Single-cell multiomics reveals the complexity of TGFß signalling to chromatin in iPSC-derived kidney organoids.

TGFß1 plays a regulatory role in the determination of renal cell fate and the progression of renal fibrosis. Here we show an association between SMAD3 and the histone methyltransferase, EZH2, during cell differentiation; ChIP-seq revealed that SMAD3 and EZH2 co-occupy the genome in iPSCs and in iPSC-derived nephron progenitors. Through integration of single cell gene expression and epigenome profiling, we identified de novo ACTA2+ve/POSTN+ve myofibroblasts in kidney organoids treated with TGFß1, characterised by increased SMAD3-dependent cis chromatin accessibility and gene expression associated with fibroblast activation. We have identified fibrosis-associated regulons characterised by enrichment of SMAD3, AP1, the ETS family of transcription factors, and NUAK1, CREB3L1, and RARG, corresponding to enriched motifs at accessible loci identified by scATACseq. Treatment with the EZH2 specific inhibitor GSK343, blocked SMAD3-dependent cis co-accessibility and inhibited myofibroblast activation. This mechanism, through which TGFß signals directly to chromatin, represents a critical determinant of fibrotic, differentiated states.

Long-term outcomes of the arthroscopic Bankart repair: a systematic review of studies at 10-year follow-up.

BACKGROUND: The purpose of this study was to systematically review the evidence in the literature to ascertain the functional outcomes and recurrences rates, as well as subsequent revision rates, following arthroscopic Bankart repair at a minimum of 10 years' follow-up. METHODS: Two independent reviewers performed a literature search based on Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines, using the Embase, MEDLINE, and Cochrane Library databases. Studies were included if they were clinical studies on arthroscopic Bankart repair with a minimum of 10 years' follow-up. Statistical analysis was performed using SPSS software. RESULTS: Our review found 9 studies including 822 shoulders meeting our inclusion criteria. The majority of patients were male patients (75.5%), the average age was 28.0 years (range, 15-73 years), and the mean follow-up period was 149.4 months. The most commonly used functional outcome score was the Rowe score, with a weighted mean of 87.0. Overall, 77.6% of athletes were able to return to sports postoperatively. The overall rate of recurrent instability was 31.2%, with 16.0% of patients having recurrent dislocations, and the overall revision rate was 17.0%. Evidence of instability arthropathy was found in 59.4% of patients, with 10.5% of patients having moderate to severe arthropathy. DISCUSSION AND CONCLUSION: Arthroscopic Bankart repair for anterior shoulder instability has been shown to result in excellent long-term functional outcomes despite a relatively high rate of recurrent instability necessitating revision surgery. In addition, the high rate of instability arthropathy is a concern following arthroscopic Bankart repair in the long term.

Defens-IN! Human α-Defensin 5 Acts as an Unwitting Double Agent to Promote Shigella Infection.

Shigella pathogenesis has confounded researchers for years because of its narrow host selectivity and extraordinary infectious capability. In this issue of Immunity, Xu et al. (2018) identify a cunning mechanism whereby Shigella hijacks human α-defensin 5 to enhance its adhesion and subsequent invasion.

Transcriptional profiling of PBMCs unravels B cell mediated immunopathogenic imprints of HCV vasculitis.

