Search

Understanding gene function and regulation in homeostasis and disease requires knowledge of the cellular and tissue contexts in which genes are expressed. Here, we applied four single-nucleus RNA sequencing methods to eight diverse, archived, frozen tissue types from 16 donors and 25 samples, generating a cross-tissue atlas of 209,126 nuclei profiles, which we integrated across tissues, donors, and laboratory methods with a conditional variational autoencoder. Using the resulting cross-tissue atlas, we highlight shared and tissue-specific features of tissue-resident cell populations; identify cell types that might contribute to neuromuscular, metabolic, and immune components of monogenic diseases and the biological processes involved in their pathology; and determine cell types and gene modules that might underlie disease mechanisms for complex traits analyzed by genome-wide association studies.

Understanding the function of genes and their regulation in tissue homeostasis and disease requires knowing the cellular context in which genes are expressed in tissues across the body. Single cell genomics allows the generation of detailed cellular atlases in human tissues, but most efforts are focused on single tissue types. Here, we establish a framework for profiling multiple tissues across the human body at single-cell resolution using single nucleus RNA-Seq (snRNA-seq), and apply it to 8 diverse, archived, frozen tissue types (three donors per tissue). We apply four snRNA-seq methods to each of 25 samples from 16 donors, generating a cross-tissue atlas of 209,126 nuclei profiles, and benchmark them vs. scRNA-seq of comparable fresh tissues. We use a conditional variational autoencoder (cVAE) to integrate an atlas across tissues, donors, and laboratory methods. We highlight shared and tissue-specific features of tissue-resident immune cells, identifying tissue-restricted and non-restricted resident myeloid populations. These include a cross-tissue conserved dichotomy between LYVE1- and HLA class II-expressing macrophages, and the broad presence of LAM-like macrophages across healthy tissues that is also observed in disease. For rare, monogenic muscle diseases, we identify cell types that likely underlie the neuromuscular, metabolic, and immune components of these diseases, and biological processes involved in their pathology. For common complex diseases and traits analyzed by GWAS, we identify the cell types and gene modules that potentially underlie disease mechanisms. The experimental and analytical frameworks we describe will enable the generation of large-scale studies of how cellular and molecular processes vary across individuals and populations.

BACKGROUND: Rejection is the primary barrier to broader implementation of vascularized composite allografts (VCAs), including face and limb transplants. The immunologic pathways activated in face transplant rejection have not been fully characterized. METHODS: Using skin biopsies prospectively collected over 9 years from 7 face transplant patients, we studied rejection by gene expression profiling, histology, immunostaining, and T cell receptor sequencing. RESULTS: Grade 1 rejection did not differ significantly from nonrejection, suggesting that it does not represent a pathologic state. In grade 2, there was a balanced upregulation of both proinflammatory T cell activation pathways and antiinflammatory checkpoint and immunomodulatory pathways, with a net result of no tissue injury. In grade 3, IFN-γ–driven inflammation, antigen-presenting cell activation, and infiltration of the skin by proliferative T cells bearing markers of antigen-specific activation and cytotoxicity tipped the balance toward tissue injury. Rejection of VCAs and solid organ transplants had both distinct and common features. VCA rejection was uniquely associated with upregulation of immunoregulatory genes, including SOCS1; induction of lipid antigen–presenting CD1 proteins; and infiltration by T cells predicted to recognize CD1b and CD1c. CONCLUSION: Our findings suggest that the distinct features of VCA rejection reflect the unique immunobiology of skin and that enhancing cutaneous immunoregulatory networks may be a useful strategy in combatting rejection. TRIAL REGISTRATION: ClinicalTrials.gov NCT01281267. FUNDING: Assistant Secretary of Defense and Health Affairs, through Reconstructive Transplant Research (W81XWH-17-1-0278, W81XWH-16-1-0647, W81XWH-16-1-0689, W81XWH-18-1-0784, W81XWH-1-810798); American Society of Transplantation’s Transplantation and Immunology Research Network Fellowship Research Grant; Plastic Surgery Foundation Fellowship from the American Society of Plastic Surgeons; Novo Nordisk Foundation (NNF15OC0014092); Lundbeck Foundation; Aage Bangs Foundation; A.P. Moller Foundation for the Advancement of Medical Science; NIH UL1 RR025758.

