Competitive facilitation of drug-resistant Plasmodium falciparum malaria parasites in pregnant women who receive preventive treatment.

Intermittent preventive treatment in pregnancy (IPTp) is used to prevent Plasmodium falciparum malaria. However, parasites resistant to the IPTp drug sulfadoxine-pyrimethamine (SP) have emerged worldwide, and infections with mixed resistant and susceptible parasites are exacerbated by pyrimethamine in mice. In a prospective delivery cohort in Muheza, Tanzania, we examined the effects of SP IPTp on parasite resistance alleles, parasite diversity, level of parasitemia, and inflammation in the placenta. IPTp use was associated with an increased fraction of parasites carrying the resistance allele at DHPS codon 581, an increase in the level of parasitemia, and more intense placental inflammation. The lowest mean level of parasite diversity and highest mean level of parasitemia occurred in women after recent IPTp use. These findings support a model of parasite release and facilitation, whereby the most highly resistant parasites out-compete less fit parasite populations and overgrow under drug pressure. Use of partially effective anti-malarial agents for IPTp may exacerbate malaria infections in the setting of widespread drug resistance.

Pgs28 belongs to a family of epidermal growth factor-like antigens that are targets of malaria transmission-blocking antibodies.

Although Pgs28, a 28-kD surface protein of Plasmodium gallinaceum oökinetes, was previously thought not to be a target of transmission-blocking antibodies, we found that polyclonal antisera to Pgs28 completely blocked parasite infectivity to Aedes aegypti mosquitoes. Antisera raised against reduced Pgs28 were less effective in blocking transmission than were antisera to nonreduced Pgs28; thus, the target epitope(s) of transmission-blocking antibodies appears to be conformation dependent. In stage-specific assays, polyclonal antisera impaired the in vitro transformation of zygotes to mature oökinetes, as well as the in vivo development of mature oökinetes to oöcysts. Using microsequence of immunoaffinity-purified Pgs28, we cloned the 666-bp open reading frame of the Pgs28 gene. The deduced amino acid sequence of Pgs28 is strikingly similar to that of a P. gallinaceum zygote surface protein, Pgs25, and its P. falciparum analogue, Pfs25. Pgs28, like Pgs25 and Pfs25, has a presumptive secretory signal sequence, followed by four epidermal growth factor-like domains, and a terminal hydrophobic region.

ACETYLCHOLINESTERASE IN FROG SYMPATHETIC AND DORSAL ROOT GANGLIA : A Study by Electron Microscope Cytochemistry and Microgasometric Analysis with the Magnetic Diver.

The localization and chemical determination of acetylcholin esterase in the frog sympathetic and dorsal root ganglia were studied by a combination of the methods of electron microscopy, histochemistry, and microgasometric analysis with the magnetic diver. The Koelle-Friedenwald copper thiocholine histochemical method was modified by eliminating the sulfide conversion and by treatment of the tissue with potassium permanganate. In fixed tissue, enzymatic activity was demonstrated on the inner surface of the endoplasmic reticulum, nuclear envelope, subsurface cisternae, and agranular reticulum of the perikaryon and axon. In briefly fixed tissue, end product appeared also at the axon-sheath and the sheath-sheath interface. Activity at the synaptic junction was most readily obtained in unfixed tissue. Isolated neurons recovered from the diver following chemical analysis were studied with the electron microscope. Cells having a high enzyme activity showed a badly ruptured or absent neural plasmalemma and sheath. In this case the measured activity was apparently due to the enzyme present in the endoplasmic reticulum. Neurons having low activity exhibited an intact plasmalemma and sheath. This may reflect the effectiveness of the neural plasmalemma and sheath as a penetration barrier. The effects of fixation on enzyme activity are discussed. Electron microscopic examination of cells following microgasometric analysis is shown to be essential for the interpretation of the biochemical data.

Adherence of Plasmodium falciparum to chondroitin sulfate A in the human placenta.

