Natural immunity to malaria preferentially targets the endothelial protein C receptor-binding regions of PfEMP1s.

Antibody responses to variant surface antigens (VSAs) produced by the malaria parasite Plasmodium falciparum may contribute to age-related natural immunity to severe malaria. One VSA family, P. falciparum erythrocyte membrane protein-1 (PfEMP1), includes a subset of proteins that binds endothelial protein C receptor (EPCR) in human hosts and potentially disrupts the regulation of inflammatory responses, which may lead to the development of severe malaria. We probed peptide microarrays containing segments spanning five PfEMP1 EPCR-binding domain variants with sera from 10 Malian adults and 10 children to determine the differences between adult and pediatric immune responses. We defined serorecognized peptides and amino acid residues as those that elicited a significantly higher antibody response than malaria-naïve controls. We aimed to identify regions consistently serorecognized among adults but not among children across PfEMP1 variants, potentially indicating regions that drive the development of immunity to severe malaria. Adult sera consistently demonstrated broader and more intense serologic responses to constitutive PfEMP1 peptides than pediatric sera, including peptides in EPCR-binding domains. Both adults and children serorecognized a significantly higher proportion of EPCR-binding peptides than peptides that do not directly participate in receptor binding, indicating a preferential development of serologic responses at functional residues. Over the course of a single malaria transmission season, pediatric serological responses increased between the start and the peak of the season, but waned as the transmission season ended. IMPORTANCE Severe malaria and death related to malaria disproportionately affect sub-Saharan children under 5 years of age, commonly manifesting as cerebral malaria and/or severe malarial anemia. In contrast, adults in malaria-endemic regions tend to experience asymptomatic or mild disease. Our findings indicate that natural immunity to malaria targets specific regions within the EPCR-binding domain, particularly peptides containing EPCR-binding residues. Epitopes containing these residues may be promising targets for vaccines or therapeutics directed against severe malaria. Our approach provides insight into the development of natural immunity to a binding target linked to severe malaria by characterizing an "adult-like" response as recognizing a proportion of epitopes within the PfEMP1 protein, particularly regions that mediate EPCR binding. This "adult-like" response likely requires multiple years of malaria exposure, as increases in pediatric serologic response over a single malaria transmission season do not appear significant.

Antenatal, intrapartum and infant azithromycin to prevent stillbirths and infant deaths: study protocol for SANTE, a 2×2 factorial randomised controlled trial in Mali.

INTRODUCTION: In high mortality settings, prophylactic azithromycin has been shown to improve birth weight and gestational age at birth when administered antenatally, to reduce the incidence of neonatal infections when administered intrapartum, and to improve survival when administered in infancy. Questions remain regarding whether azithromycin can prevent stillbirths, and regarding the optimal strategy for the delivery of azithromycin to pregnant women and their infants. METHODS AND ANALYSIS: Sauver avec l'Azithromycine en Traitant les Femmes Enceintes et les Enfants (SANTE) is a 2×2 factorial, individually randomised, placebo-controlled, double-masked trial in rural Mali. The primary aims are: (1A) to assess the efficacy of antenatal and intrapartum azithromycin on a composite outcome of stillbirths and infant mortality through 6-12 months and (1B) to assess the efficacy of azithromycin administered concurrently with the first and third doses of pentavalent vaccines (Penta-1/3) on infant mortality through 6-12 months. Pregnant participants (n=49 600) and their infants are randomised 1:1:1:1 to one of four treatment arms: (1) mother and infant receive azithromycin, (2) mother and infant receive placebo, (3) mother receives azithromycin and infant receives placebo or (4) mother receives placebo and infant receives azithromycin. Pregnant participants receive three single 2 g doses: two antepartum and one intrapartum. Infants receive a single 20 mg/kg dose at the Penta-1 and 3 visits. An additional cohort of 12 000 infants is recruited at the Penta-1 visit and randomised 1:1 to receive azithromycin or placebo at the same time points. The SANTE trial will inform guidelines and policies regarding the administration of antenatal and infant azithromycin using routine healthcare delivery platforms. ETHICS AND DISSEMINATION: This trial was approved by the Institutional Review Board at the University of Maryland School of Medicine (Protocol #HP-00084242) and the Faculté de Médecine et d'Odonto-Stomatologie in Mali. The findings of this trial will be published in open access peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT03909737.

