A pilot study to understand feasibility and acceptability of stool and cord blood sample collection for a large-scale longitudinal birth cohort.

BACKGROUND: Few data are available to guide biological sample collection around the time of birth for large-scale birth cohorts. We are designing a large UK birth cohort to investigate the role of infection and the developing immune system in determining future health and disease. We undertook a pilot to develop methodology for the main study, gain practical experience of collecting samples, and understand the acceptability of sample collection to women in late pregnancy. METHODS: Between February-July 2014, we piloted the feasibility and acceptability of collecting maternal stool, baby stool and cord blood samples from participants recruited at prolonged pregnancy and planned pre-labour caesarean section clinics at University College London Hospital. Participating women were asked to complete acceptability questionnaires. RESULTS: Overall, 265 women were approached and 171 (65%) participated, with ≥1 sample collected from 113 women or their baby (66%). Women had a mean age of 34 years, were primarily of white ethnicity (130/166, 78%), and half were nulliparous (86/169, 51%). Women undergoing planned pre-labour caesarean section were more likely than those who delivered vaginally to provide ≥1 sample (98% vs 54%), but less likely to provide maternal stool (10% vs 43%). Pre-sample questionnaires were completed by 110/171 women (64%). Most women reported feeling comfortable with samples being collected from their baby (<10% uncomfortable), but were less comfortable about their own stool (19% uncomfortable) or a vaginal swab (24% uncomfortable). CONCLUSIONS: It is possible to collect a range of biological samples from women around the time of delivery, and this was acceptable for most women. These data inform study design and protocol development for large-scale birth cohorts.

Influence of infection on malaria-specific antibody dynamics in a cohort exposed to intense malaria transmission in northern Uganda.

The role of submicroscopic infections in modulating malaria antibody responses is poorly understood and requires longitudinal studies. A cohort of 249 children ≤5 years of age, 126 children between 6 and 10 years and 134 adults ≥20 years was recruited in an area of intense malaria transmission in Apac, Uganda and treated with artemether/lumefantrine at enrolment. Parasite carriage was determined at enrolment and after 6 and 16 weeks using microscopy and PCR. Antibody prevalence and titres to circumsporozoite protein, apical membrane antigen-1 (AMA-1), merozoite surface protein-1 (MSP-119 ), merozoite surface protein-2 (MSP-2) and Anopheles gambiae salivary gland protein 6 (gSG6) were determined by ELISA. Plasmodium falciparum infections were detected in 38·1% (194/509) of the individuals by microscopy and in 57·1% (284/493) of the individuals by PCR at enrolment. Antibody prevalence and titre against AMA-1, MSP-119 , MSP-2 and gSG6 were related to concurrent (sub-)microscopic parasitaemia. Responses were stable in children who were continuously infected with malaria parasites but declined in children who were never parasitaemic during the study or were not re-infected after treatment. These findings indicate that continued malaria infections are required to maintain antibody titres in an area of intense malaria transmission.

Endogenous galectin-3 controls experimental malaria in a species-specific manner.

Galectins are evolutionarily conserved glycan-binding proteins with pleiotropic roles in innate and adaptive immune responses. Galectin-3 has been implicated in several immunological processes as well as in pathogen recognition through specific binding to glycosylated receptors on the surface of host cells or microorganisms. In spite of considerable evidence supporting a role for galectin-3 in host-pathogen interactions, the relevance of this lectin in the regulation of the host defence mechanisms in vivo is poorly understood. In this study, we analysed the impact of galectin-3 deficiency during infection with three distinct species of rodent malaria parasites, Plasmodium yoelii 17XNL, Plasmodium berghei ANKA and Plasmodium chabaudi AS. We found that galectin-3 deficiency showed a marginal effect on the course of parasitaemia during P. chabaudi infection, but did not alter the course of parasitaemia during P. berghei infection. However, lack of galectin-3 significantly reduced P. yoelii parasitaemia. This reduced parasitaemia in Lgals3(-/-) mice was consistent with higher titres of anti-P. yoelii MSP1(19) IgG2b isotype antibodies when compared with their wild-type counterparts. Our results reflect the complexity and singularity of host-pathogen interactions, indicating a species-specific role of endogenous galectin-3 in the control of parasite infections and the modulation of antibody responses.