Women are particularly susceptible to malaria during first and second pregnancies, even though they may have developed immunity over years of residence in endemic areas. Plasmodium falciparum-infected red blood cells (IRBCs) were obtained from human placentas. These IRBCs bound to purified chondroitin sulfate A (CSA) but not to other extracellular matrix proteins or to other known IRBC receptors. IRBCs from nonpregnant donors did not bind to CSA. Placental IRBCs adhered to sections of fresh-frozen human placenta with an anatomic distribution similar to that of naturally infected placentas, and this adhesion was competitively inhibited by purified CSA. Thus, adhesion to CSA appears to select for a subpopulation of parasites that causes maternal malaria.

Antigens for pre-erythrocytic malaria vaccines: building on success.

Immunization with attenuated pre-erythrocytic malaria parasites can confer sterile protection against malaria in humans and rodents, and a single pre-erythrocytic antigen incorporated in a subunit vaccine has substantially reduced clinical Plasmodium falciparum malaria episodes in African infants during phase 2 trials. Building upon this success has been hindered by technical obstacles that limit research on pre-erythrocytic parasites, especially the liver stage (LS) parasites, and by an incomplete understanding of the immune mechanisms that confer protection in humans. Recent improvements in growing and isolating LS parasites have allowed progress in defining the transcriptome and proteome of the LS parasite, although more work remains to be done particularly for the early LS parasite of P. falciparum. Next generation pre-erythrocytic antigens can be assessed and prioritized based on immunization studies in animals, and on models of immunity such as attenuated parasite vaccines that confer sterile protection or naturally acquired LS-specific immune responses that correlate with protection in endemic areas. Although mechanisms of protection in humans remain poorly understood, the availability of a human malaria challenge model for early clinical testing of candidate vaccines is a valuable tool to confirm which immunogens should move forward to larger field trials.

Developing a novel risk prediction model for severe malarial anemia.

As a pilot study to investigate whether personalized medicine approaches could have value for the reduction of malaria-related mortality in young children, we evaluated questionnaire and biomarker data collected from the Mother Offspring Malaria Study Project birth cohort (Muheza, Tanzania, 2002-2006) at the time of delivery as potential prognostic markers for pediatric severe malarial anemia. Severe malarial anemia, defined here as a Plasmodium falciparum infection accompanied by hemoglobin levels below 50 g/L, is a key manifestation of life-threatening malaria in high transmission regions. For this study sample, a prediction model incorporating cord blood levels of interleukin-1ß provided the strongest discrimination of severe malarial anemia risk with a C-index of 0.77 (95% CI 0.70-0.84), whereas a pragmatic model based on sex, gravidity, transmission season at delivery, and bed net possession yielded a more modest C-index of 0.63 (95% CI 0.54-0.71). Although additional studies, ideally incorporating larger sample sizes and higher event per predictor ratios, are needed to externally validate these prediction models, the findings provide proof of concept that risk score-based screening programs could be developed to avert severe malaria cases in early childhood.

Malaria in the pregnant woman.

Women become more susceptible to Plasmodium falciparum malaria during pregnancy, and the risk of disease and death is high for both the mother and her fetus. In low transmission areas, women of all parities are at risk for severe syndromes like cerebral malaria, and maternal and fetal mortality are high. In high transmission areas, where women are most susceptible during their first pregnancies, severe syndromes like cerebral malaria are uncommon, but severe maternal anemia and low birth weight are frequent sequelae and account for an enormous loss of life. P. falciparum-infected red cells sequester in the intervillous space of the placenta, where they adhere to chondroitin sulfate A but not to receptors like CD36 that commonly support adhesion of parasites infecting nonpregnant hosts. Poor pregnancy outcomes due to malaria are related to the macrophage-rich infiltrates and pro-inflammatory cytokines such as tumor necrosis factor-alpha that accumulate in the intervillous space. Women who acquire antibodies against chrondroitin sulfate A (CSA)-binding parasites are less likely to have placental malaria, and are more likely to deliver healthy babies. In areas of stable transmission, women acquire antibodies against CSA-binding parasites over successive pregnancies, explaining the high susceptibility to malaria during first pregnancy, and suggesting that a vaccine to prevent pregnancy malaria should target placental parasites. Prevention and treatment of malaria are essential components of antenatal care in endemic areas, but require special considerations during pregnancy. Recrudescence after drug treatment is more common during pregnancy, and the spread of drug-resistant parasites has eroded the usefulness of the few drugs known to be safe for the woman and her fetus. Determining the safety and effectiveness of newer antimalarials in pregnant women is an urgent priority. A vaccine that prevents pregnancy malaria due to P. falciparum could be delivered before first pregnancy, and would have an enormous impact on mother-child health in tropical areas.

