Probiotic Bacillus spp. enhance TLR3-mediated TNF signalling in macrophages.

Probiotics have been reported to have immunomodulatory properties in the context of infectious disease and inflammation, although the underlying mechanisms are not fully understood. Here, we aimed to determine how different probiotic bacterial strains modulated macrophage function during TLR3 stimulation mimicking viral infection. We screened 14 different strains for their ability to modulate TNF-α, IL-6 IL-10, IFN-α, IFN-ß and IFN-γ secretion in RAW 264.7 macrophages with or without poly(I:C) stimulation. Seven strains were selected for further analysis using primary porcine alveolar macrophages. In-depth transcriptomic analysis on alveolar macrophages was conducted for two strains. Most strains induced a synergistic effect when co-incubated with poly(I:C) resulting in increased levels of IL-6 and TNF-α secretion from RAW 264.7 cells. This synergistic effect was found to be TLR2 independent. Only strains of Bacillus spp. could induce this effect in alveolar macrophages. Transcriptomic analysis indicated that the increased TNF-α secretion in alveolar macrophages after co-incubation with poly(I:C) correlated with significant upregulation of TNF and IL23A-related pathways. Collectively, our data show that probiotic bacteria possess strain-dependent immunomodulatory properties that may be harnessed to enhance innate immune responses to pathogens.

Dietary non-starch polysaccharides impair immunity to enteric nematode infection.

BACKGROUND: The influence of diet on immune function and resistance to enteric infection and disease is becoming ever more established. Highly processed, refined diets can lead to inflammation and gut microbiome dysbiosis, whilst health-promoting dietary components such as phytonutrients and fermentable fibres are thought to promote a healthy microbiome and balanced mucosal immunity. Chicory (Cichorium intybus) is a leafy green vegetable rich in fibres and bioactive compounds that may promote gut health. RESULTS: Unexpectedly, we here show that incorporation of chicory into semisynthetic AIN93G diets renders mice susceptible to infection with enteric helminths. Mice fed a high level of chicory leaves (10% dry matter) had a more diverse gut microbiota, but a diminished type-2 immune response to infection with the intestinal roundworm Heligmosomoides polygyrus. Furthermore, the chicory-supplemented diet significantly increased burdens of the caecum-dwelling whipworm Trichuris muris, concomitant with a highly skewed type-1 immune environment in caecal tissue. The chicory-supplemented diet was rich in non-starch polysaccharides, particularly uronic acids (the monomeric constituents of pectin). In accordance, mice fed pectin-supplemented AIN93G diets had higher T. muris burdens and reduced IgE production and expression of genes involved in type-2 immunity. Importantly, treatment of pectin-fed mice with exogenous IL-25 restored type-2 responses and was sufficient to allow T. muris expulsion. CONCLUSIONS: Collectively, our data suggest that increasing levels of fermentable, non-starch polysaccharides in refined diets compromises immunity to helminth infection in mice. This diet-infection interaction may inform new strategies for manipulating the gut environment to promote resistance to enteric parasites.

Whipworm Infection in Mice Increases Coinfection of Enteric Pathogens but Promotes Clearance of Ascaris Larvae From the Lungs.

Infection with intestinal whipworms (Trichuris spp.) causes widespread morbidity and may alter responses to enteric and extraintestinal coinfections. Here, we show that Trichuris muris infection in mice increases coinfection with 2 evolutionary divergent enteric pathogens, the bacterium Citrobacter rodentium and the helminth Heligmosomoides polygyrus. Coinfection caused reduced weight gain and promoted type 1-biased inflammation. In contrast, T. muris-infected mice were more resistant to migrating Ascaris suum larvae in the lungs. Our results highlight the divergent nature of pathogen interactions and suggest that whipworm infection is a risk factor for coinfections with other pathogens within the gastrointestinal tract.

Dietary proanthocyanidins promote localized antioxidant responses in porcine pulmonary and gastrointestinal tissues during Ascaris suum-induced type 2 inflammation.

