Placental histopathologic changes associated with subclinical malaria infection and its impact on the fetal environment.

Microscopic examination of placental tissue can provide an accurate assessment of malaria infection during pregnancy. In this cross-sectional study of 193 women in Iquitos, Peru, 1.0% and 6.6% had parasites in the peripheral blood as detected by microscopy and polymerase chain reaction, respectively. However, 22% had placental malaria pigment indicating past, subclinical infections. Placental tissues with pigment from 24 cases were matched by gravidity and month of delivery to 24 controls and histopathologically examined. Cases had significantly higher number of monocytes in the intervillous space (44.7 versus 25.5; P = 0.012). Pigmented monocytes in fetal vessels were present in 33.3% of cases. This study demonstrated that subclinical malarial infection occurred frequently in pregnant women and is associated with increased presence of monocytes in the placenta. Pigmented monocytes in fetal vessels suggest parasites can breach the placental barrier and enter the fetal circulation.

Altered immune responses in rhesus macaques co-infected with SIV and Plasmodium cynomolgi: an animal model for coincident AIDS and relapsing malaria.

BACKGROUND: Dual epidemics of the malaria parasite Plasmodium and HIV-1 in sub-Saharan Africa and Asia present a significant risk for co-infection in these overlapping endemic regions. Recent studies of HIV/Plasmodium falciparum co-infection have reported significant interactions of these pathogens, including more rapid CD4+ T cell loss, increased viral load, increased immunosuppression, and increased episodes of clinical malaria. Here, we describe a novel rhesus macaque model for co-infection that supports and expands upon findings in human co-infection studies and can be used to identify interactions between these two pathogens. METHODOLOGY/PRINCIPAL FINDINGS: Five rhesus macaques were infected with P. cynomolgi and, following three parasite relapses, with SIV. Compared to macaques infected with SIV alone, co-infected animals had, as a group, decreased survival time and more rapid declines in markers for SIV progression, including peripheral CD4+ T cells and CD4+/CD8+ T cell ratios. The naïve CD4+ T cell pool of the co-infected animals was depleted more rapidly than animals infected with SIV alone. The co-infected animals also failed to generate proliferative responses to parasitemia by CD4+ and CD8+ T cells as well as B cells while also having a less robust anti-parasite and altered anti-SIV antibody response. CONCLUSIONS/SIGNIFICANCE: These data suggest that infection with both SIV and Plasmodium enhances SIV-induced disease progression and impairs the anti-Plasmodium immune response. These data support findings in HIV/Plasmodium co-infection studies. This animal model can be used to further define impacts of lentivirus and Plasmodium co-infection and guide public health and therapeutic interventions.

The role of soluble tumor necrosis factor receptor types I and II and tumor necrosis factor-alpha in malaria during pregnancy.

In a prospective study of rhesus monkeys inoculated with Plasmodium coatneyi or saline on an infection/gestational timeline, we determined the serum levels of tumor necrosis factor-alpha (TNF-alpha), soluble tumor necrosis factor receptor type I (sTNFR-I), and soluble tumor necrosis factor receptor type II (sTNFR-II) in peripheral blood throughout primigravid pregnancy, malaria infection, and a combination of the two. Our goal was to determine the association between levels of TNF-alpha and of its 2 soluble receptors and the course of pregnancy and/or malaria and infant outcome. We found that any detectable level of TNF-alpha was always associated with fetal death and that the sTNFRs may be important for fetal protection, possibly through neutralizing the toxic effects of TNF-alpha. Our findings also showed that increased levels of sTNFR-II were associated specifically with malaria and not with normal pregnancy or even pregnancy with low birth weight due to other causes. In contrast, increases in sTNFR-I levels during the later half of normal pregnancies indicate that sTNFR-I may be important in regulating TNF-alpha levels in preparation for normal labor and delivery.

Birth of rhesus macaque (Macaca mulatta) infants after in vitro fertilization and gestation in female rhesus or pigtailed (Macaca nemestrina) macaques.

A study was conducted to assess the possibility of using pigtailed macaques (Macaca nemestrina) as recipients for rhesus macaque (Macaca mulatta) embryos. A total of 250 oocytes were collected from 11 rhesus monkeys during 12 follicular aspirations. We performed 15 embryo transfers with two embryos each into rhesus recipients, which resulted in eight pregnancies, of which two were lost during the second trimester. Among the remaining six pregnant rhesus macaques, two were carrying twins, resulting in the birth of eight infants. Twelve transfers of rhesus embryos into pigtailed macaques resulted in one pregnancy and the birth of one infant. Fetal growth and development were monitored by monthly ultrasound examinations, during which biparietal measurements were taken and compared with those derived from 22 pregnant control monkeys. In vitro fertilization-derived singletons tended to develop faster than did twins and naturally conceived control singletons during the initial months of pregnancy and weighed more at birth than did twins. There were pronounced morphologic changes in the placenta of the rhesus that developed in the female pigtailed macaque. These included an irregular shape, elevated placenta-to-birth-weight ratio, and an abnormal length and diameter of the umbilical cord. Histologic analyses of the rhesus-pigtailed placenta showed evidence of maternal-placental floor infarction and thrombosis of the spiral artery with resulting infarction of the villi. These results demonstrate that pigtailed macaques can carry rhesus fetuses to term, but further studies are necessary to determine the cause of the decreased pregnancy rates and observed placental abnormalities.

Alterations in the profile of blood cell types during malaria in previously unexposed primigravid monkeys.