Transforming growth factor beta production is inversely correlated with severity of murine malaria infection.

We have examined the role of the immunomodulatory cytokine transforming growth factor (TGF)-beta in the resolution and pathology of malaria in BALB/c mice. Circulating levels of TGF-beta, and production of bioactive TGF-beta by splenocytes, were found to be low in lethal infections with Plasmodium berghei. In contrast, resolving infections with P. chabaudi chabaudi or P. yoelii were accompanied by significant TGF-beta production. A causal association between the failure to produce TGF-beta and the severity of malaria infection was demonstrated by treatment of infected mice with neutralizing antibody to TGF-beta, which exacerbated the virulence of P. berghei and transformed a resolving P. chabaudi chabaudi infection into a lethal infection, but had little effect on the course of P. yoelii infection. Parasitemia increased more rapidly in anti-TGF-beta-treated mice but this did not seem to be the explanation for the increased pathology of infection as peak parasitemias were unchanged. Treatment of P. berghei-infected mice with recombinant TGF-beta (rTGF-beta) slowed the rate of parasite proliferation and prolonged their survival from 15 to up to 35 d. rTGF-beta treatment was accompanied by a significant decrease in serum tumor necrosis factor alpha and an increase in interleukin 10. Finally, we present evidence that differences in TGF-beta responses in different malaria infections are due to intrinsic differences between species of malaria parasites in their ability to induce production of TGF-beta. Thus, TGF-beta seems to induce protective immune responses, leading to slower parasite growth, early in infection, and, subsequently, appears to downregulate pathogenic responses late in infection. This duality of effect makes TGF-beta a prime candidate for a major immunomodulatory cytokine associated with successful control of malaria infection.

Glycosylphosphatidylinositols in malaria pathogenesis and immunity: potential for therapeutic inhibition and vaccination.

Glycosylphosphatidylinositols (GPIs) are found in the outer cell membranes of all eukaryotes. GPIs anchor a diverse range of proteins to the surface of Plasmodium falciparum, but may also exist free of protein attachment. In vitro and in vivo studies have established GPIs as likely candidate toxins in malaria, consistent with the prevailing paradigm that attributes induction of inflammatory cytokines, fever and other pathology to parasite toxins released when schizonts rupture. Although evolutionarily conserved, sufficient structural differences appear to exist that impart upon plasmodial GPIs the ability to activate second messengers in mammalian cells and elicit immune responses. In populations exposed to P. falciparum, the antibody response to purified GPIs is characterised by a predominance of immunoglobulin (Ig)G over IgM and an increase in the prevalence, level and persistence of responses with increasing age. It remains unclear, however, if these antibodies or other cellular responses to GPIs mediate anti-toxic immunity in humans; anti-toxic immunity may comprise either reduction in the severity of disease or maintenance of the malaria-tolerant state (i.e. persistent asymptomatic parasitaemia). P. falciparum GPIs are potentially amenable to specific therapeutic inhibition and vaccination; more needs to be known about their dual roles in malaria pathogenesis and protection for these strategies to succeed.

Unique Distal Enhancers Linked to the Mouse Tnfsf11 Gene Direct Tissue-Specific and Inflammation-Induced Expression of RANKL.

