TNFα rs1799964 TT genotype may be a susceptibility factor for vertical transmission of Toxoplasma gondii and clinical signs in newborns from pregnant women with acute toxoplasmosis.

BACKGROUND: One of the main impacts of Toxoplasma gondii infection occurs during pregnancy and is related to the vertical transmission of the parasite (congenital toxoplasmosis), which can cause severe clinical outcomes and fetal death. During acute infection, in order to control the rapid replication of tachyzoites, different host immune response genes are activated, and these include cytokine-encoding genes. Considering that polymorphisms in cytokine genes may increase susceptibility to vertical transmission of T. gondii by determining the immune status of the pregnant woman, this study evaluated the influence of polymorphisms of tumor necrosis factor alpha (TNFα) rs1799964 (- 1031) and interleukin 1 beta (IL1ß) rs16944 (- 511) genes on gestational toxoplasmosis and on the vertical transmission of the parasite and verified the allele and genotype frequency of these polymorphisms in pregnant patients whose respective newborn did or did not present clinical abnormalities suggestive of congenital toxoplasmosis. METHODS AND RESULTS: A total of 204 pregnant patients with (n = 114) or without (n = 90) infection by T. gondii were enrolled. No associations were found involving the polymorphisms rs1799964 (- 1031) of the TNFα gene and rs16944 (- 511) of the IL1ß gene with the increased chance of T. gondii infection during pregnancy. However, it was observed that the maternal TT genotype referring to the polymorphism of the TNFα gene seems to influence the vertical transmission of the parasite (P = 0.01; χ2 = 6.05) and the presence of clinical manifestation in newborns from pregnancies with acute toxoplasmosis (P = 0.007; χ2 = 9.68). CONCLUSION: The TNFα rs1799964 TT genotype may act as a susceptibility factor for the vertical transmission of parasite and for the presence of clinical signs in newborns from pregnant women with acute toxoplasmosis.

Peripheral Plasmodium falciparum Infection in Early Pregnancy Is Associated With Increased Maternal Microchimerism in the Offspring.

BACKGROUND: Placental malaria has been associated with increased cord blood maternal microchimerism (MMc), which in turn may affect susceptibility to malaria in the offspring. We sought to determine the impact of maternal peripheral Plasmodium falciparum parasitemia during pregnancy on MMc and to determine whether maternal cells expand during primary parasitemia in the offspring. METHODS: We conducted a nested cohort study of maternal-infant pairs from a prior pregnancy malaria chemoprevention study. Maternal microchimerism was measured by quantitative polymerase chain reaction targeting a maternal-specific marker in genomic DNA from cord blood, first P falciparum parasitemia, and preparasitemia. Logistic and negative binomial regression were used to assess the impact of maternal peripheral parasitemia, symptomatic malaria, and placental malaria on cord blood MMc. Generalized estimating equations were used to assess predictors of MMc during infancy. RESULTS: Early maternal parasitemia was associated with increased detection of cord blood MMc (adjusted odds ratio = 3.91, P = .03), whereas late parasitemia, symptomatic malaria, and placental malaria were not. The first parasitemia episode in the infant was not associated with increased MMc relative to preparasitemia. CONCLUSIONS: Maternal parasitemia early in pregnancy may increase the amount of MMc acquired by the fetus. Future work should investigate the impact of this MMc on immune responses in the offspring.

Macrophage migration inhibitory factor (MIF) and pregnancy may impact the balance of intestinal cytokines and the development of intestinal pathology caused by Toxoplasma gondii infection.