Proanthocyanidins (PAC) are dietary polyphenols with putative anti-inflammatory and immunomodulatory effects. However, whether dietary PAC can regulate type-2 immune function and inflammation at mucosal surfaces remains unclear. Here, we investigated if diets supplemented with purified PAC modulated pulmonary and intestinal mucosal immune responses during infection with the helminth parasite Ascaris suum in pigs. A. suum infection induced a type-2 biased immune response in lung and intestinal tissues, characterized by pulmonary granulocytosis, increased Th2/Th1 T cell ratios in tracheal-bronchial lymph nodes, intestinal eosinophilia, and modulation of genes involved in mucosal barrier function and immunity. Whilst PAC had only minor effects on pulmonary immune responses, RNA-sequencing of intestinal tissues revealed that dietary PAC significantly enhanced transcriptional responses related to immune function and antioxidant responses in the gut of both naïve and A. suum-infected animals. A. suum infection and dietary PAC induced distinct changes in gut microbiota composition, primarily in the jejunum and colon, respectively. Notably, PAC consumption substantially increased the abundance of Limosilactobacillus reuteri. In vitro experiments with porcine macrophages and intestinal epithelial cells supported a role for both PAC polymers and PAC-derived microbial metabolites in regulating oxidative stress responses in host tissues. Thus, dietary PAC may have distinct beneficial effects on intestinal health during infection with mucosal pathogens, while having a limited activity to modulate naturally-induced type-2 pulmonary inflammation. Our results shed further light on the mechanisms underlying the health-promoting properties of PAC-rich foods, and may aid in the design of novel dietary supplements to regulate mucosal inflammatory responses in the gastrointestinal tract.

Diet-microbiota crosstalk and immunity to helminth infection.

Helminths are large multicellular parasites responsible for widespread chronic disease in humans and animals. Intestinal helminths live in close proximity with the host gut microbiota and mucosal immune network, resulting in reciprocal interactions that closely influence the course of infections. Diet composition may strongly regulate gut microbiota composition and intestinal immune function and therefore may play a key role in modulating anti-helminth immune responses. Characterizing the multitude of interactions that exist between different dietary components (e.g., dietary fibres), immune cells, and the microbiota, may shed new light on regulation of helminth-specific immunity. This review focuses on the current knowledge of how metabolism of dietary components shapes immune response during helminth infection, and how this information may be potentially harnessed to design new therapeutics to manage parasitic infections and associated diseases.

Alkaline oxidization can increase the in vitro antiparasitic activity of proanthocyanidin-rich plant extracts against Ascarissuum.

Proanthocyanidins (PAs) are a class of plant specialized metabolites with well-documented bioactivities such as antiparasitic effects. However, little is known about how the modification of PAs influences their bioactivity. The objective of this study was to investigate a wide range of PA-containing plant samples to determine if extracts containing PAs modified by oxidation had altered antiparasitic activities, compared to the original extracts that had not been modified in alkaline conditions. We extracted and analyzed samples from 61 proanthocyanidin-rich plants. The extracts were then oxidized under alkaline conditions. We used these non-oxidized and oxidized proanthocyanidin-rich extracts to conduct a detailed analysis of direct antiparasitic effects against the intestinal parasite Ascaris suum in vitro. These tests showed that the proanthocyanidin-rich extracts had antiparasitic activity. Modification of these extracts significantly increased the antiparasitic activity for the majority the extracts, suggesting that the oxidation procedure enhanced the bioactivity of the samples. Some samples that showed no antiparasitic activity before oxidation showed very high activity after the oxidation. High levels of other polyphenols in the extracts, such as flavonoids, was found to be associated with increased antiparasitic activity following oxidation. Thus, our in vitro screening opens up the opportunity for future research to better understand the mechanism of action how alkaline treatment of PA-rich plant extracts increases their biological activity and potential as novel anthelmintics.

Antigens from the parasitic nematode Trichuris suis induce metabolic reprogramming and trained immunity to constrain inflammatory responses in macrophages.

