Single-cell RNA sequencing distinguishes dendritic cell subsets in the rat, allowing advanced characterization of the effects of FMS-like tyrosine kinase 3 ligand.

Tissue-resident dendritic cells (DCs) are essential for immunological homeostasis and hold promise for a variety of therapeutic interventions. The rare nature of tissue-resident DCs and their suboptimal description in the lab rat model has limited their characterization. To address this limitation, FMS-like tyrosine kinase 3 ligand (FLT3L) has been utilized to expand these population in vitro and in vivo for investigative or therapeutic purposes. However, conflicting reports have suggested that FLT3L can either promote immune tolerance or enhance immunogenicity, necessitating clarification of the effects of FLT3L on DC phenotype and functionality. We first paired single-cell RNA sequencing with multicolour spectral flow cytometry to provide an updated strategy for the identification of tissue-resident classical and plasmacytoid DCs in the rat model. We then administered FLT3L to Lewis rats in vivo to investigate its effect on tissue-resident DC enumeration and phenotype in the liver, spleen, and mesenteric lymph nodes. We found that FLT3L expands classical DCs (cDCs) 1 and 2 in a dose-dependent manner and that cDC1 and cDC2 in secondary lymphoid organs had altered MHC I, MHC II, CD40, CD80, CD86, and PD-L1 cell-surface expression levels following FLT3L administration. These changes were accompanied by an increase in gene expression levels of toll-like receptors 2, 4, 7, and 9 as well as inflammatory cytokines IL-6 and TNF-α. In conclusion, FLT3L administration in vivo increases cDC enumeration in the liver, spleen, and mesenteric lymph nodes accompanied by a tissue-restricted alteration in expression of antigen presentation machinery and inflammatory mediators.

Interleukin-10 and Transforming Growth Factor-ß Cytokines Decrease Immune Activation During Normothermic Ex Vivo Machine Perfusion of the Rat Liver.

Normothermic ex vivo liver perfusion (NEVLP) is a novel system for organ preservation that may improve over static cold storage clinically and offers the chance for graft modification prior to transplantation. Although recent studies have shown the presence of inflammatory molecules during perfusion, none have yet shown the effects of NEVLP on liver-resident immune cell activation. We investigated the effects of NEVLP on liver-resident immune cell activation and assessed the ability of anti-inflammatory cytokines interleukin 10 (IL10) and transforming growth factor ß (TGF-ß) to improve organ function and reduce immune activation during perfusion. Rat livers were perfused for 4 hours at 37°C with or without the addition of 20 ng/mL of each IL10 and TGF-ß (n = 7). Naïve and cold storage (4 hours at 4°C) livers served as controls (n = 4). Following preservation, gene expression profiles were assessed through single-cell RNA sequencing; dendritic cell and macrophage activation was measured by flow cytometry; and cytokine production was assessed by enzyme-linked immunosorbent assay. NEVLP induced a global inflammatory gene expression signature, most notably in liver-resident macrophages and dendritic cells, which was accompanied by an increase in cell-surface levels of major histocompatibility complex (MHC) II, CD40, and CD86. Immune activation was partially ameliorated by IL10 and TGF-ß treatment, but no changes were observed in inflammatory cytokine production. Overall levels of liver damage and cellular apoptosis from perfusion were low, and liver function was improved with IL10 and TGF-ß treatment. This is the first study to demonstrate that liver-resident immune cells gain an activated phenotype during NEVLP on both the gene and protein level and that this activation can be reduced through therapeutic intervention with IL10 and TGF-ß.

Rh2(II,III) Catalysts with Chelating Carboxylate and Carboxamidate Supports: Electronic Structure and Nitrene Transfer Reactivity.

