A panel of recombinant Leishmania donovani cell surface and secreted proteins identifies LdBPK_323600.1 as a serological marker of symptomatic infection.

Visceral leishmaniasis is a deadly infectious disease and is one of the world's major neglected health problems. Because the symptoms of infection are similar to other endemic diseases, accurate diagnosis is crucial for appropriate treatment. Definitive diagnosis using splenic or bone marrow aspirates is highly invasive, and so, serological assays are preferred, including the direct agglutination test (DAT) or rK39 strip test. These tests, however, are either difficult to perform in the field (DAT) or lack specificity in some endemic regions (rK39), making the development of new tests a research priority. The availability of Leishmania spp. genomes presents an opportunity to identify new diagnostic targets. Here, we use genome data and a mammalian protein expression system to create a panel of 93 proteins consisting of the extracellular ectodomains of the Leishmania donovani cell surface and secreted proteins. We use these panel and sera from murine experimental infection models and natural human and canine infections to identify new candidates for serological diagnosis. We observed a concordance between the most immunoreactive antigens in different host species and transmission settings. The antigen encoded by the LdBPK_323600.1 gene can diagnose Leishmania infections with high sensitivity and specificity in patient cohorts from different endemic regions including Bangladesh and Ethiopia. In longitudinal sampling of treated patients, we observed reductions in immunoreactivity to LdBPK_323600.1 suggesting it could be used to diagnose treatment success. In summary, we have identified new antigens that could contribute to improved serological diagnostic tests to help control the impact of this deadly tropical infectious disease. IMPORTANCE: Visceral leishmaniasis is fatal if left untreated with patients often displaying mild and non-specific symptoms during the early stages of infection making accurate diagnosis important. Current methods for diagnosis require highly trained medical staff to perform highly invasive biopsies of the liver or bone marrow which pose risks to the patient. Less invasive molecular tests are available but can suffer from regional variations in their ability to accurately diagnose an infection. To identify new diagnostic markers of visceral leishmaniasis, we produced and tested a panel of 93 proteins identified from the genome of the parasite responsible for this disease. We found that the pattern of host antibody reactivity to these proteins was broadly consistent across naturally acquired infections in both human patients and dogs, as well as experimental rodent infections. We identified a new protein called LdBPK_323600.1 that could accurately diagnose visceral leishmaniasis infections in humans.

Tissue-resident B cells orchestrate macrophage polarisation and function.

B cells play a central role in humoral immunity but also have antibody-independent functions. Studies to date have focused on B cells in blood and secondary lymphoid organs but whether B cells reside in non-lymphoid organs (NLO) in homeostasis is unknown. Here we identify, using intravenous labeling and parabiosis, a bona-fide tissue-resident B cell population in lung, liver, kidney and urinary bladder, a substantial proportion of which are B-1a cells. Tissue-resident B cells are present in neonatal tissues and also in germ-free mice NLOs, albeit in lower numbers than in specific pathogen-free mice and following co-housing with 'pet-store' mice. They spatially co-localise with macrophages and regulate their polarization and function, promoting an anti-inflammatory phenotype, in-part via interleukin-10 production, with effects on bacterial clearance during urinary tract infection. Thus, our data reveal a critical role for tissue-resident B cells in determining the homeostatic 'inflammatory set-point' of myeloid cells, with important consequences for tissue immunity.

A drug repurposing screen for whipworms informed by comparative genomics.

