Donor Pretreatment Research Articles

Dimethyloxalylglycine pretreatment of living donor alleviates both donor and graft liver ischemia-reperfusion injury in rats.

Background: Under the circumstance of the increasing waiting list for liver transplantation, living donor liver transplantation (LDLT) can alleviate the shortage of liver donors to some extent. However, how to reduce both donor and graft ischemia-reperfusion injury (IRI) is still an unsolved problem in LDLT. Hypoxia-induced transcription factor 1 (HIF1) activation is considered an important mechanism of cellular adaptation to hypoxia, and early activation of HIF1 may be a new way to alleviate liver IRI. Therefore, we aimed to investigate the impact of the HIF1 stabilizer dimethyloxalylglycine (DMOG) on IRI and the survival rate of donors and recipients of rat LDLT. Methods: Seventy percent partial liver resection and 30% partial liver transplantation were used to simulate donor and recipient of clinical LDLT. Rats were treated with DMOG (40mg/kg) or with an equivalent amount of saline. The expression of HIF1 and downstream targets was analyzed after 2h of reperfusion. Liver function and histopathology, apoptosis and oxidative stress levels were detected 6h after reperfusion. At the same time, the 7-day survival rate of rats was calculated. Results: DMOG pretreatment significantly reduced IR-induced injury in the donor and recipient, which was manifested by reducing liver function damage and promoting tissue recovery. Meanwhile, compared with the untreated group, the oxidative stress level and the cell apoptosis rate were decreased in the group pretreated with DMOG. In addition, the transcription and expression of HIF1 target genes in the DMOG group were significantly enhanced. Remarkably, DMOG also increased the survival rate of the recipient. Conclusion: This study provides the first evidence that DMOG pretreatment of donors significantly alleviates liver IRI in both donors and recipients and increases the survival rate of recipients in LDLT. Therefore, DMOG may be a promising strategy for improving LDLT in the future.

High-dose atorvastatin reduces oxidative stress of ischemia/reperfusion injury after isogeneic kidney transplantation in rats: in vivo, preclinical, case–control, open-label study

BackgroundRenal ischemia/reperfusion injury is an unavoidable event in transplantation in which free radical-mediated injury determines release of pro-inflammatory cytokines and activation of innate immunity. In addition to their cholesterol-lowering action, statins have shown dose-dependent pleiotropic effects on inflammatory pathways and oxidative stress. We investigated the effects of high-dose atorvastatin (atorvastatin 40 mg/kg) in preventing ischemia/reperfusion injury in an animal model of kidney transplant.MethodsForty female rats underwent left nephrectomy and orthotopic autotransplantation. Animals were divided in four groups: A = Transplant only; B = high-dose atorvastatin + Transplant; C = right nephrectomy + Transplant; D = high-dose atorvastatin + right nephrectomy + Transplant. Bilateral nephrectomy was performed 24 h post-transplant. Oxidative stress was assessed measuring malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPx) and myeloperoxidase (MPO) activity on renal tissue; ischemia/reperfusion injury was also evaluated by histology. Donor pre-treatment with high-dose atorvastatin improved oxidative stress.ResultsMDA levels were lower in group B versus A (p = 0.002) and D (p = 0.004). High-dose atorvastatin pre-treated rats displayed higher GPx activity in group B versus A (p = 0.009) and D (p = 0.005). SOD scavenger activity was also higher in group B versus A (p < 0.001) D (p < 0.001) and C (p = 0.003). MPO activity was lower in group B versus A (p = 0.02), C (p = 0.007) and D (p = 0.03). Histology revealed significantly lower rate of intratubular casts and luminal congestion in Group D versus C (p = 0.02 and p = 0.008, respectively).ConclusionsHigh-dose atorvastatin pre-treatment reduces oxidative stress and inflammation in a model of kidney transplant in the rat.

Pre-Treatment of Transplant Donors with Hydrogen Sulfide to Protect against Warm and Cold Ischemia-Reperfusion Injury in Kidney and Other Transplantable Solid Organs

Ischemia-reperfusion injury (IRI), a pathological condition resulting from prolonged cessation and subsequent restoration of blood flow to a tissue, is an inevitable consequence of solid organ transplantation. Current organ preservation strategies, such as static cold storage (SCS), are aimed at reducing IRI. However, prolonged SCS exacerbates IRI. Recent research has examined pre-treatment approaches to more effectively attenuate IRI. Hydrogen sulfide (H2S), the third established member of a family of gaseous signaling molecules, has been shown to target the pathophysiology of IRI and thus appears to be a viable candidate that can overcome the transplant surgeon's enemy. This review discusses pre-treatment of renal grafts and other transplantable organs with H2S to mitigate transplantation-induced IRI in animal models of transplantation. In addition, ethical principles of pre-treatment and potential applications of H2S pre-treatment in the prevention of other IRI-associated conditions are discussed.