Serologic and Cytokine Profiles of Children with Concurrent Cerebral Malaria and Severe Malarial Anemia Are Distinct from Other Subtypes of Severe Malaria.

We used a protein microarray featuring Plasmodium falciparum field variants of a merozoite surface antigen to examine malaria exposure in Malian children with different severe malaria syndromes. Unlike children with cerebral malaria alone or severe malarial anemia alone, those with concurrent cerebral malaria and severe malarial anemia had serologic responses demonstrating a broader prior parasite exposure pattern than matched controls with uncomplicated disease. Comparison of levels of malaria-related cytokines revealed that children with the concurrent phenotype had elevated levels of interleukin (IL)-6, IL-8, and IL-10. Our results suggest that the pathophysiology of this severe subtype is unique and merits further investigation.

Malian adults maintain serologic responses to virulent PfEMP1s amid seasonal patterns of fluctuation.

Plasmodium falciparum erythrocyte membrane protein-1s (PfEMP1s), diverse malaria proteins expressed on the infected erythrocyte surface, play an important role in pathogenesis, mediating adhesion to host vascular endothelium. Antibodies to particular non-CD36-binding PfEMP1s are associated with protection against severe disease. We hypothesized that given lifelong P. falciparum exposure, Malian adults would have broad PfEMP1 serorecognition and high seroreactivity levels during follow-up, particularly to non-CD36-binding PfEMP1s such as those that attach to endothelial protein C receptor (EPCR) and intercellular adhesion molecule-1 (ICAM-1). Using a protein microarray, we determined serologic responses to 166 reference PfEMP1 fragments during a dry and subsequent malaria transmission season in Malian adults. Malian adult sera had PfEMP1 serologic responses throughout the year, with decreased reactivity to a small subset of PfEMP1 fragments during the dry season and increases in reactivity to a different subset of PfEMP1 fragments during the subsequent peak malaria transmission season, especially for intracellular PfEMP1 domains. For some individuals, PfEMP1 serologic responses increased after the dry season, suggesting antigenic switching during asymptomatic infection. Adults were more likely to experience variable serorecognition of CD36-binding PfEMP1s than non-CD36-binding PfEMP1s that bind EPCR or ICAM-1, which remained serorecognized throughout the year. Sustained seroreactivity to non-CD36-binding PfEMP1s throughout adulthood amid seasonal fluctuation patterns may reflect underlying protective severe malaria immunity and merits further investigation.

Epitope-Specific Antibody Responses to a Plasmodium falciparum Subunit Vaccine Target in a Malaria-Endemic Population.

Circumsporozoite protein (CSP) coats the Plasmodium falciparum sporozoite surface and is a major malaria subunit vaccine target. We measured epitope-specific reactivity to field-derived CSP haplotypes in serum samples from Malian adults and children on a custom peptide microarray. Compared to children, adults showed greater antibody responses and responses to more variants in regions proximal to and within the central repeat region. Children acquired short-lived immunity to an epitope proximal to the central repeat region but not to the central repeat region itself. This approach has the potential to differentiate immunodominant from protective epitope-specific responses when combined with longitudinal infection data.

Microarray analyses reveal strain-specific antibody responses to Plasmodium falciparum apical membrane antigen 1 variants following natural infection and vaccination.

Vaccines based on Plasmodium falciparum apical membrane antigen 1 (AMA1) have failed due to extensive polymorphism in AMA1. To assess the strain-specificity of antibody responses to malaria infection and AMA1 vaccination, we designed protein and peptide microarrays representing hundreds of unique AMA1 variants. Following clinical malaria episodes, children had short-lived, sequence-independent increases in average whole-protein seroreactivity, as well as strain-specific responses to peptides representing diverse epitopes. Vaccination resulted in dramatically increased seroreactivity to all 263 AMA1 whole-protein variants. High-density peptide analysis revealed that vaccinated children had increases in seroreactivity to four distinct epitopes that exceeded responses to natural infection. A single amino acid change was critical to seroreactivity to peptides in a region of AMA1 associated with strain-specific vaccine efficacy. Antibody measurements using whole antigens may be biased towards conserved, immunodominant epitopes. Peptide microarrays may help to identify immunogenic epitopes, define correlates of vaccine protection, and measure strain-specific vaccine-induced antibodies.