Dirhodium-catalyzed C-H amination is hypothesized to proceed via Rh2-nitrene intermediates in either the Rh2(II,II) or Rh2(II,III) redox state. Herein, we report joint theoretical and experimental studies of the ground electronic state (GES), redox potentials, and C-H amination of [Rh2(II,III)(O2CCH3)4(L)n](+) (1_L) (L = none, Cl(-), and H2O), [Rh2(esp)2](+) (2), and Rh2(espn)2Cl (3) (esp = α,α,α',α'-tetramethyl-1,3-benzenedipropanoate and espn = α,α,α',α'-tetramethyl-1,3-benzenedipropanamidate). CASSCF calculations on 1_L yield a wave function with two closely weighted configurations, (δ*)(2)(π1*)(2)(π2*)(1) and (δ*)(2)(π1*)(1)(π2*)(2), consistent with reported EPR g values [Chem. Phys. Lett. 1986, 130, 20-23]. In contrast, EPR spectra of 2 show g values consistent with the DFT-computed (π*)(4)(δ*)(1) GES. EPR spectra and Cl K-edge XAS for 3 are consistent with a (π*)(4)(δ*)(1) GES, as supported by DFT. Nitrene intermediates 2N_L and 3N_L are also examined by DFT (the nitrene is an NSO3R species). DFT calculations suggest a doublet GES for 2N_L and a quartet GES for 3N_L. CASSCF calculations describe the GES of 2N as Rh2(II,II) with a coordinated nitrene radical cation, (π*)(4)(δ*)(2)(π(nitrene,1))(1)(π(nitrene,2))(0). Conversely, the GES of 3N is Rh2(II,III) with a coordinated triplet nitrene, (π*)(4)(δ*)(1)(π(nitrene,1))(1)(π(nitrene,2))(1). Quartet transition states ((4)TSs) are found to react via a stepwise radical mechanism, whereas (2)TSs are found to react via a concerted mechanism that is lower in energy compared to (4)TSs for both 2N_L and 3N_L. The experimental (determined by intramolecular competition) and (2)TS-calculated kinetic isotopic effect (KIE) shows a KIE ∼ 3 for both 2N and 3N, which is consistent with a concerted mechanism.

Heterotopic Auxiliary Whole Liver Rat Transplant Model Utilizing a Hepaticoureterostomy for Allograft Rejection Studies.

Small animal transplant models are indispensable for organ tolerance studies investigating feasible therapeutic interventions in preclinical studies. Rat liver transplantation (LTx) protocols typically use an orthotopic model where the recipients' native liver is removed and replaced with a donor liver. This technically demanding surgical procedure requires advanced micro-surgical skills and is further complicated by lengthy anhepatic and lower body ischemia times. This prompted the development of a less complicated heterotopic method that can be performed faster with no anhepatic or lower body ischemia time, reducing post-surgery stress for the recipient animal. This heterotopic LTx protocol includes two main steps: excising the liver from the donor rat and transplanting the whole liver into the recipient rat. During the excision of the donor liver, the surgeon ligates the supra-hepatic vena cava (SHVC) and hepatic artery (HA). On the recipient side, the surgeon removes the left kidney and positions the donor liver with the portal vein (PV), infra-hepatic vena cava (IHVC), and bile duct facing the renal vessels. Further, the surgeon anastomoses the recipient's renal vein end to end with the IHVC of the liver and arterializes the PV with the renal artery using a stent. A hepaticoureterostomy is utilized for biliary drainage by anastomosing the bile duct to the recipient's ureter, permitting the discharge of bile via the bladder. The average duration of the transplantation was 130 min, cold ischemia duration was around 35 min, and warm ischemia duration was less than 25 min. Hematoxylin and eosin histology of the auxiliary liver from syngeneic transplants showed normal hepatocyte structure with no significant parenchymal alterations 30 days post-transplant. In contrast, 8-day post-transplant allogeneic graft specimens demonstrated extensive lymphocytic infiltration with a Banff Schema rejection activity index score of 9. Therefore, this LTx method facilitates a low morbidity rejection model alternative to orthotopic LTx.

Normothermic liver perfusion derived extracellular vesicles have concentration-dependent immunoregulatory properties.

