Maternal Psychosocial Stress Is Associated with Reduced Diversity in the Early Infant Gut Microbiome.

The developing infant gut microbiome is highly sensitive to environmental exposures, enabling its evolution into an organ that supports the immune system, confers protection from infection, and facilitates optimal gut and central nervous system function. In this study, we focus on the impact of maternal psychosocial stress on the infant gut microbiome. Forty-seven mother-infant dyads were recruited at the HEAL Africa Hospital in Goma, Democratic Republic of Congo. Extensive medical, demographic, and psychosocial stress data were collected at birth, and infant stool samples were collected at six weeks, three months, and six months. A composite maternal psychosocial stress score was created, based on eight questionnaires to capture a diverse range of stress exposures. Full-length 16S rRNA gene sequences were generated. Infants of mothers with high composite stress scores showed lower levels of gut microbiome beta diversity at six weeks and three months, as well as higher levels of alpha diversity at six months compared to infants of low stress mothers. Longitudinal analyses showed that infants of high stress mothers had lower levels of health-promoting Lactobacillus gasseri and Bifidobacterium pseudocatenulatum at six weeks compared to infants of low stress mothers, but the differences largely disappeared by three to six months. Previous research has shown that L. gasseri can be used as a probiotic to reduce inflammation, stress, and fatigue, as well as to improve mental state, while B. pseudocatenulatum is important in modulating the gut-brain axis in early development and in preventing mood disorders. Our finding of reduced levels of these health-promoting bacteria in infants of high stress mothers suggests that the infant gut microbiome may help mediate the effect of maternal stress on infant health and development.

Polymorphic Molecular Signatures in Variable Regions of the Plasmodium falciparum var2csa DBL3x Domain Are Associated with Virulence in Placental Malaria.

The Plasmodium falciparum protein VAR2CSA allows infected erythrocytes to accumulate within the placenta, inducing pathology and poor birth outcomes. Multiple exposures to placental malaria (PM) induce partial immunity against VAR2CSA, making it a promising vaccine candidate. However, the extent to which VAR2CSA genetic diversity contributes to immune evasion and virulence remains poorly understood. The deep sequencing of the var2csa DBL3X domain in placental blood from forty-nine primigravid and multigravid women living in malaria-endemic western Kenya revealed numerous unique sequences within individuals in association with chronic PM but not gravidity. Additional analysis unveiled four distinct sequence types that were variably present in mixed proportions amongst the study population. An analysis of the abundance of each of these sequence types revealed that one was inversely related to infant gestational age, another was inversely related to placental parasitemia, and a third was associated with chronic PM. The categorization of women according to the type to which their dominant sequence belonged resulted in the segregation of types as a function of gravidity: two types predominated in multigravidae whereas the other two predominated in primigravidae. The univariate logistic regression analysis of sequence type dominance further revealed that gravidity, maternal age, placental parasitemia, and hemozoin burden (within maternal leukocytes), reported a lack of antimalarial drug use, and infant gestational age and birth weight influenced the odds of membership in one or more of these sequence predominance groups. Cumulatively, these results show that unique var2csa sequences differentially appear in women with different PM exposure histories and segregate to types independently associated with maternal factors, infection parameters, and birth outcomes. The association of some var2csa sequence types with indicators of pathogenesis should motivate vaccine efforts to further identify and target VAR2CSA epitopes associated with maternal morbidity and poor birth outcomes.

A novel murine model of post-implantation malaria-induced preterm birth.

Despite major advances made in malaria treatment and control over recent decades, the development of new models for studying disease pathogenesis remains a vital part of malaria research efforts. The study of malaria infection during pregnancy is particularly reliant on mouse models, as a means of circumventing many challenges and costs associated with pregnancy studies in endemic human populations. Here, we introduce a novel murine model that will further our understanding of how malaria infection affects pregnancy outcome. When C57BL/6J (B6) mice are infected with Plasmodium chabaudi chabaudi AS on either embryonic day (E) 6.5, 8.5, or 10.5, preterm birth occurs in all animals by E16.5, E17.5, or E18.5 respectively, with no evidence of intrauterine growth restriction. Despite having the same outcome, we found that the time to delivery, placental inflammatory and antioxidant transcript upregulation, and the relationships between parasitemia and transcript expression prior to preterm birth differed based on the embryonic day of infection. On the day before preterm delivery, E6.5 infected mice did not experience significant upregulation of the inflammatory or antioxidant gene transcripts examined; however, peripheral and placental parasitemia correlated positively with Il1ß, Cox1, Cat, and Hmox1 placental transcript abundance. E8.5 infected mice had elevated transcripts for Ifnγ, Tnf, Il10, Cox1, Cox2, Sod1, Sod2, Cat, and Nrf2, while Sod3 was the only transcript that correlated with parasitemia. Finally, E10.5 infected mice had elevated transcripts for Ifnγ only, with a tendency for Tnf transcripts to correlate with peripheral parasitemia. Tumor necrosis factor deficient (TNF-/-) and TNF receptor 1 deficient (TNFR1-/-) mice infected on E8.5 experienced preterm birth at the same time as B6 controls. Further characterization of this model is necessary to discover the mechanism(s) and/or trigger(s) responsible for malaria-driven preterm birth caused by maternal infection during early pregnancy.