Toxoplasma gondii (T. gondii) is an intracellular parasite responsible for causing toxoplasmosis. When infection occurs during pregnancy, it can produce severe congenital infection with ocular and neurologic damage to the infant. From the oral infection parasite reaches the intestine, causing inflammatory response, damage in tissue architecture and systemic dissemination. Macrophage migration inhibition factor (MIF) is a cytokine secreted from both immune and non-immune cells, including gut epithelial cells. MIF is described to promote inflammatory responses, to be associated in colitis pathogenesis and also to play role in maintaining the intestinal barrier. The aim of the present study was to evaluate the influence of the pregnancy and MIF deficiency on T. gondii infection in the intestinal microenvironment and to address how these factors can impact on the intestinal architecture and local cytokine profile. For this purpose, small intestine of pregnant and non-pregnant C57BL/6 MIF deficient mice (MIF-/-) and Wild-type (WT) orally infected with 5 cysts of ME-49 strain of T. gondii were collected on day 8th of infection. Intestines were processed for morphological and morphometric analyses, parasite quantification and for cytokines mensuration. Our results showed that the absence of MIF and pregnancy caused an increase in T. gondii infection index. T. gondii immunolocalization demonstrated that segments preferentially infected with T. gondii were duodenum and ileum. The infection caused a reduction in the size of the intestinal villi, whereas, infection associated with pregnancy caused an increase in villi size due to edema caused by the infection. Also, the goblet cell number was increased in the ileum of MIF-/- mice, when compared to the corresponding WT group. Analyses of cytokine production in the small intestine showed that MIF was up regulated in the gut of pregnant WT mice due to infection. Also, infection provoked an intense Th1 response that was more exacerbated in pregnant MIF-/- mice. We also detected that the Th2/Treg response was more pronounced in MIF-/- mice. Altogether, our results demonstrated that pregnancy and MIF deficiency interferes in the balance of the intestinal cytokines and favors a Th1-immflamatory profile, which in turn, impact in the development of pathology caused by T. gondii infection in the intestinal microenvironment.

Pregnancy and CYP3A5 Genotype Affect Day 7 Plasma Lumefantrine Concentrations.

Pregnancy and pharmacogenetics variation alter drug disposition and treatment outcome. The objective of this study was to investigate the effect of pregnancy and pharmacogenetics variation on day 7 lumefantrine (LF) plasma concentration and therapeutic responses in malaria-infected women treated with artemether-lumefantrine (ALu) in Tanzania. A total of 277 (205 pregnant and 72 nonpregnant) women with uncomplicated Plasmodium falciparum malaria were enrolled. Patients were treated with ALu and followed up for 28 days. CYP3A4, CYP3A5, and ABCB1 genotyping were done. Day 7 plasma LF concentration and the polymerase chain reaction (PCR) - corrected adequate clinical and parasitological response (ACPR) at day 28 were determined. The mean day 7 plasma LF concentrations were significantly lower in pregnant women than nonpregnant women [geometric mean ratio = 1.40; 95% confidence interval (CI) of geometric mean ratio (1.119-1.1745), P < 0.003]. Pregnancy, low body weight, and CYP3A5*1/*1 genotype were significantly associated with low day 7 LF plasma concentration (P < 0.01). PCR-corrected ACPR was 93% (95% CI = 89.4-96.6) in pregnant women and 95.7% (95% CI = 90.7-100) in nonpregnant women. Patients with lower day 7 LF concentration had a high risk of treatment failure (mean 652 vs. 232 ng/ml, P < 0.001). In conclusion, pregnancy, low body weight, and CYP3A5*1 allele are significant predictors of low day 7 LF plasma exposure. In turn, lower day 7 LF concentration is associated with a higher risk of recrudescence. SIGNIFICANCE STATEMENT: This study reports a number of factors contributing to the lower day 7 lumefantrine (LF) concentration in women, which includes pregnancy, body weight, and CYP3A5*1/*1 genotype. It also shows that day 7 LF concentration is a main predictor of malaria treatment. These findings highlight the need to look into artemether-LF dosage adjustment in pregnant women so as to be able to maintain adequate drug concentration, which is required to reduce treatment failure rates in pregnant women.

MiRNA-146a polymorphism increases the odds of malaria in pregnancy.