Hundreds of millions of people worldwide are infected with the whipworm Trichuris trichiura. Novel treatments are urgently needed as current drugs, such as albendazole, have relatively low efficacy. We have investigated whether drugs approved for other human diseases could be repurposed as novel anti-whipworm drugs. In a previous comparative genomics analysis, we identified 409 drugs approved for human use that we predicted to target parasitic worm proteins. Here we tested these ex vivo by assessing motility of adult worms of Trichuris muris, the murine whipworm, an established model for human whipworm research. We identified 14 compounds with EC50 values of ≤50 µM against T. muris ex vivo, and selected nine for testing in vivo. However, the best worm burden reduction seen in mice was just 19%. The high number of ex vivo hits against T. muris shows that we were successful at predicting parasite proteins that could be targeted by approved drugs. In contrast, the low efficacy of these compounds in mice suggest challenges due to their chemical properties (e.g. lipophilicity, polarity, molecular weight) and pharmacokinetics (e.g. absorption, distribution, metabolism, and excretion) that may (i) promote absorption by the host gastrointestinal tract, thereby reducing availability to the worms embedded in the large intestine, and/or (ii) restrict drug uptake by the worms. This indicates that identifying structural analogues that have reduced absorption by the host, and increased uptake by worms, may be necessary for successful drug development against whipworms.

Inhibitory IL-10-producing CD4+ T cells are T-bet-dependent and facilitate cytomegalovirus persistence via coexpression of arginase-1.

Inhibitory CD4+ T cells have been linked with suboptimal immune responses against cancer and pathogen chronicity. However, the mechanisms that underpin the development of these regulatory cells, especially in the context of ongoing antigen exposure, have remained obscure. To address this knowledge gap, we undertook a comprehensive functional, phenotypic, and transcriptomic analysis of interleukin (IL)-10-producing CD4+ T cells induced by chronic infection with murine cytomegalovirus (MCMV). We identified these cells as clonally expanded and highly differentiated TH1-like cells that developed in a T-bet-dependent manner and coexpressed arginase-1 (Arg1), which promotes the catalytic breakdown of L-arginine. Mice lacking Arg1-expressing CD4+ T cells exhibited more robust antiviral immunity and were better able to control MCMV. Conditional deletion of T-bet in the CD4+ lineage suppressed the development of these inhibitory cells and also enhanced immune control of MCMV. Collectively, these data elucidated the ontogeny of IL-10-producing CD4+ T cells and revealed a previously unappreciated mechanism of immune regulation, whereby viral persistence was facilitated by the site-specific delivery of Arg1.

ANTT® standardisation facilitates new efficiencies with a novel partially-sterile Standard-ANTT PIVC Pack.

Introduction: The widespread adoption of the ANTT®Clinical Practice Framework as a single standard for aseptic technique, has highlighted that many clinical procedures do not require a sterile procedure pack to be performed safely and aseptically. This study explores the utilisation of a partially-sterile procedure pack that is specifically tailored to Standard-ANTT. Methods: A prospective project improvement evaluation, using a non-paired sample (pre: n=41; post: n =33) of emergency department staff in an NHS hospital. Staff were evaluated performing peripheral intravenous cannulations (PIVC) using Standard-ANTT and the B. Braun Standard-ANTT peripheral cannulation pack. Findings: Significant improvements were observed in practice following the implementation of the pack and training in Standard-ANTT, including: Key-Part protection significantly improved (pre: n=28, 68.2%; post: n=33, 100%), and reduction in the Key-Site being touched after disinfection (pre: n=17; 41.4%; post n=5; 15.1%). Conclusions: In conjunction with appropriate education and training, this study provides proof of concept that due to the widespread use of the ANTT Clinical Practice Framework as a single standard aseptic technique, procedure packs that are specifically tailored to Standard-ANTT, can help to promote best practice and improve efficiencies. DEFINITIONS: Partially-sterile procedure pack - all items required to be sterile remain in their individual blister wrapper. The final assembled pack itself is not then subjected to a further round of sterilisation as it is not needed. Sterile procedure pack - often contains a mixture of non-sterile and sterile items that have been stripped from their individual blister wrapper requiring the sterilisation of the final assembled pack.

Prolidase Deficiency Causes Spontaneous T Cell Activation and Lupus-like Autoimmunity.