Thermal biology and swimming performance of Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus).

Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) are two commercially important marine fishes impacted by both overfishing and climate change. Increasing ocean temperatures are affecting the physiology of these species and causing changes in distribution, growth, and maturity. While the physiology of cod has been well investigated, that of haddock has received very little attention. Here, we measured the metabolic response to increasing temperatures, as well as the critical thermal maximum (CTmax), of cod acclimated to 8 and 12 °C and haddock acclimated to 12 °C. We also compared the swimming performance (critical swimming speed, U crit) of cod and haddock at 12 °C, as well as the U crit of 12 °C-acclimated cod acutely exposed to a higher-than-optimal temperature (16 °C). The CTmax for cod was 21.4 and 23.0 °C for 8- and 12 °C-acclimated fish, respectively, whereas that for the 12 °C-acclimated haddock was 23.9 °C. These values were all significantly different and show that haddock are more tolerant of high temperatures. The aerobic maximum metabolic rate (MMR) of swimming cod remained high at 16 °C, suggesting that maximum oxygen transport capacity was not limited at a temperature above optimal in this species. However, signs of impaired swimming (struggling) were becoming evident at 16 °C. Haddock were found to reach a higher U crit than cod at 12 °C (3.02 vs. 2.62 body lengths s-1, respectively), and at a lower MMR. Taken together, these results suggest that haddock perform better than cod in warmer conditions, and that haddock are the superior swimmer amongst the two species.

Serologic responses to the PfEMP1 DBL-CIDR head structure may be a better indicator of malaria exposure than those to the DBL-α tag.

BACKGROUND: Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) antigens play a critical role in host immune evasion. Serologic responses to these antigens have been associated with protection from clinical malaria, suggesting that antibodies to PfEMP1 antigens may contribute to natural immunity. The first N-terminal constitutive domain in a PfEMP1 is the Duffy binding-like alpha (DBL-α) domain, which contains a 300 to 400 base pair region unique to each particular protein (the DBL-α "tag"). This DBL-α tag has been used as a marker of PfEMP1 diversity and serologic responses in malaria-exposed populations. In this study, using sera from a malaria-endemic region, responses to DBL-α tags were compared to responses to the corresponding entire DBL-α domain (or "parent" domain) coupled with the succeeding cysteine-rich interdomain region (CIDR). METHODS: A protein microarray populated with DBL-α tags, the parent DBL-CIDR head structures, and downstream PfEMP1 protein fragments was probed with sera from Malian children (aged 1 to 6 years) and adults from the control arms of apical membrane antigen 1 (AMA1) vaccine clinical trials before and during a malaria transmission season. Serological responses to the DBL-α tag and the DBL-CIDR head structure were measured and compared in children and adults, and throughout the season. RESULTS: Malian serologic responses to a PfEMP1's DBL-α tag region did not correlate with seasonal malaria exposure, or with responses to the parent DBL-CIDR head structure in either children or adults. Parent DBL-CIDR head structures were better indicators of malaria exposure. CONCLUSIONS: Larger PfEMP1 domains may be better indicators of malaria exposure than short, variable PfEMP1 fragments such as DBL-α tags. PfEMP1 head structures that include conserved sequences appear particularly well suited for study as serologic predictors of malaria exposure.

The acute and incremental thermal tolerance of Atlantic cod (Gadus morhua) families under normoxia and mild hypoxia.

