-matched controls for every case with PM. sEng levels varied over the course of gestation and were significantly higher in early and late gestation as compared to delivery (P<0.006 and P<0.0001, respectively). Circulating sEng levels were higher in primigravidae than multigravidae from both Cameroon and Malawi, irrespective of malarial infection status (p<0.046 and p<0.001, respectively). Peripheral parasitemia in Cameroonian women and PM in Malawian women were each associated with elevated sEng levels following correction for gestational age and gravidity (p = 0.006 and p = 0.033, respectively). Increased sEng was also associated with the delivery of LBW infants in primigravid Malawian women (p = 0.017); the association was with fetal growth restriction (p = 0.003) but not pre-term delivery (p = 0.286). Increased circulating maternal sEng levels are associated with P. falciparum infection in pregnancy and with fetal growth restriction in primigravidae with PM.
Placental malaria infections are caused by Plasmodium falciparum-infected red blood cells sequestering in the placenta by binding to chondroitin sulfate A, mediated by VAR2CSA, a variant of the PfEMP1 family of adhesion antigens. Recent studies have shown that many P. falciparum genomes have multiple genes coding for different VAR2CSA proteins, and parasites with >1 var2csa gene appear to be more common in pregnant women with placental malaria than in nonpregnant individuals. We present evidence that, in pregnant women, parasites containing multiple var2csa-type genes possess a selective advantage over parasites with a single var2csa gene. Accumulation of parasites with multiple copies of the var2csa gene during the course of pregnancy was also correlated with the development of antibodies involved in blocking VAR2CSA adhesion. The data suggest that multiplicity of var2csa-type genes enables P. falciparum parasites to persist for a longer period of time during placental infections, probably because of their greater capacity for antigenic variation and evasion of variant-specific immune responses.
A prospective longitudinal study of Plasmodium falciparum in pregnant women was conducted in the rural village of Ngali II, where malaria is hyperendemic and individuals receive ~0.7 infectious mosquito bites/person/day throughout the year. Pregnant women (N = 60; 19 primigravidae, 41 multigravidae) were enrolled early in pregnancy (median 14 wk) and were followed monthly, with 38 women followed through term (5.7 ± 1.1 prenatal visits and delivery). The total number of times primigravidae were slide-positive during pregnancy was higher than multigravidae (3.3 ± 1.1 versus 1.3 ± 1.3 times; P < 0.001), but no difference in the number of polymerase chain reaction-positive cases (4.6 ± 1.7 and 3.4 ± 1.7 times, P = 0.106) or total genotypes they harbored (8.9 ± 3.2 and 7.0 ± 2.9) was found. Only 7.9% women developed symptomatic infections. All primigravidae and 38% multigravidae were placental malaria-positive at delivery (P = 0.009). Genotyping showed that 77% of placental parasites were acquired ? 30 wks in pregnancy. These results help identify the extent of malaria-associated changes women experience during pregnancy.
Plasmodium falciparum-infected erythrocytes (IEs) sequester in the intervillous space (IVS) of the placenta causing placental malaria (PM), a condition that increases a womans chances of having a low-birth-weight baby. Because IEs sequester, they frequently are not observed in peripheral blood smears, resulting in women with PM being misdiagnosed and thus not treated. Because sequestered IEs induce inflammation in the IVS, detection of inflammatory mediators in the peripheral blood may provide an approach for diagnosing PM. Two counterregulatory molecules, TNF-?R (TNFR) 1 and TNFR2, modulate the pathological effects of TNF-?. Levels of these soluble TNFRs (sTNFRs) are reported to be elevated in children with severe malaria, but it is unclear if they are increased in the peripheral blood of PM-positive women with asymptomatic infections. In this study, sTNFR levels were measured throughout the course of pregnancy, as well as at delivery, in women with asymptomatic infections and those who remained uninfected. Results showed that both sTNFRs were significantly increased in the peripheral blood of women with asymptomatic malaria (p < 0.0001) and were positively correlated with parasitemia (p < 0.0001 for sTNFR1 and p = 0.0046 for sTNFR2). Importantly, levels of sTNFR2 were elevated in the peripheral blood of women who were PM-positive but peripheral blood-smear negative (p = 0.0017). Additionally, sTNFR2 levels were elevated in the blood of malaria-positive women who delivered low-birth-weight babies. In vitro studies demonstrated that syncytiotrophoblasts were not a major source of sTNFR. These data suggest that sTNFR2 may be a valuable biomarker for detection of malaria-associated inflammation.
