CMT Publications Publications 2018

Publications 2018

High production of pro-inflammatory cytokines by maternal blood mononuclear cells is associated with reduced maternal malaria but increased cord blood infection.Malaria Journal, 17.

Increased susceptibility to malaria during pregnancy is not completely understood. Cellular immune responses mediate both pathology and immunity but the effector responses involved in these processes have not been fully characterized. Maternal and fetal cytokine and chemokine responses to malaria at delivery, and their association with pregnancy and childhood outcomes, were investigated in 174 samples from a mother and child cohort from Mozambique. Peripheral and cord mononuclear cells were stimulated with Plasmodium falciparum lysate and secretion of IL-12p70, IFN-γ, IL-2, IL-10, IL-8, IL-6, IL-4, IL-5, IL-1β, TNF, TNF-β was quantified in culture supernatants by multiplex flow cytometry while cellular mRNA expression of IFN-γ, TNF, IL-2, IL-4, IL-6, IL-10 and IL-13 was measured by quantitative PCR. Higher concentrations of IL-6 and IL-1β were associated with a reduced risk of P. falciparum infection in pregnant women (p < 0.049). Pro-inflammatory cytokines IL-6, IL-1β and TNF strongly correlated among themselves (ρ > 0.5, p < 0.001). Higher production of IL-1β was significantly associated with congenital malaria (p < 0.046) and excessive TNF was associated with peripheral infection and placental lesions (p < 0.044). Complex network of immuno-pathological cytokine mechanisms in the placental and utero environments showed a potential trade-off between positive and negative effects on mother and newborn susceptibility to infection.

Anomalies in T cell function are associated with individuals at risk of Mycobacterium abscessus complex infection.Frontiers in Immunology, 9.

The increasing global incidence and prevalence of non-tuberculous mycobacteria (NTM) infection is of growing concern. New evidence of person-to-person transmission of multidrug-resistant NTM adds to the global concern. The reason why certain individuals are at risk of NTM infections is unknown. Using high definition flow cytometry, we studied the immune profiles of two groups that are at risk of Mycobacterium abscessuscomplex infection and matched controls. The first group was cystic fibrosis (CF) patients and the second group was elderly individuals. CF individuals with active M. abscessuscomplex infection or a history of M. abscessuscomplex infectionexhibited a unique surface T cell phenotype with a marked global deficiency in TNFα production during mitogen stimulation. Importantly, immune-based signatures were identified that appeared to predict at baseline the subset of CF individuals who were at risk of M. abscessuscomplex infection. In contrast, elderly individuals with M. abscessuscomplex infection exhibited a separate T cell phenotype underlined by the presence of exhaustion markers and dysregulation in type 1 cytokine release during mitogen stimulation. Collectively, these data suggest an association between T cell signatures and individuals at risk of M. abscessuscomplex infection, however, validation of these immune anomalies as robust biomarkers will require analysis on larger patient cohorts.

Patterns of inter-individual variability in the antibody repertoire targeting proteins across the Epstein-Barr virus proteome.Journal of Infectious Diseases, 217. pp. 1923-1931.

Little is known about variation in antibody responses targeting the full spectrum of Epstein-Barr virus (EBV)proteins and how such patterns inform disease risk. Methods: We used a microarray to measure immunoglobulin G (IgG) and immunoglobulin A (IgA) antibody responses against 199 EBV protein sequences from 5 EBV strains recovered from 289 healthy adults from Taiwan. We described positivity patterns, estimated the correlation between antibodies, and investigated the associations between environmental and genetic risk factors and variations in antibody responses. Healthy adults were more likely to mount IgG antibody responses to EBV proteins (median positivity frequency, 46.5% for IgG and 17.3% for IgA; P = 1.6 × 10–46, by the Wilcoxon rank sum test). Responses against glycoproteins were particularly prevalent. The correlations between antibody responses of the same class were higher than correlations across classes. The mucosal exposure to proteins involved in EBV reactivation (as determined by the IgA response) was associated with smoking (P = .002, by the sequence kernel association test–combined), and approximately one quarter of adults displayed antibody responses associated with EBV-related cancer risk. These data comprehensively define the variability in human IgG and IgA antibody responses to the EBV proteome. Patterns observed can serve as the foundation for elucidating which individuals are at highest risk of EBV-associated clinical conditions and for identifying targets for effective immunodiagnostic tests.