B cell depletion therapy using rituximab has been shown to be effective in achieving remission in patients with HCV-mixed cryoglobulinemic (MC) vasculitis. Previously, we have demonstrated abnormalities in peripheral immune cells involving neutrophils, chemotaxis, and innate immune activation among patients with HCV-MC vasculitis when compared to HCV patients without vasculitis. In this study, we evaluated the effect of B cell depletion therapy on transcriptional profiles of peripheral blood mononuclear cells before and after riruximab therapy, in order to unravel the pathogenic mechanism involved in HCV-MC vasculitis induced by abnormal B cell proliferation. DNA microarray analysis was performed using RNA from PBMCs from seven patients with HCV-MC vasculitis and seven normal volunteers. DNA was hybridized to Affymetrix U133A chips. After normalization, differentially expressed gene list with treatment was generated using partitional clustering. RT-PCR, flow cytometry, and enzyme immunoassay (EIA) was used to validate DNA microarray findings. Differentially expressed genes included B cells and non-B cell genes. Validation of genes using purified cell subsets demonstrated distinct effect of B cell depletion therapy on non-B cells, such as monocytes, T cells, and NK cells. Notably, B lymphocyte stimulator (BLyS) levels were persistently elevated in patients who subsequently relapsed. In conclusion, pathogenesis of HCV-MC vasculitis is mediated by abnormal proliferation of B cells, driven by BLyS, leading to significant effects on non-B cells in mediating symptomatology. Future therapeutics using a combination approach of B cell depletion and proliferation may be desired to achieve long-term remission.

Epithelial Cell Inflammasomes in Intestinal Immunity and Inflammation.

Pattern recognition receptors (PRR), such as NOD-like receptors (NLRs), sense conserved microbial signatures, and host danger signals leading to the coordination of appropriate immune responses. Upon activation, a subset of NLR initiate the assembly of a multimeric protein complex known as the inflammasome, which processes pro-inflammatory cytokines and mediates a specialized form of cell death known as pyroptosis. The identification of inflammasome-associated genes as inflammatory bowel disease susceptibility genes implicates a role for the inflammasome in intestinal inflammation. Despite the fact that the functional importance of inflammasomes within immune cells has been well established, the contribution of inflammasome expression in non-hematopoietic cells remains comparatively understudied. Given that intestinal epithelial cells (IEC) act as a barrier between the host and the intestinal microbiota, inflammasome expression by these cells is likely important for intestinal immune homeostasis. Accumulating evidence suggests that the inflammasome plays a key role in shaping epithelial responses at the host-lumen interface with many inflammasome components highly expressed by IEC. Recent studies have exposed functional roles of IEC inflammasomes in mucosal immune defense, inflammation, and tumorigenesis. In this review, we present the main features of the predominant inflammasomes and their effector mechanisms contributing to intestinal homeostasis and inflammation. We also discuss existing controversies in the field and open questions related to their implications in disease. A comprehensive understanding of the molecular basis of intestinal inflammasome signaling could hold therapeutic potential for clinical translation.

Identifying patient-related information problems: A study of information use by patient-care teams during morning rounds.

OBJECTIVE: This study identifies the types of patient-related information problems (PIPs) that patient-care teams encounter during morning rounds, and how those PIPs are identified and managed. PIPs are any issues related to patient information (e.g., wrong, missing, incomplete information) that affect the patient-care team's ability to perform their work. Not addressing PIPs can lead to workflow challenges, delayed patient-care decisions, and negative impacts to the patient. MATERIALS AND METHODS: We employed qualitative data collection methods by shadowing patient-care teams during 29 morning rounds resulting in 155h of observation. We observed the interactions between the rounding physicians and other patient-care team members, including: nurses, consulting physicians, care coordinators, pharmacists, social workers, and therapists. RESULTS: This study resulted in identifying seven types of PIPs that occur during morning rounds. Additionally, the study presents the different ways that participants identified and managed the PIPs. DISCUSSION: We discuss the potential negative effects of PIPs on the patient-care workflow. We also discuss socio-technical recommendations for organizational policies and training, as well as electronic health record (EHR) design improvements that could help patient-care teams more effectively identify and manage PIPs. CONCLUSION: Hospital teams rely on accurate, available, and up-to-date information in order to make informed decisions on patient care. However, PIPs exist in EHR systems, paper documents, and verbal conversations. This study identifies a set of PIPs and how they are currently being identified and managed.

Mitral valve prolapse is associated with altered extracellular matrix gene expression patterns.