Lipopolysaccharide (LPS) is a major component on the surface of Gram negative bacteria and is composed of lipid A-core and the O antigen polysaccharide. O polysaccharides of the gastric pathogen Helicobacter pylori contain Lewis antigens, mimicking glycan structures produced by human cells. The interaction of Lewis antigens with human dendritic cells induces a modulation of the immune response, contributing to the H. pylori virulence. The amount and position of Lewis antigens in the LPS varies among H. pylori isolates, indicating an adaptation to the host. In contrast to most bacteria, the genes for H. pylori O antigen biosynthesis are spread throughout the chromosome, which likely contributed to the fact that the LPS assembly pathway remained uncharacterized. In this study, two enzymes typically involved in LPS biosynthesis were found encoded in the H. pylori genome; the initiating glycosyltransferase WecA, and the O antigen ligase WaaL. Fluorescence microscopy and analysis of LPS from H. pylori mutants revealed that WecA and WaaL are involved in LPS production. Activity of WecA was additionally demonstrated with complementation experiments in Escherichia coli. WaaL ligase activity was shown in vitro. Analysis of the H. pylori genome failed to detect a flippase typically involved in O antigen synthesis. Instead, we identified a homolog of a flippase involved in protein N-glycosylation in other bacteria, although this pathway is not present in H. pylori. This flippase named Wzk was essential for O antigen display in H. pylori and was able to transport various glycans in E. coli. Whereas the O antigen mutants showed normal swimming motility and injection of the toxin CagA into host cells, the uptake of DNA seemed to be affected. We conclude that H. pylori uses a novel LPS biosynthetic pathway, evolutionarily connected to bacterial protein N-glycosylation.
Placental malaria (PM) is associated with adverse pregnancy outcomes including low birth weight (LBW). However, the precise mechanisms by which PM induces LBW are poorly defined. Based on the essential role of angiopoietin (ANG)-1 and -2 in normal placental vascular development, we hypothesized that PM may result in the dysregulation of angiopoietins and thereby contribute to LBW outcomes.
Plasmodium falciparum merozoite surface protein-1 (MSP1) has been extensively studied as a blood-stage malaria vaccine candidate, with most work focused on the conserved 19 kDa and semi-conserved 42 kDa C-terminal regions (blocks 16-17) and the hypervariable N-terminal repeat region (block 2). However, recent genotyping studies suggest that additional regions of MSP1 may be under selective pressure, including a locus of intragenic recombination designated as block 4 within the 3 region of the gene.
During pregnancy, women are more susceptible to Plasmodium falciparum infections and frequently have a higher parasitaemia than non-pregnant women. Several mechanisms are responsible for their increased susceptibility, including down-modulation of immune responses that aid in parasite clearance and sequestration of infected erythrocytes in the placenta. Early in pregnancy, a third mechanism may contribute to higher parasitaemia, since it has been reported that addition of human chorionic gonadotropin (hCG) to in vitro cultures of the NF54-strain of P. falciparum results in increased parasite growth rates. The goal of this study was to further examine the effect of hCG on P. falciparum growth.
Gastrointestinal disease caused by Campylobacter jejuni is characterized by localized inflammation and the destruction of the epithelial cell barrier that forms host innate protection against pathogens. This can lead to an imbalance in fluid transport across the gastrointestinal tract, resulting in severe diarrhea. The mechanisms of host cell receptor recognition of C. jejuni and downstream immune signaling pathways leading to this inflammatory disease, however, remain unclear. The aim of this study was to analyze the mechanisms involved in C. jejuni induction of the acute-phase inflammatory response regulator interleukin-6 (IL-6). Polarized intestinal epithelial Caco-2 monolayers responded to infections with Salmonella enterica serovar Typhimurium and eight isolates of C. jejuni by an increase in levels of expression and secretion of IL-6. No such IL-6 response, however, was produced upon infection with the human commensal organism Lactobacillus rhamnosus GG. The IL-6 signaling pathway was further characterized using short interfering RNA complexes to block gene expression. The inhibition of myeloid differentiation primary response protein 88 (MyD88) expression in this manner did not affect C. jejuni-induced IL-6 secretion, suggesting a MyD88-independent route to IL-6 signal transduction in C. jejuni-infected human epithelial cells. However, a significant reduction in levels of IL-6 was evident in the absence of Toll-like receptor 2 (TLR-2) expression, implying a requirement for TLR-2 in C. jejuni recognition. Caco-2 cells were also treated with heat-inactivated and purified membrane components of C. jejuni to isolate the factor responsible for triggering IL-6 signaling. The results demonstrate that C. jejuni surface polysaccharides induce IL-6 secretion from intestinal epithelial cells via TLR-2 in a MyD88-independent manner.