Host defense peptides (HDPs) represents a class of ubiquitous and rapid responding immune molecules capable of direct inactivation of a wide range of pathogens. Recent research has shown HDPs to be promising candidates for development as a novel class of broad-spectrum chemotherapeutic agent that is effective against both pathogenic microbes and malignant neoplasm. This study aims to quantitatively explore the relationship between easy-to-interpret amino acid composition descriptors of HDPs with their respective bioactivities. Classification models were constructed using the C4.5 decision tree and random forest classifiers. Good predictive performance was achieved as deduced from the accuracy, sensitivity and specificity in excess of 90% and Matthews correlation coefficient in excess of 0.5 for all three evaluated data subsets (e.g. training, 10-fold cross-validation and external validation sets). The source code and data set used for the construction of classification models are available on GitHub at https://github.com/chaninn/pepbio/.

Psoralen plus UVA (PUVA) is an effective therapy for mycosis fungoides (MF), the skin-limited variant of cutaneous T cell lymphoma (CTCL). In low-burden patients, PUVA reduced or eradicated malignant T cells and induced clonal expansion of CD8(+) T cells associated with malignant T cell depletion. High-burden patients appeared to clinically improve but large numbers of malignant T cells persisted in skin. Clinical improvement was linked to turnover of benign T cell clones but not to malignant T cell reduction. Benign T cells were associated with the Th2-recruiting chemokine CCL18 before therapy and with the Th1-recruiting chemokines CXCL9, CXCL10, and CXCL11 after therapy, suggesting a switch from Th2 to Th1. Inflammation was correlated with OX40L and CD40L gene expression; immunostaining localized these receptors to CCL18-expressing c-Kit(+) dendritic cells that clustered together with CD40(+)OX40(+) benign and CD40(+)CD40L(+) malignant T cells, creating a proinflammatory synapse in skin. Our data suggest that visible inflammation in CTCL results from the recruitment and activation of benign T cells by c-Kit(+)OX40L(+)CD40L(+) dendritic cells and that this activation may provide tumorigenic signals. Targeting c-Kit, OX40, and CD40 signaling may be novel therapeutic avenues for the treatment of MF.

Aim: Hypertension is associated with development of cardiovascular disease and has become a significant health problem worldwide. Naturally-derived antihypertensive peptides have emerged as promising alternatives to synthetic drugs. Materials & methods: This study introduces predictor of antihypertensive activity of peptides constructed using random forest classifier as a function of various combinations of amino acid, dipeptide and pseudoamino acid composition descriptors. Results: Classification models were assessed via independent test set that demonstrated accuracy of 84.73%. Feature importance analysis revealed the preference of proline and hydrophobic amino acids at the C-terminal as well as the preference of short peptides for robust activity. Conclusion: Model presented herein serves as a useful tool for predicting and analysis of antihypertensive activity of peptides.

Rejection affects greater than 80% of face transplants, yet no diagnostic criteria for antibody-mediated rejection (AMR) following face transplantation have been established. Given that different treatment strategies are required to address AMR and T cell-mediated rejection (TCMR), there is a critical need to delineate the features that can differentiate these two alloimmune responses. Here, we report the longitudinal immunological examination of what we believe to be the first and only highly sensitized recipient of a crossmatch-positive face transplant up to 4 years following transplantation. We conducted gene expression profiling on allograft biopsies collected during suspected AMR and TCMR episodes as well as during 5 nonrejection time points. Our data suggest that there are distinctive molecular features in AMR, characterized by overexpression of endothelial-associated genes, including ICAM1, VCAM1, and SELE. Although our findings are limited to a single patient, these findings highlight the potential importance of developing and implementing molecular markers to differentiate AMR from TCMR to guide clinical management. Furthermore, our case illustrates that molecular assessment of allograft biopsies offers the potential for new insights into the mechanisms underlying rejection. Finally, our medium-term outcomes demonstrate that face transplantation in a highly sensitized patient with a positive preoperative crossmatch is feasible and manageable.