Regulation of macrophage (Mɸ) function can maintain tissue homeostasis and control inflammation. Parasitic worms (helminths) are potent modulators of host immune and inflammatory responses. They have evolved various strategies to promote immunosuppression, including redirecting phagocytic cells toward a regulatory phenotype. Although soluble products from the whipworm Trichuris suis (TSPs) have shown significant effects on Mɸ function, the mechanisms underlying these modulatory effects are still not well understood. In this study, we find that TSPs suppressed inflammatory cytokines (TNF and IL-6) in Mɸs stimulated with a broad panel of TLR agonists, whilst inducing IL-10. Moreover, M1 markers such as MHCII, CD86, iNOS, and TNF were downregulated in TSP-treated Mɸs, without polarizing them towards an M2-like phenotype. We showed that TSPs could establish a suppressed activation state of Mɸs lasting at least for 72 h, indicating an anti-inflammatory innate training. Moreover, we found that TSPs, via repression of intracellular TNF generation, decreased its secretion rather than interfering with the release of surface-bound TNF. Metabolic analysis showed that TSPs promote oxidative phosphorylation (OXPHOS) without affecting glycolytic rate. Collectively, these findings expand our knowledge on helminth-induced immune modulation and support future investigations into the anti-inflammatory properties of TSPs for therapeutic purposes.

Fermentable Dietary Fiber Promotes Helminth Infection and Exacerbates Host Inflammatory Responses.

Fermentable dietary fibers promote the growth of beneficial bacteria, can enhance mucosal barrier integrity, and reduce chronic inflammation. However, effects on intestinal type 2 immune function remain unclear. In this study, we used the murine whipworm Trichuris muris to investigate the effect of the fermentable fiber inulin on host responses to infection regimes that promote distinct Th1 and Th2 responses in C57BL/6 mice. In uninfected mice, dietary inulin stimulated the growth of beneficial bacteria, such as Bifidobacterium (Actinobacteria) and Akkermansia (Verrucomicrobia). Despite this, inulin prevented worm expulsion in normally resistant mice, instead resulting in chronic infection, whereas mice fed an equivalent amount of nonfermentable fiber (cellulose) expelled worms normally. Lack of expulsion in the mice fed inulin was accompanied by a significantly Th1-skewed immune profile characterized by increased T-bet+ T cells and IFN-γ production in mesenteric lymph nodes, increased expression of Ido1 in the cecum, and a complete absence of mast cell and IgE production. Furthermore, the combination of dietary inulin and high-dose T. muris infection caused marked dysbiosis, with expansion of the Firmicutes and Proteobacteria phyla, near elimination of Bacteroidetes, and marked reductions in cecal short-chain fatty acids. Neutralization of IFN-γ during infection abrogated Ido1 expression and was sufficient to restore IgE production and worm expulsion in inulin-fed mice. Our results indicate that, whereas inulin promoted gut health in otherwise healthy mice, during T. muris infection, it exacerbated inflammatory responses and dysbiosis. Thus, the positive effects of fermentable fiber on gut inflammation appear to be context dependent, revealing a novel interaction between diet and infection.

Oxycodone induced euphoria in ED patients with acute musculoskeletal pain. A secondary analysis of data from a randomized trial.

OBJECTIVES: Some opioid-naïve patients with acute musculoskeletal pain who are treated with opioids develop persistent opioid use. The impact of opioid-induced euphoria on this transition to persistent use has not been explored. We determined whether opioid-induced euphoria could be measured as a phenomenon distinct from relief of pain. METHODS: Patients with acute pain were randomized to receive oxycodone/acetaminophen (Oxy) or acetaminophen (APAP). We measured pain using a 0-10 verbal scale. To assess euphoria, participants provided a 0-10 response to each of these: 1) How good did the medication make you feel?; 2) How high did the medication make you feel?; 3) How blissful did the medication make you feel? We analyzed these data using successive multivariable linear regression models, in which each of these items was the dependent variable, and improvement in pain and medication were the independent variables, while controlling for age and sex. RESULTS: 75 were randomized to Oxy, 76 to APAP. Mean "how good" scores were 6.3 (SD 3.3) in the Oxy group and 4.8 (3.3) in the APAP group. Mean "how high" scores were 3.8 (3.7) in the Oxy group and 2.0 (3.0) in the APAP group. Mean "how blissful" scores were 4.9 (3.7) in the Oxy group and 3.1 (3.4) in the APAP group. After controlling for improvement in pain, age, and sex, the between-group difference in "how good" was 1.0 (95%CI: -0.1, 2.0), "how high" 1.5 (95% CI 0.4, 2.6), and "how blissful" 1.5 (95%CI: 0.4, 2.7). DISCUSSION: "How high" and "how blissful" but not "how good" were associated with opioid use after controlling for improvement in pain.