Prolidase deficiency (PD) is a multisystem disorder caused by mutations in the PEPD gene, which encodes a ubiquitously expressed metallopeptidase essential for the hydrolysis of dipeptides containing C-terminal proline or hydroxyproline. PD typically presents in childhood with developmental delay, skin ulcers, recurrent infections, and, in some patients, autoimmune features that can mimic systemic lupus erythematosus. The basis for the autoimmune association is uncertain, but might be due to self-antigen exposure with tissue damage, or indirectly driven by chronic infection and microbial burden. In this study, we address the question of causation and show that Pepd-null mice have increased antinuclear autoantibodies and raised serum IgA, accompanied by kidney immune complex deposition, consistent with a systemic lupus erythematosus-like disease. These features are associated with an accumulation of CD4 and CD8 effector T cells in the spleen and liver. Pepd deficiency leads to spontaneous T cell activation and proliferation into the effector subset, which is cell intrinsic and independent of Ag receptor specificity or antigenic stimulation. However, an increase in KLRG1+ effector CD8 cells is not observed in mixed chimeras, in which the autoimmune phenotype is also absent. Our findings link autoimmune susceptibility in PD to spontaneous T cell dysfunction, likely to be acting in combination with immune activators that lie outside the hemopoietic system but result from the abnormal metabolism or loss of nonenzymatic prolidase function. This knowledge provides insight into the role of prolidase in the maintenance of self-tolerance and highlights the importance of treatment to control T cell activation.

Intelectin-1 binds and alters the localization of the mucus barrier-modifying bacterium Akkermansia muciniphila.

Intelectin-1 (ITLN1) is a lectin secreted by intestinal epithelial cells (IECs) and upregulated in human ulcerative colitis (UC). We investigated how ITLN1 production is regulated in IECs and the biological effects of ITLN1 at the host-microbiota interface using mouse models. Our data show that ITLN1 upregulation in IECs from UC patients is a consequence of activating the unfolded protein response. Analysis of microbes coated by ITLN1 in vivo revealed a restricted subset of microorganisms, including the mucolytic bacterium Akkermansia muciniphila. Mice overexpressing intestinal ITLN1 exhibited decreased inner colonic mucus layer thickness and closer apposition of A. muciniphila to the epithelial cell surface, similar to alterations reported in UC. The changes in the inner mucus layer were microbiota and A. muciniphila dependent and associated with enhanced sensitivity to chemically induced and T cell-mediated colitis. We conclude that by determining the localization of a select group of bacteria to the mucus layer, ITLN1 modifies this critical barrier. Together, these findings may explain the impact of ITLN1 dysregulation on UC pathogenesis.

EROS is a selective chaperone regulating the phagocyte NADPH oxidase and purinergic signalling.

EROS (essential for reactive oxygen species) protein is indispensable for expression of gp91phox, the catalytic core of the phagocyte NADPH oxidase. EROS deficiency in humans is a novel cause of the severe immunodeficiency, chronic granulomatous disease, but its mechanism of action was unknown until now. We elucidate the role of EROS, showing it acts at the earliest stages of gp91phox maturation. It binds the immature 58 kDa gp91phox directly, preventing gp91phox degradation and allowing glycosylation via the oligosaccharyltransferase machinery and the incorporation of the heme prosthetic groups essential for catalysis. EROS also regulates the purine receptors P2X7 and P2X1 through direct interactions, and P2X7 is almost absent in EROS-deficient mouse and human primary cells. Accordingly, lack of murine EROS results in markedly abnormal P2X7 signalling, inflammasome activation, and T cell responses. The loss of both ROS and P2X7 signalling leads to resistance to influenza infection in mice. Our work identifies EROS as a highly selective chaperone for key proteins in innate and adaptive immunity and a rheostat for immunity to infection. It has profound implications for our understanding of immune physiology, ROS dysregulation, and possibly gene therapy.

The gut microbial metabolic capacity of microbiome-humanized vs. wild type rodents reveals a likely dual role of intestinal bacteria in hepato-intestinal schistosomiasis.

