AnnoSpat annotates cell types and quantifies cellular arrangements from spatial proteomics.

Cellular composition and anatomical organization influence normal and aberrant organ functions. Emerging spatial single-cell proteomic assays such as Image Mass Cytometry (IMC) and Co-Detection by Indexing (CODEX) have facilitated the study of cellular composition and organization by enabling high-throughput measurement of cells and their localization directly in intact tissues. However, annotation of cell types and quantification of their relative localization in tissues remain challenging. To address these unmet needs for atlas-scale datasets like Human Pancreas Analysis Program (HPAP), we develop AnnoSpat (Annotator and Spatial Pattern Finder) that uses neural network and point process algorithms to automatically identify cell types and quantify cell-cell proximity relationships. Our study of data from IMC and CODEX shows the higher performance of AnnoSpat in rapid and accurate annotation of cell types compared to alternative approaches. Moreover, the application of AnnoSpat to type 1 diabetic, non-diabetic autoantibody-positive, and non-diabetic organ donor cohorts recapitulates known islet pathobiology and shows differential dynamics of pancreatic polypeptide (PP) cell abundance and CD8+ T cells infiltration in islets during type 1 diabetes progression.

Three-Dimensional Imaging of the Enteric Nervous System in Human Pediatric Colon Reveals New Features of Hirschsprung's Disease.

BACKGROUND & AIMS: Hirschsprung's disease is defined by the absence of the enteric nervous system (ENS) from the distal bowel. Primary treatment is "pull-through" surgery to remove bowel that lacks ENS, with reanastomosis of "normal" bowel near the anal verge. Problems after pull-through are common, and some may be due to retained hypoganglionic bowel (ie, low ENS density). Testing this hypothesis has been difficult because counting enteric neurons in tissue sections is unreliable, even for experts. Tissue clearing and 3-dimensional imaging provide better data about ENS structure than sectioning. METHODS: Regions from 11 human colons and 1 ileal specimen resected during Hirschsprung's disease pull-through surgery were cleared, stained with antibodies to visualize the ENS, and imaged by confocal microscopy. Control distal colon from people with no known bowel problems were similarly cleared, stained, and imaged. RESULTS: Quantitative analyses of human colon, ranging from 3 days to 60 years old, suggest age-dependent changes in the myenteric plexus area, ENS ganglion area, percentage of myenteric plexus occupied by ganglia, neurons/mm2, and neuron Feret's diameter. Neuron counting using 3-dimensional images was highly reproducible. High ENS density in neonatal colon allowed reliable neuron counts using 500-µm2 × 500-µm2 regions (36-fold smaller than in adults). Hirschsprung's samples varied 8-fold in proximal margin enteric neuron density and had diverse ENS architecture in resected bowel. CONCLUSIONS: Tissue clearing and 3-dimensional imaging provide more reliable information about ENS structure than tissue sections. ENS structure changes during childhood. Three-dimensional ENS anatomy may provide new insight into human bowel motility disorders, including Hirschsprung's disease.

Myotubularin-related proteins regulate KRAS function by controlling plasma membrane levels of polyphosphoinositides and phosphatidylserine.

KRAS is a small GTPase, ubiquitously expressed in mammalian cells, that functions as a molecular switch to regulate cell proliferation and differentiation. Oncogenic mutations that render KRAS constitutively active occur frequently in human cancers. KRAS must localize to the plasma membrane (PM) for biological activity. KRAS PM binding is mediated by interactions of the KRAS membrane anchor with phosphatidylserine (PtdSer), therefore, depleting PM PtdSer content abrogates KRAS PM binding and oncogenic function. From a genome-wide siRNA screen to search for genes that regulate KRAS PM localization, we identified a set of phosphatidylinositol (PI) 3-phosphatase family members: myotubularin-related (MTMR) proteins 2, 3, 4 and 7. Here we show that knockdown of MTMR 2/3/4/7 expression disrupts KRAS PM interactions. The molecular mechanism involves depletion of PM PI 4-phosphate (PI4P) levels, which in turn disrupts the subcellular localization and operation of oxysterol-binding protein related protein (ORP) 5, a PtdSer lipid transfer protein that maintains PM PtdSer content. Concomitantly, silencing MTMR 2/3/4/7 expression elevates PM levels of PI3P and reduces PM and total cellular levels of PtdSer. In summary we propose that the PI 3-phosphatase activity provided by MTMR proteins is required to generate PM PI for the synthesis of PM PI4P, which in turn, promotes the PM localization of PtdSer and KRAS.