BACKGROUND: Plasmodium falciparum infection during pregnancy is a major cause of poor maternal health, adverse foetal outcome and infant mortality in sub-Saharan Africa. Genetic disposition is involved in susceptibility to malaria in pregnancy and its manifestation. MicroRNAs (miRNAs) influence gene regulation including that of innate immune responses. A miRNA-146a rs2910164 G > C single nucleotide polymorphism (SNP) has been associated with increased risks of several diseases, but no data as to malaria are available. METHODS: The association between miRNA-146a rs2910164 and P. falciparum infection among 509 Ghanaian women attending antenatal care (ANC) and 296 delivering Ghanaian primiparae was investigated. Malaria parasites were diagnosed by microscopy and PCR. Leukocyte-associated hemozoin in placental samples was recorded as well. Proportions were compared between groups by Fisher's exact test, and logistic regression models were used to adjust for possible confounders. RESULTS: By PCR, P. falciparum infection was detected in 63% and 67% of ANC attendees and delivering primiparae, respectively. In both groups, two in three women were either heterozygous or homozygous for miRNA-146a rs2910164. Among ANC attendees, homozygosity conferred increased odds of infection (adjusted odds ratio (aOR), 2.3; 95% CI, 1.3-4.0), which was pronounced among primigravidae (aOR, 5.8; 95% CI, 1.6-26) but only marginal in multigravidae. Likewise, homozygosity for miRNA-146a rs2910164 in primiparae increased the odds of past or present placental P. falciparum infection almost six-fold (aOR, 5.9; 95% CI, 2.1-18). CONCLUSIONS: These results indicate that SNP rs2910164 G > C is associated with increased odds for P. falciparum infection in first-time pregnant women who are considered to lack sufficient acquired immune responses against pregnancy-specific strains of P. falciparum. These findings suggest that miRNA-146a is involved in protective malarial immunity, and specifically in the innate component.

Decidual macrophage M1 polarization contributes to adverse pregnancy induced by Toxoplasma gondii PRU strain infection.

Recent evidence indicates that macrophages at the maternal-fetal interface adapt to a phenotype characterized by alternative activation (M2 polarization) and exhibit immunosuppressive functions that favor the maintenance of pregnancy. The bias of M2 decidual macrophages toward M1 has been clinically linked to pregnancy-related complications, such as preeclampsia and preterm delivery. The aim of this study was to investigate the effect of Toxoplasma gondii PRU strain infection on the bias of decidual macrophage polarization and its contribution to adverse pregnancy outcomes. A mouse model with adverse pregnancy outcome was established by infection with T. gondii PRU strain and the expression levels of functional molecules in decidual macrophages of mice were measured. The results showed that T. gondii infection caused seriously adverse pregnancy outcome in mice. The placentae of infected mice showed obvious congestion and inflammatory cell infiltration. The expression of CD206, MHC-II, and arginase-1 considered as M2 markers was decreased in decidual macrophages after T. gondii infection, whereas the expression of CD80, CD86, iNOS, and cytokines TNF-α and IL-12 considered as M1 markers was increased. Furthermore, iNOS-positive expression was observed in the decidua basalis of infected mice. Our results indicated that T. gondii infection was responsible for the bias of M2 decidual macrophages toward M1, which changes the immunosuppressive microenvironment at the maternal-fetal interface and contributes to adverse pregnancy outcomes.

Genetic modifications of cytokine genes and Toxoplasma gondii infections in pregnant women.

PURPOSE: Toxoplasma gondii causes one of the most common intrauterine infections worldwide, thus being a severe threat during pregnancy. IL1, IL6, IL10, IL12, and TNF-α cytokines were reported to be involved in immune responses to infections with T. gondii. The research was aimed to reveal relationships between genetic changes within the polymorphisms of these cytokine genes and the incidence of T. gondii infection among pregnant women, as well as congenital transmission of the parasite to the foetuses of their infected mothers. METHODS: The primary study was performed in 148 Polish pregnant women, including 74 T. gondii-infected patients and 74 age-matched uninfected individuals; and further analysis - among the additional 142 pregnant women. Genotypes within IL1A -889 C>T, IL1B +3954 C>T, IL6 -174 G>C, IL10 -1082 G>A, IL12B -1188 A>C and TNFA -308 G>A single nucleotide polymorphisms (SNPs) were determined, using self-designed nested PCR-RFLP assays. Randomly selected PCR products, representing distinct genotypes in the analyzed polymorphisms, were confirmed by sequencing, using the Sanger method. A statistical analysis was carried out of relationships between genetic alterations within studied SNPs and the occurrence of T. gondii infection, using the following tools: cross-tabulation, Pearson's Chi-square test and the logistic regression model to estimate genetic models of inheritance. A power analysis of statistically significant outcomes was performed by Cramér's V test. RESULTS: A multiple-SNP analysis showed TC haplotype for IL1A and IL1B SNPs to be significantly associated with a decreased risk of the parasitic infection (OR 0.41, P≤0.050). The association remained important after power analysis (Cramér's V = 0.39, χ2 = 7.73, P≤0.050), and the additional analysis with larger groups of patients (OR 0.47, P≤0.050). Moreover, the CCCAGA complex variants were for all the studied polymorphisms at an increased risk of T. gondii infection (OR 8.14, P≤0.050), although this strong relationship was not significant in the further analysis (Cramér's V = 0.76, χ2 = 26.81, P = 0.310). Regarding the susceptibility to congenital transmission of T. gondii from mothers to their foetuses among the infected pregnant women, the presence of GA heterozygotic status within IL10 polymorphism significantly increased the risk of parasitic transmission (OR 5.73 in the codominant model and OR 5.18 in the overdominant model; P≤0.050). The correlation stayed important in the power analysis (Cramér's V = 0.29, χ2 = 6.03, P≤0.050), although it was non-significant in larger groups of patients. Important relationships specific for the first study cohort remained non-significant in the second group of studied pregnant women. CONCLUSIONS: Within the analyzed cohort of Polish pregnant women, the genetic modifications from SNPs of genes, encoding both the proinflammatory IL1α, IL1ß, IL6, IL12 and TNF-α, and anti-inflammatory IL10 cytokines, may have been associated with susceptibility to T. gondii infection. It is the first study on the contribution of cytokine genes polymorphisms to the occurrence of T. gondii infection during pregnancy. Further studies for other populations of pregnant women would be justified to reveal a detailed role of the analyzed polymorphisms for the occurrence of T. gondii infections during pregnancy.