A balanced pro-inflammatory and regulatory cytokine signature in young African children is associated with lower risk of clinical malaria.Clinical Infectious Diseases. (In Press)

The effect of timing of exposure to first Plasmodium falciparuminfections during early childhood on the induction of innate and adaptive cytokine responses and their contribution to the development of clinical malaria immunity is not well established. As part of a double-blind randomized placebo-controlled trial in Mozambique using monthly chemoprophylaxis with sulfadoxine-pyrimethamine plus artesunate to selectively control timing of malaria exposure during infancy, peripheral blood mononuclear cells collected at ages 2.5, 5.5, 10.5, 15 and 24 months were stimulated ex vivowith parasite schizont and erythrocyte lysates. Cytokine mRNA expressed in cell pellets and proteins secreted in supernatants were quantified by real time quantitative PCR and multiplex flow cytometry, respectively. Children were followed up for clinical malaria from birth until 4 years of age. Higher pro-inflammatory (IL-1, IL-6, TNF) and regulatory (IL-10) cytokine concentrations during the second year of life were associated with reduced incidence of clinical malaria up to 4 years of age, adjusting by chemoprophylaxis and prior malaria exposure. Significantly lower concentrations of antigen-specific TH1 (IL-2, IL-12, IFN-g) and TH2 (IL-4, IL-5) cytokines by 2 years of age were measured in children under chemoprophylaxis compared to children receiving placebo (p<0.03). Selective chemoprophylaxis altering early natural exposure to malaria blood stage antigens during infancy had a significant effect on THlymphocyte cytokine production more than one year later. Importantly, a balanced pro-inflammatory and anti-inflammatory cytokine signature probably by innate cells around age 2 years was associated with protective clinical immunity during childhood.

Identification of Cytauxzoon felis antigens via protein microarray and assessment of expression library immunization against cytauxzoonosis.Clinical Proteomics, 15.

Cytauxzoonosis is a disease of felids in North America caused by the tick-transmitted apicomplexan parasite Cytauxzoon felis. Cytauxzoonosis is particularly virulent for domestic cats, but no vaccine currently exists. The parasite cannot be cultivated in vitro, presenting a significant limitation for vaccine development. Recent sequencing of the C. felisgenome has identified over 4300 putative protein-encoding genes. From this pool we constructed a protein microarray containing 673 putative C. felisproteins. This microarray was probed with sera from C. felis-infected and naïve cats to identify differentially reactive antigens which were incorporated into two expression library vaccines, one polyvalent and one monovalent. We assessed the efficacy of these vaccines to prevent of infection and/or disease in a tick-challenge model. Probing of the protein microarray resulted in identification of 30 differentially reactive C. felisantigens that were incorporated into the two expression library vaccines. However, expression library immunization failed to prevent infection or disease in cats challenged with C. felis. Protein microarray facilitated high-throughput identification of novel antigens, substantially increasing the pool of characterized C. felisantigens. These antigens should be considered for development of C. felisvaccines, diagnostics, and therapeutics.

Identification of a novel, EBV-based antibody risk stratification signature for early detection of nasopharyngeal carcinoma in Taiwan.Clinical Cancer Research, 24 (6). pp. 1305-1314.

Epstein-Barr virus (EBV) is necessary for the development of nasopharyngeal carcinoma (NPC). By adulthood, approximately 90% of individuals test EBV-positive, but only a fraction develop cancer. Factors that identify which individuals are most likely to develop disease, including differential antibody response to the virus, could facilitate detection at early stages when treatment is most effective. We measured anti-EBV IgG and IgA antibody responses in 607 Taiwanese individuals. Antibodies were measured using a custom protein microarray targeting 199 sequences from 86 EBV proteins. Variation in response patterns between NPC cases and controls was used to develop an antibody-based risk score for predicting NPC. The overall accuracy [area under the curve (AUC)] of this risk score, and its performance relative to currently used biomarkers, was evaluated in two independent Taiwanese cohorts. Levels of 60 IgA and 73 IgG anti-EBV antibodies differed between stage I/IIa NPC cases and controls (P< 0.0002). Risk prediction analyses identified antibody targets that best discriminated NPC status-BXLF1, LF2,BZLF1, BRLF1, EAd, BGLF2, BPLF1, BFRF1, and BORF1. When combined with currently used VCA/EBNA1 IgA biomarkers, the resulting risk score predicted NPC with 93% accuracy (95% CI, 87%-98%) in the general Taiwanese population, a significant improvement beyond current biomarkers alone (82%; 95% CI, 75%-90%, P≤ 0.01). This EBV-based risk score also improved NPC prediction in genetically high-risk families (89%; 95% CI, 82%-96%) compared with current biomarkers (78%; 95% CI, 66%-90%, P≤ 0.03). We identified NPC-related differences in 133 anti-EBV antibodies and developed a risk score using this microarray dataset that targeted immune responses against EBV proteins from all stages of the viral life cycle, significantly improving the ability to predict NPC. Clin Cancer Res; 24(6); 1305-14. ©2017 AACR.