The immunology and pathogenesis of malaria during pregnancy.

Women in endemic areas become highly susceptible to malaria during first and second pregnancies, despite immunity acquired after years of exposure. Recent insights have advanced our understanding of pregnancy malaria caused by Plasmodium falciparum, which is responsible for the bulk of severe disease and death. Accumulation of parasitized erythrocytes in the blood spaces of the placenta is a key feature of maternal infection with P. falciparum. Placental parasites express surface ligands and antigens that differ from those of other P. falciparum variants, facilitating evasion of existing immunity, and mediate adhesion to specific molecules, such as chondroitin sulfate A, in the placenta. The polymorphic and clonally variant P. falciparum erythrocyte membrane protein 1, encoded by var genes, binds to placental receptors in vitro and may be the target of protective antibodies. An intense infiltration of immune cells, including macrophages, into the placental intervillous spaces, and the production of pro-inflammatory cytokines often occur in response to infection, and are associated with low birth weight and maternal anemia. Expression of alpha and beta chemokines may initiate or facilitate this cellular infiltration during placental malaria. Specific immunity against placental-binding parasites may prevent infection or facilitate clearance of parasites prior to the influx of inflammatory cells, thereby avoiding a cascade of events leading to disease and death. Much less is known about pathogenic processes in P. vivax infections, and corresponding immune responses. Emerging knowledge of the pathogenesis and immunology of malaria in pregnancy will increasingly lead to new opportunities for the development of therapeutic and preventive interventions and new tools for diagnosis and monitoring.

Correction: Identification of Novel Pre-Erythrocytic Malaria Antigen Candidates for Combination Vaccines with Circumsporozoite Protein.

[This corrects the article DOI: 10.1371/journal.pone.0159449.].

Maternal malaria and parasite adhesion.

Malaria during pregnancy continues to be a major health problem in endemic countries, with clinical consequences, including death, for both mother and child. Just as cerebral malaria results from parasite sequestration in the brain, maternal malaria results from parasite sequestration in the placenta, and a distinct subpopulation of parasites which bind chondroitin sulfate A but not CD36 causes the syndrome. Women have little or no immunological experience with this parasite prior to first pregnancy, making primigravid women particularly vulnerable to infection. Parasites adhere to the surface of trophoblastic villi, eliciting the accumulation of inflammatory leukocytes in the intervillous space, and the necrosis of adjacent placental tissue. Maternal malaria results in poor pregnancy outcomes, although the responsible mechanisms have not been defined. In holoendemic areas both placental infection and poor outcome decrease in frequency with successive pregnancies; protection may result from control of parasite adhesion, suggesting an attractive target for new therapies.

Identification of glial fibrillary acidic protein by the immunoperoxidase method in human brain tumors.

The immunocytochemical localization of glial fibrillary acidic portein within glioma cell bodies and their processes by the immunoperoxidase method is demonstrated to be of diagnostic value. This method has advantages over "special" stains because it is not so dependent upon color alone, and because it identifies a specific protein in the cells. The immunoperoxidase method using antiserum to glial fibrillary acidic protein is shown to be useful for the differentiation of mixed glial and mesenchymal tumors, and for the diagnosis of tumors in which a glial or mesenchymal cell origin is in doubt.

Immunocytochemistry of pineal astrocytes: species differences and functional implications.

Immunohistochemical demonstration of glial fibrillary acidic protein (GFAP) was performed in human, sheep, rat and guinea pig pineal bodies to determine if there were species differences. Specialized "basket-like" arrangements of many GFAP-positive astrocytic processes were shown around sheep pinealocytes. Human pineals contained scattered astrocytic cell bodies and a moderate number of GFAP-positive astrocytic processes which, as in sheep, also surrounded pinealocytes, but without the dense basket-like arrangements. In both species GFAP-positive fibers were concentrated at the periphery of pseudolobules and around blood vessels. Rat and guinea pig pineals contained only rare astrocytic cell bodies and few GFAP-positive fibers throughout the glands, but had a concentration of parallel GFAP-positive fibers at the stalk. GFAP-positive fibers in human and sheep pineals may be derived from both intra- and extraglandular sites, whereas in rodents only rare processes appear to be derived from within the gland. Astrocytes may play a role in modulation of pineal indoleamines and norepinephrine, and the species differences observed suggest that this effect may be important in sheep and human pineals but not in rodents.