Receptor activator of nuclear factor κB ligand (RANKL) is expressed by a number of cell types to participate in diverse physiological functions. We have previously identified 10 distal RANKL enhancers. Earlier studies have shown that RL-D5 is a multifunctional RANKL enhancer. Deletion of RL-D5 from the mouse genome leads to lower skeletal and lymphoid tissue RANKL, causing a high bone mass phenotype. Herein, we determine the physiological role and lineage specificity of 2 additional RANKL enhancers, RL-D6 and RL-T1, which are located 83 and 123 kb upstream of the gene's transcriptional start site, respectively. Lack of RL-D6 or RL-T1 did not alter skeletal RANKL or bone mineral density up to 48 weeks of age. Although both RL-D5 and RL-T1 contributed to activation induction of T-cell RANKL, RL-T1 knockout mice had drastically low lymphocyte and lymphoid tissue RANKL levels, indicating that RL-T1 is the major regulator of lymphocyte RANKL. Moreover, RL-T1 knockout mice had lower circulating soluble RANKL, suggesting that lymphocytes are important sources of circulating soluble RANKL. Under physiological conditions, lack of RL-D6 did not alter RANKL expression. However, lack of RL-D5 or RL-D6, but not of RL-T1, blunted the oncostatin M and lipopolysaccharide induction of RANKL ex vivo and in vivo, suggesting that RL-D5 and RL-D6 coregulate the inflammation-mediated induction of RANKL in osteocytes and osteoblasts while lack of RL-D6 did not alter secondary hyperparathyroidism or lactation induction of RANKL or bone loss. These results suggest that although RL-D5 mediates RANKL expression in multiple lineages, other cell type- or factor-specific enhancers are required for its appropriate control, demonstrating the cell type-specific and complex regulation of RANKL expression.

Does apolipoprotein E polymorphism influence susceptibility to malaria?

Outcome of infection varies greatly among people, and in the case of three very different viruses, it is determined by apolipoprotein E (APOE) genotype. APOE might affect outcome of malaria infection also, since apoE protein and the protozoon (like the viruses) share cell entry mediators (heparan sulphate proteoglycans and/or specific apoE receptors). APOE polymorphisms give rise to protein variants that differ in binding strength to these mediators; thus, the extent of competition between apoE and protozoon for cell entry, and hence magnitude of protozoan damage, might depend on apoE isoform. Genotypes of infants infected with malaria were examined. It was found that APOE epsilon 2 homozygotes became infected at an earlier age than those carrying the other genotypes, the difference being statistically significant. Parasite densities, all of which were low, did not differ significantly. This effect, although based on small numbers, suggests that APOE epsilon 2 may be a risk factor for early infection.

The statistical analysis of data from immunoepidemiological studies.

Immune responses encountered in human populations display a higher level of heterogeneity than is typically seen with laboratory animals, and the expected clear distinction between responders and non-responders to particular antigens may not be apparent. In such a situation, the methods of statistical analysis traditionally used in laboratory studies may no longer be appropriate, and there is no consensus among immunologists about how such data should be analysed. Using data from a large study of lymphocyte proliferation responses to candidate antigens for a vaccine against Plasmodium falciparum malaria, we discuss questions such as the appropriateness of logarithmic transformation of the data, the use of the stimulation index versus delta--cpm, the use of the proportion of responders for comparing two groups of individuals, and the choice of threshold for defining such a responder.

Isolation of maternal mononuclear cells from placentas for use in in vitro functional assays.

Interest in immunoregulatory mechanisms within uteroplacental tissues, particularly in malarial infection during pregnancy, prompted us to develop a technique to extract maternal mononuclear cells from human term placentas. This method is described. The phenotypes of isolated cells were characterised for expression of CD45, CD3, CD4, CD8, CD14, CD15, CD68, CD22, CAM 5.2 and class II MHC antigens and compared with those in situ in frozen sections of the same placentas. Isolated mononuclear cell preparations were examined for contamination by fetal trophoblasts. Fetal leukocyte contamination appeared unlikely since histological sections of placental tissue, after the extraction of maternal leukocytes, showed intact chorionic villi with no disruption of fetal stem vessels. This technique produces preparations of maternal placental mononuclear cells which are representative of cells in situ, show minimal fetal cell contamination and are suitable for functional studies.

T cell reactivity of defined peptides from a major Plasmodium falciparum vaccine candidate: the Pf155/RESA antigen.

