Long-term outcomes after hypothermic oxygenated machine perfusion and transplantation of 1,202 donor livers in a real-world setting (HOPE-REAL study).

BACKGROUND & AIM: We aimed to assess long-term outcome after transplantation of HOPE-treated donor livers based on real-world data (i.e., IDEAL-D stage 4). METHODS: In this international, multicentre, observational cohort study, we collected data from adult recipients of a HOPE-treated liver transplanted between January 2012 and December 2021. Analyses were stratified for brain-dead (DBD) and circulatory-dead (DCD) donor livers, sub-divided by their respective risk categories. The primary outcome was death-censored graft survival. Secondary outcomes included the incidence of primary non-function (PNF) and ischemic cholangiopathy (IC). RESULTS: We report on 1202 liver transplantations (64% DBD) performed at 22 European centres. For DBD, a total number of 99 benchmark (8%), 176 standard (15%), and 493 extended-criteria (41%) cases were included. For DCD, 117 transplants were classified as low-risk (10%), 186 as high-risk (16%), and 131 as futile (11%), with significant risk profile variations among centres. Actuarial 1-, 3-, and 5-year death-censored graft survival for DBD and DCD was 95%, 92%, and 91%, vs. 92%, 87%, and 81%, respectively (logrank p=0.003). Within DBD and DCD-strata, death-censored graft survival was similar among risk groups (logrank p=0.26, p=0.99). Graft loss due to PNF or IC was 2.3% and 0.4% (DBD), and 5% and 4.1% (DCD). CONCLUSIONS: This study shows excellent 5-year survival after transplantation of HOPE-treated DBD and DCD livers with low rates of graft loss due to PNF or IC, irrespective of their individual risk profile. HOPE-treatment has now reached IDEAL-D stage 4, which further supports the implementation of HOPE in routine clinical practice. IMPACT AND IMPLICATIONS: This study demonstrates the excellent long-term performance of HOPE-treatment of DCD and DBD liver grafts irrespective of their individual risk profile in a real-world setting, outside the evaluation of randomized controlled trials. While previous studies have established safety, feasibility, and efficacy against the current standard, according to the IDEAL-D evaluation framework, HOPE-treatment has now reached the final IDEAL-D Stage 4, which further supports the implementation of HOPE in routine clinical practice. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT05520320.

Machine perfusion organ preservation: Highlights from the American Transplant Congress 2022.

The American Transplant Congress 2022, which took place between June 4th and June 8th of 2022, was a hybrid meeting with in-person attendance in Boston-MA and a real-time virtual experience via an online platform. First, we identified abstracts discussing machine perfusion preservation for all organs, a hot topic and approach that may develop into the new gold standard of organ preservation in the near future. A total of 39 abstracts on organ machine preservation were presented at the meeting. Next, we selected abstracts which focus on advances including new approaches to organ preservation, promising biomarkers, ex-situ treatment including cellular therapies, and novel research areas. Here, we summarized the latest developments on machine perfusion preservation in both experimental and clinical studies.

A single-center, open-label, randomized cross-over study to evaluate the pharmacokinetics and bioavailability of once-daily prolonged-release formulations of tacrolimus in de novo liver transplant recipients.

BACKGROUND: The narrow therapeutic window of tacrolimus (Tac) requires intense drug monitoring to achieve adequate efficacy while minimizing dose-related toxicities. Once-daily formulations of Tac (LCP-Tac and PR-Tac) have been recently designed for higher bioavailability and a more consistent exposure over time, as opposed to the twice-daily, administered immediate-release formulation of Tac (IR-Tac). METHODS: This single-center, open-label, randomized cross-over pharmacokinetic (PK) study compares extended-release LCP-Tac with the prolonged-release formulation of tacrolimus (PR-Tac) in adult de novo liver transplant recipients. Eligible patients were screened and randomized 1:1 to the two treatment arms up to 30 days after liver transplantation. Patients were administered either LCP-Tac or PR-Tac for 14 days followed by another 14-day time interval of the other once-daily Tac medication. A 24hr-PK profile was obtained at the end of each time interval. RESULTS: Nine patients (45%) completed the study resulting in a total of 18 Tac PK profiles. Overall, the profile of the mean concentrations indicated a flattened kinetic of LCP-Tac compared to PR-Tac, especially in the first 3 h after drug intake. The average cumulative dose per day to achieve equivalent trough levels was approximately 25% lower for LCP-Tac (8.7 mg) than for PR-Tac (11.7 mg). LCP-Tac resulted in a longer tmax and fewer peak-to-trough fluctuations compared to PR-Tac. CONCLUSION: Despite methodological weaknesses that limit the conclusions, we have found a more consistent drug exposure for LCP-Tac in de novo LT recipients. LCP-Tac demonstrated a greater bioavailability compared to PR-Tac.