Malaria in nonimmune, primigravid women threatens both mother and fetus. We used the Plasmodium coatneyi/rhesus monkey model to examine factors associated with this. Clinical and immunologic responses during the blood stage of chronic malaria (4 months) were evaluated in 8 malaria-naive primigravid (PMI) and 8 age-matched nulligravid (NMI) infected monkeys, compared with those in 8 primigravid, noninfected control monkeys. Although parasitemia levels were similar, recrudescence was more frequent and prolonged, and anemia was more severe in PMI than in NMI monkeys. During infection, CD2+, CD4+, and CD8+ lymphocyte levels were higher in NMI than in PMI monkeys. Monocyte and neutrophil levels were lower in PMI than in NMI monkeys. During chronic, untreated malaria, NMI monkeys had a B lymphocyte count 23 times greater than that of PMI monkeys. Pregnancy-induced immunomodulation, defined as a lack of appropriate cellular responses to malaria, was indiscernible until the immune system was challenged by a pathogen.

Hematologic and lymphocyte immunophenotypic reference values for normal rhesus monkey (Macaca mulatta) umbilical cord blood; gravidity may play a role in study design.

Hematology and flow cytometry reference values for rhesus umbilical cord blood (UCB) were established in 17 healthy infant rhesus monkeys delivered by elective cesarean section 10 days preterm. The infants were born to age matched, singly caged primigravid or secundigravid dams. The hematology and flow cytometry values were determined by automated cell counter and by FACS. No significant differences were observed with respect to infant gender. With respect to gravida, the primigravid infants had a significantly higher percentage (P= 0.05) of CD20(+) B lymphocytes in UCB. These results provide useful reference values for future studies of maternal - fetal disease transmission, vaccine and drug evaluation in non-human primate pregnancy, as well as fetal programming and immune modulation, gene therapy and the use of UCB as a source of stem cells for research and transplantation. Importantly, our results suggest that maternal gravidity may be an important variable to consider.

The effects of Plasmodium falciparum and P. vivax infections on placental histopathology in an area of low malaria transmission.

Placental histopathology was studied in a cohort of 204 women living in an area of low Plasmodium falciparum and P. vivax malaria transmission. Detection of malaria antenatally was active, by weekly peripheral blood smears, and all infections were treated. Significant histopathologic placental malaria changes (increased malaria pigment, cytotrophoblastic prominence, and presence of parasites) were found only in a minority of women who had P. falciparum infections in pregnancy. These changes were significantly more frequent in women with evidence of peripheral blood infection close to delivery and only in these cases were placental inflammatory cells increased. Antenatal P. vivax infection was associated only with the presence of malaria pigment in the placenta. All placental infections diagnosed by blood smear and 32.4% (12 of 37) diagnosed by histopathology were associated with patent peripheral parasitemia. This study indicates that prompt treatment of peripheral parasitemias during pregnancy limits placental pathology. The effect on birth weight reduction may not result from irreversible placental changes but from the acute insult of infection. These findings emphasize the importance of treating malaria in pregnancy promptly with effective antimalarial drugs.

A preliminary evaluation of recombinant adeno-associated virus biodistribution in rhesus monkeys after intrahepatic inoculation in utero.

The ability to deliver genes to fetuses in utero may prove crucial for those genetic diseases that are associated with severe fetal morbidity and for which there is no effective postnatal therapy. In utero therapy may be especially useful in diseases that affect the central nervous system because the immature blood-brain barrier may facilitate gene delivery to neural target cells. We investigated whether in utero inoculation of recombinant adeno-associated virus (rAAV) into rhesus monkey fetuses would be a useful method of gene delivery, especially to the central nervous system. When the monkeys were sacrificed after birth, we found vector genomes distributed in many tissues, including the brain and peripheral blood. Pericapillary astrocytes expressing transgene products were detected by immunohistochemistry. In addition, we occasionally found vector genomes in the maternal blood. No adverse clinical or pathologic effects were observed in the inoculated monkeys. We concluded that (1) in utero intrahepatic inoculation of rAAV is a potentially safe and useful method of delivering genes to many fetal tissues; (2) astrocytes may be the cell type most easily targeted in the central nervous system (CNS) after systemic administration; and (3) the potential of inadvertent gene transfer to the mother must be considered.

Procedure for Successfully Introducing Cesarean-delivered Rhesus Monkey (Macaca mulatta) Infants to Their Mothers.

To reduce the high rate of maternal rejection after cesarean delivery, we developed a protocol for introducing cesarean-delivered rhesus monkey (Macaca mulatta) infants to their mothers. The new protocol was used with 14 pregnant females and the resulting infants. After an early morning cesarean section, the mother was covered with a clean towel and returned to her cage. Infants received standard nursery care until the first introduction attempt (after the mother had fully recovered from general anesthesia). The infant was rubbed with maternal blood-soaked laparotomy pads that were saved from surgery, then the infant was placed into the mother's cage. If the mother did not allow the infant to suckle within an hour of introduction, the towel placed with the mother after surgery and the infant were placed together in a nursery cage overnight. Daily attempts to introduce the infant (and the towel) continued until it was accepted or maternal behavior precluded further attempts. Within 3 days after surgery, 72% of the females had accepted their infants. All the females who accepted their infants had previous vaginal deliveries and had successfully reared at least one other infant. Our experience suggests that initial maternal rejection does not mean the mother will never accept the infant and that multiple introduction attempts should be made before nursery-rearing an infant.