The influence of physicochemical properties of lipopeptide adjuvants on immune response: a rationale for engineering a potent vaccine.Chemistry: a European journal, 24. pp. 9892-9902.

Adjuvant development and understanding the physicochemical properties of particles and interpreting the subsequent immunological responses is a challenge faced by many researchers in the vaccine field. We synthesized and investigated the physicochemicalproperties and immunogenicity of a library of multiple epitope self-adjuvant lipopeptides in a novel asymmetric arrangement. Vaccinecandidates were synthesized using a combination of solid-phase peptide synthesis and copper-mediated click chemistry. In vivo studies showed that vaccine constructs containing a single OVA CD8+T-cell epitope and two N-terminally located C16 lipid moieties were more effective at generating robust cellular immune responses compared to the same molecule containing multiple copies of the OVA CD8+T-cell epitope with or without the C16 moieties. Furthermore, attachment of the two C16 lipids to the N-terminus provoked formation of long β-sheet fibrils and was shown to induce a higher CD8+donor T-cell frequency and IFN-γ secretion, compared to vaccine constructs with an internal lipid placement. A regression analysis indicated that particle secondary structure had a significant impact on CD8+donor T-cell frequency and cytolytic activity. In addition, IFN-γ production was influenced significantly by particle shape. The findings of this research will impact the future design of a vaccine intended to elicit cellular immune responses.

Group G streptococcus induces an autoimmune carditis mediated by interleukin 17A and interferon γ in the Lewis rat model of rheumatic heart disease.Journal of Infectious Diseases, 218 (2). pp. 324-335.

Acute rheumatic fever and rheumatic heart disease (ARF/RHD) have long been described as autoimmune sequelae of Streptococcuspyogenes or group A streptococcal (GAS) infection. Both antibody and T-cell responses against immunodominant GAS virulence factors, including M protein, cross-react with host tissue proteins, triggering an inflammatory response leading to permanent heart damage. However, in some ARF/RHD-endemic regions, throat carriage of GAS is low. Because Streptococcus dysgalactiae subspecies equisimilis organisms, also known as β-hemolytic group C streptococci and group G streptococci (GGS), also express M protein, we postulated that streptococci other than GAS may have the potential to initiate or exacerbate ARF/RHD. Using a model initially developed to investigate the uniquely human disease of ARF/RHD, we have discovered that GGS causes interleukin 17A/interferon γ-induced myocarditis and valvulitis, hallmarks of ARF/RHD. Remarkably the histological, immunological, and functional changes in the hearts of rats exposed to GGS are identical to those exposed to GAS. Furthermore, antibody cross-reactivity to cardiac myosin was comparable in both GGS- and GAS-exposed animals, providing additional evidence that GGS can induce and/or exacerbate ARF/RHD.

Selective protein unfolding: a universal mechanism of action for the development of irreversible inhibitors.Chemical Communications, 54 (14). pp. 1738-1741.

High-throughput differential scanning fluorimetry of GFP-tagged proteins (HT-DSF-GTP) was applied for the identification of novel enzyme inhibitors acting by a mechanism termed: selective protein unfolding (SPU). Four different protein targets were interrogated with the same library to identify target-selective hits. Several hits selectively destabilized bacterial biotin protein ligase. Structure-activity relationship data confirmed a structure-dependent mechanism of protein unfolding. Simvastatin and altenusin were confirmed to irreversibly inactivate biotin protein ligase. The principle of SPU combined with HT-DSF-GTP affords an invaluable and innovative workflow for the identification of new inhibitors with potential applications as antimicrobials and other biocides.

Characterization of Trichuris muris secreted proteins and extracellular vesicles provides new insights into host–parasite communication.Journal of Extracellular Vesicles, 7 (1).