Extracellular vesicles (EVs) are major contributors to immunological responses following solid organ transplantation. Donor derived EVs are best known for their role in transplant rejection through transferring donor major histocompatibility complex proteins to recipient antigen presenting cells, a phenomenon known as ?cross-decoration'. In contrast, donor liver-derived EVs are associated with organ tolerance in small animal models. Therefore, the cellular source of EVs and their cargo could influence their downstream immunological effects. To investigate the immunological effects of EVs released by the liver in a physiological and transplant-relevant model, we isolated EVs being produced during normothermic ex vivo liver perfusion (NEVLP), a novel method of liver storage prior to transplantation. We found EVs were produced by the liver during NEVLP, and these EVs contained multiple anti-inflammatory miRNA species. In terms of function, liver-derived EVs were able to cross-decorate allogeneic cells and suppress the immune response in allogeneic mixed lymphocyte reactions in a concentration-dependent fashion. In terms of cytokine response, the addition of 1 × 109 EVs to the mixed lymphocyte reactions significantly decreased the production of the inflammatory cytokines TNF-α, IL-10 and IFN-γ. In conclusion, we determined physiologically produced liver-derived EVs are immunologically regulatory, which has implications for their role and potential modification in solid organ transplantation.

Single cell RNA-sequencing identifies the effect of Normothermic ex vivo liver perfusion on liver-resident T cells.

BACKGROUND: Normothermic ex vivo liver perfusion (NEVLP) is an exciting strategy to preserve livers prior to transplant, however, the effects of NEVLP on the phenotype of tissue-resident immune cells is largely unknown. The presence of tissue-resident memory T cells (TRM) in the liver may protect against acute rejection and decrease allograft dysfunction. Therefore, we investigated the effects of NEVLP on liver TRMs and assessed the ability of anti-inflammatory cytokines to reduce TRM activation during NEVLP. METHODS: Rat livers underwent NEVLP with or without the addition of IL-10 and TGF-ß. Naïve and cold storage livers served as controls. Following preservation, TRM T cell gene expression profiles were assessed through single cell RNA sequencing (scRNA-seq). Differential gene expression analysis was performed with Wilcoxon rank sum test to identify differentially expressed genes (DEGs) associated with a specific treatment group. Using the online Database for Annotation, Visualization and Integrated Discovery (DAVID), gene set enrichment was then conducted with Fisher's exact test on DEGs to highlight differentially regulated pathways and functional terms associated with treatment groups. RESULTS: Through scRNA-seq analysis, an atlas of liver-resident memory T cell subsets was created for all livers. TRM T cells could be identified in all livers, and through scRNA-seq, DEG was identified with Wilcoxon rank sum test at FDR < 0.05. Based on the gene set enrichment analysis of DEGs using Fisher's exact test, NEVLP is associated with downregulation of multiple gene enrichment pathways associated with surface proteins. Furthermore, NEVLP with anti-inflammatory cytokines was associated with down regulation of 52 genes in TRM T cells when compared to NEVLP alone (FDR <0.05), most of which are pro-inflammatory. CONCLUSION: This is the first study to create an atlas of liver TRM T cells in the rat liver undergoing NEVLP and demonstrate the effects of NEVLP on liver TRM T cells at the single cell gene expression level.

The Immunological Effect of Oxygen Carriers on Normothermic Ex Vivo Liver Perfusion.