Donor Pretreatment with Agonistic Anti-DR3 Antibody Prevents Chronic GvHD in a Murine Sclerodermatous Model

Background: DR3 is a cell surface receptor of the TNF superfamily and is expressed preferentially by activated T cells. The agonistic antibody of DR3 (aDR3) can stimulate regulatory T cell (Treg) expansion and ameliorate acute GVHD in a major-mismatch murine model. However, it is still unclear whether the aDR3 is effective in other GVHD models. A murine sclerodermatous GVHD model features skin fibrosis and shares many clinical characteristics with human chronic GVHD, for which currently available therapies have demonstrated limited efficacy. Methods: The αDR3 or isotype control antibody (0.5 mg/kg) was intraperitoneally injected into B10.D2 mice four days before transplantation. To generate a sclerodermatous GVHD model, lethally irradiated BALB/c recipient mice were transplanted with T cell-depleted bone marrow cells from non-treated B10.D2 mice and splenocytes isolated from the αDR3 or isotype-treated B10.D2 mice. Some Balb/c mice were transplanted with only the T cell-depleted bone marrow cells (omitting splenocytes) and served as the non-GVHD control (the control recipients). Recipient mice were monitored for six weeks to assess body weight, GVHD score, and survival or sacrificed to harvest skin, spleen, and colon on 7, 14, and 42 days after transplantation. Bronchoalveolar lavage (BAL) was performed on days 7 and 14. Results: Recipients of the aDR3-treated splenocytes (the aDR3 recipients) experienced significantly less weight loss and showed decreased GVHD scores than recipients of isotype-treated splenocytes (the isotype recipients). The severity of cutaneous GVHD was considerably reduced in the aDR3 recipients than in the isotype recipients. Pathologic examinations such as the ratio of the dermis to subcutaneous tissue in the skin and histologic scores of the colon performed on days 7, 14, and 42 post-transplant confirmed the clinical results. Flow cytometric analysis suggested that the reconstitution of B and T cells in the spleen of isotype recipients was severely disrupted during the later phase of the transplant. Although the number of CD4 T cells and B cells in the control recipients and the aDR3 recipients had been increasing until day 42, those in the isotype recipients slightly increased on day 14 but markedly decreased on day 42. Therefore, the number of CD4 T cells in the isotype recipients was significantly lower than that of the other groups on day 42, and the number of B cells in the isotype showed a significant difference on days 14 and 42. In contrast, the reconstitution of CD11b monocytes was not disrupted in the spleen of the isotype recipients. The reconstitution of splenic Treg showed a similar pattern to CD4 T cells. Interestingly, the number of Treg in the aDR3 recipients was consistently higher than that of the other groups. These findings suggest that GVHD may disrupt lymphocyte reconstitution after hematopoietic cell transplantation but treating donors with αDR3 can prevent it. Next, we performed qPCR analysis to quantitate the mRNA levels of key cytokines and chemokines in the skin and colon. The expression level of TGF-β was remarkably higher in the skin than in the colon, and the reduction of TGF-β in the αDR3 recipients was more pronounced in the skin than in the colon, confirming its primary role in skin fibrosis. In addition, the αDR3 recipients had decreased levels of IFNg, IL-6, and IL-10 in the skin and the colon than the isotype recipients. Moreover, the αDR3 recipients had diminished expression of various chemokines (CCL1, CCL2, CCL3, CCL4, CCL8, CCL17, and CCL22) in the skin compared to the isotype recipients. Chemokine expression profile in the colon was almost identical to the skin except for CCL17 and CCL22. These two cytokines were similarly reduced in the colon of the αDR3 recipients and the isotype recipients. Next, we examined inflammatory cell content in the BAL fluid. The numbers of total cells and macrophages were significantly lower in the αDR3 recipients than in the isotype recipients on days 7 and 14, but the numbers of lymphocytes and neutrophils were different between these two groups only on day 14. The αDR3 recipients had significantly fewer CD4 T cells, B cells, and CD11b monocytes in BAL fluid than the isotype recipients. Conclusions: Treating donors with αDR3 before transplantation could alleviate sclerodermatous GVHD by preventing disruption of hematopoiesis, fostering immune reconstitution, and reducing skin fibrosis and lung inflammation.