Maternal Microchimerism Predicts Increased Infection but Decreased Disease due to Plasmodium falciparum During Early Childhood.

Background: A mother's infection with placental malaria (PM) can affect her child's susceptibility to malaria, although the mechanism remains unclear. The fetus acquires a small amount of maternal cells and DNA known as maternal microchimerism (MMc), and we hypothesized that PM increases MMc and that MMc alters risk of Plasmodium falciparum malaria during infancy. Methods: In a nested cohort from Muheza, Tanzania, we evaluated the presence and level of cord blood MMc in offspring of women with and without PM. A maternal-specific polymorphism was identified for each maternal-infant pair, and MMc was assayed by quantitative polymerase chain reaction. The ability of MMc to predict malaria outcomes during early childhood was evaluated in longitudinal models. Results: Inflammatory PM increased the detection rate of MMc among offspring of primigravidae and secundigravidae, and both noninflammatory and inflammatory PM increased the level of MMc. Detectable MMc predicted increased risk of positive blood smear but, interestingly, decreased risk of symptomatic malaria and malaria hospitalization. Conclusions: The acquisition of MMc may result in increased malaria infection but protection from malaria disease. Future studies should be directed at the cellular component of MMc, with attention to its ability to directly or indirectly coordinate anti-malarial immune responses in the offspring.

Effect of pharmacogenetics on plasma lumefantrine pharmacokinetics and malaria treatment outcome in pregnant women.

BACKGROUND: Pregnancy has considerable effects on the pharmacokinetic properties of drugs used to treat uncomplicated Plasmodium falciparum malaria. The role of pharmacogenetic variation on anti-malarial drug disposition and efficacy during pregnancy is not well investigated. The study aimed to examine the effect of pharmacogenetics on lumefantrine (LF) pharmacokinetics and treatment outcome in pregnant women. METHODS: Pregnant women with uncomplicated falciparum malaria were enrolled and treated with artemether-lumefantrine (ALu) at Mkuranga and Kisarawe district hospitals in Coast Region of Tanzania. Day-7 LF plasma concentration and genotyping forCYP2B6 (c.516G>T, c.983T>C), CYP3A4*1B, CYP3A5 (*3, *6, *7) and ABCB1 c.4036A4G were determined. Blood smear for parasite quantification by microscopy, and dried blood spot for parasite screening and genotyping using qPCR and nested PCR were collected at enrolment up to day 28 to differentiate between reinfection from recrudescence. Treatment response was recorded following the WHO protocol. RESULTS: In total, 92 pregnant women in their second and third trimester were included in the study and 424 samples were screened for presence of P. falciparum. Parasites were detected during the follow up period in 11 (12%) women between day 7 and 28 after treatment and PCR genotyping confirmed recrudescent infection in 7 (63.3%) women. The remaining four (36.4%) pregnant women had reinfection: one on day 14 and three on day 28. The overall PCR-corrected treatment failure rate was 9.0% (95% CI 4.4-17.4). Day 7 LF concentration was not significantly influenced by CYP2B6, CYP3A4*1B and ABCB1 c.4036A>G genotypes. Significant associations between CYP3A5 genotype and day 7 plasma LF concentrations was found, being higher in carriers of CYP3A5 defective variant alleles than CYP3A5*1/*1 genotype. No significant influence of CYP2B6, CYP3A5 and ABCB1 c.4036A>Genotypes on malaria treatment outcome were observed. However, CYP3A4*1B did affect malaria treatment outcome in pregnant women followed up for 28 days (P = 0.018). CONCLUSIONS: Genetic variations in CYP3A4 and CYP3A5may influence LF pharmacokinetics and treatment outcome in pregnant women.