Whipworms are parasitic nematodes that live in the gut of more than 500 million people worldwide. Owing to the difficulty in obtaining parasite material, the mouse whipworm Trichuris murishas been extensively used as a model to study human whipworm infections. These nematodes secrete a multitude of compounds that interact with host tissues where they orchestrate a parasitic existence. Herein we provide the first comprehensive characterization of the excretory/secretory products of T. muris. We identify 148 proteins secreted by T. murisand show for the first time that the mouse whipworm secretes exosome-like extracellular vesicles (EVs) that can interact with host cells. We use an Optiprep® gradient to purify the EVs, highlighting the suitability of this method for purifying EVs secreted by a parasitic nematode. We also characterize the proteomic and genomic content of the EVs, identifying >350 proteins, 56 miRNAs (22 novel) and 475 full-length mRNA transcripts mapping to T. murisgene models. Many of the miRNAs putatively mapped to mouse genes are involved in regulation of inflammation, implying a role in parasite-driven immunomodulation. In addition, for the first time to our knowledge, colonic organoids have been used to demonstrate the internalization of parasite EVs by host cells. Understanding how parasites interact with their host is crucial to develop new control measures. This first characterization of the proteins and EVs secretedby T. murisprovides important information on whipworm-host communication and forms the basis for future studies.

Defined small molecules produced by Himalayan medicinal plants display immunomodulatory properties.International Journal of Molecular Sciences, 19 (11).

Plant-derived compounds that modulate the immune responses are emerging as frontline treatment agents for cancer, infectious diseases and autoimmunity. Herein we have isolated 40 phytochemicals from five Bhutanese Sowa Rigpamedicinal plants-Aconitum laciniatumAjania nubeginaCorydalis crispaCorydalis dubiaand Pleurospermum amabile-and tested 14 purified compounds for their immunomodulatory properties using a murine dendritic cell (DC) line, and cytotoxicity against a human cholangiocyte cell line using xCELLigence real time cell monitoring. These compounds were: pseudaconitine, 14-veratryolpseudaconitine, 14-O-acetylneoline, linalool oxide acetate, (E)-spiroether, luteolin, luteolin-7-O-β-d-glucopyranoside, protopine, ochrobirine, scoulerine, capnoidine, isomyristicin, bergapten, and isoimperatorin. Of the 14 compounds tested here, scoulerine had adjuvant-like properties and strongly upregulated MHC-Igene and protein expression whereas bergapten displayed immunosuppressive properties and strongly down-regulated gene and protein expression of MHC-I and other co-stimulatory molecules. Both scoulerine and bergapten showed low cytotoxicity against normal healthy cells that were consistent with their immunoregulatory properties. These findings highlight the breadth of immunomodulatory properties of defined compounds from Bhutanese medicinal plants and show that some of these compounds exert their mechanisms of action by modulating DC activity.

Cobos, Claudia, Bansal, Paramjit S., Jones, Linda, Wangchuk, Phurpa, Wilson, David, Loukas, Alex, and Daly, Norelle L. (2018) Engineering of an anti-inflammatory peptide based on the disulfide-rich linaclotide scaffold.Biomedicines, 6 (4).

Inflammatory bowel diseases are a set of complex and debilitating diseases, for which there is no satisfactory treatment. Peptides as small as three amino acids have been shown to have anti-inflammatory activity in mouse models of colitis, but they are likely to be unstable, limiting their development as drug leads. Here, we have grafted a tripeptide from the annexin A1 protein into linaclotide, a 14-amino-acid peptide with three disulfide bonds, which is currently in clinical use for patients with chronic constipation or irritable bowel syndrome. This engineered disulfide-rich peptide maintained the overall fold of the original synthetic guanylate cyclase C agonist peptide, and reduced inflammation in a mouse model of acute colitis. This is the first study to show that this disulfide-rich peptide can be used as a scaffold to confer a new bioactivity.

Hookworm secreted extracellular vesicles interact with host cells and prevent inducible colitis in mice.Frontiers in Immunology, 9.