Objective The aims of this study are: firstly, to investigate if admission to specialized burn critical care units leads to better clinical outcomes; secondly, to elucidate if the multidisciplinary critical care contributes to this superior outcome. Methods A multi-centre cohort analysis of a prospectively collected national database of 1759 adult burn patients admitted to 13 critical care units in England and Wales between 2005 and 2011. Units were contacted via telephone to establish frequency and constitution of daily ward rounds. Critical care units were categorized into 3 settings: specialized burns critical care units, generalized critical care units and ‘visiting’ critical care units. Multivariate logistic regression analysis and propensity dose–response analysis were used to calculate risk adjusted mortality. Results Multivariate logistic regression analysis shows that admission to a specialized burn critical care service is independently associated with significant survival benefit compared to generalized critical care unit (adjusted OR for in-hospital death 1.81, [95% CI, 1.24, 2.66]) and ‘visiting’ critical care services (adjusted OR for in-hospital death 2.24 [95% CI, 1.49, 3.38]). Further analysis using propensity dose–response analysis demonstrates that risk-adjusted in-hospital mortality rate decreased as the dose of multidisciplinary care increased, with an adjusted odds ratio of 1 (specialized burn critical care units), 1.81 (generalized critical care units) and 2.24 (‘visiting’ critical care units). Conclusions Admission to a specialized burn critical care service is independently associated with significant survival benefit. This is, at least in part, due to care being provided by a fully integrated multidisciplinary team.

Aim: Toxicity arising from hemolytic activity of peptides hinders its further progress as drug candidates. Materials & methods: This study describes a sequence-based predictor based on a random forest classifier using amino acid composition, dipeptide composition and physicochemical descriptors (named HemoPred). Results: This approach could outperform previously reported method and typical classification methods (e.g., support vector machine and decision tree) verified by fivefold cross-validation and external validation with accuracy and Matthews correlation coefficient in excess of 95% and 0.91, respectively. Results revealed the importance of hydrophobic and Cys residues on α-helix and β-sheet, respectively, on the hemolytic activity. Conclusion: A sequence-based predictor which is publicly available as the web service of HemoPred, is proposed to predict and analyze the hemolytic activity of peptides.

Summary Background Facial self-inflicted gunshot wounds (SIGSWs) cause a devastating midfacial defect and pose a challenging problem to the reconstructive surgeon. Face transplantation (FT) has the potential for near-normal restoration in otherwise non-reconstructible defects. Two out of 7 FT recipients at Brigham and Women's Hospital (BWH) sustained SIGSWs. In this study, we illustrate the role of FT in the management of SIGSWs through an aesthetic, functional, and psychosocial examination of outcomes. Methods We performed a retrospective analysis of individuals with SIGSWs who were screened at BWH between 2008 and 2015. We then collected data of the injuries, modes of conventional reconstruction (CR), and deficits. For the FT recipients, we critically reviewed the psychosocial screening process and post-transplantation aesthetic, functional, and psychosocial outcomes. Results A total of six individuals post-SIGSWs were screened for FT. All of them had undergone CR, with five receiving loco-regional flaps and free tissue transfers, and one undergoing serial debridement and primary soft-tissue repair. Following CR, all suffered from residual functional and aesthetic deficits. Two underwent partial FT and one is currently undergoing FT screening. We describe the pre-transplant psychosocial screening process and the aesthetic, functional, and psychosocial outcomes of the SIGSW FT recipients. Conclusions We examined the facial SIGSW injury, outcomes of CR, and the mechanism of FT to offer a potential solution to the shortcomings of CR. More importantly, we highlight the critical nature of the psychosocial component of the multidisciplinary evaluation given the history of mental illness and suicidal behavior in this subset of patients.

Fcγ receptors (FcγR) provide important immunoregulation. Targeting inhibitory FcγRIIb may therefore prolong allograft survival, but its role in transplantation has not been addressed. FcγRIIb signaling was examined in murine models of acute or chronic cardiac allograft rejection by transplanting recipients that either lacked FcγRIIb expression (FcγRIIb−/−) or overexpressed FcγRIIb on B cells (B cell transgenic [BTG]). Acute heart allograft rejection occurred at the same tempo in FcγRIIb−/− C57BL/6 (B6) recipients as wild type recipients, with similar IgG alloantibody responses. In contrast, chronic rejection of MHC class II–mismatched bm12 cardiac allografts was accelerated in FcγRIIb−/− mice, with development of more severe transplant arteriopathy and markedly augmented effector autoantibody production. Autoantibody production was inhibited and rejection was delayed in BTG recipients. Similarly, whereas MHC class I–mismatched B6.Kd hearts survived indefinitely and remained disease free in B6 mice, much stronger alloantibody responses and progressive graft arteriopathy developed in FcγRIIb−/− recipients. Notably, FcγRIIb-mediated inhibition of B6.Kd heart graft rejection was abrogated by increasing T cell help through transfer of additional H2.Kd-specific CD4 T cells. Thus, inhibitory FcγRIIb signaling regulates chronic but not acute rejection, most likely because the supra-optimal helper CD4 T cell response in acute rejection overcomes FcγRIIb-mediated inhibition of the effector B cell population. Immunomodulation of FcγRIIb in clinical transplantation may hold potential for inhibiting progression of transplant arteriopathy and prolonging transplant survival.