The Relative Efficacy of Seven Skeletal Muscle Relaxants. An Analysis of Data From Randomized Studies.

BACKGROUND: Low back pain (LBP) causes 2.6 million visits to U.S. emergency departments (EDs) annually. These patients are often treated with skeletal muscle relaxants (SMRs). OBJECTIVES: The goal of this study was to determine whether efficacy of SMRs is associated with age, sex, or baseline LBP severity. METHODS: This was a planned analysis of data from 4 randomized studies of patients with acute nonradicular LBP. Patients were enrolled during an ED visit and followed-up 1 week later. The primary outcome was improvement in the Roland-Morris Disability Questionnaire (RMDQ) between ED discharge and the 1-week follow-up. We compared the change in RMDQ among 8 groups: placebo, baclofen, metaxalone, tizanidine, diazepam, orphenadrine, methocarbamol, and cyclobenzaprine. All patients also received a nonsteroidal anti-inflammatory drug. We performed analysis of variance to determine statistically significant differences between medications and linear regression to determine the association of age, sex, and baseline severity with the primary outcome. RESULTS: The mean improvement in RMDQ per group was placebo 10.5 (95% confidence interval [CI] 9.5-11.5), baclofen 10.6 (95% CI 8.6-12.7), metaxalone 10.3 (95% CI 8.1-12.4), tizanidine 11.5 (95% CI 9.5-13.4), diazepam 11.1 (95% CI 9-13.2), orphenadrine 9.5 (95% CI 7.4-11.5), methocarbamol 8.1 (95% CI 6.1-10.1), and cyclobenzaprine 10.1 (95% CI 8.3-12). The between-group differences were not statistically significantly different. Results were similar regardless of age, sex, and baseline severity. Higher baseline RMDQ was associated with greater clinical improvement (B coefficient 5.7, p < 0.01). Adverse medication effects were more common with cyclobenzaprine than with placebo (p < 0.01). CONCLUSIONS: Among patients in the ED with acute LBP treated with a nonsteroidal anti-inflammatory drug, SMRs do not improve outcomes more than placebo. Neither age, sex, nor baseline impairment impacts these results.

Pre-weaning adaptation responses in piglets fed milk replacer with gradually increasing amounts of wheat.

Hyperprolific sows rear more piglets than they have teats, and to accommodate this, milk replacers are often offered as a supplement. Milk replacers are based on bovine milk, yet components of vegetable origin are often added. This may reduce growth, but could also accelerate maturational changes. Therefore, we investigated the effect of feeding piglets a milk replacer with gradually increasing levels of wheat flour on growth, gut enzyme activity and immune function compared with a diet based entirely on bovine milk. The hypothesis tested was that adding a starch component (wheat flour) induces maturation of the mucosa as measured by higher digestive activity and improved integrity and immunity of the small intestines (SI). To test this hypothesis, piglets were removed from the sow at day 3 and fed either a pure milk replacer diet (MILK) or from day 11 a milk replacer diet with increasing levels of wheat (WHEAT). The WHEAT piglets had an increased enzyme activity of maltase and sucrase in the proximal part of the SI compared with the MILK group. There were no differences in gut morphology, histopathology and gene expression between the groups. In conclusion, the pigs given a milk replacer with added wheat displayed immunological and gut mucosal enzyme maturational changes, indicatory of adaptation towards a vegetable-based diet. This was not associated with any clinical complications, and future studies are needed to show whether this could improve responses in the subsequent weaning process.

Emerging interactions between diet, gastrointestinal helminth infection, and the gut microbiota in livestock.

Increasing evidence suggests that nutritional manipulation of the commensal gut microbiota (GM) may play a key role in maintaining animal health and production in an era of reduced antimicrobial usage. Gastrointestinal helminth infections impose a considerable burden on animal performance, and recent studies suggest that infection may substantially alter the composition and function of the GM. Here, we discuss the potential interactions between different bioactive dietary components (prebiotics, probiotics and phytonutrients) and helminth infection on the GM in livestock. A number of recent studies suggest that host diet can strongly influence the nature of the helminth-GM interaction. Nutritional manipulation of the GM may thus impact helminth infection, and conversely infection may also influence how the GM responds to dietary interventions. Moreover, a dynamic interaction exists between helminths, the GM, intestinal immune responses, and inflammation. Deciphering the mechanisms underlying the diet-GM-helminth axis will likely inform future helminth control strategies, as well as having implications for how health-promoting feed additives, such as probiotics, can play a role in sustainable animal production.