Gastrointestinal (GI) parasites, hookworms in particular, have evolved to cause minimal harm to their hosts, allowing them to establish chronic infections. This is mediated by creating an immunoregulatory environment. Indeed, hookworms are such potent suppressors of inflammation that they have been used in clinical trials to treat inflammatory bowel diseases (IBD) and celiac disease. Since the recent description of helminths (worms) secreting extracellular vesicles (EVs), exosome-like EVs from different helminths have been characterized and their salient roles in parasite-host interactions have been highlighted. Here, we analyze EVs from the rodent parasite Nippostrongylus brasiliensis, which has been used as a model for human hookworm infection. N. brasiliensisEVs (Nb-EVs) are actively internalized by mouse gut organoids, indicating a role in driving parasitism. We used proteomics and RNA-Seq to profile the molecular composition of Nb-EVs. We identified 81 proteins, including proteins frequently present in exosomes (like tetraspanin, enolase, 14-3-3 protein, and heat shock proteins), and 27 sperm-coating protein-like extracellular proteins. RNA-Seq analysis revealed 52 miRNA species, many of which putatively map to mouse genes involved in regulation of inflammation. To determine whether GI nematode EVs had immunomodulatory properties, we assessed their potential to suppress GI inflammation in a mouse model of inducible chemical colitis. EVs from N. brasiliensisbut not those from the whipworm Trichuris murisor control vesicles from grapes protected against colitic inflammation in the gut of mice that received a single intraperitoneal injection of EVs. Key cytokines associated with colitic pathology (IL-6, IL-1β, IFNγ, and IL-17a) were significantly suppressed in colon tissues from EV-treated mice. By contrast, high levels of the anti-inflammatory cytokine IL-10 were detected in Nb-EV-treated mice. Proteins and miRNAs contained within helminth EVs hold great potential application in development of drugs to treat helminth infections as well as chronic non-infectious diseases resulting from a dysregulated immune system, such as IBD.

Of dogs and hookworms: man's best friend and his parasites as a model for translational biomedical research.Parasites & Vectors, 11.

We present evidence that the dog hookworm (Ancylostoma caninum) is underutilised in the study of host-parasite interactions, particularly as a proxy for the human-hookworm relationship. The inability to passage hookworms through all life stages in vitro means that adult stage hookworms have to be harvested from the gut of their definitive hosts for ex vivo research. This makes study of the human-hookworm interface difficult for technical and ethical reasons. The historical association of humans, dogs and hookwormspresents a unique triad of positive evolutionary pressure to drive the A. caninum-canine interaction to reflect that of the human-hookworm relationship. Here we discuss A. caninum as a proxy for human hookworm infection and situate this hookworm model within the current research agenda, including the various 'omics' applications and the search for next generation biologics to treat a plethora of human diseases. Historically, the dog hookworm has been well described on a physiological and biochemical level, with an increasing understanding of its role as a human zoonosis. With its similarity to human hookworm, the recent publications of hookworm genomes and other omics databases, as well as the ready availability of these parasites for ex vivo culture, the dog hookworm presents itself as a valuable tool for discovery and translational research.

Polypyridylruthenium(II) complexes exert in vitro and in vivo nematocidal activity and show significant inhibition of parasite acetylcholinesterases.International Journal for Parasitology: Drugs and Drugs Resistance, 8 (1). pp. 1-7.

Over 4.5 billion people are at risk of infection with soil transmitted helminths and there are concerns about the development of resistance to the handful of frontline nematocides in endemic populations. We investigated the anti-nematode efficacy of a series of polypyridylruthenium(II) complexes and showed they were active against L3 and adult stages of Trichuris muris, the rodent homologue of the causative agent of human trichuriasis, T. trichiura. One of the compounds, Rubb12-mono, which was among the most potent in its ability to kill L3 (IC50= 3.1 ± 0.4 μM) and adult (IC50= 5.2 ± 0.3 μM) stage worms was assessed for efficacy in a mouse model of trichuriasis by administering 3 consecutive daily oral doses of the drug 3 weeks post infection with the murine whipworm Trichuris muris. Mice treated with Rubb12-mono showed an average 66% reduction (P = 0.015) in faecal egg count over two independent trials. The drugs partially exerted their activity through inhibition of acetylcholinesterases, as worms treated in vitro and in vivo showed significantdecreases in the activity of this class of enzymes. Our data show that ruthenium complexes are effective against T. muris, a model gastro-intestinal nematode and soil-transmitted helminth. Further, knowledge of the target of ruthenium drugs can facilitate modification of current compounds to identify analogues which are even more effective and selective against Trichuris and other helminths of human and veterinary importance.

Sertraline, paroxetine, and chlorpromazine are rapidly acting anthelmintic drugs capable of clinical repurposing.Scientific Reports, 8.

