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Greenhouse gas (GHG) emissions from livestock production must be urgently tackled to substantially reduce their contribution to global warming. Simply reducing livestock numbers to this end risks impacting negatively on food security, rural livelihoods and climate change adaptation. We argue that significant mitigation of livestock emissions can be delivered immediately by improving animal health and hence production efficiency, but this route is not prioritized because its benefits, although intuitive, are poorly quantified. Rigorous methodology must be developed to estimate emissions from animal disease and hence achievable benefits from improved health through interventions. If, as expected, climate change is to affect the distribution and severity of health conditions, such quantification becomes of even greater importance. We have therefore developed a framework and identified data sources for robust quantification of the relationship between animal health and greenhouse gas emissions, which could be applied to drive and account for positive action. This will not only help mitigate climate change but at the same time promote cost-effective food production and enhanced animal welfare, a rare win–win in the search for a sustainable planetary future. [ABSTRACT FROM AUTHOR]

Patients with decompensated liver cirrhosis, in particular those classified as Childs-Pugh class C, are at increased risk of severe coronavirus disease-2019 (COVID-19) upon infection with severe acute respiratory coronavirus 2 (SARS-CoV-2). The biological mechanisms underlying this are unknown. We aimed to examine the levels of serum intrinsic antiviral proteins as well as alterations in the innate antiviral immune response in patients with decompensated liver cirrhosis. Serum from 53 SARS-CoV-2 unexposed and unvaccinated individuals, with decompensated liver cirrhosis undergoing assessment for liver transplantation, were screened using SARS-CoV-2 pseudoparticle and SARS-CoV-2 virus assays. The ability of serum to inhibit interferon (IFN) signalling was assessed using a cell-based reporter assay. Severity of liver disease was assessed using two clinical scoring systems, the Child-Pugh class and the MELD-Na score. In the presence of serum from SARS-CoV-2 unexposed patients with decompensated liver cirrhosis there was no association between SARS-CoV-2 pseudoparticle infection or live SARS-CoV-2 virus infection and severity of liver disease. Type I IFNs are a key component of the innate antiviral response. Serum from patients with decompensated liver cirrhosis contained elevated levels of auto-antibodies capable of binding IFN-α2b compared to healthy controls. High MELD-Na scores were associated with the ability of these auto-antibodies to neutralize type I IFN signalling by IFN-α2b but not IFN-β1a. Our results demonstrate that neutralizing auto-antibodies targeting IFN-α2b are increased in patients with high MELD-Na scores. The presence of neutralizing type I IFN-specific auto-antibodies may increase the likelihood of viral infections, including severe COVID-19, in patients with decompensated liver cirrhosis. Serum from patients with decompensated liver cirrhosis inhibits type I interferon (IFN) signalling due to the presence of auto-antibodies capable of directly binding recombinant IFN-α2b. Depletion of IgG reverses the inhibition of type I IFN signalling. Our results demonstrate that type I IFN-neutralizing auto-antibodies are increased in a proportion of end-stage liver disease patients, which may increase the risk of viral infection in this patient cohort. [ABSTRACT FROM AUTHOR]

Liver-resident CD56brightCD16- natural killer (NK) cells are enriched in the human liver and are phenotypically distinct from their blood counterparts. Although these cells are capable of rapid cytotoxic effector activity, their functional role remains unclear. We hypothesise that they may contribute to immune tolerance in the liver during transplantation. RNA sequencing was carried out on FACS sorted NK cell subpopulations from liver perfusates (n=5) and healthy blood controls (n=5). Liver-resident CD56brightCD16+/- NK cells upregulate genes associated with tissue residency. They also upregulate expression of CD160 and LY9, both of which encode immune receptors capable of activating NK cells. Co-expression of CD160 and Ly9 on liver-resident NK cells was validated using flow cytometry. Hepatic NK cell cytotoxicity against allogenic T cells was tested using an in vitro co-culture system of liver perfusate-derived NK cells and blood T cells (n=10-13). In coculture experiments, hepatic NK cells but not blood NK cells induced significant allogenic T cell death (p=0.0306). Allogenic CD8+ T cells were more susceptible to hepatic NK cytotoxicity than CD4+ T cells (p<0.0001). Stimulation of hepatic CD56bright NK cells with an anti-CD160 agonist mAb enhanced this cytotoxic response (p=0.0382). Our results highlight a role for donor liver NK cells in regulating allogenic CD8+ T cell activation, which may be important in controlling recipient CD8+ T cell-mediated rejection post liver-transplant. [ABSTRACT FROM AUTHOR]