Body fluid from the parasitic worm Ascaris suum inhibits broad-acting pro-inflammatory programs in dendritic cells.

Dendritic cells (DCs) are essential for generating T-cell-based immune responses through sensing of potential inflammatory and metabolic cues in the local environment. However, there is still limited insight into the processes defining the resultant DC phenotype, including the type of early transcriptional changes in pro-inflammatory cues towards regulatory or type 2 immune-based cues induced by a variety of exogenous and endogenous molecules. Here we compared the ability of a selected number of molecules to modulate the pro-inflammatory phenotype of lipopolysaccharide (LPS) and interferon-γ (IFN-γ)-stimulated human monocyte-derived DCs towards an anti-inflammatory or regulatory phenotype, including Ascaris suum body fluid [helminth pseudocoelomic fluid (PCF)], the metabolites succinate and butyrate, and the type 2 cytokines thymic stromal lymphopoietin and interleukin-25. Our data show that helminth PCF and butyrate treatment suppress the T helper type 1 (Th1)-inducing pro-inflammatory DC phenotype through induction of different transcriptional programs in DCs. RNA sequencing indicated that helminth PCF treatment strongly inhibited the Th1 and Th17 polarizing ability of LPS + IFN-γ-matured DCs by down-regulating myeloid differentiation primary response gene 88 (MyD88)-dependent and MyD88-independent pathways in Toll-like receptor 4 signaling. By contrast, butyrate treatment had a strong Th1-inhibiting action, and transcripts encoding important gut barrier defending factors such as IL18, IL1B and CXCL8 were up-regulated. Collectively, our results further understanding of how compounds from parasites and gut microbiota-derived butyrate may exert immunomodulatory effects on the host immune system.

Anthelmintic resistance and novel control options in equine gastrointestinal nematodes.

Control of equine nematodes has relied on benzimidazoles (BZs), tetrahydropyrimidines and macrocyclic lactones. The intensive use of anthelmintics has led to the development of anthelmintic resistance (AR) in equine cyathostomins and Parascaris equorum. Field studies indicate that BZ and pyrantel resistance is widespread in cyathostomins and there are also increasing reports of resistance to macrocyclic lactones in cyathostomins and P. equorum. The unavailability of reliable laboratory-based techniques for detecting resistance further augments the problem of nematode control in horses. The only reliable test used in horses is the fecal egg count reduction test; therefore, more focus should be given to develop and validate improved methodologies for diagnosing AR at an early stage, as well as determining the mechanisms involved in resistance development. Therefore, equine industry and researchers should devise and implement new strategies for equine worm control, such as the use of bioactive pastures or novel feed additives, and control should increasingly incorporate alternative and evidence-based parasite control strategies to limit the development of AR. This review describes the history and prevalence of AR in equine nematodes, along with recent advances in developing resistance diagnostic tests and worm control strategies in horses, as well as giving some perspective on recent research into novel control strategies.

Ascaris Suum Infection Downregulates Inflammatory Pathways in the Pig Intestine In Vivo and in Human Dendritic Cells In Vitro.

Ascaris suum is a helminth parasite of pigs closely related to its human counterpart, A. lumbricoides, which infects almost 1 billion people. Ascaris is thought to modulate host immune and inflammatory responses, which may drive immune hyporesponsiveness during chronic infections. Using transcriptomic analysis, we show here that pigs with a chronic A. suum infection have a substantial suppression of inflammatory pathways in the intestinal mucosa, with a broad downregulation of genes encoding cytokines and antigen-processing and costimulatory molecules. A. suum body fluid (ABF) suppressed similar transcriptional pathways in human dendritic cells (DCs) in vitro. DCs exposed to ABF secreted minimal amounts of cytokines and had impaired production of cyclooxygengase-2, altered glucose metabolism, and reduced capacity to induce interferon-gamma production in T cells. Our in vivo and in vitro data provide an insight into mucosal immune modulation during Ascaris infection, and show that A. suum profoundly suppresses immune and inflammatory pathways.