Renal inflamm-aging provokes intra-graft inflammation following experimental kidney transplantation

Donor age is a major risk factor for allograft outcome in kidney transplantation. The underlying cellular mechanisms and the recipient's immune response within an aged allograft have yet not been analyzed. A comprehensive immunophenotyping of naïve and transplanted young versus aged kidneys revealed that naïve aged murine kidneys harbor significantly higher frequencies of effector/memory T cells, whereas regulatory T cells were reduced. Aged kidney-derived CD8+ T cells produced more IFNγ than their young counterparts. Senescent renal CD8+ T and NK cells upregulated the cytotoxicity receptor NKG2D and the enrichment of memory-like CD49a+ CXCR6+ NK cells was documented in aged naïve kidneys. In the C57BL/6 to BALB/c kidney transplantation model, recipient-derived T cells infiltrating an aged graft produced significantly more IFNγ, granzyme B and perforin on day 7 post-transplantation, indicating an enhanced inflammatory, cytotoxic response towards the graft. Pre-treatment of aged kidney donors with the senolytic drug ABT-263 changed the recipient-derived effector molecule profile to significantly reduced levels of IFNγ and IL-10 compared to controls. Graft function after ABT-263 pre-treatment was significantly improved 28 days post kidney transplantation. In conclusion, renal senescence also occurs at the immunological level (inflamm-aging) and aged organs provoke an altered recipient-dominated immune response in the graft.

427.1: Donor Dendritic Cell Depletion Allows Novel Insights Into the Alloimmune Response Following Murine Composite Tissue and Skin Transplantation

Background: Cell-based therapies in vascularized composite tissue allotransplantation (VCA) have demonstrated promising results with the potential to modify life-long conventional immunosuppression. Dendritic cells (DCs) promote pro-inflammatory, alloimmune responses. Of particular interest is the role of DCs as antigen-presenting cells and potential tolerogenic effects as immature DCs. Material and Methods: Diphteria Toxin Receptor (DTR) transgenic or wild-type (WT) donor mice were used to deplete subsets of DCs or APCs respectively in a fully histoincompatible murine hind limb and skin transplant models; prior to procurement, all donors were either treated with diphteria toxin (DT; at -15 hours) or with clodronate (CL; 8 days prior to Tx). DBA/2J mice served as recipients. Study endpoint was on POD 6. Alloimmune response was assessed sequentially; graft survival, intragraft structural and inflammatory changes were tested serially. Results: The total number of dendritic cells (CD11b- CD11c+ DC) had markedly increased in recipients of skin WT grafts compared to recipients of VCA WT grafts (p<0.0001). The treatment with CL or DT significantly reduced the numbers of maturated and activated DC in the spleen, blood, and LN of mice receiving a skin graft, whereas numbers of CD11b-CD11c+MHCII+ and CD11b-CD11c+CD40+ DC were exclusively reduced in the spleen of VCA recipients after donor pretreatment with DT. cDC 1 subtypes were significantly higher in VCA WT vs. skin WT recipients (p<0.001). Donor pretreatment with DT decreased cDC1 counts only in recipients of VCA grafts (p<0.001).Luminex analysis revealed significantly higher TNF-alpha serum levels in the VCA WT vs. skin WT group (p<0.01). TNF-beta serum levels were significantly decreased in VCA depletion groups vs VCA WT group (p<0.001). Clinical signs of acute rejection among the VCA groups revealed higher rejection grades in the WT compared to the DC depletion group according to the BANFF criteria (grade III vs. grade II). Additional injection of immature DCs did not show any difference in allograft survival (p=ns).Conclusion: This is to our knowledge the first systematic study delineating the role of mature and immature DCs tested across and skin transplants. Those data may help explaining split-tolerance phenomenon in VCA while providing a basic concept for the utilization of mature and immature DCs in modifying alloimmune responses in VCA transplants.

Virus-Derived Chemokine Modulating Protein Pre-Treatment Blocks Chemokine-Glycosaminoglycan Interactions and Significantly Reduces Transplant Immune Damage.

Immune cell invasion after the transplantation of solid organs is directed by chemokines binding to glycosaminoglycans (GAGs), creating gradients that guide immune cell infiltration. Renal transplant is the preferred treatment for end stage renal failure, but organ supply is limited and allografts are often injured during transport, surgery or by cytokine storm in deceased donors. While treatment for adaptive immune responses during rejection is excellent, treatment for early inflammatory damage is less effective. Viruses have developed highly active chemokine inhibitors as a means to evade host responses. The myxoma virus-derived M-T7 protein blocks chemokine: GAG binding. We have investigated M-T7 and also antisense (ASO) as pre-treatments to modify chemokine: GAG interactions to reduce donor organ damage. Immediate pre-treatment of donor kidneys with M-T7 to block chemokine: GAG binding significantly reduced the inflammation and scarring in subcapsular and subcutaneous allografts. Antisense to N-deacetylase N-sulfotransferase1 (ASONdst1) that modifies heparan sulfate, was less effective with immediate pre-treatment, but reduced scarring and C4d staining with donor pre-treatment for 7 days before transplantation. Grafts with conditional Ndst1 deficiency had reduced inflammation. Local inhibition of chemokine: GAG binding in donor organs immediately prior to transplant provides a new approach to reduce transplant damage and graft loss.