The liver fluke, Fasciola hepatica, is an obligate blood-feeder, and the gastrodermal cells of the parasite form the interface with the host's blood. Despite their importance in the host–parasite interaction, in-depth proteomic analysis of the gastrodermal cells is lacking. Here, we used laser microdissection of F. hepatica tissue sections to generate unique and biologically exclusive tissue fractions of the gastrodermal cells and tegument for analysis by mass spectrometry. A total of 226 gastrodermal cell proteins were identified, with proteases that degrade haemoglobin being the most abundant. Other detected proteins included those such as proton pumps and anticoagulants which maintain a microenvironment that facilitates digestion. By comparing the gastrodermal cell proteome and the 102 proteins identified in the laser microdissected tegument with previously published tegument proteomic datasets, we showed that one-quarter of proteins (removed by freeze–thaw extraction) or one-third of proteins (removed by detergent extraction) previously identified as tegumental were instead derived from the gastrodermal cells. Comparative analysis of the laser microdissected gastrodermal cells, tegument, and F. hepatica secretome revealed that the gastrodermal cells are the principal source of secreted proteins, as well as showed that both the gastrodermal cells and the tegument are likely to release subpopulations of extracellular vesicles (EVs). Microscopical examination of the gut caeca from flukes fixed immediately after their removal from the host bile ducts showed that selected gastrodermal cells underwent a progressive thinning of the apical plasma membrane which ruptured to release secretory vesicles en masse into the gut lumen. Our findings suggest that gut-derived EVs are released via a novel atypical secretory route and highlight the importance of the gastrodermal cells in nutrient acquisition and possible immunomodulation by the parasite. [ABSTRACT FROM AUTHOR]

Helminth pathogens express papain-like cysteine peptidases, termed cathepsins, which have important roles in virulence, including host entry, tissue migration and the suppression of host immune responses. The liver fluke Fasciola hepatica, an emerging human pathogen, expresses the largest cathepsin L cysteine protease family yet described. Recent phylogenetic, biochemical and structural studies indicate that this family contains five separate clades, which exhibit overlapping but distinct substrate specificities created by a process of gene duplication followed by subtle residue divergence within the protease active site. The developmentally regulated expression of these proteases correlates with the passage of the parasite through host tissues and its encounters with different host macromolecules.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.06.052 Byline: James A. St John (a), Christina Claxton (a), Mark W. Robinson (a), Fumiichiro Yamamoto (b), Steven E. Domino (d), Brian Key (a)(c) Keywords: Carbohydrate; Blood group A; Blood group H; Neuron; Guidance; Glomerulus; Accessory olfactory Abstract: Primary sensory neurons in the vertebrate olfactory systems are characterised by the differential expression of distinct cell surface carbohydrates. We show here that the histo-blood group H carbohydrate is expressed by primary sensory neurons in both the main and accessory olfactory systems while the blood group A carbohydrate is expressed by a subset of vomeronasal neurons in the developing accessory olfactory system. We have used both loss-of-function and gain-of-function approaches to manipulate expression of these carbohydrates in the olfactory system. In null mutant mice lacking the [alpha](1,2)fucosyltransferase FUT1, the absence of blood group H carbohydrate resulted in the delayed maturation of the glomerular layer of the main olfactory bulb. In addition, ubiquitous expression of blood group A on olfactory axons in gain-of-function transgenic mice caused mis-routing of axons in the glomerular layer of the main olfactory bulb and led to exuberant growth of vomeronasal axons in the accessory olfactory bulb. These results provide in vivo evidence for a role of specific cell surface carbohydrates during development of the olfactory nerve pathways. Author Affiliation: (a) Brain Growth and Regeneration Laboratory, School of Biomedical Sciences, The University of Queensland, Brisbane 4072, Queensland, Australia (b) The Burnham Institute, La Jolla Cancer Research Center, La Jolla, CA 92037, USA (c) Centre for Functional and Applied Genomics, The University of Queensland, Brisbane, 4072, Queensland, Australia (d) Department of Obstetrics and Gynecology, University of Michigan Medical Center, Ann Arbor, MI 48109, USA Article History: Received 7 April 2006; Revised 29 June 2006; Accepted 30 June 2006