Given climate change projections, the limited ability of fish reared in sea-cages to behaviourally thermoregulate, and that thermal tolerance may be heritable, studies that examine family-related differences in upper thermal tolerance are quite relevant to the aquaculture industry. Thus, we investigated the upper thermal tolerance of 15 Atlantic cod (Gadus morhua L.) families by challenging them with acute (2 °C h-1) and incremental (1 °C every 4 days) temperature increases (CTmax and ITmax tests, respectively) under normoxia (~ 100% air saturation) and mild hypoxia (~ 75% air sat.). The cod's CTmax was 22.5 ±â€¯0.1 °C (mean ±â€¯S.E.) during normoxia and 21.8 ±â€¯0.1 °C during hypoxia (P < 0.001); and these two CTmax values were significantly correlated across families. In both the normoxic and hypoxic ITmax tests, feed intake fell by ~50% between 17 and 18 °C, and stopped entirely by 21 °C. No mortalities were observed under 20 °C in the normoxic and hypoxic ITmax tests, and the ITmax value was ~21.7 °C in both groups. Differences in the upper thermal tolerance between families were only observed in the CTmax experiment. No correlation was found between the specific growth rate and the CTmax of the families. Further, no correlation existed between CTmax and ITmax. This study is the first to compare the thermal tolerance of fish families to both CTmax and ITmax challenges, and the data: 1) suggest that the Atlantic cod is quite tolerant of acute (i.e., hours) or short-term (i.e., weeks) exposure to high water temperatures (i.e., up to 20 °C); 2) indicate that it might be difficult to select fish with higher ITmax values; and 3) question the relevance of CTmax for selecting fish that are destined for sea-cages where temperatures slowly warm over the summer.

Antibodies to Peptides in Semiconserved Domains of RIFINs and STEVORs Correlate with Malaria Exposure.

The repetitive interspersed family (RIFIN) and the subtelomeric variable open reading frame (STEVOR) family represent two of three major Plasmodium falciparum variant surface antigen families involved in malaria pathogenesis and immune evasion and are potential targets in the development of natural immunity. Protein and peptide microarrays populated with RIFINs and STEVORs associated with severe malaria vulnerability in Malian children were probed with adult and pediatric sera to identify epitopes that reflect malaria exposure. Adult sera recognized and reacted with greater intensity to all STEVOR proteins than pediatric sera did. Serorecognition of and seroreactivity to peptides within the semiconserved domain of STEVORs increased with age and seasonal malaria exposure, while serorecognition and seroreactivity increased for the semiconserved and second hypervariable domains of RIFINs only with age. Serologic responses to RIFIN and STEVOR peptides within the semiconserved domains may play a role in natural immunity to severe malaria.IMPORTANCE Malaria, an infectious disease caused by the parasite Plasmodium falciparum, causes nearly 435,000 deaths annually worldwide. RIFINs and STEVORs are two variant surface antigen families that are involved in malaria pathogenesis and immune evasion. Recent work has shown that a lack of humoral immunity to these proteins is associated with severe malaria vulnerability in Malian children. This is the first study to have compared serologic responses of children and adults to RIFINs and STEVORs in settings of malaria endemicity and to examine such serologic responses before and after a clinical malaria episode. Using microarrays, we determined that the semiconserved domains in these two parasite variant surface antigen families harbor peptides whose seroreactivity reflects malaria exposure. A similar approach has the potential to illuminate the role of variant surface antigens in the development of natural immunity to clinical malaria. Potential vaccines for severe malaria should include consideration of peptides within the semiconserved domains of RIFINs and STEVORs.

Cross-Platform Comparison of Computer-assisted Image Analysis Quantification of In Situ mRNA Hybridization in Investigative Pathology.

Although availability of automated platforms has proliferated, there is no standard practice for computer-assisted generation of scores for mRNA in situ hybridization (ISH) visualized by brightfield microscopic imaging on tissue sections. To address this systematically, an ISH for peptidylprolyl isomerase B (PPIB) (cyclophilin B) mRNA was optimized and applied to a tissue microarray of archival non-small cell lung carcinoma cases, and then automated image analysis for PPIB was refined across 4 commercially available software platforms. Operator experience and scoring results from ImageScope, HALO, CellMap, and Developer XD were systematically compared with each other and to manual pathologist scoring. Markup images were compared and contrasted for accuracy, the ability of the platform to identify cells, and the ease of visual assessment to determine appropriate interpretation. Comparing weighted scoring approaches using H-scores (Developer XD, ImageScope, and manual scoring) a correlation was observed (R value=0.7955), and association between the remaining 2 approaches (HALO and CellMap) was of similar value. ImageScope showed the highest R value in comparison with manual scoring (0.7377). Mean-difference plots showed that HALO produced the highest relative normalized values, suggesting higher relative sensitivity. ImageScope overestimated PPIB ISH signal at the high end of the range scores; however, this tendency was not observed in other platforms. HALO emerged with the highest number of favorable observations, no apparent systematic bias in score generation compared with the other methods, and potentially higher sensitivity to detect ISH. HALO may serve as a tool to empower teams of investigative pathology laboratory scientists to assist pathologists readily with quantitative scoring of ISH.

