Opportunities for High-plex Spatial Transcriptomics in Solid Organ Transplantation.

The last 5 y have seen the development and widespread adoption of high-plex spatial transcriptomic technology. This technique detects and quantifies mRNA transcripts in situ, meaning that transcriptomic signatures can be sampled from specific cells, structures, lesions, or anatomical regions while conserving the physical relationships that exist within complex tissues. These methods now frequently implement next-generation sequencing, enabling the simultaneous measurement of many targets, up to and including the whole mRNA transcriptome. To date, spatial transcriptomics has been foremost used in the fields of neuroscience and oncology, but there is potential for its use in transplantation sciences. Transplantation has a clear dependence on biopsies for diagnosis, monitoring, and research. Transplant patients represent a unique cohort with multiple organs of interest, clinical courses, demographics, and immunosuppressive regimens. Obtaining high complexity data on the disease processes underlying rejection, tolerance, infection, malignancy, and injury could identify new opportunities for therapeutic intervention and biomarker identification. In this review, we discuss currently available spatial transcriptomic technologies and how they can be applied to transplantation.

Decoding mTOR signalling heterogeneity in the tumour microenvironment using multiplexed imaging and graph convolutional networks.

Evaluating the contribution of the tumour microenvironment (TME) in tumour progression has proven a complex challenge due to the intricate interactions within the TME. Multiplexed imaging is an emerging technology that allows concurrent assessment of multiple of these components simultaneously. Here we utilise a highly multiplexed dataset of 61 markers across 746 colorectal tumours to investigate how complex mTOR signalling in different tissue compartments influences patient prognosis. We found that the signalling of mTOR pathway can have heterogeneous activation patterns in tumour and immune compartments which correlate with patient prognosis. Using graph neural networks, we determined the most predictive features of mTOR activity in immune cells and identified relevant cellular subpopulations. We validated our observations using spatial transcriptomics data analysis in an independent patient cohort. Our work provides a framework for studying complex cell signalling and reveals important insights for developing mTOR-based therapies.

Plasma iron controls neutrophil production and function.

Low plasma iron (hypoferremia) induced by hepcidin is a conserved inflammatory response that protects against infections but inhibits erythropoiesis. How hypoferremia influences leukocytogenesis is unclear. Using proteomic data, we predicted that neutrophil production would be profoundly more iron-demanding than generation of other white blood cell types. Accordingly in mice, hepcidin-mediated hypoferremia substantially reduced numbers of granulocytes but not monocytes, lymphocytes, or dendritic cells. Neutrophil rebound after anti-Gr-1-induced neutropenia was blunted during hypoferremia but was rescued by supplemental iron. Similarly, hypoferremia markedly inhibited pharmacologically stimulated granulopoiesis mediated by granulocyte colony-stimulating factor and inflammation-induced accumulation of neutrophils in the spleen and peritoneal cavity. Furthermore, hypoferremia specifically altered neutrophil effector functions, suppressing antibacterial mechanisms but enhancing mitochondrial reactive oxygen species-dependent NETosis associated with chronic inflammation. Notably, antagonizing endogenous hepcidin during acute inflammation enhanced production of neutrophils. We propose plasma iron modulates the profile of innate immunity by controlling monocyte-to-neutrophil ratio and neutrophil activity in a therapeutically targetable system.

Hermansky-Pudlak syndrome type 1 causes impaired anti-microbial immunity and inflammation due to dysregulated immunometabolism.

Hermansky-Pudlak syndrome (HPS) types 1 and 4 are caused by defective vesicle trafficking. The mechanism for Crohn's disease-like inflammation, lung fibrosis, and macrophage lipid accumulation in these patients remains enigmatic. The aim of this study is to understand the cellular basis of inflammation in HPS-1. We performed mass cytometry, proteomic and transcriptomic analyses to investigate peripheral blood cells and serum of HPS-1 patients. Using spatial transcriptomics, granuloma-associated signatures in the tissue of an HPS-1 patient with granulomatous colitis were dissected. In vitro studies were conducted to investigate anti-microbial responses of HPS-1 patient macrophages and cell lines. Monocytes of HPS-1 patients exhibit an inflammatory phenotype associated with dysregulated TNF, IL-1α, OSM in serum, and monocyte-derived macrophages. Inflammatory macrophages accumulate in the intestine and granuloma-associated macrophages in HPS-1 show transcriptional signatures suggestive of a lipid storage and metabolic defect. We show that HPS1 deficiency leads to an altered metabolic program and Rab32-dependent amplified mTOR signaling, facilitated by the accumulation of mTOR on lysosomes. This pathogenic mechanism translates into aberrant bacterial clearance, which can be rescued with mTORC1 inhibition. Rab32-mediated mTOR signaling acts as an immuno-metabolic checkpoint, adding to the evidence that defective bioenergetics can drive hampered anti-microbial activity and contribute to inflammation.

Neutrophil Extracellular Traps, Local IL-8 Expression, and Cytotoxic T-Lymphocyte Response in the Lungs of Patients With Fatal COVID-19.