The liver fluke, Fasciola hepatica , is a global burden on the wellbeing and productivity of farmed ruminants, and a zoonotic threat to human health. Despite the clear need for accelerated discovery of new drug and vaccine treatments for this pathogen, we still have a relatively limited understanding of liver fluke biology and host interactions. Noncoding RNAs, including micro (mi)RNAs, are key to transcriptional regulation in all eukaryotes, such that an understanding of miRNA biology can shed light on organismal function at a systems level. Four previous publications have reported up to 89 mature miRNA sequences from F. hepatica , but our data show that this does not represent a full account of this species miRNome. We have expanded on previous studies by sequencing, for the first time, miRNAs from multiple life stages (adult, newly excysted juvenile (NEJ), metacercariae and adult-derived extracellular vesicles (EVs)). These experiments detected an additional 61 high-confidence miRNAs, most of which have not been described in any other species, expanding the F. hepatica miRNome to 150 mature sequences. We used quantitative (q)PCR assays to provide the first developmental profile of miRNA expression across metacercariae, NEJ, adult and adult-derived Evs. The majority of miRNAs were expressed most highly in metacercariae, with at least six distinct expression clusters apparent across life stages. Intracellular miRNAs were functionally analyzed to identify target mRNAs with inversely correlated expression in F. hepatica tissue transcriptomes, highlighting regulatory interactions with key virulence transcripts including cathepsin proteases, and neuromuscular genes that control parasite growth, development and motility. We also linked 28 adult-derived EV miRNAs with downregulation of 397 host genes in F. hepatica -infected transcriptomes from ruminant lymph node, peripheral blood mononuclear cell (PBMC) and liver tissue transcriptomes. These included genes involved in signal transduction, immune and metabolic pathways, adding to the evidence for miRNA-based immunosuppression during fasciolosis. These data expand our understanding of the F. hepatica miRNome, provide the first data on developmental miRNA regulation in this species, and provide a set of testable hypotheses for functional genomics interrogations of liver fluke miRNA biology. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression being involved in many different biological processes and play a key role in developmental timing. Additionally, recent studies have shown that miRNAs released from parasites are capable of regulating the expression of host genes. In the present work, we studied the expression patterns of ncRNAs of various intra-mammalian life-cycle stages of the liver fluke, Fasciola hepatica , as well as those packaged into extracellular vesicles and shed by the adult fluke. The miRNA expression profile of the intra-mammalian stages shows important variations, despite a set of predominant miRNAs that are highly expressed across all stages. No substantial variations in miRNA expression between dormant and activated metacercariae were detected, suggesting that they might not be central players in regulating fluke gene expression during this crucial step in the invasion of the definitive host. We generated a curated pipeline for the prediction of putative target genes that reports only sites conserved between three different prediction approaches. This pipeline was tested against an iso-seq curated database of the 3' UTR regions of F. hepatica genes to detect miRNA regulation networks within liver fluke. Several functions related to the host immune response or modulation were enriched among the targets of the most highly expressed parasite miRNAs, stressing that they might be key players during the establishment and maintenance of infection. Additionally, we detected fragments derived from the processing of tRNAs, in all developmental stages analyzed, and documented the presence of novel long tRNA fragments enriched in vesicles. We confirmed the presence of at least 5 putative vault RNAs (vtRNAs), that are expressed across different stages and enriched in vesicles. The presence of tRNA fragments and vtRNAs in vesicles raise the possibility that they could be involved in the host-parasite interaction. [ABSTRACT FROM AUTHOR]

Diverse populations of natural killer (NK) cells have been identified in circulating peripheral blood and a wide variety of different tissues and organs. These tissue-resident NK cell populations are phenotypically distinct from circulating NK cells, however, functional descriptions of their roles within tissues are lacking. Recent advances in single cell RNA sequencing (scRNA-seq) have enabled detailed transcriptional profiling of tissues at the level of single cells and provide the opportunity to explore NK cell diversity within tissues. This review explores potential novel functions of human liver-resident (lr)NK cells identified in human liver scRNA-seq studies. By comparing these datasets we identified up-regulated and down-regulated genes associated with lrNK cells clusters. These genes encode a number of activating and inhibiting receptors, as well as signal transduction molecules, which highlight potential unique pathways that lrNK cells utilize to respond to stimuli within the human liver. This unique receptor repertoire of lrNK cells may confer the ability to regulate a number of immune cell populations, such as circulating monocytes and T cells, while avoiding activation by liver hepatocytes and Kupffer cells. Validating the expression of these receptors on lrNK cells and the proposed cellular interactions within the human liver will expand our understanding of the liver-specific homeostatic roles of this tissue-resident immune cell population. [ABSTRACT FROM AUTHOR]

Parasite-released extracellular vesicles (EVs) deliver signals to the host immune system that are critical to maintaining the long-term relationship between parasite and host. In the present study, total EVs (FhEVs) released in vitro by adults of the helminth parasite Fasciola hepatica were isolated using a recently described gravity flow method that protects their structural integrity. The FhEVs molecular cargo was defined using proteomic analysis and their surface topology characterised by glycan microarrays. The proteomic analysis identified 618 proteins, 121 of which contained putative N-linked glycosylation sites while 132 proteins contained putative O-linked glycosylation sites. Glycan arrays revealed surface-exposed glycans with a high affinity for mannose-binding lectins indicating the predominance of oligo mannose-rich glycoproteins, as well as other glycans with a high affinity for complex-type N-glycans. When added to bone-marrow derived dendritic cells isolated FhEV induced a novel phenotype that was categorised by the secretion of low levels of TNF, enhanced expression of cell surface markers (CD80, CD86, CD40, OX40L, and SIGNR1) and elevation of intracellular markers (SOCS1 and SOCS3). When FhEV-stimulated BMDCs were introduced into OT-II mice by adoptive transfer, IL-2 secretion from skin draining lymph nodes (sdLN) and spleen cells was inhibited in response to both specific and non-specific antigen stimulation. Immunisation of mice with a suspension of FhEV did not elicit significant immune responses; however, in the presence of alum, FhEVs induced a mixed Th1/Th2 immune response with high antigen specific antibody titres. Thus, we have demonstrated that FhEVs induce a unique phentotype in DC capable of suppressing IL-2 secretion from T-cells. Our studies add to the growing immuno-proteomic database that will be an important source for the discovery of future parasite vaccines and immunotherapeutic biologicals. Author summary: Parasite-released extracellular vesicles (EVs) deliver signals to the host immune system that are critical to maintaining the long-term relationship between parasite and host. This study isolated total EVs (FhEVs) released in vitro by the adult stages of the parasitic worm Fasciola hepatica using a gravity flow method that protects the structural integrity of the vesicles. Proteomic analysis identified 618 proteins, 121 of which contained putative N-linked glycosylation sites while 132 proteins contained putative O-linked glycosylation sites while glycan arrays revealed surface-exposed glycans were predominantly oligo mannose-rich glycoproteins, and glycans with a high affinity for complex-type N-glycans. Since the EV molecular cargo can influence host immune cells, FhEVs were added to bone-marrow derived dendritic cells, inducing a novel cell phenotype that when adoptive transferred into OT-II mice inhibited IL-2 secretion from skin draining lymph nodes (sdLN) and spleen cells. Immunisation of mice with FhEV did not elicit significant immune responses; however, in the presence of alum, FhEVs induced a mixed Th1/Th2 immune response with high antigen specific antibody titres. This studied sheds like on the biological activity of FhEVs and added to the growing immuno-proteomic database that will be an important source for the discovery of future therapeutics. [ABSTRACT FROM AUTHOR]