Increasing evidence shows that the host gut microbiota might be involved in the immunological cascade that culminates with the formation of tissue granulomas underlying the pathophysiology of hepato-intestinal schistosomiasis. In this study, we investigated the impact of Schistosoma mansoni infection on the gut microbial composition and functional potential of both wild type and microbiome-humanized mice. In spite of substantial differences in microbiome composition at baseline, selected pathways were consistently affected by parasite infection. The gut microbiomes of infected mice of both lines displayed, amongst other features, enhanced capacity for tryptophan and butyrate production, which might be linked to the activation of mechanisms aimed to prevent excessive injuries caused by migrating parasite eggs. Complementing data from previous studies, our findings suggest that the host gut microbiome might play a dual role in the pathophysiology of schistosomiasis, where intestinal bacteria may contribute to egg-associated pathology while, in turn, protect the host from uncontrolled tissue damage.

How to improve aseptic technique to reduce bloodstream infection during vascular access procedures.

Bloodstream infections associated with vascular access procedures pose a serious risk to patients that can be reduced by better standards of aseptic technique. The objectives of this roundtable of experts were to achieve a consensus on how to improve skin antisepsis in hospital, improve training, competency, compliance and consistency in skin antisepsis, review the role of devices in improving skin antisepsis, identify methods to improve skin antisepsis integrated with the Aseptic Non Touch Technique (ANTT®) approach, and identify challenges to the implementation of the panel's recommendations. Recommendations include using MHRA-licensed 2% chlorhexidine gluconate in 70% isopropyl alcohol solution with bidirectional strokes for up to 30 seconds, then leaving the skin to air dry for 30 seconds; using the ANTT Clinical Practice Framework and terminology as the standard for skin antisepsis training and practice; standardised ANTT and skin antisepsis education with 3-yearly competency assessments for all UK health professionals; and more research to address the evidence gap on transmission of infection after skin antisepsis.

Vivaxin genes encode highly immunogenic, non-variant antigens on the Trypanosoma vivax cell-surface.

Trypanosoma vivax is a unicellular hemoparasite, and a principal cause of animal African trypanosomiasis (AAT), a vector-borne and potentially fatal livestock disease across sub-Saharan Africa. Previously, we identified diverse T. vivax-specific genes that were predicted to encode cell surface proteins. Here, we examine the immune responses of naturally and experimentally infected hosts to these unique parasite antigens, to identify immunogens that could become vaccine candidates. Immunoprofiling of host serum shows that one particular family (Fam34) elicits a consistent IgG antibody response. This gene family, which we now call Vivaxin, encodes at least 124 transmembrane glycoproteins that display quite distinct expression profiles and patterns of genetic variation. We focused on one gene (viv-ß8) that encodes one particularly immunogenic vivaxin protein and which is highly expressed during infections but displays minimal polymorphism across the parasite population. Vaccination of mice with VIVß8 adjuvanted with Quil-A elicits a strong, balanced immune response and delays parasite proliferation in some animals but, ultimately, it does not prevent disease. Although VIVß8 is localized across the cell body and flagellar membrane, live immunostaining indicates that VIVß8 is largely inaccessible to antibody in vivo. However, our phylogenetic analysis shows that vivaxin includes other antigens shown recently to induce immunity against T. vivax. Thus, the introduction of vivaxin represents an important advance in our understanding of the T. vivax cell surface. Besides being a source of proven and promising vaccine antigens, the gene family is clearly an important component of the parasite glycocalyx, with potential to influence host-parasite interactions.

Group 3 innate lymphocytes make a distinct contribution to type 17 immunity in bladder defence.

Bladder infection affects a hundred million people annually, but our understanding of bladder immunity is incomplete. We found type 17 immune response genes among the most up-regulated networks in mouse bladder following uropathogenic Escherichia coli (UPEC) challenge. Intravital imaging revealed submucosal Rorc+ cells responsive to UPEC challenge, and we found increased Il17 and IL22 transcripts in wild-type and Rag2 -/- mice, implicating group 3 innate lymphoid cells (ILC3s) as a source of these cytokines. NCR-positive and negative ILC3 subsets were identified in murine and human bladders, with local proliferation increasing IL17-producing ILC3s post infection. ILC3s made a more limited contribution to bladder IL22, with prominent early induction of IL22 evident in Th17 cells. Single-cell RNA sequencing revealed bladder NCR-negative ILC3s as the source of IL17 and identified putative ILC3-myeloid cell interactions, including via lymphotoxin-ß-LTBR. Altogether, our data provide important insights into the orchestration and execution of type 17 immunity in bladder defense.