Mitral valve prolapse (MVP) is the leading indication for isolated mitral valve surgery in the United States. Disorganization of collagens and glycosaminoglycans in the valvular extracellular matrix (ECM) are histological hallmarks of MVP. We performed a transcriptome analysis to study the alterations in ECM-related gene expression in humans with sporadic MVP. Mitral valve specimens were obtained from individuals undergoing valve repair for MVP (n=7 patients) and from non-beating heart-tissue donors (n=3 controls). Purified RNA was subjected to whole-transcriptome microarray analysis. Microarray results were validated by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Gene ontology enrichment analysis was performed. 2046 unique genes showed significant differential expression (false discovery rate <0.5%). After demonstrating appropriate sample clustering, microarray results were globally validated using a subset of 22 differentially expressed genes by RT-qPCR (Pearson's correlation r=0.65, p=0.001). Gene ontology enrichment analyses performed with ErmineJ and DAVID Bioinformatics Database demonstrated overrepresentation of ECM components (p<0.05). Functional annotation clustering calculated enrichment of ECM-related ontology groups (enrichment score=4.1). ECM-related gene expression is significantly altered in MVP. Our study is consistent with the histologically observed alterations in collagen and mucopolysaccharide profiles of myxomatous mitral valves. Furthermore, whole-transcriptome analyses suggest dysregulation of multiple pathways, including TGF-beta signaling.

Memory Th1 Cells Are Protective in Invasive Staphylococcus aureus Infection.

Mechanisms of protective immunity to Staphylococcus aureus infection in humans remain elusive. While the importance of cellular immunity has been shown in mice, T cell responses in humans have not been characterised. Using a murine model of recurrent S. aureus peritonitis, we demonstrated that prior exposure to S. aureus enhanced IFNγ responses upon subsequent infection, while adoptive transfer of S. aureus antigen-specific Th1 cells was protective in naïve mice. Translating these findings, we found that S. aureus antigen-specific Th1 cells were also significantly expanded during human S. aureus bloodstream infection (BSI). These Th1 cells were CD45RO+, indicative of a memory phenotype. Thus, exposure to S. aureus induces memory Th1 cells in mice and humans, identifying Th1 cells as potential S. aureus vaccine targets. Consequently, we developed a model vaccine comprising staphylococcal clumping factor A, which we demonstrate to be an effective human T cell antigen, combined with the Th1-driving adjuvant CpG. This novel Th1-inducing vaccine conferred significant protection during S. aureus infection in mice. This study notably advances our understanding of S. aureus cellular immunity, and demonstrates for the first time that a correlate of S. aureus protective immunity identified in mice may be relevant in humans.

Genome sequencing of the extinct Eurasian wild aurochs, Bos primigenius, illuminates the phylogeography and evolution of cattle.

BACKGROUND: Domestication of the now-extinct wild aurochs, Bos primigenius, gave rise to the two major domestic extant cattle taxa, B. taurus and B. indicus. While previous genetic studies have shed some light on the evolutionary relationships between European aurochs and modern cattle, important questions remain unanswered, including the phylogenetic status of aurochs, whether gene flow from aurochs into early domestic populations occurred, and which genomic regions were subject to selection processes during and after domestication. Here, we address these questions using whole-genome sequencing data generated from an approximately 6,750-year-old British aurochs bone and genome sequence data from 81 additional cattle plus genome-wide single nucleotide polymorphism data from a diverse panel of 1,225 modern animals. RESULTS: Phylogenomic analyses place the aurochs as a distinct outgroup to the domestic B. taurus lineage, supporting the predominant Near Eastern origin of European cattle. Conversely, traditional British and Irish breeds share more genetic variants with this aurochs specimen than other European populations, supporting localized gene flow from aurochs into the ancestors of modern British and Irish cattle, perhaps through purposeful restocking by early herders in Britain. Finally, the functions of genes showing evidence for positive selection in B. taurus are enriched for neurobiology, growth, metabolism and immunobiology, suggesting that these biological processes have been important in the domestication of cattle. CONCLUSIONS: This work provides important new information regarding the origins and functional evolution of modern cattle, revealing that the interface between early European domestic populations and wild aurochs was significantly more complex than previously thought.