A study on the metal ion binding characteristics of recombinant Plasmodium falciparum M17 leucine aminopeptidase (rPfLAP) is carried out to gain insight into the two metal-binding active sites, a readily exchangeable site (site 1) and a tight binding site (site 2). The type of metal ion present at site 1 is observed to affect not only the catalytic efficiency of the enzyme for peptide substrates but also the mode of binding by bestatin, a metal-chelating inhibitor of M17 aminopeptidases with antimalarial activity.

The adult human liver hosts a complex repertoire of liver resident and transient natural killer (NK) cell populations with diverse phenotypes and functions. Liver resident NK cells are CD56bright NK cells defined by a unique expression profile of transcription factors and cell surface markers (EomeshiTbetloTIGIT+CD69+CXCR6+CD49e−). Despite extensive characterization of the phenotype of liver resident NK cells, it remains unclear how factors within the liver microenvironment induce and maintain this unique phenotype. In this study, we have explored the factors regulating the phenotype of liver resident NK cells. Isolation of healthy liver resident NK cells from donor liver perfusate and in vitro culture results in the gradual loss of the characteristic Tbetlo phenotype, with the cells increasing Tbet expression significantly at day 7. This phenotypic loss could be halted through the dose-dependent addition of liver conditioned media (LCM), generated from the ex vivo culture of liver biopsies from healthy organ donors. TGF-β, but not IL-10, replicated the Tbet suppressive effects of LCM in both liver resident and peripheral blood NK cells. Furthermore, blocking TGF-β receptor signaling using the inhibitor SB431542, reversed the effect of LCM treatment on liver resident NK cells, causing the loss of tissue resident Eomeshi Tbetlo phenotype. Our findings identify liver-derived TGF-β as an important component of the liver microenvironment, which acts to regulate and maintain the phenotype of liver resident NK cells. [ABSTRACT FROM AUTHOR]

Helminth parasites secrete extracellular vesicles (EVs) that can be internalised by host immune cells resulting in modulation of host immunity. While the molecular cargo of EVs have been characterised in many parasites, little is known about the surface-exposed molecules that participate in ligand-receptor interactions with the host cell surface to initiate vesicle docking and subsequent internalisation. Using a membrane-impermeable biotin reagent to capture proteins displayed on the outer membrane surface of two EV sub-populations (termed 15k and 120k EVs) released by adult F. hepatica, we describe 380 surface proteins including an array of virulence factors, membrane transport proteins and molecules involved in EV biogenesis/trafficking. Proteomics and immunohistochemical analysis show that the 120k EVs have an endosomal origin and may be released from the parasite via the protonephridial (excretory) system whilst the larger 15k EVs are released from the gastrodermal epithelial cells that line the fluke gut. A parallel lectin microarray strategy was used to profile the topology of major surface oligosaccharides of intact fluorogenically-labelled EVs as they would be displayed to the host. Lectin profiles corresponding to glycoconjugates exposed on the surface of the 15 K and 120K EV sub-populations are practically identical but are distinct from those of the parasite surface tegument, although all are predominated by high mannose sugars. We found that while the F. hepatica EVs were resistant to exo- and endo-glycosidases, the glyco-amidase PNGase F drastically remodelled the surface oligosaccharides and blocked the uptake of EVs by host macrophages. In contrast, pre-treatment with antibodies obtained from infected hosts, or purified antibodies raised against the extracellular domains of specific EV surface proteins (DM9-containing protein, CD63 receptor and myoferlin), significantly enhanced their cellular internalisation. This work highlights the diversity of EV biogenesis and trafficking pathways used by F. hepatica and sheds light on the molecular interaction between parasite EVs and host cells. [ABSTRACT FROM AUTHOR]