An international multicenter study of protocols for liver transplantation during a pandemic: A case for quadripartite equipoise.

BACKGROUND & AIMS: The outbreak of COVID-19 has vastly increased the operational burden on healthcare systems worldwide. For patients with end-stage liver failure, liver transplantation is the only option. However, the strain on intensive care facilities caused by the pandemic is a major concern. There is an urgent need for ethical frameworks to balance the need for liver transplantation against the availability of national resources. METHODS: We performed an international multicenter study of transplant centers to understand the evolution of policies for transplant prioritization in response to the pandemic in March 2020. To describe the ethical tension arising in this setting, we propose a novel ethical framework, the quadripartite equipoise (QE) score, that is applicable to liver transplantation in the context of limited national resources. RESULTS: Seventeen large- and medium-sized liver transplant centers from 12 countries across 4 continents participated. Ten centers opted to limit transplant activity in response to the pandemic, favoring a "sickest-first" approach. Conversely, some larger centers opted to continue routine transplant activity in order to balance waiting list mortality. To model these and other ethical tensions, we computed a QE score using 4 factors - recipient outcome, donor/graft safety, waiting list mortality and healthcare resources - for 7 countries. The fluctuation of the QE score over time accurately reflects the dynamic changes in the ethical tensions surrounding transplant activity in a pandemic. CONCLUSIONS: This four-dimensional model of quadripartite equipoise addresses the ethical tensions in the current pandemic. It serves as a universally applicable framework to guide regulation of transplant activity in response to the increasing burden on healthcare systems. LAY SUMMARY: There is an urgent need for ethical frameworks to balance the need for liver transplantation against the availability of national resources during the COVID-19 pandemic. We describe a four-dimensional model of quadripartite equipoise that models these ethical tensions and can guide the regulation of transplant activity in response to the increasing burden on healthcare systems.

Autotransplantation of the Liver for Ex Vivo Resection of Intrahepatic Caval Leiomyosarcoma: A Case Report.

Intrahepatic caval leiomyosarcomas are rare tumors with limited therapeutic options as patients with the disease are not eligible for liver transplantation from the deceased-donor pool. Advances in surgical techniques gained in split and domino liver transplant procedures can be applied to resection of advanced tumors involving the hepatocaval confluence. Here, we describe the case of a 58-year-old white female who presented with visible abdominal wall collaterals and a palpable right subcostal tumor. Imaging revealed a 5.7 × 5.7 × 11-cm intrahepatic caval soft tissue mass extending into the hepatic veins, right renal vein, and infrarenal caval vein. The entire inferior caval vein was resected en bloc with the liver and right kidney and replaced with a blood group-identical fresh caval vein graft from a deceased donor. The splanchnic circulation was decompressed with a temporary portocaval shunt to the caval vein graft, and caudal inflow into the caval vein graft was established with a left iliac anastomosis. Ex vivo resection of the native inferior caval vein containing the intravascular tumor together with a sleeve of liver was performed under hypothermic conditions, and hepatic outflow was reconstructed with vein from the deceased donor. The liver was autotransplanted via the classical piggyback technique with uneventful portal reperfusion following a cold ischemic time of 2 hours. Histology confirmed a grade 3 leiomyosarcoma with clear resection margins. Liver function was stable, and the patient is currently alive at 2 years after resection. Follow-up imaging at 12 months was unremarkable, but local recurrence was detected on the most recent computed tomography scan. In conclusion, ex vivo resection of an intrahepatic caval leiomyosarcoma with inferior caval vein replacement by a deceased-donor caval graft and subsequent liver autotransplantation are technically demanding but provide a chance on prolonged survival.

Randomized Sirolimus-based Early Calcineurin Inhibitor Reduction in Liver Transplantation: Impact on Renal Function.