Pre-erythrocytic immunity to Plasmodium falciparum: the case for an LSA-1 vaccine.

A vaccine is urgently needed to stem the global resurgence of Plasmodium falciparum malaria. Vaccines targeting the erythrocytic stage are often viewed as an anti-disease strategy. By contrast, infection might be completely averted by a vaccine against the liver stage, a pre-erythrocytic stage during which the parasite multiplies 10000-fold within hepatocytes. Sterilizing immunity can be conferred by inoculating humans with irradiated pre-erythrocytic parasites, and a recombinant pre-erythrocytic vaccine partially protects humans from infection. Liver-stage antigen-1, one of a few proteins known to be expressed by liver-stage parasites, holds particular promise as a vaccine. Studies of naturally exposed populations have consistently related immune responses against this antigen to protection.

Glial fibrillary acidic protein and Alzheimer-type senile dementia.

Glial fibrillary acidic protein (GFAP), a protein that is associated with 9-nm filaments of astrocytes, was observed to be increased in the astrocytes surrounding senile plaques in patients with Alzheimer dementia and in aged subjects without dementia. A few GFAP-positive fibers were seen in the centers of plaques. These results emphasized the selectivity of senile changes; whereas some cells seemed to undergo degeneration or dysfunction, other cells--astrocytes--maintain their capacity for reaction and may increase the formation of at least one protein, GFAP.

Immunization with SPf66 and subsequent infection with homologous and heterologous Plasmodium falciparum parasites.

In an area of intense transmission, a malaria vaccine could reduce infection due to the parasite types represented in the vaccine, but have no detectable effect on the overall frequency of infection if it did not protect against infection with heterologous parasites. These studies were performed to determine whether immunization with SPf66 decreased infection with homologous parasites containing the 11 amino acid peptide from merozoite surface protein-1 (MSP-1) in SPf66, or increased infection due to heterologous parasites containing heterologous (alternative) MSP-1 sequences. Based on this 11 amino acid peptide (YSLFQKEKMVL), three forward primers (S,Q,V) were designed to amplify the MSP-1 sequence present in SPf66, and 3 additional forward primers (G,H,I) to amplify the alternative MSP-1 sequence (YGLFHKEKMIL). This strategy was validated by polymerase chain reaction (PCR) amplification and dideoxy sequencing with 14 cloned laboratory isolates, which demonstrated that each primer amplified one MSP-1 sequence or the other, but not both. The technique was then used to examine filter paper blots from an SPf66 vaccine study of 69 subjects in Saradidi, Kenya. In that study, the prevalence of infection with YSLFQKEKMVL or YGLFHKEKMIL type parasites was unaffected by immunization with SPf66 (based on PCR amplification with the S, Q, V, G, H and I primers, respectively). These results suggest that immunization with SPf66 does not produce a selective effect in vivo. They demonstrate a molecular method to test for selection in vivo as an indirect measure of vaccine efficacy.

Murine typhus identified as a major cause of febrile illness in a camp for displaced Khmers in Thailand.

Scrub and murine typhus have been identified as causes of illness among the 238,000 displaced Khmer people residing in temporary settlements on the Thai side of the Thai-Cambodian border. Still, the true extent of the problem and the relative frequency of infection with scrub typhus as compared to murine typhus are unknown. We evaluated consecutive patients with unexplained pyrexia (documented fever, no exclusionary diagnosis, and constitutional symptoms) in 1 temporary settlement over 1 month. Laboratory studies included culture of blood and assay of paired sera for rickettsial IgM and IgG antibody, for dengue IgM and IgG antibody, and for leptospiral IgM and IgG antibody. Among 37 patients (27 adults and 10 children), 28 (75%) had a rickettsiosis (26 cases of murine typhus and 2 cases of scrub typhus). No case of enteric fever, dengue, or leptospirosis was diagnosed. The illnesses of 9 patients were not identified. Signs and symptoms did not distinguish confirmed rickettsial infections from undiagnosed illnesses. The 1 month attack rate of rickettsial infection was 29/100,000 for children and 185/100,000 for adults. Murine typhus was a major cause of febrile illness in this settlement.