Designing for privacy management in hospitals: Understanding the gap between user activities and IT staff's understandings.

PURPOSE: We examined the role of privacy in collaborative clinical work and how it is understood by hospital IT staff. The purpose of our study was to identify the gaps between hospital IT staff members' perceptions of how electronic health record (EHR) users' protect the privacy of patient information and how users actually protect patients' private information in their daily collaborative activities. Since the IT staff play an important role in implementing and maintaining the EHR, any gaps that exist between the IT staff's perceptions of user work practices and the users' actual work practices can result in a number of problems in the configuration, implementation, or customization of the EHR, which can lead to collaboration challenges, interrupted workflow, and privacy breaches. METHODS: We used qualitative data collection methods for this study. We conducted semi-structured interviews with 20 hospital IT staff members. We also conducted observations of EHR users in the in-patient units of the same hospital. RESULTS: We identified gaps in IT staff's understandings of users' work activities, especially in regards to privacy-compromising workarounds that are used by users and why they are used. DISCUSSION: We discuss the reasons why this gap may exist between IT staff and users and ways to improve IT staff's understanding of why users perform certain privacy-compromising workarounds. CONCLUSION: A hospital's IT staff face a daunting task in ensuring users' collaborative work practices are supported by the system while providing effective privacy mechanisms. In order to achieve both goals, the IT staff must have a clear understanding of their users' practices. However, as this study highlights, there may be a mismatch between the IT staff's understandings of how users protect patient privacy and how users actually protect privacy.

Manipulation of Autophagy in Phagocytes Facilitates Staphylococcus aureus Bloodstream Infection.

The capacity for intracellular survival within phagocytes is likely a critical factor facilitating the dissemination of Staphylococcus aureus in the host. To date, the majority of work on S. aureus-phagocyte interactions has focused on neutrophils and, to a lesser extent, macrophages, yet we understand little about the role played by dendritic cells (DCs) in the direct killing of this bacterium. Using bone marrow-derived DCs (BMDCs), we demonstrate for the first time that DCs can effectively kill S. aureus but that certain strains of S. aureus have the capacity to evade DC (and macrophage) killing by manipulation of autophagic pathways. Strains with high levels of Agr activity were capable of causing autophagosome accumulation, were not killed by BMDCs, and subsequently escaped from the phagocyte, exerting significant cytotoxic effects. Conversely, strains that exhibited low levels of Agr activity failed to accumulate autophagosomes and were killed by BMDCs. Inhibition of the autophagic pathway by treatment with 3-methyladenine restored the bactericidal effects of BMDCs. Using an in vivo model of systemic infection, we demonstrated that the ability of S. aureus strains to evade phagocytic cell killing and to survive temporarily within phagocytes correlated with persistence in the periphery and that this effect is critically Agr dependent. Taken together, our data suggest that strains of S. aureus exhibiting high levels of Agr activity are capable of blocking autophagic flux, leading to the accumulation of autophagosomes. Within these autophagosomes, the bacteria are protected from phagocytic killing, thus providing an intracellular survival niche within professional phagocytes, which ultimately facilitates dissemination.

Hepatitis C-associated mixed cryoglobulinemic vasculitis induces differential gene expression in peripheral mononuclear cells.

This study examines the distinct gene expression profile of peripheral blood mononuclear cells from patients with chronic hepatitis C infection and mixed cryoglobulinemic (MC) vasculitis. Our DNA microarray analysis indicates that hepatitis C virus (HCV)-associated MC vasculitis is characterized by compromised neutrophil function, impaired chemotaxis, and increased interferon-stimulated gene (ISG) expression, contributing to overall MC pathogenesis and end-organ damage. Increased ISG expression is suggestive of an enhanced endogenous interferon gene signature. PBMC depletion assays demonstrate that this increased expression is likely due to an activation of monocytes and not a direct result of B cell expansion. Notably, this monocyte activation of ISG expression in HCV-associated MC vasculitis suggests a poor predictor status of interferon-based treatment. Further analysis of PBMC gene expression profiles before and after in vivo B cell depletion therapy is critical to completely understanding the mechanisms of MC vasculitis pathogenesis.