GIMAP6 regulates autophagy, immune competence, and inflammation in mice and humans.

Inborn errors of immunity (IEIs) unveil regulatory pathways of human immunity. We describe a new IEI caused by mutations in the GTPase of the immune-associated protein 6 (GIMAP6) gene in patients with infections, lymphoproliferation, autoimmunity, and multiorgan vasculitis. Patients and Gimap6-/- mice show defects in autophagy, redox regulation, and polyunsaturated fatty acid (PUFA)-containing lipids. We find that GIMAP6 complexes with GABARAPL2 and GIMAP7 to regulate GTPase activity. Also, GIMAP6 is induced by IFN-γ and plays a critical role in antibacterial immunity. Finally, we observed that Gimap6-/- mice died prematurely from microangiopathic glomerulosclerosis most likely due to GIMAP6 deficiency in kidney endothelial cells.

Identification of gut microbial species linked with disease variability in a widely used mouse model of colitis.

Experimental mouse models are central to basic biomedical research; however, variability exists across genetically identical mice and mouse facilities making comparisons difficult. Whether specific indigenous gut bacteria drive immunophenotypic variability in mouse models of human disease remains poorly understood. We performed a large-scale experiment using 579 genetically identical laboratory mice from a single animal facility, designed to identify the causes of disease variability in the widely used dextran sulphate sodium mouse model of inflammatory bowel disease. Commonly used treatment endpoint measures-weight loss and intestinal pathology-showed limited correlation and varied across mouse lineages. Analysis of the gut microbiome, coupled with machine learning and targeted anaerobic culturing, identified and isolated two previously undescribed species, Duncaniella muricolitica and Alistipes okayasuensis, and demonstrated that they exert dominant effects in the dextran sulphate sodium model leading to variable treatment endpoint measures. We show that the identified gut microbial species are common, but not ubiquitous, in mouse facilities around the world, and suggest that researchers monitor for these species to provide experimental design opportunities for improved mouse models of human intestinal diseases.

The Leishmania donovani Ortholog of the Glycosylphosphatidylinositol Anchor Biosynthesis Cofactor PBN1 Is Essential for Host Infection.

Visceral leishmaniasis is a deadly infectious disease caused by Leishmania donovani, a kinetoplastid parasite for which no licensed vaccine is available. To identify potential vaccine candidates, we systematically identified genes encoding putative cell surface and secreted proteins essential for parasite viability and host infection. We identified a protein encoded by LdBPK_061160 which, when ablated, resulted in a remarkable increase in parasite adhesion to tissue culture flasks. Here, we show that this phenotype is caused by the loss of glycosylphosphatidylinositol (GPI)-anchored surface molecules and that LdBPK_061160 encodes a noncatalytic component of the L. donovani GPI-mannosyltransferase I (GPI-MT I) complex. GPI-anchored surface molecules were rescued in the LdBPK_061160 mutant by the ectopic expression of both human genes PIG-X and PIG-M, but neither gene could complement the phenotype alone. From further sequence comparisons, we conclude that LdBPK_061160 is the functional orthologue of yeast PBN1 and mammalian PIG-X, which encode the noncatalytic subunits of their respective GPI-MT I complexes, and we assign LdBPK_061160 as LdPBN1. The LdPBN1 mutants could not establish a visceral infection in mice, a phenotype that was rescued by constitutive expression of LdPBN1. Although mice infected with the null mutant did not develop an infection, exposure to these parasites provided significant protection against subsequent infection with a virulent strain. In summary, we have identified the orthologue of the PBN1/PIG-X noncatalytic subunit of GPI-MT I in trypanosomatids, shown that it is essential for infection in a murine model of visceral leishmaniasis, and demonstrated that the LdPBN1 mutant shows promise for the development of an attenuated live vaccine. IMPORTANCE Visceral leishmaniasis is a deadly infectious disease caused by the parasites Leishmania donovani and Leishmania infantum. It remains a major global health problem, and there is no licensed highly effective vaccine. Molecules that are displayed on the surface of parasites are involved in host-parasite interactions and have important roles in immune evasion, making vaccine development difficult. One major way in which parasite surface molecules are tethered to the surface is via glycophosphatidylinositol (GPI) anchors; however, the enzymes required for all the biosynthetic steps in these parasites are not known. Here, we identified the enzyme required for an essential step in the GPI anchor-biosynthetic pathway in L. donovani, and we show that while parasites lacking this gene are viable in vitro, they are unable to establish infections in mice, a property we show can be exploited to develop a live genetically attenuated parasite vaccine.