Molecular dissection of placental malaria protein VAR2CSA interaction with a chemo-enzymatically synthesized chondroitin sulfate library.

Placental malaria, a serious infection caused by the parasite Plasmodium falciparum, is characterized by the selective accumulation of infected erythrocytes (IEs) in the placentas of the pregnant women. Placental adherence is mediated by the malarial VAR2CSA protein, which interacts with chondroitin sulfate (CS) proteoglycans present in the placental tissue. CS is a linear acidic polysaccharide composed of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-galactosamine that are modified by sulfate groups at different positions. Previous reports have shown that placental-adhering IEs were associated with an unusually low sulfated form of chondroitin sulfate A (CSA) and that a partially sulfated dodecasaccharide is the minimal motif for the interaction. However, the fine molecular structure of this CS chain remains unclear. In this study, we have characterized the CS chain that interacts with a recombinant minimal CS-binding region of VAR2CSA (rVAR2) using a CS library of various defined lengths and sulfate compositions. The CS library was chemo-enzymatically synthesized with bacterial chondroitin polymerase and recombinant CS sulfotransferases. We found that C-4 sulfation of the N-acetyl-D-galactosamine residue is critical for supporting rVAR2 binding, whereas no other sulfate modifications showed effects. Interaction of rVAR2 with CS is highly correlated with the degree of C-4 sulfation and CS chain length. We confirmed that the minimum structure binding to rVAR2 is a tri-sulfated CSA dodecasaccharide, and found that a highly sulfated CSA eicosasaccharide is a more potent inhibitor of rVAR2 binding than the dodecasaccharides. These results suggest that CSA derivatives may potentially serve as targets in therapeutic strategies against placental malaria.

IFN-gamma and TNF associated with severe falciparum malaria infection in Saudi pregnant women.

BACKGROUND: Tumour necrosis factor (TNF) and interferon gamma (IFN-γ), encoded by TNF-836 C/A (rs 1800630) and IFN-γ -1616 C/T (rs2069705) genes, are key immunological mediators that are believed to both play protective and pathological roles in malaria. The aim of this study was to investigate the relationship between TNF-836 C/A and IFN-γ-1616 C/T polymorphism and susceptibility to severe malaria in pregnant women. METHODS: A prospective cohort (cross-sectional) study was conducted in pregnant women attending the out-patient clinic in King Fahad Specialist Hospital in Jazan (KFSHJ), with a clinical diagnosis of malaria. A total of one hundred and eighty six pregnant women were genotyped for single nucleotide polymorphism (SNP) for TNF and IFN-γ using Taqman® MGB Probes. Serum cytokine concentrations were measured by sandwich ELISA method. RESULTS: A hospital case-control study of severe malaria in a Saudi population identified strong associations with individual single-nucleotide polymorphisms in the TNF and IFN-γ genes, and defined TNF-836 C and IFN-γ-1616 T genotypes and alleles which were statistically significantly associated with severe malaria infection. Furthermore, TNF-836 CC and IFN-γ-1616 TT genotypes were associated with higher serum concentration of TNF and IFN-γ, respectively, and with susceptibility to severe malaria. CONCLUSIONS: This data provides a starting point for functional and genetic analysis of the TNF and IFN-γ genomic region in malaria infection affecting Saudi populations.

An ATP2B4 polymorphism protects against malaria in pregnancy.