Myeloperoxidase and Other Markers of Neutrophil Activation Associate With Malaria and Malaria/HIV Coinfection in the Human Placenta.

Introduction: Placental malaria (PM) is characterized by accumulation of inflammatory leukocytes in the placenta, leading to poor pregnancy outcomes. Understanding of the underlying mechanisms remains incomplete. Neutrophils respond to malaria parasites by phagocytosis, generation of oxidants, and externalization of Neutrophil Extracellular Traps (NETs). NETs drive inflammation in malaria but evidence of NETosis in PM has not been reported. Neutrophil activity in the placenta has not been directly investigated in the context of PM and PM/HIV-co-infection. Methods: Using peripheral and placental plasma samples and placental tissue collected from Kenyan women at risk for malaria and HIV infections, we assessed granulocyte levels across all gravidities and markers of neutrophil activation, including NET formation, in primi- and secundigravid women, by ELISA, western blot, immunohistochemistry and immunofluorescence. Results: Reduced peripheral blood granulocyte numbers are observed with PM and PM/HIV co-infection in association with increasing parasite density and placental leukocyte hemozoin accumulation. In contrast, placental granulocyte levels are unchanged across infection groups, resulting in enhanced placental: peripheral count ratios with PM. Within individuals, PM- women have reduced granulocyte counts in placental relative to peripheral blood; in contrast, PM stabilizes these relative counts, with HIV coinfection tending to elevate placental counts relative to the periphery. In placental blood, indicators of neutrophil activation, myeloperoxidase (MPO) and proteinase 3 (PRTN3), are significantly elevated with PM and, more profoundly, with PM/HIV co-infection, in association with placental parasite density and hemozoin-bearing leukocyte accumulation. Another neutrophil marker, matrix metalloproteinase (MMP9), together with MPO and PRTN3, is elevated with self-reported fever. None of these factors, including the neutrophil chemoattractant, CXCL8, differs in relation to infant birth weight or gestational age. CXCL8 and MPO levels in the peripheral blood do not differ with infection status nor associate with birth outcomes. Indicators of NETosis in the placental plasma do not vary with infection, and while structures consistent with NETs are observed in placental tissue, the results do not support an association with PM. Conclusions: Granulocyte levels are differentially regulated in the peripheral and placental blood in the presence and absence of PM. PM, both with and without pre-existing HIV infection, enhances neutrophil activation in the placenta. The impact of local neutrophil activation on placental function and maternal and fetal health remains unclear. Additional investigations exploring how neutrophil activation and NETosis participate in the pathogenesis of malaria in pregnant women are needed.

Hemozoin: a Complex Molecule with Complex Activities.

PURPOSE OF REVIEW: Malaria is a disease caused by parasites that reside in host red blood cells and use hemoglobin as a nutrient source. Heme released by hemoglobin catabolism is modified by the parasite to produce hemozoin (HZ), which has toxic effects on the host. Experimentation aiming to elucidate how HZ contributes to malaria pathogenesis has utilized different preparations of this molecule, complicating interpretation and comparison of findings. We examine natural synthesis and isolation of HZ and highlight studies that have used multiple preparations, including synthetic forms, in a comparative fashion. RECENT FINDINGS: Recent work utilizing sophisticated imaging and detection techniques reveals important molecular characteristics of HZ synthesis and biochemistry. Other recent studies further refine understanding of contributions of HZ to malaria pathogenesis yet highlight the continuing need to characterize HZ preparations and contextualize experimental conditions in the in vivo infection milieu. SUMMARY: This review highlights the necessity of collectively determining what is physiologically relevant HZ. Characterization of isolated natural HZ and use of multiple preparations in each study are recommended with application of in vivo studies whenever possible. Adoption of such practices is expected to improve reproducibility of results and elucidate the myriad of ways that HZ participates in malaria pathogenesis.