Children with cerebral malaria or severe malarial anaemia lack immunity to distinct variant surface antigen subsets.

Variant surface antigens (VSAs) play a critical role in severe malaria pathogenesis. Defining gaps, or "lacunae", in immunity to these Plasmodium falciparum antigens in children with severe malaria would improve our understanding of vulnerability to severe malaria and how protective immunity develops. Using a protein microarray with 179 antigen variants from three VSA families as well as more than 300 variants of three other blood stage P. falciparum antigens, reactivity was measured in sera from Malian children with cerebral malaria or severe malarial anaemia and age-matched controls. Sera from children with severe malaria recognized fewer extracellular PfEMP1 fragments and were less reactive to specific fragments compared to controls. Following recovery from severe malaria, convalescent sera had increased reactivity to certain non-CD36 binding PfEMP1s, but not other malaria antigens. Sera from children with severe malarial anaemia reacted to fewer VSAs than did sera from children with cerebral malaria, and both of these groups had lacunae in their seroreactivity profiles in common with children who had both cerebral malaria and severe malarial anaemia. This microarray-based approach may identify a subset of VSAs that could inform the development of a vaccine to prevent severe disease or a diagnostic test to predict at-risk children.

Mother-Newborn Pairs in Malawi Have Similar Antibody Repertoires to Diverse Malaria Antigens.

Maternal antibodies may play a role in protecting newborns against malaria disease. Plasmodium falciparum parasite surface antigens are diverse, and protection from infection requires allele-specific immunity. Although malaria-specific antibodies have been shown to cross the placenta, the extent to which antibodies that respond to the full repertoire of diverse antigens are transferred from the mother to the infant has not been explored. Understanding the breadth of maternal antibody responses and to what extent these antibodies are transferred to the child can inform vaccine design and evaluation. We probed plasma from cord blood and serum from mothers at delivery using a customized protein microarray that included variants of malaria vaccine target antigens to assess the intensity and breadth of seroreactivity to three malaria vaccine candidate antigens in mother-newborn pairs in Malawi. Among the 33 paired specimens that were assessed, mothers and newborns had similar intensity and repertoire of seroreactivity. Maternal antibody levels against vaccine candidate antigens were the strongest predictors of infant antibody levels. Placental malaria did not significantly impair transplacental antibody transfer. However, mothers with placental malaria had significantly higher antibody levels against these blood-stage antigens than mothers without placental malaria. The repertoire and levels of infant antibodies against a wide range of malaria vaccine candidate antigen variants closely mirror maternal levels in breadth and magnitude regardless of evidence of placental malaria. Vaccinating mothers with an effective malaria vaccine during pregnancy may induce high and potentially protective antibody repertoires in newborns.

Expression of complement and toll-like receptor pathway genes is associated with malaria severity in Mali: a pilot case control study.

BACKGROUND: The host response to infection by Plasmodium falciparum, the parasite most often responsible for severe malaria, ranges from asymptomatic parasitaemia to death. The clinical trajectory of malaria is influenced by host genetics and parasite load, but the factors determining why some infections produce uncomplicated malaria and some proceed to severe disease remain incompletely understood. METHODS: To identify molecular markers of severe falciparum malaria, human gene expression patterns were compared between children aged 6 months to 5 years with severe and uncomplicated malaria who were enrolled in a case-control study in Bandiagara, Mali. Microarrays were used to obtain expression data on severe cases and uncomplicated controls at the time of acute disease presentation (five uncomplicated and five severe), 1 week after presentation (three uncomplicated and three severe) and treatment initiation, and in the subsequent dry season (late convalescence, four uncomplicated and four severe). This is a pilot study for the first use of microarray technology in Mali. RESULTS: Complement and toll-like receptor (TLR) pathways were differentially expressed, with severe cases showing higher expression of the C1q, TLR2, TLR4, TLR8, and CR1 genes. Other genes previously associated with malaria pathogenesis, GZMB, FOS and HSPA6, were also higher among severe cases. TLR2, TLR4, TLR8, CR1, GZMB, FOS, and HSPA6 genes were expressed at lower levels in severe cases at late convalescence. CONCLUSIONS: Overexpression of genes previously associated with uncomplicated malaria was associated with severe disease. Low baseline expression of these genes may represent candidate markers for severe malaria. Despite the small sample size, results of this pilot study offer promising targets for follow-up analyses.