Several immunodominant B-cell epitopes of the P. falciparum antigen blood stage Pf155/RESA, a major vaccine candidate antigen, are located in the molecular regions containing amino acid repeats. We started to map Pf155/RESA for T cell reactive epitopes. For this purpose, short synthetic peptides corresponding to the 3'- and 5' repeat regions of the molecule as well as to non-repeated sequences outside these regions were prepared. T cells from P. falciparum primed donors from two highly endemic areas of Africa were tested for their responsiveness to the peptides by thymidine incorporation and/or interferon gamma (IFN-gamma) release. There was a considerable variation in the response to the different peptides. However, the strongest and most frequent responses were seen with a few peptides from the 3'- and 5'-repeat regions. Thus, the immunodominant B cell epitope regions of Pf155/RESA, contain several T cell epitopes. Since the repeat regions are known to be conserved in different P. falciparum strains, the T cell epitopes reported here may be suitable constituents of a P. falciparum subunit vaccine.

Characterization of regulatory T cell responses to defined immunodominant T cell epitopes of the Plasmodium falciparum antigen Pf155/RESA.

Several immunodominant B and T cell epitopes of the P. falciparum blood stage antigen Pf155/RESA, a vaccine candidate, are located in the central (5') and C-terminal (3') invariant repeat regions of the molecule. Here we have attempted to functionally analyze human T cell responses to some of the T cell epitopes. For this purpose short synthetic peptides corresponding to these epitopes were used to study the induction of in vitro expression of IL-4 mRNA, IFN-gamma secretion, proliferation and B cell help for antibody production. In individual malaria immune donors these different T cell activities were not correlated. The findings emphasize the importance of examining multiple parameters of T cell activation when estimating the total proportion of individuals responding to a defined antigen. IL-4 mRNA was expressed in activated T cells of donors who had elevated serum concentrations of antibodies to the peptide used for T cell activation. These results suggest the involvement of IL-4 producing T helper cells in the induction of Pf155/RESA specific antibody production in individuals in which immunity has been induced by natural infection. Taken together, these findings also suggest that functionally distinct CD4+ T cells occur in humans similarly to what has been described in mice. In further experiments, we have also attempted to establish MHC class II restriction of the immune response to these epitopes at the level of the donor populations. When studying monozygotic twins, antibody responses to Pf155/RESA derived peptides and some of the T cell responses could be paired within the twin pairs, indicating a genetic regulation of their B cell responses. Whether or not this regulation reflects MHC class II restriction, or other factors needs to be elucidated.

Isolation and characterization of a soluble antigen complex of Plasmodium falciparum with pyrogenic properties.

A soluble antigen complex, previously designated antigen no. 7 (Ag7) on the basis of the pattern obtained by crossed immunoelectrophoresis of culture supernatants of P. falciparum, was isolated by affinity chromatography. It was shown to be synthesized at the schizont stage of the parasite growth cycle and to be located on the surface of the schizonts. Antibodies to Ag7 did not inhibit the growth of the parasite in vitro. Ag7 is recognized by immune human sera from many parts of the world and it stimulated the production of specific antibody in mice when incorporated into immune-stimulating complex (ISCOM) structures. It also specifically stimulated in vitro proliferation of lymphocytes from clinically immune adults. That it induced the secretion of interleukin 1 by human monocytes and was pyrogenic in rabbits was of particular interest. Thus Ag7 has endotoxin-like properties which make it a possible candidate for an antitoxic malaria vaccine.

Neuropsychological functioning in first-episode psychosis--evidence of specific deficits.