For several decades, allograft rejection was believed to be mediated almost exclusively by cellular immune responses, but it is now realized that humoral responses also play a major role. Although directed typically against donor human leukocyte antigen, it is becoming increasingly evident that the antibody response can also target autoantigens that are shared between donor and recipient and that this autoantibody may contribute to graft rejection. Many aspects of transplant-induced humoral autoimmunity remain poorly understood and key questions persist; not least what triggers the response and how autoantibody causes graft damage. Here, we collate results from recent clinical and experimental studies in transplantation and autoimmune diseases to propose answers to these questions.

Background: Renin-angiotensin-aldosterone system (RAS) blockers are commonly used for cardiovascular diseases. Currently, little information exists for the Asian population on angioedema, a rare yet serious adverse event. This study aimed to describe characteristics of RAS blockers-associated angioedema (RASBA) in Thai patients. Methods: A retrospective study using the national pharmacovigilance database of Thailand was undertaken. Cases indicating the presence of angioedema with RAS blockers uses from 1984-2011 were identified. Patient demographics, co-morbidities, concomitant drugs, information for the RAS blockers and angioedema were obtained as well as causality assessment and quality of reports. Background: Renin-angiotensin-aldosterone system (RAS) blockers are commonly used for cardiovascular diseases. Currently, little information exists for the Asian population on angioedema, a rare yet serious adverse event. This study aimed to describe characteristics of RAS blockers-associated angioedema (RASBA) in Thai patients. Methods: A retrospective study using the national pharmacovigilance database of Thailand was undertaken. Cases indicating the presence of angioedema with RAS blockers uses from 1984-2011 were identified. Patient demographics, co-morbidities, concomitant drugs, information for the RAS blockers and angioedema were obtained as well as causality assessment and quality of reports. Results: A total of 895 cases were identified. Mean age was 59.9 + 12.8 years and 66.5% being female. Most angioedema events (48.6%) occurred during the first week of treatment. Angiotensin converting enzyme inhibitors (87.7%) were the most commonly implicated agents followed by angiotensin receptor blockers (10.5%), aldosterone antagonist (2.1%) and direct renin inhibitor (0.2%). Out of the 895 cases incorporated in this study, 165 (18.4%) were classified as serious events and resulted in hospitalization. The overall case fatality rate was 0.4%. Respiratory disturbance occurred in 46 cases (5.1%). Patients with respiratory complications tended to be younger (53.4 + 13.9 vs 60.3 + 12.7 years old; p =0.002) and with higher frequency of allergy history (26.1% vs 14.7%; p =0.032) compared to those without respiratory complications. Based on multivariate logistic regression, the adjusted OR for history of allergy was 2.23 (95%CI: 1.04 - 4.78, p = 0.041). Conclusions: RASBA in Thai population occurred mostly in elderly female patients and often led to hospitalization. Since large number of patients is regularly exposed to RAS-blockers, a nationwide attempt to raise awareness of clinicians when prescribing RAS-blockers is prudent. Conclusions: RASBA in Thai population occurred mostly in elderly female patients and often led to hospitalization. Since large number of patients is regularly exposed to RAS-blockers, a nationwide attempt to raise awareness of clinicians when prescribing RAS-blockers is prudent. x

Tertiary lymphoid organs (TLOs) may develop within allografts, but their contribution to graft rejection remains unclear. Here, we study a mouse model of autoantibody-mediated cardiac allograft vasculopathy to clarify the alloimmune responses mediated by intragraft TLOs and whether blocking lymphotoxin-β-receptor (LTβR) signaling, a pathway essential for lymphoid organogenesis, abrogates TLO development. TLOs (defined as discrete lymphoid aggregates associated with high endothelial venules) were detectable in 9 of 13 heart allografts studied and were predominantly B cell in composition, harboring germinal-center activity. These are most likely manifestations of the humoral autoimmunity triggered in this model after transplantation; TLOs did not develop if autoantibody production was prevented. Treatment with inhibitory LTβR-Ig fusion protein virtually abolished allograft TLO formation (mean TLOs/heart: 0.2 vs. 2.2 in control recipients; P=0.02), with marked attenuation of the autoantibody response. Recipients primed for autoantibody before transplantation rejected grafts rapidly, but this accelerated rejection was prevented by postoperative administration of LTβR-Ig (median survival time: 18 vs. >50 d, respectively, P=0.003). Our results provide the first demonstration that TLOs develop within chronically rejecting heart allografts, are predominantly B cell in origin, and can be targeted pharmacologically to inhibit effector humoral responses.