Polymorphisms of ATP2B4 encoding an ubiquitous Ca(2+) pump protect against severe childhood malaria. We assessed the influence of a main polymorphism (rs10900585) on malaria among 834 delivering Ghanaian women. In homozygous primiparae, the odds of placental Plasmodium falciparum infection were reduced by 64%. No influence of the polymorphism on parasite density, low birth weight, or preterm delivery was discernible. However, malarial anemia was greatly reduced in primiparous carriers of the variant allele, paralleling the reduced impact of malaria on hemoglobin levels in this group. A common ATP2B4 polymorphism protects against malaria in pregnancy and related maternal anemia, suggesting ATP2B4 variant associated protection not to be limited to severe childhood malaria.

Sub-microscopic Plasmodium falciparum infections and multiple drug resistant single nucleotide polymorphic alleles in pregnant women from southwestern Nigeria.

OBJECTIVES: The study evaluated sub-microscopic malaria infections in pregnancy using two malaria Rapid Diagnostic Tests (mRDTs), microscopy and RT-PCR and characterized Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and Plasmodium falciparum dihydropteroate synthase (Pfdhps) drug resistant markers in positive samples. METHODS: This was a cross sectional survey of 121 pregnant women. Participants were finger pricked, blood drops were collected for rapid diagnosis with P. falciparum histidine-rich protein 11 rapid diagnostic test kit and the ultra-sensitive Alere Pf malaria RDT, Blood smears for microscopy and dried blood spots on Whatman filter paper for molecular analysis were made. Real time PCR targeting the var acidic terminal sequence (varATS) gene of P. falciparum was carried out on a CFX 96 real time system thermocycler (BioRad) in discriminating malaria infections. For each run, laboratory strain of P. falciparum 3D7 and nuclease free water were used as positive and negative controls respectively. Additionally, High resolution melt analyses was employed for genotyping of the different drug resistance markers. RESULTS: Out of one hundred and twenty-one pregnant women sampled, the SD Bioline™ Malaria Ag P.f HRP2-based malaria rapid diagnostic test (mRDT) detected eight (0.06%) cases, the ultra-sensitive Alere™ malaria Ag P.f rapid diagnostic test mRDT had similar outcome in the same samples as detected by the HRP2-based mRDT. Microscopy and RT-PCR confirmed four out of the eight infections detected by both rapid diagnostic tests as true positive and RT-PCR further detected three false negative samples by the two mRDTs providing a sub-microscopic malaria prevalence of 3.3%. Single nucleotide polymorphism in Pfdhps gene associated with sulphadoxine resistance revealed the presence of S613 mutant genotypes in three of the seven positive isolates and isolates with mixed wild/mutant genotype at codon A613S. Furthermore, four mixed genotypes at the A581G codon were also recorded while the other Pfdhps codons (A436G, A437G and K540E) showed the presence of wild type alleles. In the Pfdhfr gene, there were mutations in 28.6%, 28.6%, and 85.7% at the I51, R59 and N108 codons respectively. Mixed wild and mutant type genotypes were also observed in 28.6% each of the N51I, and C59R codons. For the Pfcrt, two haplotypes CVMNK and CVIET were observed. The SVMNT was altogether absent. Triple mutant CVIET 1(14.3%) and triple mutant + wild genotype CVIET + CVMNK 1(14.3%) were observed. The Pfmdr1 haplotypes were single mutants YYND 1(14.3%); NFND 1(14.3%) and double mutants YFND 4(57.1%); YYDD 1(14.3%).

Increased prevalence of dhfr and dhps mutants at delivery in Malawian pregnant women receiving intermittent preventive treatment for malaria.

In the context of an Intermittent preventive treatment (IPTp) trial for pregnant women in Malawi, Plasmodium falciparum samples from 85 women at enrollment and 35 women at delivery were genotyped for mutations associated with sulfadoxine-pyrimethamine resistance. The prevalence of the highly resistant haplotype with mutations at codons 51 and 108 of dihydrofolate reductase (dhfr) and codons 437 and 540 of dihydropteroate synthase (dhps) increased from 81% at enrollment to 100% at delivery (P = 0.01). Pregnant women who were smear-positive at enrollment were more likely to have P. falciparum parasitemia at delivery. These results lend support to concerns that IPTp use may lead to increased drug resistance in pregnant women during pregnancy and emphasise the importance of screening pregnant women for malaria parasites in areas with prevalent SP resistance even when they are already on IPTp.

The chondroitin sulfate A-binding site of the VAR2CSA protein involves multiple N-terminal domains.