The interferon lambda (IFN-λ) cytokines have well-known antiviral properties, yet their contribution to immune regulation is not well understood. Epithelial cells represent the major target cell of IFN-λ; peripheral blood mononuclear cells are generally considered nonresponsive, with the exception of plasmacytoid dendritic cells (pDCs). In this study we aimed to define the potential for discrete subpopulations of cells to directly respond to IFN-λ. Analysis of peripheral blood leukocytes reveals that, while pDCs uniformly express the highest levels of IFN-λ receptor, a small proportion of B cells and monocytes also express the receptor. Nevertheless, B cells and monocytes respond poorly to IFN-λ stimulation in vitro, with minimal STAT phosphorylation and interferon-stimulated gene (ISG) induction observed. We confirm that pDCs respond to IFN-λ in vitro, upregulating their expression of pSTAT1, pSTAT3, and pSTAT5. However, we found that pDCs do not upregulate pSTAT6 in response to IFN-λ treatment. Our results highlight unique aspects of the response to IFN-λ and confirm that while the IFN-λ receptor is expressed by a small proportion of several different circulating immune cell lineages, under normal conditions only pDCs respond to IFN-λ stimulation with robust STAT phosphorylation and ISG induction. The difference in STAT6 responsiveness of pDCs to type I and type III interferons may help explain the divergence in their biological activities. [ABSTRACT FROM AUTHOR]

The adult human liver is enriched with natural killer (NK) cells, accounting for 30-50% of hepatic lymphocytes, which include tissue-resident hepatic NK-cell subpopulations, distinct from peripheral blood NK cells. In murine liver, a subset of liver-resident hepatic NK cells have altered expression of the two highly related T-box transcription factors, T-bet and eomesodermin (Eomes). Here, we investigate the heterogeneity of T-bet and Eomes expression in NK cells from healthy adult human liver with a view to identifying human liver-resident populations. Hepatic NK cells were isolated from donor liver perfusates and biopsies obtained during orthotopic liver transplantation ( N = 28). Hepatic CD56bright NK cells were Eomeshi T-betlo, a phenotype virtually absent from peripheral blood. These NK cells express the chemokine receptor CXCR6 (chemokine (C-X-C motif) receptor 6), a marker of tissue residency, which is absent from hepatic CD56dim and blood NK cells. Compared to blood populations, these hepatic CD56bright NK cells have increased expression of activatory receptors (NKp44, NKp46, and NKG2D). They show reduced ability to produce IFN-γ but enhanced degranulation in response to challenge with target cells. This functionally distinct population of hepatic NK cells constitutes 20-30% of the total hepatic lymphocyte repertoire and represents a tissue-resident immune cell population adapted to the tolerogenic liver microenvironment. [ABSTRACT FROM AUTHOR]

To maintain a persistent infection viruses such as hepatitis C virus (HCV) employ a range of mechanisms that subvert protective T cell responses. The suppression of antigen-specific T cell responses by HCV hinders efforts to profile T cell responses during chronic infection and antiviral therapy. Conventional methods of detecting antigen-specific T cells utilize either antigen stimulation (e.g., ELISpot, proliferation assays, cytokine production) or antigen-loaded tetramer staining. This limits the ability to profile T cell responses during chronic infection due to suppressed effector function and the requirement for prior knowledge of antigenic viral peptide sequences. Recently, high-throughput sequencing (HTS) technologies have been developed for the analysis of T cell repertoires. In the present study, we have assessed the feasibility of HTS of the TCRβ complementarity determining region (CDR)3 to track T cell expansions in an antigen-independent manner. Using sequential blood samples from HCV-infected individuals undergoing antiviral therapy, we were able to measure the population frequencies of >35,000 TCRβ sequence clonotypes in each individual over the course of 12 weeks. TRBV/TRBJ gene segment usage varied markedly between individuals but remained relatively constant within individuals across the course of therapy. Despite this stable TRBV/TRBJ gene segment usage, a number of TCRβ sequence clonotypes showed dramatic changes in read frequency. These changes could not be linked to therapy outcomes in the present study; however, the TCRβ CDR3 sequences with the largest fold changes did include sequences with identical TRBV/TRBJ gene segment usage and high junction region homology to previously published CDR3 sequences from HCV-specific T cells targeting the HLA-B*0801-restricted 1395HSKKKCDEL1403 and HLAA* 0101-restricted 1435ATDALMTGY1443 epitopes. The pipeline developed in this proof of concept study provides a platform for the design of future experiments to accurately address the question of whether T cell responses contribute to SVR upon antiviral therapy. This pipeline represents a novel technique to analyze T cell dynamics in situations where conventional antigen-dependent methods are limited due to suppression of T cell functions and highly diverse antigenic sequences. [ABSTRACT FROM AUTHOR]

INTRODUCTION: White coat ceremonies (WCCs) in medical school mark the transition of students to medicine, beginning their professional identity formation as a physician. However, a literature/web search revealed a paucity of residency-focused WCCs. METHODS: A 90-minute Family Medicine Residency (FM) WCC was designed to support residents' professional identity formation as a specialty physician. Through faculty narratives and brief histories of the white coat and the specialty, the WCC concludes with new residents donning their specialty embroidered white coats. A brief e-survey was sent to attendees, and WCC leaders were debriefed to determine the value and key elements of WCC. RESULTS: Seventy-nine percent of survey respondents (34/43) agreed that the WCC is an important transition event for residents' identity while reaffirming FM values for faculty/staff. WCC leaders identified critical steps for initiating a WCC. CONCLUSION: A resident WCC formally marks the transition to specialty physician identity. LESSONS LEARNED: Ceremony structure will evolve over time. [ABSTRACT FROM AUTHOR]