Parasitic helminths infect over 1 billion people worldwide, while current treatments rely on a limited arsenal of drugs. To expedite drug discovery, we screened a small-molecule library of compounds with histories of use in human clinical trials for anthelmintic activity against the soil nematode Caenorhabditis elegans. From this screen, we found that the neuromodulatory drugs sertraline, paroxetine, and chlorpromazine kill C. elegans at multiple life stages including embryos, developing larvae and gravid adults. These drugs act rapidly to inhibit C. elegans feeding within minutes of exposure. Sertraline, paroxetine, and chlorpromazine also decrease motility of adult Trichuris muris whipworms, prevent hatching and development of Ancylostoma caninum hookworms and kill Schistosoma mansoni flatworms, three widely divergent parasitic helminth species. C. elegans mutants with resistance to known anthelmintic drugs such as ivermectin are equally or more susceptible to these three drugs, suggesting that they may act on novel targets to kill worms. Sertraline, paroxetine, and chlorpromazine have long histories of use clinically as antidepressant or antipsychotic medicines. They may represent new classes of anthelmintic drug that could be used in combination with existing front-line drugs to boost effectiveness of anti-parasite treatment as well as offset the development of parasite drug resistance.

A medicinal plant compound, capnoidine, prevents the onset of inflammation in a mouse model of colitis.Journal of Ethnopharmacology, 211. pp. 17-28.

The traditional uses of Corydalis dubia, Ajania nubigena and Pleurospermum amabile in the Bhutanese traditional medicine for treating disorders related to inflammatory conditions and the in vitro anti-inflammatory activity of their crude extracts inspired the isolation and the investigation of anticolitic properties of four pure compounds. Three medicinal plants were collected from Himalayan Mountains of Bhutan. Capnoidine and scoulerine were isolated from C. dubia, linalool oxide acetate from A. nubigena and isomyristicin from P. amabile using natural product isolation protocols. Four compounds were investigated for their anti-inflammatory activities against IBD-colitis using chemically induced (TNBS) mice model of colitis. Capnoidine conferred the best preliminary protection against TNBS-induced colitis in mice and we have conducted in-depth pharmacological investigation of this compound including clinical symptoms, pathological signs, cytokine profiles, histological structure and inflammasomes using relevant bioassay protocols. Capnoidine-treated mice had significantly: a) improved clinical symptoms (body weight loss, mobility, piloerection and faecal consistency); b) reduced colon pathology (adhesion, oedema, ulceration, and colon length); c) altered inflammatory cytokines profiles within the colons; d) reduced levels of p-IκB-α (Ser32) and p-NF-κB p65 (Ser536) and e) reduced histological inflammation in the colon when compared with mice administered TNBS only. Capnoidine presents as a potential new anti-inflammatory drug lead candidate for diseases where current standard-of-care often fails and is associated with major side effects. It also validates the traditional uses of C. dubia against inflammatory conditions and underlines the value of pursuing bioactive compounds derived from traditionally used ethnobotanical medicines.

A novel blood-feeding detoxification pathway in Nippostrongylus brasiliensis L3 reveals a potential checkpoint for arresting hookworm development.PLoS Pathogens, 14 (3).

As part of on-going efforts to control hookworm infection, the "human hookworm vaccine initiative" has recognised blood feeding as a feasible therapeutic target for inducing immunity against hookworm infection. To this end, molecular approaches have been used to identify candidate targets, such as Necator americanus (Na) haemoglobinase aspartic protease-1 (APR-1), with immunogenicity profiled in canine and hamster models. We sought to accelerate the immune analysis of these identified therapeutic targets by developing an appropriate mouse model. Here we demonstrate that Nippostrongylus brasiliensis (Nb), a phylogenetically distant strongylid nematode of rodents, begins blood feeding early in its development and that immunisation with Na-APR-1 can block its growth and completion of its life cycle. Furthermore, we identify a new haem detoxification pathway in Nb required for blood feeding that can be blocked by drugs of the quinolone family, reducing both infection burden and the associated anaemia in rodents. Collectively, our findings show that haem metabolism has potential as a checkpoint for interrupting hookworm development in early stages of the hookworm life cycle and that the Nippostrongylus brasiliensis rodent model is relevant for identifying novel therapeutic targets against human hookworm.

Circulating microRNAs as biomarkers for acute ischemic stroke: a systematic review.Journal of Stroke and Cerebrovascular Diseases, 27 (3). pp. 522-530.