BACKGROUND: Excessive inflammation is pathogenic in the pneumonitis associated with severe COVID-19. Neutrophils are among the most abundantly present leukocytes in the inflammatory infiltrates and may form neutrophil extracellular traps (NETs) under the local influence of cytokines. NETs constitute a defense mechanism against bacteria, but have also been shown to mediate tissue damage in a number of diseases. RESEARCH QUESTION: Could NETs and their tissue-damaging properties inherent to neutrophil-associated functions play a role in the respiratory failure seen in patients with severe COVID-19, and how does this relate to the SARS-CoV-2 viral loads, IL-8 (CXCL8) chemokine expression, and cytotoxic T-lymphocyte infiltrates? STUDY DESIGN AND METHODS: Sixteen lung biopsy samples obtained immediately after death were analyzed methodically as exploratory and validation cohorts. NETs were analyzed quantitatively by multiplexed immunofluorescence and were correlated with local levels of IL-8 messenger RNA (mRNA) and the density of CD8+ T-cell infiltration. SARS-CoV-2 presence in tissue was quantified by reverse-transcriptase polymerase chain reaction and immunohistochemistry analysis. RESULTS: NETs were found in the lung interstitium and surrounding the bronchiolar epithelium with interindividual and spatial heterogeneity. NET density did not correlate with SARS-CoV-2 tissue viral load. NETs were associated with local IL-8 mRNA levels. NETs were also detected in pulmonary thrombi and in only one of eight liver tissues. NET focal presence correlated negatively with CD8+ T-cell infiltration in the lungs. INTERPRETATION: Abundant neutrophils undergoing NETosis are found in the lungs of patients with fatal COVID-19, but no correlation was found with viral loads. The strong association between NETs and IL-8 points to this chemokine as a potentially causative factor. The function of cytotoxic T-lymphocytes in the immune responses against SARS-CoV-2 may be interfered with by the presence of NETs.

No significant difference between 1940 and 1470 nm in endovenous laser ablation using an in vitro porcine liver model.

Current endovenous laser ablation (EVLA) practice favours 1470 nm, as water is a major chromophore for this wavelength. Water has a greater affinity for 1940 nm, leading to claims that lower powers or linear endovenous energy densities (LEEDs) are needed. We compared the thermal spread and carbonisation of EVLA using these two wavelengths, in the porcine liver model. Using the previously validated porcine liver model, we performed 5 treatments, at each power: 2 W, 4 W, 6 W, 8 W and 10 W using a standard pullback of 8 s/cm. This gave LEEDs for each wavelength of 16, 32, 48, 64 and 80 J/cm. Digital images were given random codes and analysed by two blinded observers. Thermal spread was measured using "SketchandCalc" online software and graded carbonisation from 0 (none) to 3 (black carbon tract). There was no significant difference in thermal spread between the two wavelengths at 6 W, 8 W and 10 W. At 2 W, the 1470-nm laser had a significantly increased thermal spread over the 1940 nm. Significantly more carbonisation was found with the 1940-nm laser compared to 1470 nm. In this model, there was no significant difference in thermal spread at powers of 6 W and more. At 2 W and potentially 4 W, 1470 nm showed spread than 1940 nm, due to increased absorption at the device/tissue interface. At powers and LEEDs used for saphenous ablation, we found no evidence to support reduced power or LEED when using 1940 nm. However, 1940 nm may be more advantageous than 1470 nm when ablating small thin-walled veins, near to the skin.

Identification of LZTFL1 as a candidate effector gene at a COVID-19 risk locus.

The severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) disease (COVID-19) pandemic has caused millions of deaths worldwide. Genome-wide association studies identified the 3p21.31 region as conferring a twofold increased risk of respiratory failure. Here, using a combined multiomics and machine learning approach, we identify the gain-of-function risk A allele of an SNP, rs17713054G>A, as a probable causative variant. We show with chromosome conformation capture and gene-expression analysis that the rs17713054-affected enhancer upregulates the interacting gene, leucine zipper transcription factor like 1 (LZTFL1). Selective spatial transcriptomic analysis of lung biopsies from patients with COVID-19 shows the presence of signals associated with epithelial-mesenchymal transition (EMT), a viral response pathway that is regulated by LZTFL1. We conclude that pulmonary epithelial cells undergoing EMT, rather than immune cells, are likely responsible for the 3p21.31-associated risk. Since the 3p21.31 effect is conferred by a gain-of-function, LZTFL1 may represent a therapeutic target.

Pesticides Misused for Bed Bug Control: Comparing Professional and Nonprofessional Applications Reported to the National Pesticide Information Center, 2013-2017.