Circulating T cell specific extracellular vesicle profiles in cardiac allograft acute cellular rejection.

There is a critical need for biomarkers of acute cellular rejection (ACR) in organ transplantation. We hypothesized that ACR leads to changes in donor-reactive T cell small extracellular vesicle (sEV) profiles in transplant recipient circulation that match the kinetics of alloreactive T cell activation. In rodent heart transplantation, circulating T cell sEV quantities (P < .0001) and their protein and mRNA cargoes showed time-specific expression of alloreactive and regulatory markers heralding early ACR in allogeneic transplant recipients but not in syngeneic transplant recipients. Next generation sequencing of their microRNA cargoes identified novel candidate biomarkers of ACR, which were validated by stem loop quantitative reverse transcription polymerase chain reaction (n = 10). Circulating T cell sEVs enriched from allogeneic transplant recipients mediated targeted cytotoxicity of donor cardiomyocytes by apoptosis assay (P < .0001). Translation of the concept and EV methodologies to clinical heart transplantation demonstrated similar upregulation of circulating T cell sEV profiles at time points of grade 2 ACR (n = 3 patients). Furthermore, T cell receptor sequencing of T cell sEV mRNA cargo demonstrated expression of T cell clones with intact complementarity determining region 3 signals. These data support the diagnostic potential of T cell sEVs as noninvasive biomarker of ACR and suggest their potential functional roles.

Non-Markovian Modeling of Nonequilibrium Fluctuations and Dissipation in Active Viscoelastic Biomatter.

Based on a Hamiltonian that incorporates the elastic coupling between a tracer particle and the embedding active viscoelastic biomatter, we derive a generalized non-Markovian Langevin model for the nonequilibrium mechanical tracer response. Our analytical expressions for the frequency-dependent tracer response function and the tracer positional autocorrelation function agree quantitatively with experimental data for red blood cells and actomyosin networks with and without adenosine triphosphate over the entire frequency range and in particular reproduce the low-frequency violation of the fluctuation-dissipation theorem. The viscoelastic power laws, the elastic constants and effective friction coefficients extracted from the experimental data allow straightforward physical interpretation.

Immunotherapy targeting B cells and long-lived plasma cells effectively eliminates pre-existing donor-specific allo-antibodies.

Pre-existing anti-human leukocyte antigen (HLA) allo-antibodies constitute a major barrier to transplantation. Current desensitization approaches fail due to ineffective depletion of allo-specific memory B cells (Bmems) and long-lived plasma cells (LLPCs). We evaluate the efficacy of chimeric antigen receptor (CAR) T cells targeting CD19 and B cell maturation antigen (BCMA) to eliminate allo-antibodies in a skin pre-sensitized murine model of islet allo-transplantation. We find that treatment of allo-sensitized hosts with CAR T cells targeting Bmems and LLPCs eliminates donor-specific allo-antibodies (DSAs) and mitigates hyperacute rejection of subsequent islet allografts. We then assess the clinical efficacy of the CAR T therapy for desensitization in patients with multiple myeloma (MM) with pre-existing HLA allo-antibodies who were treated with the combination of CART-BCMA and CART-19 (ClinicalTrials.gov: NCT03549442) and observe clinically meaningful allo-antibody reduction. These findings provide logical rationale for clinical evaluation of CAR T-based immunotherapy in highly sensitized candidates to promote successful transplantation.

Computational workflow and interactive analysis of single-cell expression profiling of islets generated by the Human Pancreas Analysis Program.