Staphylococcus aureus infection of mice expands a population of memory γδ T cells that are protective against subsequent infection.

The development of vaccines against Staphylococcus aureus has consistently failed in clinical trials, likely due to inefficient induction of cellular immunity. T cell-derived IL-17 is one of the few known correlates of antistaphylococcoal immunity, conferring protection against S. aureus infections through its ability to promote phagocytic cell effector functions. A comprehensive understanding of the discrete T cell subsets critical for site-specific IL-17-mediated bacterial clearance will therefore be necessary to inform the development of vaccines that efficiently target cellular immunity. In this study, we have identified a population of CD44+ CD27- memory γδ T cells, expanded upon infection of C57BL/6 mice with S. aureus, which produce high levels of IL-17 and mediate enhanced bacterial clearance upon reinfection with the bacterium. These cells are comprised largely of the Vγ4+ subset and accumulate at the site of infection subsequent to an initial Vγ1.1+ and Vγ2+ T cell response. Moreover, these Vγ4+ T cells are retained in the peritoneum and draining mediastinal lymph nodes for a prolonged period following bacterial clearance. In contrast to its critical requirement for γδ T cell activation during the primary infection, IL-1 signaling was dispensable for activation and expansion of memory γδ T cells upon re-exposure to S. aureus. Our findings demonstrate that a γδ T cell memory response can be induced upon exposure to S. aureus, in a fashion analogous to that associated with classical αß T cells, and suggest that induction of IL-17-expressing γδ T cells may be an important property of a protective vaccine against S. aureus.

Targeted nasal vaccination provides antibody-independent protection against Staphylococcus aureus.

Despite showing promise in preclinical models, anti-Staphylococcus aureus vaccines have failed in clinical trials. To date, approaches have focused on neutralizing/opsonizing antibodies; however, vaccines exclusively inducing cellular immunity have not been studied to formally test whether a cellular-only response can protect against infection. We demonstrate that nasal vaccination with targeted nanoparticles loaded with Staphylococcus aureus antigen protects against acute systemic S. aureus infection in the absence of any antigen-specific antibodies. These findings can help inform future developments in staphylococcal vaccine development and studies into the requirements for protective immunity against S. aureus.

Identification and management of information problems by emergency department staff.

Patient-care teams frequently encounter information problems during their daily activities. These information problems include wrong, outdated, conflicting, incomplete, or missing information. Information problems can negatively impact the patient-care workflow, lead to misunderstandings about patient information, and potentially lead to medical errors. Existing research focuses on understanding the cause of these information problems and the impact that they can have on the hospital's workflow. However, there is limited research on how patient-care teams currently identify and manage information problems that they encounter during their work. Through qualitative observations and interviews in an emergency department (ED), we identified the types of information problems encountered by ED staff, and examined how they identified and managed the information problems. We also discuss the impact that these information problems can have on the patient-care teams, including the cascading effects of information problems on workflow and the ambiguous accountability for fixing information problems within collaborative teams.

Impaired HCV clearance in HIV/HCV coinfected subjects treated with PegIFN and RBV due to interference of IFN signaling by IFNαR2a.

Enhanced endogenous interferon (IFN) stimulated gene (ISG) signature has been associated with nonresponsiveness to hepatitis C treatment using pegylated-IFNα (pegIFNα) and ribavirin (RBV) in human immunodeficiency virus/hepatitis C virus (HIV/HCV) coinfected patients. Using a proteomic approach, we identified high levels of IFNα receptor 2a (IFNαR2a) in the serum of null responders to pegIFNα/RBV. IFNαR2a inhibited antiviral activity of all formulations of IFNα in JFH/Huh7.5 cells. Furthermore, serum from null responders, but not from those who achieved sustained virologic response, suppressed IFN-signaling and ISG expression in IFNα-stimulated PBMCs of healthy donors in an IFNαR2a specific fashion. An IFNαR2a transgenic mice model (C57BL/6) was generated that had significantly higher levels of IFNαR2a in the serum than the controls (P=0.001). Total ISG expression in the lymph nodes was significantly higher compared to wild-type mice (P value=0.0016). In addition, IFITM1 and SP110 had significantly increased expression in the liver, IFITM1 and ISG15 in the lymph node, and ISG15 and PLSCR1 in the spleen (P value<0.05). The underlying mechanism of resistance to hepatitis C treatment may involve transsignaling of the JAK/STAT pathway by the sIFNαR2a-IFNα/ß complex and result in the enhanced ISG signature observed in null responders. In this regard, the transgenic mice model simulated nonresponders to IFNα therapy and provides valuable insights into the role of sIFNαR2a-IFNα interactions in vivo.