Daidzein Augments Cholesterol Homeostasis via ApoE to Promote Functional Recovery in Chronic Stroke.

Stroke is the world's leading cause of physiological disability, but there are currently no available agents that can be delivered early after stroke to enhance recovery. Daidzein, a soy isoflavone, is a clinically approved agent that has a neuroprotective effect in vitro, and it promotes axon growth in an animal model of optic nerve crush. The current study investigates the efficacy of daidzein on neuroprotection and functional recovery in a clinically relevant mouse model of stroke recovery. In light of the fact that cholesterols are essential lipid substrates in injury-induced synaptic remodeling, we found that daidzein enhanced the cholesterol homeostasis genetic program, including Lxr and downstream transporters, Apoe, Abca1, and Abcg1 genes in vitro. Daidzein also elevated the cholesterol homeostasis genes in the poststroke brain with Apoe, the highest expressing transporter, but did not affect infarct volume or hemispheric swelling. Despite the absence of neuroprotection, daidzein improved motor/gait function in chronic stroke and elevated synaptophysin expression. However, the daidzein-enhanced functional benefits and synaptophysin expression were abolished in Apoe-knock-out mice, suggesting the importance of daidzein-induced ApoE upregulation in fostering stroke recovery. Dissociation between daidzein-induced functional benefits and the absence of neuroprotection further suggest the presence of nonoverlapping mechanisms underlying recovery processes versus acute pathology. With its known safety in humans, early and chronic use of daidzein aimed at augmenting ApoE may serve as a novel, translatable strategy to promote functional recovery in stroke patients without adverse acute effect. SIGNIFICANCE STATEMENT: There have been recurring translational failures in treatment strategies for stroke. One underlying issue is the disparity in outcome analysis between animal and clinical studies. The former mainly depends on acute infarct size, whereas long-term functional recovery is an important outcome in patients. In an attempt to identify agents that promote functional recovery, we discovered that an FDA-approved soy isoflavone, daidzein, improved stroke-induced behavioral deficits via enhancing cholesterol homeostasis in chronic stroke, and this occurs without causing adverse effects in the acute phase. With its known safety in humans, the study suggests that the early and chronic use of daidzein serves as a potential strategy to promote functional recovery in stroke patients.

Differential recognition of terminal extracellular Plasmodium falciparum VAR2CSA domains by sera from multigravid, malaria-exposed Malian women.

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family mediates parasite sequestration in small capillaries through tissue-specific cytoadherence. The best characterized of these proteins is VAR2CSA, which is expressed on the surface of infected erythrocytes that bind to chondroitin sulfate in the placental matrix. Antibodies to VAR2CSA prevent placental cytoadherence and protect against placental malaria. The size and complexity of the VAR2CSA protein pose challenges for vaccine development, but smaller constitutive domains may be suitable for subunit vaccine development. A protein microarray was printed to include five overlapping fragments of the 3D7 VAR2CSA extracellular region. Malian women with a history of at least one pregnancy had antibody recognition of four of these fragments and had stronger reactivity against the two distal fragments than did nulliparous women, children, and men from Mali, suggesting that the C-terminal extracellular VAR2CSA domains are a potential focus of protective immunity. With carefully chosen sera from longitudinal studies of pregnant women, this approach has the potential to identify seroreactive VAR2CSA domains associated with protective immunity against pregnancy-associated malaria.

Seroreactivity to a large panel of field-derived Plasmodium falciparum apical membrane antigen 1 and merozoite surface protein 1 variants reflects seasonal and lifetime acquired responses to malaria.

Parasite antigen diversity poses an obstacle to developing an effective malaria vaccine. A protein microarray containing Plasmodium falciparum apical membrane antigen 1 (AMA1, n = 57) and merozoite surface protein 1 19-kD (MSP119, n = 10) variants prevalent at a malaria vaccine testing site in Bandiagara, Mali, was used to assess changes in seroreactivity caused by seasonal and lifetime exposure to malaria. Malian adults had significantly higher magnitude and breadth of seroreactivity to variants of both antigens than did Malian children. Seroreactivity increased over the course of the malaria season in children and adults, but the difference was more dramatic in children. These results help to validate diversity-covering protein microarrays as a promising tool for measuring the breadth of antibody responses to highly variant proteins, and demonstrate the potential of this new tool to help guide the development of malaria vaccines with strain-transcending efficacy.