Nitric Oxide Improves Salt Tolerance of Cyclocarya paliurus by Regulating Endogenous Glutathione Level and Antioxidant Capacity.

Cyclocarya paliurus is commonly used to treat diabetes in China. However, the natural habitats of C. paliurus are typically affected by salt stress. Previous studies showed that nitric oxide (NO) level was related to salt tolerance of C. paliurus, and its synthesis was induced by exogenous hydrogen sulfide. However, the effects of different NO donors in alleviating the negative effect of salt stress are still unclear. In the present study, C. paliurus seedlings pretreated with three NO donors (S-nitroso-N-acetylpenicillamine, SNAP and S-nitrosoglutathione, GSNO and sodium nitroprusside, SNP) were exposed to salt stress, and then, the total biomass, chlorophyll fluorescence parameters, NO and glutathione levels, oxidative damage, and antioxidant enzyme activities were investigated. The results showed that pretreatment of NO donors maintained chlorophyll fluorescence and attenuated the loss of plant biomass under salt stress, and the best performance was observed in C. paliurus under SNP treatment. We also found that pretreatment of NO donors further increased the endogenous NO content and nitrate reductase (NR) activity compared with salt treatment. Moreover, pretreatment with NO donors, especially SNP, alleviated salt-induced oxidative damage, as indicated by lowered lipid peroxidation, through an enhanced antioxidant system including glutathione accumulation and increased antioxidant enzyme activities. The supply of NO donors is an interesting strategy for alleviating the negative effect of salt on C. paliurus. Our data provide new evidence contributing to the current understanding of NO-induced salt stress tolerance.

Single Donor Infusion of S-Nitroso-Human-Serum-Albumin Attenuates Cardiac Isograft Fibrosis and Preserves Myocardial Micro-RNA-126-3p in a Murine Heterotopic Heart Transplant Model.

Objectives: Cold ischemia and subsequent reperfusion injury are non-immunologic cornerstones in the development of graft injury after heart transplantation. The nitric oxide donor S-nitroso-human-serum-albumin (S-NO-HSA) is known to attenuate myocardial ischemia-reperfusion (I/R)-injury. We assessed whether donor preservation with S-NO-HSA affects isograft injury and myocardial expression of GATA2 as well as miR-126-3p, which are considered protective against vascular and endothelial injury. Methods: Donor C57BL/6 mice received intravenous (0.1 μmol/kg/h) S-NO-HSA (n = 12), or 0.9% saline (control, n = 11) for 20 min. Donor hearts were stored in cold histidine-tryptophan-α-ketoglutarate-N solution for 12 h and underwent heterotopic, isogenic transplantation, except 5 hearts of each group, which were analysed immediately after preservation. Fibrosis was quantified and expression of GATA2 and miR-126-3p assessed by RT-qPCR after 60 days or immediately after preservation. Results: Fibrosis was significantly reduced in the S-NO-HSA group (6.47% ± 1.76 vs. 11.52% ± 2.16; p = 0.0023; 12 h-S-NO-HSA-hHTX vs. 12 h-control-hHTX). Expression of miR-126-3p was downregulated in all hearts after ischemia compared to native myocardium, but the effect was significantly attenuated when donors received S-NO-HSA (1 ± 0.27 vs. 0.33 ± 0.31; p = 0.0187; 12 h-S-NO-HSA-hHTX vs. 12 h-control-hHTX; normalized expression to U6 snRNA). Conclusion: Donor pre-treatment with S-NO-HSA lead to reduced fibrosis and preservation of myocardial miR-126-3p and GATA2 levels in murine cardiac isografts 60 days after transplantation.

Antidepressant Like Effect of Ascorbic Acid in Mice: Possible Involvement of NO-sGC-cGMP Signaling.

The present study was designed to determine the antidepressant like activity of ascorbic acid (AA) in mice. Further the influence of NO-sGC-cGMP signaling in the antidepressant like effect of AA in mice was determined. Male swiss albino mice were used in the present study. Mice in the control group received saline and fluoxetine (10mg/kg, i.p.) was used as the standard antidepressant drug. AA (50, 100 and 150mg/kg, i.p.) was administered to the mice and depression related behavior were determined using tail suspension test (TST) and forced swim test (FST). Further the whole brain nitrite and serotonin levels were also determined. It was observed that the administration of AA (100mg/kg, i.p.) reversed the depression like behavior in mice in TST and FST. AA (100mg/kg, i.p.) treatment decreased the level of nitrite and increased the level of serotonin in the brain of mice significantly as compared to control. Further the behavioral and neurochemical effect of AA (50mg/kg, i.p) was studied in NO modulator [NO donor: L-Arginine (50mg/kg, i.p); NO-sGC inhibitor: methylene blue (1mg/kg, i.p.) and cGMP modulator: sildenafil (1mg/kg, i.p.)] pretreated mice. It was observed that the pretreatment of NO donor and cGMP modulator counteracted the effect conferred by AA (50mg/kg, i.p). While the pretreatment of NO-sGC inhibitor potentiated the effect conferred by AA (50mg/kg, i.p). The present study suggested that the AA confer antidepressant like effect in mice and NO-sGC-cGMP signaling pathway influence the antidepressant like effect of AA in mice.