Objectives. To compare outcomes when pesticides are used to control bed bugs by professionals and nonprofessionals. Methods. All US National Pesticide Information Center inquiries from 2013 to 2017 were assessed to identify scenarios involving bed bugs and pesticide applications. Cases were evaluated with respect to types of applicators, misapplications, and human pesticide exposures. Results. Misapplications were more than twice as likely to be reported in cases involving nonprofessional applications (14%) as in cases involving professional applications (5%). Human exposures to pesticides were reported more often when pesticides were misapplied (70%) than when there were no apparent misapplications (31%). Conclusions. Both professionals and nonprofessionals may misuse pesticides to control bed bugs, which may increase the risks of exposure and adverse outcomes. Policy interventions may reduce pesticide incidents related to bed bug control by promoting professional involvement and adherence to product label instructions.

Humanization of Immunodeficient Animals for the Modeling of Transplantation, Graft Versus Host Disease, and Regenerative Medicine.

The humanization of animals is a powerful tool for the exploration of human disease pathogenesis in biomedical research, as well as for the development of therapeutic interventions with enhanced translational potential. Humanized models enable us to overcome biologic differences that exist between humans and other species, while giving us a platform to study human processes in vivo. To become humanized, an immune-deficient recipient is engrafted with cells, tissues, or organoids. The mouse is the most well studied of these hosts, with a variety of immunodeficient strains available for various specific uses. More recently, efforts have turned to the humanization of other animal species such as the rat, which offers some technical and immunologic advantages over mice. These advances, together with ongoing developments in the incorporation of human transgenes and additional mutations in humanized mouse models, have expanded our opportunities to replicate aspects of human allotransplantation and to assist in the development of immunotherapies. In this review, the immune and tissue humanization of various species is presented with an emphasis on their potential for use as models for allotransplantation, graft versus host disease, and regenerative medicine.

Endothelial Cell Amplification of Regulatory T Cells Is Differentially Modified by Immunosuppressors and Intravenous Immunoglobulin.

Immunosuppressive treatment is a prerequisite for both organ transplantation and tolerance of the allograft. However, long-term immunosuppression has been associated with a higher incidence of malignancies and infections. Immunosuppressors mainly target circulating immune cells and little is known of their "off-target" effects, such as their impact on endothelial cells (ECs). In chronic antibody-mediated rejection (AMR), the allograft endothelium is a target of damage, histologically detected as transplant glomerulopathy, and which correlates with poor graft survival. Under inflammatory conditions, EC expression of HLA class II antigens can lead to CD4+-T lymphocyte alloactivation and selective expansion of pro-inflammatory Th17 and pro-tolerance Treg subsets. This response can be modified and preactivation of the EC by HLA-DR antibody binding promoted a proinflammatory Th17 response. However, whether or not immunosuppressors alter EC immunogenicity has not been examined. In alloimmunized patients with AMR, cyclosporine A (CsA) and mycophenolic acid (MPA) are often combined with intravenous immunoglobulins (IVIgs). This study reports changes in the microvascular EC phenotype and function after treatment with CsA, MPA, or IVIg. Both CsA and MPA decreased HLA-DR and increased CD54 expression, whereas IVIg increased HLA-DR expression. Interleukin 6 secretion was reduced by all three immunomodulators. Preincubation of ECs with CsA or MPA limited, while IVIg amplified, Treg expansion. Because CsA, MPA, and IVIg are known for their ability to act upon leukocytes, we confirmed that ECs maintained their immunoregulatory role when allogeneic leukocytes were pretreated with CsA, MPA, or IVIg. The results reveal that individual immunosuppressors, used in the induction and maintenance of renal allograft tolerance, had direct and distinct effects on ECs. Results of experiments associating IVIg with either CsA or MPA underlined the differences observed using individual immunosuppressors. Paradoxically, CsA or MPA may increase EC mediated inflammatory responses and long-term exposure may contribute to limitation of allograft tolerance. In contrast, IVIg interaction with the endothelium may mediate some of its immunosuppressive effects through promotion of Treg expansion, contributing to the maintenance of allograft tolerance.

Donor Specific Antibodies are not only directed against HLA-DR: Minding your Ps and Qs.

During solid organ transplantation, interactions between recipient and donor immune cells occur chiefly in the allograft microvasculature. All three HLA class II antigens, DR, DP and DQ, have been detected on renal EC with a markedly increased expression of HLA class II observed in renal allografts undergoing rejection. Recent studies of donor-specific antibodies (DSA) have exposed the prevalence of de novo DSA directed against HLA-DQ, as well as a strong association between these antibodies and allograft damage. The HLA-DQ molecule can be distinguished from the other class II antigens by its transcription, expression and peptide repertoire. The distinct intragraft expression and immunogenicity of HLA-DQ may contribute to the incidence of HLA-DQ DSA, as well as directing the DSA-mediated damage. The possibility of HLA class II antigen-specific signaling in EC may reveal different mechanisms of allograft damage that act in tandem with complement-dependent injury. This review addresses the features of the HLA-DQ heterodimer that may underlie the high incidence of HLA-DQ directed DSA and their association with allograft damage. We also consider existing data in hematopoietic stem cell transplantation concerning HLA directed DSA.