Sm16 is a 16 KDa protein released by Schistosoma mansoni that modulates inflammatory responses in host cells. Sm 16 is expressed by several life cycle stages of S. mansoni , including the egg stage. Schistosome eggs are known to provoke chronic schistosomiasis pathology, which involves the development of liver fibrosis. Hepatic stellate cells (HSCs), which are responsible for this fibrosis, are susceptible to immunomodulation by S. mansoni whole egg secretions. To define the effects of Sm 16 exposure on HSCs, two synthetic peptide derivatives of Sm 16, coined "KS-84″ and "KS-66″, were tested against LX-2 cells, an immortalised human HSC line, and RNA sequencing was used to assess the transcriptional changes induced by each peptide. In total, 78 and 798 genes were found to be significantly differentially expressed by KS-84 and KS-66 treatment, respectively. In silico pathway analysis of these genes revealed that KS-84 reduced LX-2 cell activation and fibrotic potential, whereas KS-66 increased both processes. Reduced transforming growth factor-β1 (TGF-β1) signalling was identified as a potential mechanism of KS-84-induced inhibition of LX-2 activation. Taken together, these findings indicate a potential role for Sm 16 in combatting fibrotic liver disease. [Display omitted] • Peptide derivatives of the S. mansoni protein Sm 16 alter the responses of HSCs. • KS-84 was predicted to inhibit HSC activation and fibrotic potential. • Reduced TGF-β signaling was identified as a possible mechanism for this inhibition. • Sm 16-derived peptides may have a role in combatting fibrotic liver disease. [ABSTRACT FROM AUTHOR]

Infections with helminth parasites prevent/attenuate auto-inflammatory disease. Here we show that molecules secreted by a helminth parasite could prevent Type 1 Diabetes (T1D) in nonobese diabetic (NOD) mice. When delivered at 4 weeks of age (coincident with the initiation of autoimmunity), the excretory/secretory products of Fasciola hepatica (FhES) prevented the onset of T1D, with 84% of mice remaining normoglycaemic and insulitis-free at 30 weeks of age. Disease protection was associated with suppression of IFN-γ secretion from autoreactive T cells and a switch to the production of a regulatory isotype (from IgG2a to IgG1) of autoantibody. Following FhES injection, peritoneal macrophages converted to a regulatory M2 phenotype, characterised by increased expression levels of Ym1, Arg-1, TGFβ and PD-L1. Expression of these M2 genetic markers increased in the pancreatic lymph nodes and the pancreas of FhES-treated mice. In vitro, FhES-stimulated M2 macrophages induced the differentiation of Tregs from splenocytes isolated from naïve NOD mice. Collectively, our data shows that FhES contains immune-modulatory molecules that mediate protection from autoimmune diabetes via the induction and maintenance of a regulatory immune environment. [ABSTRACT FROM AUTHOR]

Host defence peptides (HDPs) are expressed throughout the animal and plant kingdoms. They have multifunctional roles in the defence against infectious agents of mammals, possessing both bactericidal and immune-modulatory activities. We have identified a novel family of molecules secreted by helminth parasites (helminth defence molecules; HDMs) that exhibit similar structural and biochemical characteristics to the HDPs. Here, we have analyzed the functional activities of four HDMs derived from Schistosoma mansoni and Fasciola hepatica and compared them to human, mouse, bovine and sheep HDPs. Unlike the mammalian HDPs the helminth-derived HDMs show no antimicrobial activity and are non-cytotoxic to mammalian cells (macrophages and red blood cells). However, both the mammalian- and helminth-derived peptides suppress the activation of macrophages by microbial stimuli and alter the response of B cells to cytokine stimulation. Therefore, we hypothesise that HDMs represent a novel family of HDPs that evolved to regulate the immune responses of their mammalian hosts by retaining potent immune modulatory properties without causing deleterious cytotoxic effects. Author Summary: In mammals, secreted host defence peptides (HDPs) protect against a wide range of infectious pathogens. They also perform a range of immune modulatory functions which regulate the immune response to pathogens, ensuring that the protective inflammatory response is not exacerbated and that post-infection repair mechanisms are initiated. We identified a novel family of molecules secreted by medically-important helminth pathogens (termed helminth defence molecules; HDMs) that exhibit striking structural and biochemical similarities to the HDPs. To further investigate the extent of this similarity, we have performed a comparative functional study between several well characterized, anti-microbial, mammalian HDPs and a series of parasite-derived peptides. The parasite HDMs displayed immune modulatory properties that were similar to their HDP homologs in mammals, but possessed no antimicrobial or cytotoxic activity. We propose that HDMs of these helminth pathogens underwent specific adaptation, losing their anti-microbial activity but retaining their ability to regulate the immune responses of their mammalian hosts. This absence of cytotoxicity and retention of immune-modulatory activity offers an opportunity to design novel immunotherapeutics derived from the HDMs which could be used to combat destructive inflammatory responses associated with microbial infection and immune-related disorders. [ABSTRACT FROM AUTHOR]