Malaria during pregnancy is a major health problem for African women. The disease is caused by Plasmodium falciparum malaria parasites, which accumulate in the placenta by adhering to chondroitin sulfate A (CSA). The interaction between infected erythrocytes and the placental receptor is mediated by a parasite expressed protein named VAR2CSA. A vaccine protecting pregnant women against placental malaria should induce antibodies inhibiting the interaction between VAR2CSA and CSA. Much effort has been put into defining the part of the 350 kDa VAR2CSA protein that is responsible for binding. It has been shown that full-length recombinant VAR2CSA binds specifically to CSA with high affinity, however to date no sub-fragment of VAR2CSA has been shown to interact with CSA with similar affinity or specificity. In this study, we used a biosensor technology to examine the binding properties of a panel of truncated VAR2CSA proteins. The experiments indicate that the core of the CSA-binding site is situated in three domains, DBL2X-CIDR(PAM) and a flanking domain, located in the N-terminal part of VAR2CSA. Furthermore, recombinant VAR2CSA subfragments containing this region elicit antibodies with high parasite adhesion blocking activity in animal immunization experiments.

Mutations of complement lectin pathway genes MBL2 and MASP2 associated with placental malaria.

BACKGROUND: Innate immunity plays a crucial role in the host defense against malaria including Plasmodium falciparum malaria in pregnancy, but the roles of the various underlying genes and mechanisms predisposing to the disease are poorly understood. METHODS: 98 single-nucletoide polymorphisms were genotyped in a set of 17 functionally related genes of the complement system in 145 primiparous Ghanaian women with placental malaria, defined by placental parasitaemia or malaria pigment, and as a control, in 124 non-affected primiparae. RESULTS: Placental malaria was significantly associated with SNPs in the lectin pathway genes MBL2, MASP2, FCN2 and in properdin. In particular, the main African mannose-binding lectin deficiency variant (MBL2*G57E, rs1800451) increased the odds of placental malaria (OR 1.6; permuted p-value 0.014). In contrast, a common MASP2 mutation (R439H, rs12085877), which reduces the activity of MBL-MASP2 complexes occurred in 33% of non-affected women and in 22% primiparae with placental malaria (OR 0.55, permuted p-value 0.020). CONCLUSIONS: Excessive complement activation is of importance in the pathogenesis of placental malaria by mediating inflammation, coagulation, and endothelial dysfunction. Mutated MBL and MASP2 proteins could have direct intrinsic effects on the susceptibility to placental malaria, in addition to their roles in regulation of downstream complement activation.

A variant in the gene FUT9 is associated with susceptibility to placental malaria infection.

Malaria in pregnancy forms a substantial part of the worldwide burden of malaria, with an estimated annual death toll of up to 200 000 infants, as well as increased maternal morbidity and mortality. Studies of genetic susceptibility to malaria have so far focused on infant malaria, with only a few studies investigating the genetic basis of placental malaria, focusing only on a limited number of candidate genes. The aim of this study therefore was to identify novel host genetic factors involved in placental malaria infection. To this end we carried out a nested case-control study on 180 Mozambican pregnant women with placental malaria infection, and 180 controls within an intervention trial of malaria prevention. We genotyped 880 SNPs in a set of 64 functionally related genes involved in glycosylation and innate immunity. A single nucleotide polymorphism (SNP) located in the gene FUT9, rs3811070, was significantly associated with placental malaria infection (odds ratio = 2.31, permutation P-value=0.028). Haplotypic analysis revealed a similarly strong association of a common haplotype of four SNPs including rs3811070. FUT9 codes for a fucosyl-transferase that is catalyzing the last step in the biosynthesis of the Lewis-x antigen, which forms part of the Lewis blood group-related antigens. These results therefore suggest an involvement of this antigen in the pathogenesis of placental malaria infection.

Cytokine gene expression at the materno-foetal interface after experimental Neospora caninum infection of heifers at 110 days of gestation.