Neuropsychological impairment is ubiquitous in schizophrenia even at the first presentation of psychotic symptoms. We sought to elucidate the nature of the neuropsychological profile at the onset of the illness by examining the neuropsychological functioning of 40 patients experiencing their first episode of psychosis and 22 matched controls. All participants completed a battery of neuropsychological tasks designed to assess attention, verbal learning/memory, non-verbal memory, spatial ability, psychomotor speed, and executive function. First-episode patients showed significant impairment on tasks of executive function, including those requiring the ability to form and initiate a strategy, to inhibit prepotent responses, and to shift cognitive set, and also on tasks of verbal fluency. Memory impairments were seen on verbal learning and delayed non-verbal memory only. Impairment on tasks of psychomotor speed suggests that there may be a significant amount of cognitive slowing even at the first onset of psychosis. We suggest that our patients may be experiencing difficulty in specific aspects of executive functions, including the ability to form and execute a strategy, and these difficulties may be mediating the deficits observed on tasks of verbal learning.

Suppression of cell-mediated immune responses to malaria antigens in pregnant Gambian women.

In malaria endemic areas, pregnancy predisposes previously immune women to clinical and subclinical malaria infection. While parameters of humoral immunity do not seem to be affected by pregnancy, suppression of cellular immunity has been demonstrated for a number of antigens. In this study of women from a rural area of the Gambia where falciparum malaria is holoendemic, we show that lymphoproliferative responses to Plasmodium falciparum antigens are depressed in pregnant women compared to parity matched non-pregnant women, and that this effect is particularly marked in primigravidae. The data also indicate that malaria antigen induced gamma-interferon production may be depressed in pregnant women. There was no significant difference in antimalarial antibody titers between the 2 groups.

IgG3 antibodies to Plasmodium falciparum merozoite surface protein 2 (MSP2): increasing prevalence with age and association with clinical immunity to malaria.

In a cross-sectional survey carried out in west Africa (The Gambia), where Plasmodium falciparum malaria is endemic with seasonal transmission, 178 individuals 1-75 years of age were assessed for their antibody response to the malaria vaccine candidate, merozoite surface protein 2 (MSP2). Total IgG to recombinant antigens representing full-length, repetitive, and group-specific domains of both allelic families of MSP2 was determined by ELISA. The IgG-subclass profile of IgG-positive sera was assessed. Antibody prevalence was age-dependent, reaching a peak during adolescence. In MSP2-seropositive individuals, there was a predominance of cytophilic antibodies (IgG1 and IgG3); IgG1 antibodies were prevalent in children less than 10 years of age, whereas in adolescents and adults MSP2-specific antibodies were predominantly IgG3. In parallel, we conducted a longitudinal study of children (3-8 years of age) from the same community; sera collected before the malaria transmission season were tested for the presence of anti-MSP2 antibodies. The subsequent susceptibility of these children to clinical malaria was monitored and the association between anti-MSP2 antibodies of different IgG subclasses and resistance to clinical malaria was tested. The presence of IgG3 antibodies to MSP2 serogroup A was negatively associated with the risk of clinical malaria whereas IgG1 antibodies to MSP2 serogroup B were associated with an increased risk of clinical infection. Our data suggest that age/exposure-related acquisition of IgG3 antibodies to MSP2 may contribute to the development of clinically protective immunity to malaria.

Cellular immune responses to Plasmodium falciparum antigens in children receiving long term anti-malarial chemoprophylaxis.

Fifty-two Gambian children who had received fortnightly chemoprophylaxis with maloprim, (pyrimethamine and dapsone), and 45 receiving placebo, were studied. Cellular immune responses to malaria antigens, measured by lymphoproliferative responses and interferon production, were higher in children who had received prophylaxis than in controls, although the anti-malarial antibody levels were lower. During a one-year period after termination of prophylaxis, there was no increase in the frequency of clinical episodes of malaria in the children who had received Maloprim. These results suggest that chemoprophylaxis for 3 years may lower malaria antibody levels, but does not interfere with the development of protective immunity, perhaps by enhancing cell-mediated immune responses to malaria in protected children.

A prospective study of the influence of alpha thalassaemia on morbidity from malaria and immune responses to defined Plasmodium falciparum antigens in Gambian children.