Nitric oxide, malaria, and anemia: inverse relationship between nitric oxide production and hemoglobin concentration in asymptomatic, malaria-exposed children.

The cause of the anemia associated with chronic, intermittent, asymptomatic, low-level parasitemia in children in malaria-endemic endemic areas is not well understood. Nitric oxide (NO) decreases erythropoiesis, and it is likely an important mediator of anemia of chronic disease. Production of NO is decreased in acute uncomplicated and cerebral malaria, but it is increased in asymptomatic Tanzanian children (with or without parasitemia). We hypothesized that chronic overproduction of NO in these asymptomatic children contributes to the anemia associated with subclinical/subpatent malaria. In 44 fasting, asymptomatic, malaria-exposed, Tanzanian children, NO production (measured using fasting urine NOx excretion) was inversely associated with hemoglobin concentration (P = 0.03, controlling for age and gender). Using multiple linear regression, hemoglobin concentration was negatively associated with parasitemia (P = 0.005). After controlling for age and parasitemia, NO was no longer an independent predictor of anemia. One of the mechanisms of parasite-related anemia in such children may be through the adverse hematologic effects of parasite-induced NO production.

Effects of age and parasitemia on nitric oxide production/leukocyte nitric oxide synthase type 2 expression in asymptomatic, malaria-exposed children.

Age appears to influence not only the acquisition of clinical immunity to malaria but also the susceptibility to and clinical manifestations of severe malaria. Asymptomatic malaria-exposed Tanzanian children have high production of nitric oxide (NO) and universal expression of leukocyte NO synthase type 2 (NOS2), which may protect against disease. To determine the effects of age and parasitemia on NO production, we measured urine and plasma NO metabolites and leukocyte NOS2 expression in 45 fasting, asymptomatic, malaria-exposed children of different ages, stratifying parasitemia by thick film and polymerase chain reaction (PCR) analysis. Although NO production was significantly higher in thick film-positive children than in thick film-negative children, after adjusting for age and gender, we were unable to detect a difference in NO production in thick film-negative children between those who were PCR positive and PCR negative. The relationship between age and NO production was determined using a generalized additive model adjusted for the effects of gender and parasitemia. Production of NO using all three measures was highest in infancy, decreasing after the first year of life, and then increasing again after 5 years of age. This pattern of age-related NO production is the reverse of the pattern of age-related morbidity from cerebral malaria in coastal Tanzanian children. Elevated production of NO in both infants and older children may be related to age per se and malaria infection respectively, and may be one of the mediators of the anti-disease immunity found most commonly in these two age groups.

Phase I safety and immunogenicity testing of clinical lots of the synthetic Plasmodium falciparum vaccine SPf66 produced under good manufacturing procedure conditions in the United States.

Two clinical lots of alum-adsorbed SPf66 vaccine produced in the United States were evaluated in separate, open-label, Phase I clinical trials involving 15 healthy, malaria-naive, 18-45-year old men and women. Subjects received 2 mg doses subcutaneously in alternate arms at 0, one, and six months. Safety was assessed by monitoring local and systemic reactions and laboratory parameters. The most common side effects were erythema and local tenderness at the site of injection, which increased in frequency with subsequent doses of vaccine. These local reactions were considered mild and resolved within 24-48 hr. Eleven of 14 volunteers who received all three doses of vaccine seroconverted by enzyme-linked immunosorbent assay. The distribution of high, medium, and low nonresponders was comparable with that seen in trials of Colombian-produced vaccine. One high responder developed antibodies reactive with asexual stage parasite antigens by immunofluorescence and immunoblot. The results indicated that at full adult doses, SPf66 of U.S. origin is mildly reactogenic and induces immune responses similar to those reported for SPf66 of Colombian origin.