The whipworm (Trichuris suis) secretes prostaglandin E2 to suppress proinflammatory properties in human dendritic cells.

Clinical trials have shown that administration of the nematode Trichuris suis can be beneficial in treating various immune disorders. To provide insight into the mechanisms by which this worm suppresses inflammatory responses, an active component was purified from T. suis soluble products (TsSPs) that suppress---- TNF and IL-12 secretion from LPS-activated human dendritic cells (DCs). Analysis by liquid chromatography tandem mass spectrometry identified this compound as prostaglandin (PG)E2. The purified compound showed similar properties compared with TsSPs and commercial PGE2 in modulating LPS-induced expression of many cytokines and chemokines and in modulating Rab7B and P2RX7 expression in human DCs. Furthermore, the TsSP-induced reduction of TNF secretion from DCs is reversed by receptor antagonists for EP2 and EP4, indicating PGE2 action. T. suis secretes extremely high amounts of PGE2 (45-90 ng/mg protein) within their excretory/secretory products but few related lipid mediators as established by metabololipidomic analysis. Culture of T. suis with several cyclooxygenase (COX) inhibitors that inhibit mammalian prostaglandin synthesis affected the worm's motility but did not inhibit PGE2 secretion, suggesting that the worms can synthesize PGE2 via a COX-independent pathway. We conclude that T. suis secretes PGE2 to suppress proinflammatory responses in human DCs, thereby modulating the host's immune response.-Laan, L. C., Williams, A. R., Stavenhagen, K., Giera, M., Kooij, G., Vlasakov, I., Kalay, H., Kringel, H., Nejsum, P., Thamsborg, S. M., Wuhrer, M., Dijkstra, C. D., Cummings, R. D., van Die, I. The whipworm (Trichuris suis) secretes prostaglandin E2 to suppress proinflammatory properties in human dendritic cells.

Role of Activins in Hepcidin Regulation during Malaria.

Epidemiological observations have linked increased host iron with malaria susceptibility, and perturbed iron handling has been hypothesized to contribute to the potentially life-threatening anemia that may accompany blood-stage malaria infection. To improve our understanding of these relationships, we examined the pathways involved in regulation of the master controller of iron metabolism, the hormone hepcidin, in malaria infection. We show that hepcidin upregulation in Plasmodium berghei murine malaria infection was accompanied by changes in expression of bone morphogenetic protein (BMP)/sons of mothers against decapentaplegic (SMAD) pathway target genes, a key pathway involved in hepcidin regulation. We therefore investigated known agonists of the BMP/SMAD pathway and found that Bmp gene expression was not increased in infection. In contrast, activin B, which can signal through the BMP/SMAD pathway and has been associated with increased hepcidin during inflammation, was upregulated in the livers of Plasmodium berghei-infected mice; hepatic activin B was also upregulated at peak parasitemia during infection with Plasmodium chabaudi Concentrations of the closely related protein activin A increased in parallel with hepcidin in serum from malaria-naive volunteers infected in controlled human malaria infection (CHMI) clinical trials. However, antibody-mediated neutralization of activin activity during murine malaria infection did not affect hepcidin expression, suggesting that these proteins do not stimulate hepcidin upregulation directly. In conclusion, we present evidence that the BMP/SMAD signaling pathway is perturbed in malaria infection but that activins, although raised in malaria infection, may not have a critical role in hepcidin upregulation in this setting.

Co-operative suppression of inflammatory responses in human dendritic cells by plant proanthocyanidins and products from the parasitic nematode Trichuris suis.