Nlrp-3-driven interleukin 17 production by γδT cells controls infection outcomes during Staphylococcus aureus surgical site infection.

Recent work has identified T cells and the cytokines they produce as important correlates of immune protection during Staphylococcus aureus infections through the ability of these T cells to regulate local neutrophil responses. However, the specific T-cell subsets that are involved in coordinating protection at distinct sites of infection remains to be established. In this study, we identify for the first time an important role for γδT cells in controlling S. aureus surgical site infection (SSI). γδT cells are recruited to the wound site following S. aureus challenge, where they represent the primary source of interleukin 17 (IL-17), with a small contribution from other non-γδT cells. The IL-17 response is entirely dependent upon IL-1 receptor signaling. Using IL-17 receptor-deficient mice, we demonstrate that IL-17 is required to control bacterial clearance during S. aureus SSI. However, we demonstrate a strain-dependent requirement for γδT cells in this process due to the differential abilities of individual strains to activate IL-1ß production. IL-1ß processing relies upon activation of the Nlrp3 inflammasome complex, and we demonstrate that Nlrp3-deficient and IL-1 receptor-deficient mice have an impaired ability to control S. aureus SSI due to reduced production of IL-17 by γδT cells at the site of infection. Given that IL-17 has been identified as an important correlate of immune protection during S. aureus infection, it is vital that the unique cellular sources of this cytokine and mechanisms inducing its activation are identified at distinct sites of infection. Our study demonstrates that while IL-17 may be critically important for mediating immune protection during S. aureus SSI, the relative contribution of γδT cells to these protective effects may be strain dependent.

Outcome of pure mucocele-like lesions diagnosed on breast core biopsy.

AIM: Current guidelines recommend that mucocele-like lesions (MLL) of the breast diagnosed on needle core biopsy (NCB) should be categorized as a lesion of uncertain malignant potential (B3). However, data on the outcome of MLL diagnosed on NCB remains limited due to the rarity of this lesion. The aim of this study was to assess the outcome of pure MLL without atypia diagnosed on NCB using a large series of cases and a review of the literature to provide evidence that can guide management. METHODS AND RESULTS: Patients who underwent diagnostic excision biopsy after a core biopsy diagnosis of MLL without atypia were identified from several centres. Two of 54 patients (4%) with MLL without atypia on core biopsy had ductal carcinoma in situ in the subsequent excision specimen. This is similar to the rate in previous studies of 4% (four of 106). If there is atypia in the core biopsy, previous studies found that the frequency of malignancy is much higher at 21% (seven of 33). CONCLUSIONS: Our results provide evidence that pure MLL without atypia diagnosed on NCB is usually associated with a benign outcome.

A survey of rural hospitals' perspectives on health information technology outsourcing.

A survey of rural hospitals was conducted in the spring of 2012 to better understand their perspectives on health information technology (HIT) outsourcing and the role that hospital-to-hospital HIT partnerships (HHPs) can play as an outsourcing mechanism. The survey sought to understand how HHPs might be leveraged for HIT implementation, as well as the challenges with forming them. The results suggest that HHPs have the potential to address rural hospitals' slow rate of HIT adoption, but there are also challenges to creating these partnerships. These issues, as well as avenues for further research, are then discussed.