A novel murine model for assessing fetal and birth outcomes following transgestational maternal malaria infection.

Plasmodium falciparum infection during pregnancy is a major cause of severe maternal illness and neonatal mortality. Mouse models are important for the study of gestational malaria pathogenesis. When infected with Plasmodium chabaudi chabaudi AS in early gestation, several inbred mouse strains abort at midgestation. We report here that outbred Swiss Webster mice infected with P. chabaudi chabaudi AS in early gestation carry their pregnancies to term despite high parasite burden and malarial hemozoin accumulation in the placenta at midgestation, with the latter associated with induction of heme oxygenase 1 expression. Infection yields reduced fetal weight and viability at term and a reduction in pup number at weaning, but does not influence postnatal growth prior to weaning. This novel model allows for the exploration of malaria infection throughout pregnancy, modeling chronic infections observed in pregnant women prior to the birth of underweight infants and enabling the production of progeny exposed to malaria in utero, which is critical for understanding the postnatal repercussions of gestational malaria. The use of outbred mice allows for the exploration of gestational malaria in a genetically diverse model system, better recapitulating the diversity of infection responses observed in human populations.

Composition of the gut microbiota transcends genetic determinants of malaria infection severity and influences pregnancy outcome.

BACKGROUND: Malaria infection in pregnancy is a major cause of maternal and foetal morbidity and mortality worldwide. Mouse models for gestational malaria allow for the exploration of the mechanisms linking maternal malaria infection and poor pregnancy outcomes in a tractable model system. The composition of the gut microbiota has been shown to influence susceptibility to malaria infection in inbred virgin mice. In this study, we explore the ability of the gut microbiota to modulate malaria infection severity in pregnant outbred Swiss Webster mice. METHODS: In Swiss Webster mice, the composition of the gut microbiota was altered by disrupting the native gut microbes through broad-spectrum antibiotic treatment, followed by the administration of a faecal microbiota transplant derived from mice possessing gut microbes reported previously to confer susceptibility or resistance to malaria. Female mice were infected with P. chabaudi chabaudi AS in early gestation, and the progression of infection and pregnancy were tracked throughout gestation. To assess the impact of maternal infection on foetal outcomes, dams were sacrificed at term to assess foetal size and viability. Alternatively, pups were delivered by caesarean section and fostered to assess neonatal survival and pre-weaning growth in the absence of maternal morbidity. A group of dams was also euthanized at mid-gestation to assess infection and pregnancy outcomes. FINDINGS: Susceptibility to infection varied significantly as a function of source of transplanted gut microbes. Parasite burden was negatively correlated with the abundance of five specific OTUs, including Akkermansia muciniphila and OTUs classified as Allobaculum, Lactobacillus, and S24-7 species. Reduced parasite burden was associated with reduced maternal morbidity and improved pregnancy outcomes. Pups produced by dams with high parasite burdens displayed a significant reduction in survival in the first days of life relative to those from malaria-resistant dams when placed with foster dams. At midgestation, plasma cytokine levels were similar across all groups, but expression of IFNγ in the conceptus was elevated in infected dams, and IL-10 only in susceptible dams. In the latter, transcriptional and microscopic evidence of monocytic infiltration was observed with high density infection; likewise, accumulation of malaria haemozoin was enhanced in this group. These responses, combined with reduced vascularization of the placenta in this group, may contribute to poor pregnancy outcomes. Thus, high maternal parasite burden and associated maternal responses, potentially dictated by the gut microbial community, negatively impacts term foetal health and survival in the early postnatal period. INTERPRETATION: The composition of the gut microbiota in Plasmodium chabaudi chabaudi AS-infected pregnant Swiss Webster mice transcends the outbred genetics of the Swiss Webster mouse stock as a determinant of malaria infection severity, subsequently influencing pregnancy outcomes in malaria-exposed progeny. FUND: Research reported in this manuscript was supported by the University of Florida College of Veterinary Medicine (JMM, MM, and MG), the National Institute of Allergy and Infectious Diseases, the National Institute of Diabetes and Digestive and Kidney Diseases, and the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under award numbers T32AI060546 (to CDMS), R01HD46860 and R21AI111242 (to JMM), and R01 DK109560 (to MM). MG was supported by Department of Infectious Diseases and Immunology and University of Florida graduate assistantships. AA was supported by the 2017-2019 Peach State LSAMP Bridge to the Doctorate Program at the University of Georgia (National Science Foundation, Award # 1702361). The content is solely the responsibility of the authors and does not necessarily represent official views of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, the National Institute of Allergy and Infectious Diseases, the National Institute of Diabetes and Digestive and Kidney Diseases, or the National Institutes of Health.