Current status of glucocorticoid usage in solid organ transplantation.

Glucocorticoids (GCs) have been the mainstay of immunosuppressive therapy in solid organ transplantation (SOT) for decades, due to their potent effects on innate immunity and tissue protective effects. However, some SOT centers are reluctant to administer GCs long-term because of the various related side effects. This review summarizes the advantages and disadvantages of GCs in SOT. PubMed and Scopus databases were searched from 2011 to April 2021 using search syntaxes covering “transplantation” and “glucocorticoids”. GCs are used in transplant recipients, transplant donors, and organ perfusate solution to improve transplant outcomes. In SOT recipients, GCs are administered as induction and maintenance immunosuppressive therapy. GCs are also the cornerstone to treat acute antibody- and T-cell-mediated rejections. Addition of GCs to organ perfusate solution and pretreatment of transplant donors with GCs are recommended by some guidelines and protocols, to reduce ischemia-reperfusion injury peri-transplant. GCs with low bioavailability and high potency for GC receptors, such as budesonide, nanoparticle-mediated targeted delivery of GCs to specific organs, and combination use of dexamethasone with inducers of immune-regulatory cells, are new methods of GC application in SOT patients to reduce side effects or induce immune-tolerance instead of immunosuppression. Various side effects involving different non-targeted organs/tissues, such as bone, cardiovascular, neuromuscular, skin and gastrointestinal tract, have been noted for GCs. There are also potential drug-drug interactions for GCs in SOT patients.

Possible involvement of NO-sGC-cGMP signaling in the antidepressant like effect of pyridoxine in mice

The present study was designed to determine the antidepressant like effect of pyridoxine in mice. Pyridoxine (12.5, 25 and 50mg/kg, i.p.) was administered to the mice and depression related behavioral and neurochemical alterations were determined. It was observed that pyridoxine (50mg/kg, i.p.) treatment decreased the immobility period in tail suspension test (TST) and forced swim test (FST) significantly as compared to control. Pyridoxine (50mg/kg, i.p.) treatment increased the level of serotonin (5-HT) and decreased the level of nitrite in the brain of mice significantly as compared to control. Pyridoxine thus confer antidepressant like effect by increasing the level of 5-HT and by decreasing the level of nitrite in the brain of mice. Further the influence of nitric oxide (NO)/ soluble guanylate cyclase (sGC)/ cyclic guanosine monophosphate (cGMP) in antidepressant-like effect of pyridoxine was studied. It was observed that the pretreatment of NO donor (i.e. L-Arginine) and cGMP modulator (i.e. sildenafil) counteracted while the pretreatment of NO/sGC inhibitor (i.e. methylene blue) potentiated the effect of pyridoxine in TST and FST. Pretreatment of NO donor did not influence, pretreatment of NO/sGC inhibitor decreased while the pretreatment of cGMP modulator increased the level of brain nitrite in pyridoxine treated mice. Further the pretreatment of NO donor and cGMP modulator decreased while the pretreatment of NO/sGC inhibitor increased the level of brain serotonin in pyridoxine treated mice. Pyridoxine thus exerted antidepressant like effect and NO-sGC-cGMP signaling modulated the antidepressant like effect of pyridoxine in mice.

Letter to the Editor: The Concept of Real‐Time Spectroscopy for Liver Viability Assessment