Neospora caninum is a major cause of abortion in cattle, but the reasons why only some animals abort remain unclear. The immunological control of the parasite in the placenta or by the foetus could be the key to determining the mechanism of abortion and/or transplacental transmission to the foetus. In this study, cytokine gene expression, analysed by real-time RT-PCR, at the maternal (caruncle) and foetal placenta (cotyledon) of heifers infected at 110 days of gestation by intravenous inoculation of N. caninum tachyzoites was compared with the responses in uninfected heifers. Animals were euthanized 3 weeks after infection. Upregulated Th1, Th2 and T-regulatory (Treg) cytokine gene expression was observed in both the maternal and the foetal placenta in the infected group. In the caruncle of infected animals, the main changes included upregulation of IFN-γ, IL-12p40, IL-6 and IL-10. In the cotyledon, the main changes included upregulation of IFN-γ and downregulation of TGF-ß, being the later the only cytokine downregulated in the infected group. The observed cytokine expression pattern was associated with alive but transplacentally infected foetuses, suggesting that such cytokine pattern is beneficial to foetal survival, but could have a role in the transplacental transmission of the parasite.

Natural selection of FLT1 alleles and their association with malaria resistance in utero.

Placental malaria (PM) caused by Plasmodium falciparum contributes significantly to infant mortality in sub-Saharan Africa and is associated with pregnancy loss. We hypothesized that fetal genes that modify PM would be associated with fetal fitness. During PM, placental trophoblasts produce soluble fms-like tyrosine kinase 1 (sFlt1), also known as soluble VEGF receptor 1, an angiogenesis inhibitor associated with preeclampsia. Here we present a study examining the genotype of the fms-related tyrosine kinase 1 (FLT1) 3' UTR in Tanzanian mother-infant pairs. First-time mothers suffer the most PM, and newborn FLT1 genotype distribution differed by birth order, with newborns of first-time mothers outside of Hardy-Weinberg equilibrium (HWE) during peak PM season. Among first-time but not other mothers, maternal FLT1 genotype was associated with a history of prior pregnancy loss. During PM, newborn FLT1 genotype was associated with low birth weight and placental inflammatory gene expression. FLT1 genotype was also associated with Flt1 levels among study subjects and in vitro. Thus, FLT1 variants confer fetal fitness in utero and are associated with the maternal immune response during PM. This indicates that FLT1 is under natural selection in a malaria endemic area and that human exposure to malaria can influence the evolutionary genetics of the maternal-fetal relationship.

Toxoplasma gondii GRA28 Is Required for Placenta-Specific Induction of the Regulatory Chemokine CCL22 in Human and Mouse.

Toxoplasma gondii is an intracellular protozoan pathogen of humans that can cross the placenta and result in adverse pregnancy outcomes and long-term birth defects. The mechanisms used by T. gondii to cross the placenta are unknown, but complex interactions with the host immune response are likely to play a role in dictating infection outcomes during pregnancy. Prior work showed that T. gondii infection dramatically and specifically increases the secretion of the immunomodulatory chemokine CCL22 in human placental cells during infection. Given the important role of this chemokine during pregnancy, we hypothesized that CCL22 induction was driven by a specific T. gondii-secreted effector. Using a combination of bioinformatics and molecular genetics, we have now identified T. gondii GRA28 as the gene product required for CCL22 induction. GRA28 is secreted into the host cell, where it localizes to the nucleus, and deletion of the GRA28 gene results in reduced CCL22 placental cells as well as a human monocyte cell line. The impact of GRA28 on CCL22 production is also conserved in mouse immune and placental cells both in vitro and in vivo. Moreover, parasites lacking GRA28 are impaired in their ability to disseminate throughout the animal, suggesting a link between CCL22 induction and the ability of the parasite to cause disease. Overall, these data demonstrate a clear function for GRA28 in altering the immunomodulatory landscape during infection of both placental and peripheral immune cells and show a clear impact of this immunomodulation on infection outcome. IMPORTANCE Toxoplasma gondii is a globally ubiquitous pathogen that can cause severe disease in HIV/AIDS patients and can also cross the placenta and infect the developing fetus. We have found that placental and immune cells infected with T. gondii secrete significant amounts of a chemokine (called CCL22) that is critical for immune tolerance during pregnancy. In order to better understand whether this is a response by the host or a process that is driven by the parasite, we have identified a T. gondii gene that is absolutely required to induce CCL22 production in human cells, indicating that CCL22 production is a process driven almost entirely by the parasite rather than the host. Consistent with its role in immune tolerance, we also found that T. gondii parasites lacking this gene are less able to proliferate and disseminate throughout the host. Taken together, these data illustrate a direct relationship between CCL22 levels in the infected host and a key parasite effector and provide an interesting example of how T. gondii can directly modulate host signaling pathways in order to facilitate its growth and dissemination.