The Exported Chaperone PfHsp70x Is Dispensable for the Plasmodium falciparum Intraerythrocytic Life Cycle.

Export of parasite proteins into the host erythrocyte is essential for survival of Plasmodium falciparum during its asexual life cycle. While several studies described key factors within the parasite that are involved in protein export, the mechanisms employed to traffic exported proteins within the host cell are currently unknown. Members of the Hsp70 family of chaperones, together with their Hsp40 cochaperones, facilitate protein trafficking in other organisms, and are thus likely used by P. falciparum in the trafficking of its exported proteins. A large group of Hsp40 proteins is encoded by the parasite and exported to the host cell, but only one Hsp70, P. falciparum Hsp70x (PfHsp70x), is exported with them. PfHsp70x is absent in most Plasmodium species and is found only in P. falciparum and closely related species that infect apes. Herein, we have utilized clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome editing in P. falciparum to investigate the essentiality of PfHsp70x. We show that parasitic growth was unaffected by knockdown of PfHsp70x using both the dihydrofolate reductase (DHFR)-based destabilization domain and the glmS ribozyme system. Similarly, a complete gene knockout of PfHsp70x did not affect the ability of P. falciparum to proceed through its intraerythrocytic life cycle. The effect of PfHsp70x knockdown/knockout on the export of proteins to the host red blood cell (RBC), including the critical virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1), was tested, and we found that this process was unaffected. These data show that although PfHsp70x is the sole exported Hsp70, it is not essential for the asexual development of P. falciparum. IMPORTANCE Half of the world's population lives at risk for malaria. The intraerythrocytic life cycle of Plasmodium spp. is responsible for clinical manifestations of malaria; therefore, knowledge of the parasite's ability to survive within the erythrocyte is needed to combat the deadliest agent of malaria, P. falciparum. An outstanding question in the field is how P. falciparum undertakes the essential process of trafficking its proteins within the host cell. In most organisms, chaperones such as Hsp70 are employed in protein trafficking. Of the Plasmodium species causing human disease, the chaperone PfHsp70x is unique to P. falciparum, and it is the only parasite protein of its kind exported to the host (S. Külzer et al., Cell Microbiol 14:1784-1795, 2012). This has placed PfHsp70x as an ideal target to inhibit protein trafficking and kill the parasite. However, we show that PfHsp70x is not required for export of parasite effectors and it is not essential for parasite survival inside the RBC.

Oxidative Stress: A Potential Therapeutic Target in Placental Malaria.

Placental malaria, characterized by sequestration of Plasmodium falciparum in the maternal placental blood space and associated inflammatory damage, contributes to poor birth outcomes and ~200,000 infant deaths annually. Specific mechanisms that contribute to placental damage and dysfunction during malaria are not completely understood. To investigate a potential role for oxidative stress, antioxidant genes and markers for oxidative damage were assessed by quantitative PCR and immunohistochemistry in Plasmodium chabaudi AS-infected pregnant mice. Widespread evidence of lipid peroxidation was observed and was associated with higher antioxidant gene expression in conceptuses of infected mice. To assess the extent to which this oxidative damage might contribute to poor birth outcomes and be amenable to therapeutic intervention, infected pregnant mice were treated with N-acetylcysteine, a free radical scavenger, or tempol, an intracellular superoxide dismutase mimetic. The results show that mice treated with N-acetylcysteine experienced malaria induced-pregnancy loss at the same rate as control animals and failed to mitigate placental oxidative damage. In contrast, tempol-treated mice exhibited subtle improvement in embryo survival at gestation day 12. Although lipid peroxidation was not consistently reduced in the placentas of these mice, it was inversely related to embryo viability. Moreover, reduced IFN-γ and CCL2 plasma levels in treated mice were associated with midgestational embryo viability. Thus, although oxidative stress is remarkable in placental malaria and its mitigation by antioxidant therapy may improve pregnancy outcomes, the underlying mechanistic basis and potential therapeutic strategies require additional investigation.