We previously reported the identification of a novel family of immunomodulatory proteins, termed helminth defense molecules (HDMs), that are secreted by medically important trematode parasites. Since HDMs share biochemical, structural, and functional characteristics with mammalian cathelicidin-like host defense peptides (HDPs), we proposed that HDMs modulate the immune response v/a molecular mimicry of host molecules. In the present study, we report the mechanism by which HDMs influence the function of macrophages. We show that the HDM secreted by Fasciola hepatica (FhHDM-1) binds to macrophage plasma membrane lipid rafts via selective interaction with phospholipids and/or cholesterol before being internalized by endocytosis. Following inter nalization, FhHDM-1 is rapidly processed by lysosomal cathepsin L to release a short C-terminal peptide (containing a conserved amphipathic helix that is a key to HDM function), which then prevents the acidification of the endolysosomal compartments by inhibiting vacuolar ATPase activity. The resulting endolysosomal alkalization impedes macrophage antigen processing and prevents the transport of peptides to the cell surface in conjunction with MHC class II for presentation to CD4+ T cells. Thus, we have elucidated a novel mechanism by which helminth pathogens alter innate immune cell function to assist their survival in the host. [ABSTRACT FROM AUTHOR]

Host defense peptides (HDPs) are an evolutionarily conserved component of the innate immune response found in all living species. They possess antimicrobial activities against a broad range of organisms including bacteria, fungi, eukaryotic parasites, and viruses. HDPs also have the ability to enhance immune responses by acting as immunomodulators. We discovered a new family of HDPs derived from pathogenic helminth (worms) that cause enormous disease in animals and humans worldwide. The discovery of these peptides was based on their similar biochemical and functional characteristics to the human defense peptide LL-37. We propose that these new peptides modulate the immune response via molecular mimicry of mammalian HDPs thus providing a mechanism behind the anti-inflammatory properties of helminth infections. [ABSTRACT FROM AUTHOR]

A review of the article "Role of female sex hormones, estradiol and progesterone, in mast cell behavior," by Oliver Zierau, Ana C. Zenclussen, and Federico Jensen, which appeared in the periodical "Frontiers in Immunology" on June 19, 2012, is presented.

Malaria caused by several species of Plasmodium is major parasitic disease of humans, causing 1-3 million deaths worldwide annually. The widespread resistance of the human parasite to current drug therapies is of major concern making the identification of new drug targets urgent. While the parasite grows and multiplies inside the host erythrocyte it degrades the host cell hemoglobin and utilizes the released amino acids to synthesize its own proteins. The P. falciparum malarial M1 alanyl-aminopeptidase (PfA-M1) is an enzyme involved in the terminal stages of hemoglobin digestion and the generation of an amino acid pool within the parasite. The enzyme has been validated as a potential drug target since inhibitors of the enzyme block parasite growth in vitro and in vivo. In order to gain further understanding of this enzyme, molecular dynamics simulations using data from a recent crystal structure of PfA-M1 were performed. The results elucidate the pentahedral coordination of the catalytic Zn in these metallo-proteases and provide new insights into the roles of this cation and important active site residues in ligand binding and in the hydrolysis of the peptide bond. Based on the data, we propose a two-step catalytic mechanism, in which the conformation of the active site is altered between the Michaelis complex and the transition state. In addition, the simulations identify global changes in the protein in which conformational transitions in the catalytic domain are transmitted at the opening of the N-terminal 8 Å-long channel and at the opening of the 30 Å-long C-terminal internal chamber that facilitates entry of peptides to the active site and exit of released amino acids. The possible implications of these global changes with regard to enzyme function are discussed. [ABSTRACT FROM AUTHOR]

Over the last decade a significant number of studies have highlighted the central role of host antimicrobial (or defence) peptides in modulating the response of innate immune cells to pathogen-associated ligands. In humans, the most widely studied antimicrobial peptide is LL-37, a 37-residue peptide containing an amphipathic helix that is released via proteolytic cleavage of the precursor protein CAP18. Owing to its ability to protect against lethal endotoxaemia and clinically-relevant bacterial infections, LL-37 and its derivatives are seen as attractive candidates for anti-sepsis therapies. We have identified a novel family of molecules secreted by parasitic helminths (helminth defence molecules; HDMs) that exhibit similar biochemical and functional characteristics to human defence peptides, particularly CAP18. The HDM secreted by Fasciola hepatica (FhHDM-1) adopts a predominantly α-helical structure in solution. Processing of FhHDM-1 by F. hepatica cathepsin L1 releases a 34-residue C-terminal fragment containing a conserved amphipathic helix. This is analogous to the proteolytic processing of CAP18 to release LL-37, which modulates innate cell activation by classical toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS). We show that full-length recombinant FhHDM-1 and a peptide analogue of the amphipathic C- terminus bind directly to LPS in a concentration-dependent manner, reducing its interaction with both LPS-binding protein (LBP) and the surface of macrophages. Furthermore, FhHDM-1 and the amphipathic C-terminal peptide protect mice against LPS-induced inflammation by significantly reducing the release of inflammatory mediators from macrophages. We propose that HDMs, by mimicking the function of host defence peptides, represent a novel family of innate cell modulators with therapeutic potential in anti-sepsis treatments and prevention of inflammation. [ABSTRACT FROM AUTHOR]