Type 1 and Type 2 diabetes are distinct genetic diseases of the pancreas which are defined by the abnormal level of blood glucose. Understanding the initial molecular perturbations that occur during the pathogenesis of diabetes is of critical importance in understanding these disorders. The inability to biopsy the human pancreas of living donors hampers insights into early detection, as the majority of diabetes studies have been performed on peripheral leukocytes from the blood, which is not the site of pathogenesis. Therefore, efforts have been made by various teams including the Human Pancreas Analysis Program (HPAP) to collect pancreatic tissues from deceased organ donors with different clinical phenotypes. HPAP is designed to define the molecular pathogenesis of islet dysfunction by generating detailed datasets of functional, cellular, and molecular information in pancreatic tissues of clinically well-defined organ donors with Type 1 and Type 2 diabetes. Moreover, data generated by HPAP continously become available through a centralized database, PANC-DB, thus enabling the diabetes research community to access these multi-dimensional data prepublication. Here, we present the computational workflow for single-cell RNA-seq data analysis of 258,379 high-quality cells from the pancreatic islets of 67 human donors generated by HPAP, the largest existing scRNA-seq dataset of human pancreatic tissues. We report various computational steps including preprocessing, doublet removal, clustering and cell type annotation across single-cell RNA-seq data from islets of four distintct classes of organ donors, i.e. non-diabetic control, autoantibody positive but normoglycemic, Type 1 diabetic, and Type 2 diabetic individuals. Moreover, we present an interactive tool, called CellxGene developed by the Chan Zuckerberg initiative, to navigate these high-dimensional datasets. Our data and interactive tools provide a reliable reference for singlecell pancreatic islet biology studies, especially diabetes-related conditions.

AnnoSpat annotates cell types and quantifies cellular arrangements from spatial proteomics.

Cellular composition and anatomical organization influence normal and aberrant organ functions. Emerging spatial single-cell proteomic assays such as Image Mass Cytometry (IMC) and Co-Detection by Indexing (CODEX) have facilitated the study of cellular composition and organization by enabling high-throughput measurement of cells and their localization directly in intact tissues. However, annotation of cell types and quantification of their relative localization in tissues remain challenging. To address these unmet needs, we developed AnnoSpat (Annotator and Spatial Pattern Finder) that uses neural network and point process algorithms to automatically identify cell types and quantify cell-cell proximity relationships. Our study of data from IMC and CODEX show the superior performance of AnnoSpat in rapid and accurate annotation of cell types compared to alternative approaches. Moreover, the application of AnnoSpat to type 1 diabetic, non-diabetic autoantibody-positive, and non-diabetic organ donor cohorts recapitulated known islet pathobiology and showed differential dynamics of pancreatic polypeptide (PP) cell abundance and CD8+ T cells infiltration in islets during type 1 diabetes progression.

Exocrine pancreas regeneration modifies original pancreas to alleviate diabetes in mouse models.

Diabetes is a major public health issue because of its widely epidemic nature and lack of cure. Here, we show that pancreas-derived mesenchymal stem cells (PMSCs) are capable of regenerating exocrine pancreas when implanted into the kidney capsule of mice with streptozotocin (STZ)-induced diabetes. Mechanistically, we found that the regenerated exocrine pancreas elevated interleukin-6 (IL-6) in PMSC implants, which transiently activated tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) to inhibit IL-17, thereby rescuing damaged exocrine pancreas and islet ß cells. In addition, we used knockout mouse models to show that global lack of IL-6, TNF-α, or IFN-γ resulted in increased severity of STZ-induced diabetes and resistance to PMSC implantation therapy, confirming the roles of these factors in safeguarding pancreatic ß cells. Furthermore, removal of the kidney capsule PMSC implants at 28 days after implantation did not affect the PMSC-initiated therapeutic effect on diabetic mice. This study reveals a previously unknown role of exocrine pancreas regeneration in safeguarding ß cells and demonstrates a "soil-rescues-seed" strategy for type 1 diabetes therapy.

KRAS Inhibitor that Simultaneously Inhibits Nucleotide Exchange Activity and Effector Engagement.

We describe a small molecule ligand ACA-14 (2-hydroxy-5-{[(2-phenylcyclopropyl) carbonyl] amino} benzoic acid) as an initial lead for the development of direct inhibitors of KRAS, a notoriously difficult anticancer drug target. We show that the compound binds to KRAS near the switch regions with affinities in the low micromolar range and exerts different effects on KRAS interactions with binding partners. Specifically, ACA-14 impedes the interaction of KRAS with its effector Raf and reduces both intrinsic and SOS-mediated nucleotide exchange rates. Likely as a result of these effects, ACA-14 inhibits signal transduction through the MAPK pathway in cells expressing mutant KRAS and inhibits the growth of pancreatic and colon cancer cells harboring mutant KRAS. We thus propose compound ACA-14 as a useful initial lead for the development of broad-acting inhibitors that target multiple KRAS mutants and simultaneously deplete the fraction of GTP-loaded KRAS while abrogating the effector-binding ability of the already GTP-loaded fraction.