Helicobacter Infection Significantly Alters Pregnancy Success in Laboratory Mice.

Helicobacter spp. are gram-negative, helically shaped bacteria that cause gastric and enterohepatic infections in mammalian species. Although Helicobacter infection frequently is implicated to interfere with reproductive success, few experimental data support these claims. We therefore retrospectively investigated the effect of Helicobacter infection on murine pregnancy outcome after the identification of endemic Helicobacter infection in an animal research facility. Multiplex conventional PCR analysis was used to characterize Helicobacter infection status in one inbred and 2 transgenic strains of mice in 2 self-contained rooms assigned to the same investigator. Outcomes of timed-mating experiments were compared among Helicobacter spp.-infected and uninfected mice of the same strain; Helicobacter infection was eradicated from the colony through fostering with uninfected dams. Although Helicobacter infection affected fecundity in only one strain of transgenic mouse, the total number of embryos per gravid uterus was significantly reduced in C57BL/6J mice that were infected with a single Helicobacter species, H. typhlonius. Helicobacter infection was also associated with a significant increase in the number of resorbing embryos per uterus and significant decreases in pregnancy-associated weight gain relative to uninfected mice in C57BL6/J mice and one transgenic strain. Helicobacter spp.-infected mice of all tested strains exhibited higher frequency of intrauterine hemorrhaging relative to uninfected mice. These results indicate that naturally-acquired Helicobacter infection not only reduces the productivity of a research animal breeding colony, but also negatively impacts embryo health. Despite these deleterious effects, these data suggest that colonies can be rederived to be Helicobacter-free by Cesarean section and fostering with uninfected dams. This paper provides the first evidence that H. typhlonius infection is sufficient to interfere with reproductive success and embryo health of C57BL/6J mice. Animal research facilities should therefore implement Helicobacter spp. surveillance and control practices to avoid confounding experimental results and to improve breeding colony efficiency.

Zika virus infection disrupts neurovascular development and results in postnatal microcephaly with brain damage.

Zika virus (ZIKV) infection of pregnant women can result in fetal brain abnormalities. It has been established that ZIKV disrupts neural progenitor cells (NPCs) and leads to embryonic microcephaly. However, the fate of other cell types in the developing brain and their contributions to ZIKV-associated brain abnormalities remain largely unknown. Using intracerebral inoculation of embryonic mouse brains, we found that ZIKV infection leads to postnatal growth restriction including microcephaly. In addition to cell cycle arrest and apoptosis of NPCs, ZIKV infection causes massive neuronal death and axonal rarefaction, which phenocopy fetal brain abnormalities in humans. Importantly, ZIKV infection leads to abnormal vascular density and diameter in the developing brain, resulting in a leaky blood-brain barrier (BBB). Massive neuronal death and BBB leakage indicate brain damage, which is further supported by extensive microglial activation and astrogliosis in virally infected brains. Global gene analyses reveal dysregulation of genes associated with immune responses in virus-infected brains. Thus, our data suggest that ZIKV triggers a strong immune response and disrupts neurovascular development, resulting in postnatal microcephaly with extensive brain damage.

Parasite accumulation in placenta of non-immune baboons during Plasmodium knowlesi infection.

BACKGROUND: Placental malaria (PM) causes adverse pregnancy outcomes in the mother and her foetus. It is difficult to study PM directly in humans due to ethical challenges. This study set out to bridge this gap by determining the outcome of PM in non-immune baboons in order to develop a non-human primate model for the disease. METHODS: Ten pregnant baboons were acquired late in their third trimester (day 150) and randomly grouped as seven infected and three non-infected. Another group of four nulligravidae (non-pregnant) infected was also included in the analysis of clinical outcome. Malaria infection was intravenously initiated by Plasmodium knowlesi blood-stage parasites through the femoral vein on 160(th) day of gestation (for pregnant baboons). Peripheral smear, placental smear, haematological samples, and histological samples were collected during the study period. Median values of clinical and haematological changes were analysed using Kruskal-Wallis and Dunn's Multiple Comparison Test. Parasitaemia profiles were analysed using Mann Whitney U test. A Spearman's rank correlation was run to determine the relationship between the different variables of severity scores. Probability values of P <0.05 were considered significant. RESULTS: Levels of white blood cells increased significantly in pregnant infected (34%) than in nulligravidae infected baboons (8%). Placental parasitaemia levels was on average 19-fold higher than peripheral parasitaemia in the same animal. Infiltration of parasitized erythrocytes and inflammatory cells were also observed in baboon placenta. Malaria parasite score increased with increase in total placental damage score (rs = 0.7650, P <0.05) and inflammatory score (rs = 0.8590, P <0.05). Although the sample size was small, absence of parasitized erythrocytes in cord blood and foetal placental region suggested lack of congenital malaria in non-immune baboons. CONCLUSION: This study has demonstrated accumulation of parasitized red blood cells and infiltration of inflammatory cells in the placental intravillous space (IVS) of baboons that are non-immune to malaria. This is a key feature of placental falciparum malaria in humans. This presents the baboon as a new model for the characterization of malaria during pregnancy.