Potential conflict of interest: Nothing to report. TO THE EDITOR: We read with interest the article by Ember et al., where the investigators explored the value of Raman spectroscopy to assess liver viability in a porcine model donated after circulator death (DCD) with subsequent normothermic regional perfusion (NRP).(1) The microvascular environment was analyzed through real‐time stimulated Raman spectroscopy (SRS) and histology. Trapped erythrocytes were quantified in ischemic liver areas through the intrinsic Raman signal of hemoglobin (excitation wavelength, 532 nm).(1) The investigators found a reduction of liver congestion with less red blood cells, stuck in the sinusoids after NRP. Removal of 10% of donor blood before treatment withdrawal and circulatory death was found to be protective and resulted in healthier sinusoids with less congestion.(1) Although the article targets the hot topic of viability assessment, a few points require clarification. The investigators suggest a reduction of donor blood volume before organ donation, to decrease organ injury for transplantation. However, any donor pretreatment, including heparin administration, before withdrawal of all supportive measures, is strictly prohibited in many countries. Following treatment withdrawal, DCD donors deteriorate until circulatory death occurs, with a subsequent mandatory stand‐off period (2‐30 minutes) worldwide. Super‐rapid donor laparotomy and cannulation with cold flush or NRP are only started after donor death confirmation. Any measures, which may accelerate natural donor deterioration, including a reduction of blood volume, is therefore a violation of the donation protocol. During combined thoracic‐abdominal DCD organ procurement, the donor blood, used for normothermic ex situ heart perfusion, is obtained after circulatory death.(2) Next, although we agree that a healthy microvasculature is important, it is only one contributor to liver viability, which should target the key players of liver function: mitochondria. Ischemia leads here to metabolite accumulation and energy loss.(3) At reperfusion, reactive oxygen species and flavin mononucleotides (FMNs) are released from mitochondrial complex‐I.(4) Additionally, this autofluoresecent, mitochondrial FMN can be easily quantified from various perfusates by real‐time fluorescence spectroscopy, as recently demonstrated during hypothermic oxygenated perfusion (HOPE; Fig. 1).(4) FMN concentration in HOPE perfusates has a high predictive accuracy for graft loss and posttransplant complications and was validated by mass spectometry.(5)FIG. 1: Real‐time spectroscopy for viability assessment before liver transplantation. Real‐time viability tests are currently a hot topic in solid organ transplantation. Various spectroscopic techniques are explored on tissues and perfusates. Donor and graft assessment is a staged procedure and involves multiple unspecific tests performed from donor to recipient center.( 1‐3 ) Fluorescence spectroscopy is a valuable tool for viability assessment from perfusates, obtained during machine perfusion. Mitochondrial molecules, including FMN, are released during reoxygenation of human tissue and represent mitochondrial function and injury to predict outcomes after transplantation of high‐risk donor organs.( 4 )In summary, various spectroscopic techniques are currently tested as important tools for future viability assessment. Author Contributions M.F.C., A.S.: Conception and design. M.F.C.: Acquisition of data. M.F.C., A.S.: Analysis and interpretation of data. M.F.C.: Drafting of the article. P.M., P.D., A.S.: Critical revision for important intellectual content. M.F.C., M.M., P.M., P.D., A.S.: Final approval of the published version.

Stable colony-stimulating factor 1 fusion protein treatment increases hematopoietic stem cell pool and enhances their mobilisation in mice

BackgroundPrior chemotherapy and/or underlying morbidity commonly leads to poor mobilisation of hematopoietic stem cells (HSC) for transplantation in cancer patients. Increasing the number of available HSC prior to mobilisation is a potential strategy to overcome this deficiency. Resident bone marrow (BM) macrophages are essential for maintenance of niches that support HSC and enable engraftment in transplant recipients. Here we examined potential of donor treatment with modified recombinant colony-stimulating factor 1 (CSF1) to influence the HSC niche and expand the HSC pool for autologous transplantation.MethodsWe administered an acute treatment regimen of CSF1 Fc fusion protein (CSF1-Fc, daily injection for 4 consecutive days) to naive C57Bl/6 mice. Treatment impacts on macrophage and HSC number, HSC function and overall hematopoiesis were assessed at both the predicted peak drug action and during post-treatment recovery. A serial treatment strategy using CSF1-Fc followed by granulocyte colony-stimulating factor (G-CSF) was used to interrogate HSC mobilisation impacts. Outcomes were assessed by in situ imaging and ex vivo standard and imaging flow cytometry with functional validation by colony formation and competitive transplantation assay.ResultsCSF1-Fc treatment caused a transient expansion of monocyte-macrophage cells within BM and spleen at the expense of BM B lymphopoiesis and hematopoietic stem and progenitor cell (HSPC) homeostasis. During the recovery phase after cessation of CSF1-Fc treatment, normalisation of hematopoiesis was accompanied by an increase in the total available HSPC pool. Multiple approaches confirmed that CD48−CD150+ HSC do not express the CSF1 receptor, ruling out direct action of CSF1-Fc on these cells. In the spleen, increased HSC was associated with expression of the BM HSC niche macrophage marker CD169 in red pulp macrophages, suggesting elevated spleen engraftment with CD48−CD150+ HSC was secondary to CSF1-Fc macrophage impacts. Competitive transplant assays demonstrated that pre-treatment of donors with CSF1-Fc increased the number and reconstitution potential of HSPC in blood following a HSC mobilising regimen of G-CSF treatment.ConclusionThese results indicate that CSF1-Fc conditioning could represent a therapeutic strategy to overcome poor HSC mobilisation and subsequently improve HSC transplantation outcomes.

Efficacy and safety of fecal microbiota transplantation for treating patients with ulcerative colitis: A systematic review and meta-analysis.