Interactions between dendritic cells (DCs) and environmental, dietary and pathogen antigens play a key role in immune homeostasis and regulation of inflammation. Dietary polyphenols such as proanthocyanidins (PAC) may reduce inflammation, and we therefore hypothesized that PAC may suppress lipopolysaccharide (LPS) -induced responses in human DCs and subsequent T helper type 1 (Th1) -type responses in naive T cells. Moreover, we proposed that, because DCs are likely to be exposed to multiple stimuli, the activity of PAC may synergise with other bioactive molecules that have anti-inflammatory activity, e.g. soluble products from the helminth parasite Trichuris suis (TsSP). We show that PAC are endocytosed by monocyte-derived DCs and selectively induce CD86 expression. Subsequently, PAC suppress the LPS-induced secretion of interleukin-6 (IL-6) and IL-12p70, while enhancing secretion of IL-10. Incubation of DCs with PAC did not affect lymphocyte proliferation; however, subsequent interferon-γ production was markedly suppressed, while IL-4 production was unaffected. The activity of PAC was confined to oligomers (degree of polymerization ≥ 4). Co-pulsing DCs with TsSP and PAC synergistically reduced secretion of tumour necrosis factor-α, IL-6 and IL-12p70 while increasing IL-10 secretion. Moreover, both TsSP and PAC alone induced Th2-associated OX40L expression in DCs, and together synergized to up-regulate OX40L. These data suggest that PAC induce an anti-inflammatory phenotype in human DCs that selectively down-regulates Th1 response in naive T cells, and that they also act cooperatively with TsSP. Our results indicate a novel interaction between dietary compounds and parasite products to influence immune function, and may suggest that combinations of PAC and TsSP can have therapeutic potential for inflammatory disorders.

Targets and Mechanisms Associated with Protection from Severe Plasmodium falciparum Malaria in Kenyan Children.

Severe malaria (SM) is a life-threatening complication of infection with Plasmodium falciparum Epidemiological observations have long indicated that immunity against SM is acquired relatively rapidly, but prospective studies to investigate its immunological basis are logistically challenging and have rarely been undertaken. We investigated the merozoite targets and antibody-mediated mechanisms associated with protection against SM in Kenyan children aged 0 to 2 years. We designed a unique prospective matched case-control study of well-characterized SM clinical phenotypes nested within a longitudinal birth cohort of children (n= 5,949) monitored over the first 2 years of life. We quantified immunological parameters in sera collected before the SM event in cases and their individually matched controls to evaluate the prospective odds of developing SM in the first 2 years of life. Anti-AMA1 antibodies were associated with a significant reduction in the odds of developing SM (odds ratio [OR] = 0.37; 95% confidence interval [CI] = 0.15 to 0.90; P= 0.029) after adjustment for responses to all other merozoite antigens tested, while those against MSP-2, MSP-3, Plasmodium falciparum Rh2 [PfRh2], MSP-119, and the infected red blood cell surface antigens were not. The combined ability of total IgG to inhibit parasite growth and mediate the release of reactive oxygen species from neutrophils was associated with a marked reduction in the odds of developing SM (OR = 0.07; 95% CI = 0.006 to 0.82;P= 0.03). Assays of these two functional mechanisms were poorly correlated (Spearman rank correlation coefficient [rs] = 0.12;P= 0.07). Our data provide epidemiological evidence that multiple antibody-dependent mechanisms contribute to protective immunity via distinct targets whose identification could accelerate the development of vaccines to protect against SM.

Neutralization of Plasmodium falciparum merozoites by antibodies against PfRH5.

There is intense interest in induction and characterization of strain-transcending neutralizing Ab against antigenically variable human pathogens. We have recently identified the human malaria parasite Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) as a target of broadly neutralizing Abs, but there is little information regarding the functional mechanism(s) of Ab-mediated neutralization. In this study, we report that vaccine-induced polyclonal anti-PfRH5 Abs inhibit the tight attachment of merozoites to erythrocytes and are capable of blocking the interaction of PfRH5 with its receptor basigin. Furthermore, by developing anti-PfRH5 mAbs, we provide evidence of the following: 1) the ability to block the PfRH5-basigin interaction in vitro is predictive of functional activity, but absence of blockade does not predict absence of functional activity; 2) neutralizing mAbs bind spatially related epitopes on the folded protein, involving at least two defined regions of the PfRH5 primary sequence; 3) a brief exposure window of PfRH5 is likely to necessitate rapid binding of Ab to neutralize parasites; and 4) intact bivalent IgG contributes to but is not necessary for parasite neutralization. These data provide important insight into the mechanisms of broadly neutralizing anti-malaria Abs and further encourage anti-PfRH5-based malaria prevention efforts.