Acute ischemic stroke is a leading cause of death and disability worldwide. Unlike myocardial infarction, there is no current blood test to diagnose acute ischemic stroke. MicroRNAs (miRNAs) are very stable in the blood and have been suggested as potential diagnostic markers. This review aimed to systematically assess case-control studies investigating the association of circulatingmiRNAs with acute ischemic stroke. Medline, CINAHL, Cochrane Library, Web of Science, Scopus, and PubMed were searched for studies that examined the association of circulating miRNAs in patients with acute ischemic stroke. Studies meeting specific inclusion and exclusion criteria (such as blood samples obtained within 24 hours of an acute ischemic stroke) were selected for data extraction. Two authors extracted data from the included studies relevant to the study design, the patient characteristics, and the relative miRNA expression. Eight studies were included involving 572 cases and 431 healthy controls. Twenty-two miRNAs (12 upregulated and 10 downregulated) were reported as differentially expressed. Only 1 miRNA, miR-106b, was reported as differentially expressed in at least 2 studies. Significant heterogeneity in the design and methods of the included studies was noted. Differential expression of a large number of miRNAs has been reported early following acute ischemic stroke. More research is required in larger patient populations to further evaluate the diagnostic potential of the reported miRNAs.

Prescription of pharmacotherapy and the incidence of stroke in patients with symptoms of peripheral artery disease.Stroke. (In Press)

Background and Purpose- Current guidelines recommend prescription of a number of medications to prevent cardiovascular events in patients with peripheral artery disease (PAD). The impact that these medications have on the incidence of stroke in PAD patients has not been thoroughly investigated. This study aimed to investigate the association of prescription of antihypertensive drugs, antiplatelet medications, and statins, as well as cardiovascular disease risk factors, with stroke incidence in patients with symptoms of PAD. Methods- A database search was completed to identify studies reporting the incidence of stroke and prescription of antihypertensive drugs, antiplatelet medications, and statins in patients with PAD symptoms. A random-effects model was used to meta-analyze the incidence of stroke in patients with symptoms of PAD and in subgroups with intermittent claudication and critical limb ischemia. Metaregression was performed to explore the association between the incidence of stroke and the prescription of medications and the presence of cardiovascular disease risk factors. Results- Twelve studies including 67 915 patients with symptoms of PAD were included. A meta-analysis of data from 7 studies demonstrated an incidence of stroke of 1.31 per 100 patient-years. Patients with critical limb ischemia experienced stroke 2.3× more frequently than those with intermittent claudication (95% CI, 1.58-3.36; P<0.01). The reported prescription of antihypertensive agents varied between 10% and 71%, antiplatelet drugs between 49% and 90%, and statins between 11% and 79% in different studies. Metaregression suggested an association between a lower incidence of stroke and the prescription ofantiplatelet drugs ( R2=0.81, P<0.01), and statins ( R2=0.85, P<0.01), but not antihypertensives medications. A prior history of cerebrovascular events was associated with a higher incidence of stroke ( R2=0.58, P<0.05). Conclusions- This review supports previous research which suggests the need for more effective means of ensuring more widespread prescription of preventative medications in patients with PAD.

Prevalence of low anterior resection syndrome at a regional Australian centre.ANZ Journal of Surgery. (In Press)

With decreasing indication for abdominoperineal resection and an increase in sphincter preserving surgery, there is a growing population of patients who suffer from low anterior resection syndrome (LARS). The aim of this study is to use the LARS score to determine the prevalence of LARS at a regional centre in Australia and determine the effect of short- and long-course neoadjuvant therapy, anastomotic technique and interval from surgery will also be assessed. Patients who had undergone an anterior resection (high, low or ultralow) at a regional centre over an 11-year period were identified. Eligible patients were contacted to complete a LARS score questionnaire. Results were analysed to determine the rate of major LARS and possible causative roles of certain patient and treatment-related variables. A total of 64 of 76 patients (84%) returned completed questionnaires. The prevalence of major LARS was 37.5%. Short-course neoadjuvant therapy appeared to be more likely to be associated with major LARS compared to long course (odds ratio (OR) = 2.4, 95% confidence interval (CI) 0.37-15.3, P = 0.35); however, this did not reach statistical significance. Rates of major LARS appear to decrease slowly over time and J-pouch colonic anastomosis appears to be slightly protective against major LARS (OR = 0.7, 95% CI 0.12-3.9, P = 0.70); however, neither results were statistically significant. The rate of major LARS at this regional centre is 37.5%. Larger prospective multicentre studies are required to determine impact of variables such as type of neoadjuvant therapy, anastomotic techniques and progression of LARS over time.