To assess the effect of donor selection, stool procedures and pretreatment with antibiotics on the efficacy and safety of fecal microbiota transplantation (FMT)-treated ulcerative colitis (UC). A systematic review and meta-analysis was conducted including studies on UC treated with FMT as the primary therapeutic agent published up to June 30, 2020. Primary end-point data included clinical remission (CR) or CR combined with endoscopic remission. A total of 37 studies (seven random controlled trials [RCTs], five controlled and 25 uncontrolled cohort studies) and 959 patients with UC were enrolled. In controlled cohort studies and RCTs, FMT had a significantly greater benefit than placebo (pooled odds ratio [P-OR] 3.392, 95% CI 2.196-5.240, P < 0.001), with no heterogeneity (I2 = 0%). Furthermore, administration of FMT via the lower gastrointestinal (GI) tract was more effective in achieving CR than via the upper GI tract (44.3% vs 31.7%). The remission rate was also higher when the total stool dosage was over 275 g compared with less than 275 g (51.9% vs 29.5%). Overall, the incidence of serious adverse events of FMT was 5.9%. There was no significant difference between single and multiple donors, fresh and frozen stool sample used, and whether or not antibiotic pretreatment was administered before FMT. FMT administration via the lower GI tract and using higher dosage appear to be effective and safe in inducing remission of active UC.

Cell Therapy for Idiopathic Pulmonary Fibrosis: Rationale and Progress to Date.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by progressive lung scarring due to unknown injurious stimuli ultimately leading to respiratory failure. Diagnosis is complex and requires a combination of clinical, laboratory, radiological, and histological investigations, along with exclusion of known causes of lung fibrosis. The current understanding of the disease etiology suggests an interaction between genetic factors and epigenetic alterations in susceptible, older individuals. Prognosis is dismal and current treatment options include anti-fibrotic agents that only slow down disease progression and carry considerable side effects that hamper patients' quality of life. Therefore, the need for new, more effective treatments, alone or in combination with existing pharmacotherapy, is sorely needed. Regenerative medicine, the potential use of cell therapies to treat destructive diseases that cause architectural distortion to the target organ, has also emerged as an alternative therapeutic for lung diseases with unfavorable prognosis such as IPF. Mesenchymal stem cells (MSCs) and type II alveolar epithelial cells (AEC2s) have been used and their safety has been demonstrated. In the case of MSCs, both homogenic and allogeneic sources have been used and both are considered viable options without immunosuppressive therapy, taking into consideration the absence of immunogenicity and HLA response. AEC2s have been used in one trial with promising results but their use requires a deceased donor and immunosuppressive pre-treatment. In this review, we briefly summarize the current state of knowledge regarding the pathogenesis of IPF, and the background and rationale for using MSCs or AEC2s as potential treatment options. We list and describe the clinical trials completed to date and provide a comparison of their methods and results as well as a possible way forward.

Extended Pig-to-Baboon Lung Xenograft Survival

In baboons, lungs from galactosyl transferase knock-out (GTKO) pigs expressing human complement regulatory proteins (hCPRP: hCD46, hCD55) exhibit elevated pulmonary vascular resistance and loss of vascular barrier function within hours. Here we summarize efforts over ten years to understand and prevent these phenomena. Orthotopic left single lung transplantation was performed using GTKO.hCPRP lungs with additional expression of one or more thromboregulatory (hEPCR, hTBM, hTFPI, hCD39) and anti-inflammatory genes (HO-1, HLA-E, hCD47), and β4Gal carbohydrate xenoantigen knock-down (KD), in various combinations. Anti-inflammatory drugs (steroids, sC1Inh, anti-histamine, anti-thromboxane, A1AT, sTNFR, αIL-6, αIL-8, PSGL-1, αMac1), antibody adsorption (AbAds) using donor pig kidneys, and donor pretreatment with chlodronate liposomes were progressively added in recent years. Transient life-supporting GTKO.hCD46 lung function was consistently observed with either hEPCR (alone [n=6] or with hCD55 [6], HO-1 [2], or hCD47 [1]); with hTBM (alone [3] or with HO-1 [1] or CD47 [1]); or with hCD55.hTFPI.CD47 [11]. Although recipient survival was occasionally extended to 2 [n=6] or even 8 days [1], loss of xenograft vascular barrier function and systemic inflammation typically occurred within 24-48 hours. Consistent recipient survival beyond 12 hours was only observed with hEPCR and hTBM co-expression [n=12] in combination with one or more additional genetic modifications (HO-1 [2], CD47.HO-1 [3], hCD55 [2], hCD55.hCD39 [3], or β4GalKD.hCD47 [2]), including 7 and 9 day survivals with selectin/integrin blockade, A1AT, and liposomes. β4GalKD.hCD47, liposome pretreatment, recipient splenectomy, and AbAds were associated with prolonged recipient survival to 31 days in one instance. Graft demise beyond 2 days was association with elaboration of anti-non-Gal antibody whether using conventional or αCD40-based immunosuppression. Consistent life-supporting lung xenograft function and increasingly frequent recipient survival >2d reflect significant progress in understanding the remaining barriers to clinical translation. We identify a pivotal role for dysregulated coagulation and multiple proinflammatory mechanisms in the loss of vascular barrier function which is associated with lung xenograft failure, and the need for more effective immunosuppression.