Vertical transmission of Babesia microti, United States.

Babesiosis is usually acquired from a tick bite or through a blood transfusion. We report a case of babesiosis in an infant for whom vertical transmission was suggested by evidence of Babesia spp. antibodies in the heel-stick blood sample and confirmed by detection of Babesia spp. DNA in placenta tissue.

Maternal malaria induces a procoagulant and antifibrinolytic state that is embryotoxic but responsive to anticoagulant therapy.

Low birth weight and fetal loss are commonly attributed to malaria in endemic areas, but the cellular and molecular mechanisms that underlie these poor birth outcomes are incompletely understood. Increasing evidence suggests that dysregulated hemostasis is important in malaria pathogenesis, but its role in placental malaria (PM), characterized by intervillous sequestration of Plasmodium falciparum, proinflammatory responses, and excessive fibrin deposition is not known. To address this question, markers of coagulation and fibrinolysis were assessed in placentae from malaria-exposed primigravid women. PM was associated with significantly elevated placental monocyte and proinflammatory marker levels, enhanced perivillous fibrin deposition, and increased markers of activated coagulation and suppressed fibrinolysis in placental plasma. Submicroscopic PM was not proinflammatory but tended to be procoagulant and antifibrinolytic. Birth weight trended downward in association with placental parasitemia and high fibrin score. To directly assess the importance of coagulation in malaria-induced compromise of pregnancy, Plasmodium chabaudi AS-infected pregnant C57BL/6 mice were treated with the anticoagulant, low molecular weight heparin. Treatment rescued pregnancy at midgestation, with substantially decreased rates of active abortion and reduced placental and embryonic hemorrhage and necrosis relative to untreated animals. Together, the results suggest that dysregulated hemostasis may represent a novel therapeutic target in malaria-compromised pregnancies.

Sequence polymorphism, segmental recombination and toggling amino acid residues within the DBL3X domain of the VAR2CSA placental malaria antigen.

Plasmodium falciparum malaria remains one of the world's foremost health problems, primarily in highly endemic regions such as Sub-Saharan Africa, where it is responsible for substantial morbidity, mortality and economic losses. Malaria is a significant cause of severe disease and death in pregnant women and newborns, with pathogenesis being associated with expression of a unique variant of the multidomain Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) called VAR2CSA. Here, we characterize the polymorphism of the DBL3X domain of VAR2CSA and identify regions under selective pressure among placental parasites from women living in endemic western Kenya. In addition to significant levels of polymorphism, our analysis reveals evidence for diversification through intra-segmental recombination and novel mutations that likely contributed to the high number of unique VAR2CSA sequence types identified in this study. Interestingly, we also identified a number of critical residues that may be implicated in immune evasion through switching (or toggling) to alternative amino acids, including an arginine residue within the predicted binding pocket in subdomain III, which was previously implicated in binding to placental CSA. Overall, these findings are important for understanding parasite diversity in pregnant women and will be useful for identifying epitopes and variants of DBL3X to be included in a vaccine against placental malaria.

Malaria-induced murine pregnancy failure: distinct roles for IFN-gamma and TNF.