A meta-analysis of the prevalence of Low Anterior Resection Syndrome and systematic review of risk factors.International Journal of Surgery, 56. pp. 234-241.

To summarize the reported prevalence and causative factors of Low Anterior Resection Syndrome (LARS) from studies using the LARS score. A systematic literature search was conducted using Pubmed, Ovid Medline and the Cochrane database. Searches were performed using a combination of MeSH (medical subject headings) terms and key terms. Studies that were included used the LARS score as their primary collection tool. Studies were excluded if initial surgery was not for malignancy, or if the majority of LARS scores were from patients less than 1 year post initial surgery or closure of diverting stoma. Eligible studies were assessed with a validated quality assessment tool prior to performing a meta-analysis with quality effects model. Meta-analysis was conducted with prevalenceestimates that had been transformed using the double arcsine method. Following the initial search and implementation of inclusion and exclusion criteria 11 studies were deemed suitable for meta-analysis. Meta-analysis found the estimated prevalence of major LARS was 41% (95% CI 34 -48). Where possible outlier studies were excluded, the prevalence was 42% (95%CI 35-48). Radiotherapy and tumour height were the most consistently assessed variables, both showing a consistent negative effect on bowel function. Defunctioning ileostomy was found to have a statically significant negative impact on bowel function in 4 of 11 studies. The majority of reported data has been produced by groups in Denmark and the United Kingdom with limited numbers provided by other locations. Available data is heterogenous with some variables having limited numbers, making meta-analysis of certain variables impossible. There is significant prevalence of Low Anterior Resection Syndrome following oncological rectal resection. A lowanastomotic height or history of radiotherapy are major risk factors.

The NLRP3 inflammasome suppresses protective immunity to gastrointestinal helminth infection.Cell Reports, 23 (4). pp. 1085-1098.

Inflammasomes promote immunity to microbial pathogens by regulating the function of IL-1-family cytokines such as IL-18 and IL-1β. However, the roles for inflammasomes during parasitic helminth infections remain unclear. We demonstrate that mice and humans infected with gastrointestinal nematodes display increased IL-18 secretion, which in Trichuris-infected or worm antigen-treated mice and in macrophages co-cultured with Trichuris antigens or exosome-like vesicles was dependent on the NLRP3 inflammasome. NLRP3-deficient mice displayed reduced pro-inflammatory type 1 cytokine responses and augmented protective type 2 immunity, which was reversed by IL-18 administration. NLRP3-dependent suppression of immunity partially required CD4+cells but was apparent even in Rag1-/-mice that lack adaptive immune cells, suggesting that NLRP3 influences both innate and adaptive immunity. These data highlight a role for NLRP3 in limiting protective immunity to helminths, suggesting that targeting the NLRP3 inflammasome may be an approach for limiting the disease burden associated with helminth infections.

A systematic review: the role of resident memory T cells in infectious diseases and their relevance for vaccine development.Frontiers in Immunology, 9. pp. 1-21.

Resident memory T cells have emerged as key players in the immune response generated against a number of pathogens. Their ability to take residence in non-lymphoid peripheral tissues allows for the rapid deployment of secondary effector responses at the site of pathogen entry. This ability to provide enhanced regional immunity has gathered much attention, with the generation of resident memory T cells being the goal of many novel vaccines. This review aimed to systematically analyze published literature investigating the role of resident memory T cells in human infectious diseases. Known effector responses mounted by these cells are summarized and key strategies that are potentially influential in the rational design of resident memory T cell inducing vaccines have also been highlighted. A Boolean search was applied to Medline, SCOPUS, and Web of Science. Studies that investigated the effector response generated by resident memory T cells and/or evaluated strategies for inducing these cells were included irrespective of published date. Studies must have utilized an established technique for identifying resident memory T cells such as T cell phenotyping. While over 600 publications were revealed by the search, 147 articles were eligible for inclusion. The reference lists of included articles were also screened for other eligible publications. This resulted in the inclusion of publications that studied residentmemory T cells in the context of over 25 human pathogens. The vast majority of studies were conducted in mouse models and demonstrated that resident memory T cells mount protective immune responses. Although the role resident memory T cells play in providing immunity varies depending on the pathogen and anatomical location they resided in, the evidence overall suggests that these cells are vital for the timely and optimal protection against a number of infectious diseases. The induction of resident memory T cells should be further investigated and seriously considered when designing new vaccines.