BLyS neutralization results in selective anti-HLA alloantibody depletion without successful desensitization.

Pre-existing anti-HLA allo-antibodies (allo-Abs) are a major barrier to successful kidney transplantation, resulting in an elevated risk for antibody-mediated rejection (AMR) and eventual graft loss. The cytokine B lymphocyte stimulator (BLyS) promotes B cell maturation and plasma cell survival; consequently, anti-BLyS therapy represents a potential therapeutic opportunity in diminishing pre-existing allo-Abs. Here we report that in our 1-year pilot trial, BLyS neutralization failed to reduce total anti-HLA allo-Ab levels in highly sensitized candidates awaiting kidney transplant in a clinically meaningful way. Additionally, we performed a post hoc analysis using sera from trial candidates which revealed selective depletion of anti-HLA class I and class II Abs in response to belimumab treatment, restricted to certain allele specificities and IgG subclasses. Altogether, we observed that BLyS blockade only results in selective depletion of anti-HLA Abs recognizing a few discrete HLA allele specificities.

A nanofibrous encapsulation device for safe delivery of insulin-producing cells to treat type 1 diabetes.

Transplantation of stem cell-derived ß (SC-ß) cells represents a promising therapy for type 1 diabetes (T1D). However, the delivery, maintenance, and retrieval of these cells remain a challenge. Here, we report the design of a safe and functional device composed of a highly porous, durable nanofibrous skin and an immunoprotective hydrogel core. The device consists of electrospun medical-grade thermoplastic silicone-polycarbonate-urethane and is soft but tough (~15 megapascal at a rupture strain of >2). Tuning the nanofiber size to less than ~500 nanometers prevented cell penetration while maintaining maximum mass transfer and decreased cellular overgrowth on blank (cell-free) devices to as low as a single-cell layer (~3 micrometers thick) when implanted in the peritoneal cavity of mice. We confirmed device safety, indicated as continuous containment of proliferative cells within the device for 5 months. Encapsulating syngeneic, allogeneic, or xenogeneic rodent islets within the device corrected chemically induced diabetes in mice and cells remained functional for up to 200 days. The function of human SC-ß cells was supported by the device, and it reversed diabetes within 1 week of implantation in immunodeficient and immunocompetent mice, for up to 120 and 60 days, respectively. We demonstrated the scalability and retrievability of the device in dogs and observed viable human SC-ß cells despite xenogeneic immune responses. The nanofibrous device design may therefore provide a translatable solution to the balance between safety and functionality in developing stem cell-based therapies for T1D.

Menin-regulated Pbk controls high fat diet-induced compensatory beta cell proliferation.

Pancreatic beta cells undergo compensatory proliferation in the early phase of type 2 diabetes. While pathways such as FoxM1 are involved in regulating compensatory beta cell proliferation, given the lack of therapeutics effectively targeting beta cell proliferation, other targetable pathways need to be identified. Herein, we show that Pbk, a serine/threonine protein kinase, is essential for high fat diet (HFD)-induced beta cell proliferation in vivo using a Pbk kinase deficiency knock-in mouse model. Mechanistically, JunD recruits menin and HDAC3 complex to the Pbk promoter to reduce histone H3 acetylation, leading to epigenetic repression of Pbk expression. Moreover, menin inhibitor (MI) disrupts the menin-JunD interaction and augments Pbk transcription. Importantly, MI administration increases beta cell proliferation, ameliorating hyperglycemia, and impaired glucose tolerance (IGT) in HFD-induced diabetic mice. Notably, Pbk is required for the MI-induced beta cell proliferation and improvement of IGT. Together, these results demonstrate the repressive role of the menin/JunD/Pbk axis in regulating HFD-induced compensatory beta cell proliferation and pharmacologically regulating this axis may serve as a novel strategy for type 2 diabetes therapy.

Ex-Vivo Repair of Complex Hilar Renal Artery Aneurysms and Auto-Transplantation of Solitary Kidney.