Is ANTT Achievable in the Home Healthcare Setting?

At the heart of infection prevention in the home care setting is aseptic technique, a generic term for the clinical competency of ensuring asepsis during invasive clinical procedures and the maintenance of invasive medical devices. Variable terminology has resulted in confused education and clinical practice. Ambiguity has played out in all care environments, but especially in the home, where the challenge of providing effective aseptic technique is complicated by unique environmental and logistical factors. This has led some to conclude that aseptic technique is not possible in the home, or that it is not required at all in certain situations. This article challenges these assumptions and outlines the Aseptic Non Touch Technique (ANTT®) Clinical Practice Framework, that is used widely internationally. Through a thematic analysis of focus group discussions with home care nurses (n = 107) in the United Kingdom and the United States, opinions about aseptic practice in the home care setting were examined. Three main polarizing themes were identified and used to discuss theory and practice applications of aseptic technique for home healthcare nurses. Specific challenges in performing aseptic technique in home-based settings are exacerbated by ambiguity between what constitutes "clean," "aseptic," or so-called "sterile" technique, and are perpetuated by a lack of competency-based training that doesn't reflect the critical importance of aseptic technique to patient safety. Rather than continuing an endless debate about the definition of aseptic technique, ANTT has redefined aseptic technique education and clinical practice, with a comprehensive clinical practice framework and associated clinical governance better supporting patient safety.

Systematic identification of genes encoding cell surface and secreted proteins that are essential for in vitro growth and infection in Leishmania donovani.

Leishmaniasis is an infectious disease caused by protozoan parasites belonging to the genus Leishmania for which there are no approved human vaccines. Infections localise to different tissues in a species-specific manner with the visceral form of the disease caused by Leishmania donovani and L. infantum being the most deadly in humans. Although Leishmania spp. parasites are predominantly intracellular, the visceral disease can be prevented in dogs by vaccinating with a complex mixture of secreted products from cultures of L. infantum promastigotes. With the logic that extracellular parasite proteins make good subunit vaccine candidates because they are directly accessible to vaccine-elicited host antibodies, here we attempt to discover proteins that are essential for in vitro growth and host infection with the goal of identifying subunit vaccine candidates. Using an in silico analysis of the Leishmania donovani genome, we identified 92 genes encoding proteins that are predicted to be secreted or externally anchored to the parasite membrane by a single transmembrane region or a GPI anchor. By selecting a transgenic L. donovani parasite that expresses both luciferase and the Cas9 nuclease, we systematically attempted to target all 92 genes by CRISPR genome editing and identified four that were required for in vitro growth. For fifty-five genes, we infected cohorts of mice with each mutant parasite and by longitudinally quantifying parasitaemia with bioluminescent imaging, showed that nine genes had evidence of an attenuated infection although all ultimately established an infection. Finally, we expressed two genes as full-length soluble recombinant proteins and tested them as subunit vaccine candidates in a murine preclinical infection model. Both proteins elicited significant levels of protection against the uncontrolled development of a splenic infection warranting further investigation as subunit vaccine candidates against this deadly infectious tropical disease.