Background: The temporal expression and secretion of distinct members of a family of virulence-associated cathepsin L cysteine peptidases (FhCL) correlates with the entry and migration of the helminth pathogen Fasciola hepatica in the host. Thus, infective larvae traversing the gut wall secrete cathepsin L3 (FhCL3), liver migrating juvenile parasites secrete both FhCL1 and FhCL2 while the mature bile duct parasites, which are obligate blood feeders, secrete predominantly FhCL1 but also FhCL2. Methodology/Principal Findings: Here we show that FhCL1, FhCL2 and FhCL3 exhibit differences in their kinetic parameters towards a range of peptide substrates. Uniquely, FhCL2 and FhCL3 readily cleave substrates with Pro in the P2 position and peptide substrates mimicking the repeating Gly-Pro-Xaa motifs that occur within the primary sequence of collagen. FhCL1, FhCL2 and FhCL3 hydrolysed native type I and II collagen at neutral pH but while FhCL1 cleaved only non-collagenous (NC, non-Gly-X-Y) domains FhCL2 and FhCL3 exhibited collagenase activity by cleaving at multiple sites within the α1 and α2 triple helix regions (Col domains). Molecular simulations created for FhCL1, FhCL2 and FhCL3 complexed to various seven-residue peptides supports the idea that Trp67 and Tyr67 in the S2 subsite of the active sites of FhCL3 and FhCL2, respectively, are critical to conferring the unique collagenase-like activity to these enzymes by accommodating either Gly or Pro residues at P2 in the substrate. The data also suggests that FhCL3 accommodates hydroxyproline (Hyp)-Gly at P3-P2 better than FhCL2 explaining the observed greater ability of FhCL3 to digest type I and II collagens compared to FhCL2 and why these enzymes cleave at different positions within the Col domains. Conclusions/Significance: These studies further our understanding of how this helminth parasite regulates peptidase expression to ensure infection, migration and establishment in host tissues. Author Summary: Fasciola hepatica is a helminth parasite that causes liver fluke disease (fasciolosis) in domestic animals (sheep and cattle) and humans worldwide. In order to infect their mammalian hosts, F. hepatica larvae must penetrate and traverse the intestinal wall of the duodenum, move through the peritoneum and penetrate the liver. After migrating through the liver, causing extensive tissue damage, the parasites move to their final niche in the bile ducts where they mature and feed on host haemoglobin to support the production of eggs. To achieve these tasks, F. hepatica secretes a number of distinct cathepsin L cysteine peptidases (FhCL). Thus, the infective larvae that penetrate the host gut secrete cathepsin L3 (FhCL3), the migrating liver-stage juvenile parasites secrete both FhCL1 and FhCL2 while mature bile duct parasites that feed on host blood secrete predominantly FhCL1 but also FhCL2. Here we show that the major cathepsin L peptidases secreted by F. hepatica (FhCL1, FhCL2 and FhCL3) display differential ability to degrade host collagen (an important component of host tissues) and investigate this phenomenon at the molecular level. [ABSTRACT FROM AUTHOR]

Opisthorchis viverrini is an important helminth pathogen of humans that is endemic in Thailand and Laos. Adult flukes reside within host bile ducts and feed on epithelial tissue and blood cells. Chronic opisthorchiasis is associated with severe hepatobiliary diseases such as cholangiocarcinoma. Here we report that adult O. viverrini secrete two major cysteine proteases: cathepsin F ( Ov-CF-1) and cathepsin B1 ( Ov-CB-1). Ov-CF-1 is secreted as an inactive zymogen that autocatalytically processes and activates to a mature enzyme at pH 4.5 via an intermolecular cleavage at the prosegment–mature domain junction. Ov-CB-1 is also secreted as a zymogen but, in contrast to Ov-CF-1, is fully active against peptide and macromolecular substrates despite retaining the N-terminal prosegment. The active Ov-CB-1 zymogen was capable of trans-activating Ov-CF-1 by proteolytic removal of its prosegment at pH 5.5, a pH at which the Ov-CF-1 zymogen cannot autocatalytically activate. Both cathepsins hydrolyse human haemoglobin but their combined action more efficiently degrades haemoglobin to smaller peptides than each enzyme alone. Ov-CF-1 degraded extracellular matrix proteins more effectively than Ov-CB-1 at physiological pH. We propose that Ov-CB-1 regulates Ov-CF-1 activity and that both enzymes work together to degrade host tissue contributing to the development of liver fluke-associated cholangiocarcinoma. [ABSTRACT FROM AUTHOR]