The protective effect of alpha thalassaemia (-alpha/alpha alpha) against morbidity from falciparum malaria was assessed in a prospective study of rural Gambian children. The gene frequency for single alpha-globin gene deletions was 0.12. Malariometric indices measured during cross-sectional surveys and morbidity from malaria determined by weekly surveillance were similar in children with alpha thalassaemia and in those with a normal alpha-globin genotype. However, the small number of children who carried both alpha thalassaemia and the sickle cell trait had fewer clinical episodes of malaria than children with the sickle cell trait alone. Specific antibody responses and cell-mediated immune responses in vitro to defined Plasmodium falciparum antigens were measured in children participating in the study. In general, there was no evidence of an increased prevalence or intensity of humoral or cell-mediated immune responses to the malaria antigens studied in children heterozygous for alpha thalassaemia compared with children with a normal alpha-globin genotype.

Human leucocyte antigen (HLA) and malaria morbidity in a Gambian community.

Human leucocyte antigen (HLA) class I and class II typing was performed on 177 children in a rural area of The Gambia who were followed for 2 years in a longitudinal study of malaria morbidity. A comparison was made between those who experienced an episode of clinical malaria in one or both years and those who showed no evidence of infection in either year. No convincing association was found between morbidity and class I phenotype. An overall association of morbidity with the distribution of class II haplotypes was seen, but association with individual DR-DQ haplotypes were not conclusive.

Differential antibody response of Gambian donors to soluble Plasmodium falciparum antigens.

A seroepidemiological and clinical study was performed in an area of West Africa (The Gambia) where Plasmodium falciparum is endemic with seasonal transmission. Plasma samples were tested by intermediate gel immunoelectrophoresis for antibodies against 7 soluble P. falciparum antigens. There were marked differences in the age-related pattern of antibody response to the different antigens. Antibodies to 4 of the antigens were acquired slowly with a maximum prevalence reached after 25-35 years of age. Antibodies against the 3 remaining antigens, including the endotoxin-like antigen, Ag7, were acquired earlier with a plateau of maximum prevalence reached after 5-11 years, i.e. at the time when morbidity due to malaria decreased. Children who had not appeared to be infected with malaria during the preceding transmission season had lower levels of antibodies to soluble antigens than did children who had had a documented attack of clinical malaria or parasitaemia. There was no difference in antibody profiles to soluble antigens between children with sickle cell trait and children with normal haemoglobin.

Aflatoxin exposure, malaria and hepatitis B infection in rural Gambian children.

Aflatoxin-albumin adduct levels were measured in serum samples obtained from a group of Gambian children. The relationships between exposure to aflatoxin and the prevalence of malaria, between exposure and humoral and cellular responses in vitro to defined malaria antigens and, amongst children with evidence of exposure to hepatitis B infection, between aflatoxin and carriage of the hepatitis B surface antigen (HBsAg), were assessed. Aflatoxin-albumin adduct was found in nearly all serum samples collected during a survey performed at the end of the dry season and levels of adduct were generally high (up to 720 pg aflatoxin-lysine equivalent/mg albumin). Higher levels of aflatoxin-albumin adduct were detected in Wollof children than in children of other ethnic groups and marked variation in mean adduct levels between villages was observed. Aflatoxin-albumin adduct levels were higher in children who were HbsAg positive and in children with Plasmodium falciparum parasitaemia than in controls. However, levels of adduct had no consistent effect on either malaria-specific antibody responses, lymphoproliferative responses in vitro, or morbidity from malaria during the subsequent rainy season. Much lower levels of aflatoxin-albumin adduct were detected in repeat samples obtained at the end of the rainy season. There was poor correlation between dry and rainy season levels of adduct in individual children. We have shown that Gambian children are exposed to high levels of aflatoxin. The seasonal variation of aflatoxin-albumin adduct and marked fluctuation of adduct with time in individual children need to be considered in the future planning of epidemiological studies using this marker of exposure.(ABSTRACT TRUNCATED AT 250 WORDS)