Introduction: Normothermic ex vivo liver perfusion (NEVLP) is an organ preservation method that allows liver graft functional assessment prior to transplantation. One key component of normothermic perfusion solution is an oxygen carrier to provide oxygen to the liver to sustain metabolic activities. Oxygen carriers such as red blood cells (RBCs) or hemoglobin-based oxygen carriers have an unknown effect on the liver-resident immune cells during NEVLP. In this study, we assessed the effects of different oxygen carriers on the phenotype and function of liver-resident immune cells. Methods: Adult Lewis rat livers underwent NEVLP using three different oxygen carriers: human packed RBCs (pRBCs), rat pRBCs, or Oxyglobin (a synthetic hemoglobin-based oxygen carrier). Hourly perfusate samples were collected for downstream analysis, and livers were digested to isolate immune cells. The concentration of common cytokines was measured in the perfusate, and the immune cells underwent phenotypic characterization with flow cytometry and quantitative reverse transcription polymerase chain reaction (qRT-PCR). The stimulatory function of the liver-resident immune cells was assessed using mixed lymphocyte reactions. Results: There were no differences in liver function, liver damage, or histology between the three oxygen carriers. qRT-PCR revealed that the gene expression of nuclear factor κ light chain enhancer of activated B cells (NF-kB), Interleukin (IL-1ß), C-C motif chemokine ligand 2 (CCL2), C-C motif chemokine ligand 7 (CCL7), and CD14 was significantly upregulated in the human pRBC group compared with that in the naive, whereas the rat pRBC and Oxyglobin groups were not different from that of naive. Flow cytometry demonstrated that the cell surface expression of the immune co-stimulatory protein, CD86, was significantly higher on liver-resident macrophages and plasmacytoid dendritic cells perfused with human pRBC compared to Oxyglobin. Mixed lymphocyte reactions revealed increased allogeneic T-cell proliferation in the human and rat pRBC groups compared to that in the Oxyglobin group. Conclusions: Liver-resident immune cells are important mediators of rejection after transplantation. In this study, we show that the oxygen carrier used in NEVLP solutions can affect the phenotype of these liver-resident immune cells. The synthetic hemoglobin-based oxygen carrier, Oxyglobin, showed the least amount of liver-resident immune cell activation and the least amount of allogeneic proliferation when compared to human or rat pRBCs. To mitigate liver-resident immune cell activation during NEVLP (and subsequent transplantation), Oxyglobin may be an optimal oxygen carrier.

Pharmacy information systems in Canada.

The goal of Canada Health Infoway is to provide at least 50% of all Canadians with an electronic health record (EHR) by 2010. The goal of the Infoway Drug Information Systems Program is to develop an interoperable drug information system that will keep each patient's medication history: prescribed and dispensed drugs, allergies, ongoing drug treatment, etc. Drug and drug-interaction checks will be performed automatically and added to the patients' drug profiles. Physicians and pharmacists will be supplied with data to support appropriate and accurate prescribing and dispensing, thereby avoiding adverse drug interactions and drug-related deaths [1]. This paper describes Canadian developments in pharmacy eHealth. It presents the results of the Pharmacy Informatics Pharmacy Special Networks (PSN) survey about computer systems used in hospital pharmacies across Canada including information concerning Computerized Provider Order Entry (CPOE) systems deployed; which may reduce the number of errors in orders.

Exploring the experiences of parents caring for infants with developmental dysplasia of the hip attending a dedicated clinic.

Specialised DDH (developmental dysplasion of the hip) clinics are developing around Ireland but are, however, variable in how they are operated. A DDH clinic was set up in the South-east of Ireland in 2002 with the goal of achieving an integrated care pathway between the orthopaedic surgical team and nursing team, working to an explicit protocol while also fostering a strong collaboration with the ultrasound department. This paper aims to explore the effectiveness of this dedicated clinic in the Southeast of Ireland.

Effect of Citrus Byproducts on Survival of O157:H7 and Non-O157 Escherichia coli Serogroups within In Vitro Bovine Ruminal Microbial Fermentations.

Citrus byproducts (CBPs) are utilized as a low cost nutritional supplement to the diets of cattle and have been suggested to inhibit the growth of both Escherichia coli O157:H7 and Salmonella. The objective of this study was to examine the effects in vitro that varying concentrations of CBP in the powdered or pelleted variety have on the survival of Shiga-toxin Escherichia coli (STEC) serotypes O26:H11, O103:H8, O111:H8, O145:H28, and O157:H7 in bovine ruminal microorganism media. The O26:H11, O111:H8, O145:H28, and O157:H7 serotypes did not exhibit a change in populations in media supplemented with CBP with either variety. The O103:H8 serotype displayed a general trend for an approximate 1log(10) reduction in 5% powdered CBP and 20% pelleted CBP over 6 h. There was a trend for reductions in populations of a variant form of O157:H7 mutated in the stx1 and stx2 genes in higher concentrations of CBP. These results suggest that variations exist in the survival of these serotypes of STEC within mixed ruminal microorganism fluid media when supplemented with CBP. Further research is needed to determine why CBPs affect STEC serotypes differently.