Background—The development of autoantibody after heart transplantation is increasingly associated with poor graft outcome, but what triggers its development and whether it has a direct causative role in graft rejection is not clear. Here, we study the development of antinuclear autoantibody in an established mouse model of heart allograft vasculopathy.Methods and Results—Humoral vascular changes, including endothelial complement staining, were present in bm12 heart grafts, explanted 50 days after transplantation. Alloantibody was not detectable, but long-lasting autoantibody responses developed in C57BL/6 recipients from the third week after transplantation. No autoantibody was generated if donor CD4 T cells were depleted before heart graft retrieval or in recipients that lacked B-cell major histocompatibility complex class II expression, indicating that humoral autoimmunity is a consequence of donor CD4 T-cell allorecognition of the major histocompatibility complex class II complex on recipient autoreactive B cells. An effector role for autoantibody in graft rejection was confirmed by abrogation of humoral vascular rejection, and attenuation of vasculopathy, in B-cell deficient recipients and by development of vascular obliteration and accelerated rejection in recipients primed for autoantibody before transplantation.Conclusions—Passenger CD4 T cells within heart transplants can contribute to allograft vasculopathy by providing help to recipient B cells for autoantibody generation.

In 11 healthy volunteers, we evaluated, in a double-blind crossover study, whether the vasodilation that follows isometric contraction is mediated by prostaglandins (PGs) and/or is O2dependent. Subjects performed isometric handgrip for 2 min at 60% maximal voluntary contraction (MVC), after pretreatment with placebo or aspirin (600 mg orally), when breathing air or 40% O2. Forearm blood flow was measured in the dominant forearm by venous occlusion plethysmography. Arterial blood pressure was also recorded, allowing calculation of forearm vascular conductance (FVC; forearm blood flow/arterial blood pressure). During air breathing, aspirin significantly reduced the increase in FVC that followed contraction at 60% MVC: from a baseline of 0.09 ± 0.011 [mean ± SE, conductance units (CU)], the peak value was reduced from 0.24 ± 0.03 to 0.14 ± 0.01 CU. Breathing 40% O2similarly reduced the increase in FVC relative to that evoked when breathing air; the peak value was 0.24 ± 0.03 vs. 0.15 ± 0.02 CU. However, after aspirin, breathing 40% O2had no further effect on the contraction-evoked increase in FVC (the peak value was 0.15 ± 0.02 vs. 0.16 ± 0.02 CU). Thus the present study indicates that prostaglandins make a substantial contribution to the peak of the vasodilation that follows isometric contraction of forearm muscles at 60% MVC. Given that hyperoxia similarly reduced the vasodilation and attenuated the effect of aspirin, we propose that the stimulus for prostaglandin synthesis and release is hypoxia of the endothelium.

Summary points Traumatic hand or digit amputations can be catastrophic injuries, and often occur in young productive patients.1 2 3 Figures from the US national database have shown that amputation injuries represent 1% of all trauma attendances. Finger and thumb amputations were most common (69%), and more proximal amputations of the upper limb contributed a further 9%.3 Most patients with amputations are initially managed by non-specialists, before referral to microsurgical units. Good initial management is a key determinant of outcome, and expertise in the management of amputation is only available in specialist centres in most countries.4 We provide a summary of the initial management of amputation, with emphasis on amputate preservation, indications for replantation (reattachment surgery), and potential outcomes, to help non-specialists manage these emergencies and refer them appropriately. #### Sources and selection criteria Few large scale, randomised controlled trials have looked at upper extremity amputation. Data mostly come from multiple case series, case reports, or small trials. We have combined our knowledge with that published in articles identified by PubMed searches, using the terms “amputation” and “replantation” and selecting English language articles relevant to upper extremity replantation. ### Assessment of patient Patients are assessed according to the Advanced Trauma Life Support (ATLS) protocol, and resuscitated as necessary.4 5 6 Multiple amputation is an independent risk factor for death.3 If bleeding vessels are clamped …