Although an important role for excessive proinflammatory cytokines in compromise of pregnancy has been established, an immunological basis for malaria-induced fetal loss remains to be demonstrated. In this study, the roles of IFN-gamma and TNF in Plasmodium chabaudi AS-induced fetal loss in mice were directly investigated. Pregnant IFN-gamma(-/-) mice experienced a more severe course of infection compared with intact C57BL/6 mice, characterized by high parasitemia, severe anemia, and marked weight loss. However, fetal loss was delayed in these mice relative to intact controls. Because IFN-gamma(-/-) mice exhibited sustained levels of plasma TNF, the role of this cytokine was examined. Whereas splenic tnf expression in C57BL/6 mice was highest 3 days before peak parasitemia, increased placental expression relative to uninfected mice was sustained, indicating that locally produced TNF may be important in malaria-induced pregnancy failure. Indeed, Ab neutralization of TNF resulted in preservation of embryos until day 12 of gestation, at which point all embryos were lost in untreated mice. Histological analysis revealed that TNF ablation preserved placental architecture whereas placentae from untreated infected mice had widespread hemorrhage and placental disruption, with fibrin thrombi in some maternal blood sinusoids. Consistent with a role for cytokine-driven thrombosis in fetal loss, expression of procoagulant tissue factor was significantly increased in the placentae of infected C57BL/6 mice but was reduced in mice treated with anti-TNF Ab. Together, these results suggest that IFN-gamma contributes to malaria-induced fetal loss and TNF is a critical factor that acts by inducing placental coagulopathy.

Association of malaria-induced murine pregnancy failure with robust peripheral and placental cytokine responses.

Malarial infection in nonimmune pregnant women is a major risk factor for pregnancy failure. The biological mechanisms that underlie malaria-associated fetal loss, however, are poorly understood. Plasmodium chabaudi AS infection during early pregnancy results in midgestational embryonic loss in naive C57BL/6 mice. To define the immunopathogenesis of this malaria-induced pregnancy compromise, cytokine production in plasma, spleen, and placenta cell culture supernatants during the first 11 days of infection and gestation was studied. In infected pregnant mice, systemic interleukin-1beta and both systemic and splenic gamma interferon levels were elevated relative to those in uninfected pregnant mice, and gamma interferon was also robustly produced within the placenta 1 to 2 days before malaria-induced fetal loss. Although circulating tumor necrosis factor production was not affected by pregnancy or infection, circulating soluble tumor necrosis factor receptor II was highest in infected pregnant mice, particularly those undergoing abortion, but decreased at the placental level preceding abortion. Systemic levels of interleukin-10 were also high in infected mice at this time point, but this cytokine was not detected at the placental level. Histological examination revealed that trophoblast giant cells of aborting mice phagocytosed infected red blood cells and hemozoin. Furthermore, in vitro-cultured trophoblast cells isolated from embryos on day 7 of gestation phagocytosed P. chabaudi AS-infected red blood cells and secreted tumor necrosis factor. These results suggest that systemic and placenta-level proinflammatory antimalarial immune responses, in the absence of adequate and sustained counterregulatory mechanisms, contribute to pregnancy loss in this model.

C5a enhances dysregulated inflammatory and angiogenic responses to malaria in vitro: potential implications for placental malaria.

BACKGROUND: Placental malaria (PM) is a leading cause of maternal and infant mortality. Although the accumulation of parasitized erythrocytes (PEs) and monocytes within the placenta is thought to contribute to the pathophysiology of PM, the molecular mechanisms underlying PM remain unclear. Based on the hypothesis that excessive complement activation may contribute to PM, in particular generation of the potent inflammatory peptide C5a, we investigated the role of C5a in the pathogenesis of PM in vitro and in vivo. METHODOLOGY AND PRINCIPAL FINDINGS: Using primary human monocytes, the interaction between C5a and malaria in vitro was assessed. CSA- and CD36-binding PEs induced activation of C5 in the presence of human serum. Plasmodium falciparum GPI (pfGPI) enhanced C5a receptor expression (CD88) on monocytes, and the co-incubation of monocytes with C5a and pfGPI resulted in the synergistic induction of cytokines (IL-6, TNF, IL-1beta, and IL-10), chemokines (IL-8, MCP-1, MIP1alpha, MIP1beta) and the anti-angiogenic factor sFlt-1 in a time and dose-dependent manner. This dysregulated response was abrogated by C5a receptor blockade. To assess the potential role of C5a in PM, C5a plasma levels were measured in malaria-exposed primigravid women in western Kenya. Compared to pregnant women without malaria, C5a levels were significantly elevated in women with PM. CONCLUSIONS AND SIGNIFICANCE: These results suggest that C5a may contribute to the pathogenesis of PM by inducing dysregulated inflammatory and angiogenic responses that impair placental function.