During pregnancy, Plasmodium falciparum-infected erythrocytes (IE) sequester in the placenta where they induce pathology and increase the risk of low-birth-weight (LBW) babies. The innate immune mediator, mannose-binding lectin (MBL), enhances phagocytosis of pathogens. Since MBL is reported to bind to IE, we hypothesized that it might aid in clearance of IE from the placenta, thereby reducing the risk of LBW babies. To test this hypothesis, molecular genotyping was used to detect polymorphisms at codon 57 (A/C) in exon 1 of MBL2 in 401 pregnant Cameroonian women, with or without placental malaria, who had LBW and normal-weight babies. Polymorphisms in the promoter region at positions -550 (H/L), -221 (X/Y), and +4 (P/Q) were also determined, and plasma MBL levels were measured during pregnancy and at delivery. The expected correlation between genotype and plasma MBL levels was confirmed. However, asymptomatic infections were not associated with an increase in MBL levels in the peripheral blood, and MBL levels were similar in the placental and cord blood of women with or without placental malaria at delivery. There was no evidence that MBL levels at delivery were associated with malaria-related poor pregnancy outcomes. Women with the LXPA haplotype, however, were more likely to have LBW babies, but the risk was not related to malaria. These results do not support the hypothesis that MBL aids in the clearance of parasites from the placenta but suggest that Cameroonian women with LXPA are at risk of having LBW babies due to other causes.
VAR2CSA mediates sequestration of Plasmodium falciparum-infected erythrocytes in the placenta, increasing the risk of poor pregnancy outcomes. Naturally acquired antibodies (Ab) to placental parasites at delivery have been associated with improved pregnancy outcomes, but Ab levels and how early in pregnancy Ab must be present in order to eliminate placental parasites before delivery remains unknown. Antibodies to individual Duffy-binding like domains of VAR2CSA have been studied, but the domains lack many of the conformational epitopes present in full-length VAR2CSA (FV2). Thus, the purpose of this study was to describe the acquisition of Ab to FV2 in women residing in high and low transmission areas and determine how Ab levels during pregnancy correlate with clearance of placental parasites. Plasma samples collected monthly throughout pregnancy from pregnant women living in high and low transmission areas in Cameroon were evaluated for Ab to FV2 and the proportion of high avidity Ab (i.e., Ab that remain bound in the presence of 3M NH(4)SCN) was assessed. Ab levels and proportion of high avidity Ab were compared between women with placental malaria (PM(+)) and those without (PM(-)) at delivery. Results showed that PM(-) women had significantly higher Ab levels (p = 0.0047) and proportion of high avidity Ab (p = 0.0009) than PM(+) women throughout pregnancy. Specifically, women with moderate to high Ab levels (>5,000 MFI) and those with ? 35% high avidity Ab at 5-6 months were found to have 2.3 (95% CI, 1.0-4.9) and 7.6-fold (p = 0.0013, 95% CI: 1.2-50.0) reduced risk of placental malaria, respectively. These data show that high levels of Ab to FV2, particularly those with high avidity for FV2, produced by mid-pregnancy are important in clearing parasites from the placenta. Both high Ab levels and proportion of high avidity Ab to FV2 may serve as correlates of protection for assessing immunity against placental malaria.
Placental malaria, caused by sequestration of Plasmodium falciparum-infected erythrocytes in the placenta, is associated with increased risk of maternal morbidity and poor birth outcomes. The parasite antigen VAR2CSA (variant surface antigen 2-chondroitin sulfate A) is expressed on infected erythrocytes and mediates binding to chondroitin sulfate A, initiating inflammation and disrupting homeostasis at the maternal-fetal interface. Although antibodies can prevent sequestration, it is unclear whether parasite clearance is due to antibodies to a single Duffy binding-like (DBL) domain or to an extensive repertoire of antibodies to multiple DBL domains and allelic variants. Accordingly, plasma samples collected longitudinally from pregnant women were screened for naturally acquired antibodies against an extensive panel of VAR2CSA proteins, including 2 to 3 allelic variants for each of 5 different DBL domains. Analyses were performed on plasma samples collected from 3 to 9 months of pregnancy from women living in areas in Cameroon with high and low malaria transmission. The results demonstrate that high antibody levels to multiple VAR2CSA domains, rather than a single domain, were associated with the absence of placental malaria when antibodies were present from early in the second trimester until term. Absence of placental malaria was associated with increasing antibody breadth to different DBL domains and allelic variants in multigravid women. Furthermore, the antibody responses of women in the lower-transmission site had both lower magnitude and lesser breadth than those in the high-transmission site. These data suggest that immunity to placental malaria results from high antibody levels to multiple VAR2CSA domains and allelic variants and that antibody breadth is influenced by malaria transmission intensity.
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