Donor pretreatment and machine perfusion: current views.

To summarise recently published studies of donor pretreatment and machine perfusion strategies in kidney transplantation. The sparsity of donor pretreatment trials has resulted in the re-analysis of already existing data, and RCTs are urgently needed to reinvigorate this aspect of donor research. Uncontrolled donation after circulatory death kidney transplantation has the highest risk of delayed graft function and graft failure, and recent studies have reported that normothermic regional perfusion improves graft function and survival in this setting. Hypothermic machine perfusion reduces delayed graft function following deceased donor kidney transplantation across donor types but unanswered questions still remain regarding its use. The use of oxygenated hypothermic machine perfusion appears to improve graft function in controlled donation after circulatory death mediated by a reduction in acute rejection. Ex-situ normothermic perfusion is emerging and while technically challenging it may facilitate the delivery of pretreatments. RCTs are urgently needed to reinvigorate research into donor pretreatment and to establish the place of specific preservation techniques in deceased donor kidney transplantation.

Oligonucleotide-based Preconditioning of DCD Cardiac Donors and Its Impact on Cardiac Viability.

While clinical donation after circulatory death (DCD) cardiac transplantation is being implemented with increasing frequency to address the supply/demand mismatch of donor grafts, no research to date has examined a strategy of donor preconditioning to optimize the viability of DCD hearts for transplantation. In our rat model of the DCD protocol, we investigate the impact of pretreating donors with phosphorothioate-linked cytosine and guanine rich oligodeoxynucleotides (CpG ODN) and their effects on cardiac function, injury, and a novel left ventricular (LV) mRNA biomarker panel. DCD rats were subjected to a withdrawal protocol, followed by 20 minutes of warm acirculatory standoff, representing a group of severely injured hearts as previously demonstrated. Beating heart controls and DCD rats were pretreated with vehicle or stimulatory CpG ODN (beating heart control and DCD stimulated with CpG ODN, BST and DST). Hearts were harvested for ex situ heart perfusion (ESHP), where LV function, histochemical injury, and differences in gene expression were characterized between groups. Donor pretreatment with CpG ODN doubled the number of functional DCD hearts at ESHP. Pretreatment was associated with improved systolic and diastolic LV function, a reduction in histological injury, and markedly reduced elaboration of cardiac troponin-I in coronary effluent during ESHP. Pretreatment was also associated with a reduction in mRNA biomarkers associated with myocardial injury. A single dose of CpG ODN was associated with reduced biomarkers of cardiac injury and a 100% increase in cardiac viability in this rodent model of marginal DCD cardiac donation.

Blocking Complement Factor B Activation Reduces Renal Injury and Inflammation in a Rat Brain Death Model.

Introduction: The majority of kidneys used for transplantation are retrieved from brain-dead organ donors. In brain death, the irreversible loss of brain functions results in hemodynamic instability, hormonal changes and immunological activation. Recently, brain death has been shown to cause activation of the complement system, which is adversely associated with renal allograft outcome in recipients. Modulation of the complement system in the brain-dead donor might be a promising strategy to improve organ quality before transplantation. This study investigated the effect of an inhibitory antibody against complement factor B on brain death-induced renal inflammation and injury.Method: Brain death was induced in male Fischer rats by inflating a balloon catheter in the epidural space. Anti-factor B (anti-FB) or saline was administered intravenously 20 min before the induction of brain death (n = 8/group). Sham-operated rats served as controls (n = 4). After 4 h of brain death, renal function, renal injury, and inflammation were assessed.Results: Pretreatment with anti-FB resulted in significantly less systemic and local complement activation than in saline-treated rats after brain death. Moreover, anti-FB treatment preserved renal function, reflected by significantly reduced serum creatinine levels compared to saline-treated rats after 4 h of brain death. Furthermore, anti-FB significantly attenuated histological injury, as seen by reduced tubular injury scores, lower renal gene expression levels (>75%) and renal deposition of kidney injury marker-1. In addition, anti-FB treatment significantly prevented renal macrophage influx and reduced systemic IL-6 levels compared to saline-treated rats after brain death. Lastly, renal gene expression of IL-6, MCP-1, and VCAM-1 were significantly reduced in rats treated with anti-FB.Conclusion: This study shows that donor pretreatment with anti-FB preserved renal function, reduced renal damage and inflammation prior to transplantation. Therefore, inhibition of factor B in organ donors might be a promising strategy to reduce brain death-induced renal injury and inflammation.