Seroreactivity to Plasmodium falciparum erythrocyte membrane protein 1 intracellular domain in malaria-exposed children and adults.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigens mediate parasite sequestration and host immune evasion. Reactivity to 21 PfEMP1 fragments on a protein microarray was measured in serum samples from Malian children aged 1-6 years and adults. Seroreactivity to PfEMP1 fragments was higher in adults than in children; intracellular conserved fragments were more widely recognized than were extracellular hypervariable fragments. Over a malaria season, children maintained this differential seroreactivity and recognized additional intracellular PfEMP1 fragments. This approach has the potential to identify conserved, seroreactive extracellular PfEMP1 domains critical for protective immunity to malaria.

Alcohol drinking and deprivation alter basal extracellular glutamate concentrations and clearance in the mesolimbic system of alcohol-preferring (P) rats.

The present study determined the effects of voluntary ethanol drinking and deprivation on basal extracellular glutamate concentrations and clearance in the mesolimbic system and tested the hypothesis that chronic ethanol drinking would persistently increase basal glutamate neurotransmission. Three groups of alcohol-preferring (P) rats were used: 'water group (WG),' 'ethanol maintenance group (MG; 24-hour free choice water versus 15% ethanol)' and 'ethanol deprivation group (DG; 2 weeks of deprivation).' Quantitative microdialysis and Western blots were conducted to measure basal extracellular glutamate concentrations, clearance and proteins associated with glutamate clearance. Chronic alcohol drinking produced a 70-100% increase of basal extracellular glutamate concentrations in the posterior ventral tegmental area (4.0 versus 7.0 µM) and nucleus accumbens shell (3.0 versus 6.0 µM). Glutamate clearances were reduced by 30-40% in both regions of MG rats compared with WG rats. In addition, Western blots revealed a 40-45% decrease of excitatory amino transporter 1 (EAAT1) protein, but no significant changes in the levels of EAAT2 or cystine-glutamate antiporter in these regions of MG versus WG rats. The enhanced glutamate concentrations returned to control levels, accompanied by a recovery of glutamate clearance following deprivation. These results indicated that chronic alcohol drinking enhanced extracellular glutamate concentrations in the mesolimbic system, as a result, in part, of reduced clearance, suggesting that enhanced glutamate neurotransmission may contribute to the maintenance of alcohol drinking. However, because the increased glutamate levels returned to normal after deprivation, elevated glutamate neurotransmission may not contribute to the initiation of relapse drinking.

High-affinity choline uptake (HACU) and choline acetyltransferase (ChAT) activity in neuronal cultures for mechanistic and drug discovery studies.

Acetylcholine (ACh) is the neurotransmitter used by cholinergic neurons at the neuromuscular junction, in parasympathetic peripheral nerve terminals, and in important memory-related circuits in the brain, and takes part in other critical functions. ACh is synthesized from choline and acetyl coenzyme A by the enzyme choline acetyltransferase (ChAT). The formation of ACh in cholinergic nerve terminals requires the transport of choline into cells from the extracellular space and the activity of ChAT. High-affinity choline uptake (HACU) represents the majority of choline uptake into the nerve terminal and is the acutely regulated, rate-limiting step in ACh synthesis. HACU can be differentiated from nonspecific choline uptake by inhibition of the choline transporter with hemicholinium. Several methods have been described previously to measure HACU and ChAT activity simultaneously in synaptosomes, but a well-documented protocol for cultured cells is lacking. We describe a procedure for simultaneous measurement of HACU and ChAT in cultured cells by simple radionuclide-based techniques. Using this procedure, we have quantitatively determined HACU and ChAT activity in cholinergically differentiated human neuroblastoma (SK-N-SH) cells. These simple methods can be used for neurochemical and drug discovery studies relevant to several disorders, including Alzheimer's disease, myasthenia gravis, and cardiovascular disease.