Renal artery aneurysms (RAA) are rare and challenging to repair. We present a case of a 48-year-old female with solitary right kidney who had complex aneurysms near the renal hilum. CT angiogram showed fibromuscular dysplasia (FMD) features of the renal artery and 2 saccular aneurysms measuring 2.3 cm and 1 cm. An additional small lower pole renal artery added to the complexity of the case. Ex-vivo aneurysm repair was performed after the kidney was removed and flushed with preservation solution. This was followed by auto-transplantation to the right external iliac vessels. The patient did well postoperatively without need for dialysis with serum creatinine returning to normal 5 weeks after.

Reciprocal Learning Between Military and Civilian Surgeons: Past and Future Paths for Medical Innovation.

Numerous surgical advances have resulted from exchanges between military and civilian surgeons. As part of the U.S. National Library of Medicine Michael E. DeBakey Fellowship in the History of Medicine, we conducted archival research to shed light on the lessons that civilian surgery has learned from the military system and vice-versa. Several historical case studies highlight the need for immersive programs where surgeons from the military and civilian sectors can gain exposure to the techniques, expertise, and institutional knowledge the other domain provides. Our findings demonstrate the benefits and promise of structured programs to promote reciprocal learning between military and civilian surgery.

The Identity of Human Tissue-Emigrant CD8+ T Cells.

Lymphocyte migration is essential for adaptive immune surveillance. However, our current understanding of this process is rudimentary, because most human studies have been restricted to immunological analyses of blood and various tissues. To address this knowledge gap, we used an integrated approach to characterize tissue-emigrant lineages in thoracic duct lymph (TDL). The most prevalent immune cells in human and non-human primate efferent lymph were T cells. Cytolytic CD8+ T cell subsets with effector-like epigenetic and transcriptional signatures were clonotypically skewed and selectively confined to the intravascular circulation, whereas non-cytolytic CD8+ T cell subsets with stem-like epigenetic and transcriptional signatures predominated in tissues and TDL. Moreover, these anatomically distinct gene expression profiles were recapitulated within individual clonotypes, suggesting parallel differentiation programs independent of the expressed antigen receptor. Our collective dataset provides an atlas of the migratory immune system and defines the nature of tissue-emigrant CD8+ T cells that recirculate via TDL.

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans.

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in several diseases including cancer. The free-living nematode C. elegans possesses an evolutionary and functionally conserved EGFR-RAS-ERK MAP signal cascade which is central for the development of the vulva. Gain of function mutations in RAS homolog LET-60 and EGFR homolog LET-23 induce the generation of visible nonfunctional ectopic pseudovulva along the ventral body wall of these worms. Previously, the multivulval (Muv) phenotype in these worms has been shown to be inhibited by small chemical molecules. Here we describe a protocol for using the worm in a liquid-based assay to identify inhibitors that abolish the activities of EGFR and RAS proteins. Using this assay, we show R-fendiline, an indirect inhibitor of K-RAS, suppresses the Muv phenotype expressed in the let-60(n1046) and let-23(sa62) mutant worms. The assay is simple, inexpensive, is not time consuming to setup, and can be used as an initial platform for the discovery of anticancer therapeutics.

Kidney transplantation and donation in the transgender population: A single-institution case series.

The medical needs of the transgender population are increasingly recognized within the US health care system. Hormone therapy and gender-affirming surgery present distinct anatomic, hormonal, infectious, and psychosocial issues among transgender kidney transplant donors and recipients. We present the first reported experience with kidney transplantation and donation in transgender patients. A single-center case series (January 2014-December 2018) comprising 4 transgender kidney transplant recipients and 2 transgender living donors was constructed and analyzed. Experts in transplant surgery, transplant psychiatry, transplant infectious disease, pharmacy, and endocrinology were consulted to discuss aspects of care for these patients. Four transgender patients identified as male-to-female and 2 as female-to-male. Three of 6 had gender-affirming surgeries prior to transplant surgery, 1 of whom had further procedures posttransplant. Additionally, 4 patients were on hormone therapy. All 6 had psychiatric comorbidities. The 4 grafts have done well, with an average serum creatinine of 1.45 mg/dL at 2 years (range 1.01-1.85 mg/dL). However, patients encountered various postoperative complications, 1 of which was attributable to modified anatomy. Thus, transgender kidney transplant patients can present novel challenges in regard to surgical considerations as well as pre- and posttransplant care. Dedicated expertise is needed to optimize outcomes for this population.