BACKGROUND: The long-term use of calcineurin inhibitors (CNIs) after liver transplantation (LT) is associated with nephrotoxicity. METHODS: Five-year follow-up data were retrieved from the randomized controlled multicenter SiLVER trial. Standard CNI-based mammalian target of rapamycin-free immunosuppression (group A, n = 264) was compared with a 50% reduction of CNI and introduction of the mammalian target of rapamycin inhibitor Sirolimus (SIR) within 4-6 weeks after LT (group B, n = 261). RESULTS: Median MELD at LT was low with 10 (7-15) (group A) and 11 (8-15) (group B) in the intention-to-treat approach. CNI dose and CNI trough were reduced by 20% and 8% (group A) versus 55% and 56% (group B) at 3 months posttransplantation. Renal function was preserved at 3 months after LT in the SIR arm (estimated glomerular filtration rate 74 [57-95] versus 67 [55-85] mL/min/1.73m2 P = 0.004) but was similarly impaired thereafter compared with group A. The per protocol analysis identified LT recipients in group B with concomitant early CNI minimization and SIR treatment ≥ year 1 with significantly superior estimated glomerular filtration rate and lowest rate of chronic kidney disease (≥stage 3) from year 1 onwards until study end. Competing risk factors for renal disease (arterial hypertension, fat metabolism disorder, and hyperglycemia) were not associated with worse kidney function. CONCLUSIONS: Prevention of CNI nephrotoxicity by SIR-based early CNI minimization protects renal function only short-term after LT in the intention-to-treat analysis of this low MELD cohort. Yet, selected LT recipients compliant with early CNI minimization and SIR maintenance achieved better long-term renal outcomes compared with real-world practice.

Chronic rejection after intestinal transplantation: A systematic review of experimental models.

Intestinal transplantation (ITX) constitutes a salvage treatment for irreversible intestinal failure and failure of parenteral nutrition. Chronic rejection (CR) remains the key obstacle for long-term intestinal graft survival but the pathomechanisms are incompletely understood. This study systematically reviews experimental models addressing CR after ITX in order to summarize current knowledge on CR pathogenesis and identify promising experimental strategies. A systematic literature search was conducted in line with the PRISMA guidelines, and 68 out of 677 articles qualified for the final analysis. The average methodological quality of the studies was suboptimal with 7 out of 11 points as assessed by a modified Oxford Centre for Evidence-Based Medicine score. Histology of the chronically rejected graft was almost universally integrated as outcome parameter but we found significant heterogeneity in utilized transplant techniques, organ preservation, immunosuppression and time points of CR-assessment. Several studies identified cellular and humoral immunologic mechanisms in chronic intestinal rejection. Yet, neither preventive nor therapeutic strategies against CR have been successfully introduced into human intestinal transplantation highlighting the persistent need for optimized experimental models. In this review, we aim to improve the translational value of forthcoming investigations on CR by discussing the experimental status quo and potential innovative approaches.

Role of colectomy in preventing recurrent primary sclerosing cholangitis in liver transplant recipients.

AIM: To study the published evidence on the impact of colectomy in preventing recurrent primary sclerosing cholangitis (rPSC). METHODS: An unrestricted systematic literature search in PubMed, EMBASE, Medline OvidSP, ISI Web of Science, Lista (EBSCO) and the Cochrane library was performed on clinical studies investigating colectomy in liver transplantation (LT) recipients with and without rPSC in the liver allograft. Study quality was evaluated according to a modification of the methodological index for non-randomized studies (MINORS) criteria. Primary endpoints were the impact of presence, timing and type of colectomy on rPSC. Overall presence of inflammatory bowel disease (IBD), time of IBD diagnosis, posttransplant IBD and immunosuppressive regimen were investigated as secondary outcome. RESULTS: The literature search yielded a total of 180 publications. No randomized controlled trial was identified. Six retrospective studies met the inclusion criteria of which 5 studies were graded as high quality articles. Reporting of IBD was heterogenous but in four publications, either inflammatory bowel disease, ulcerative colitis or in particular active colitis post-LT significantly increased the risk of rPSC. The presence of an intact (i.e., retained) colon at LT was identified as risk factor for rPSC in two of the high quality studies while four studies found no effect. Type of colectomy was not associated with rPSC but this endpoint was underreported (only in 33% of included studies). Neither tacrolimus nor cyclosporine A yielded a significant benefit in disease recurrence of primary sclerosing cholangitis (PSC). CONCLUSION: The data favours a protective role of pre-/peri-LT colectomy in rPSC but the current evidence is not strong enough to recommend routine colectomy for rPSC prevention.

Revascularization Time in Liver Transplantation: Independent Prediction of Inferior Short- and Long-term Outcomes by Prolonged Graft Implantation.

BACKGROUND: Strategies for successful transplantation are much needed in the era of organ shortage, and there has been a resurgence of interest on the impact of revascularization time (RT) on outcomes in liver transplantation (LT). METHODS: All primary LT performed in Birmingham between 2009 and 2014 (n = 678) with portal reperfusion first were stratified according to RT (<44 minutes vs ≥44 minutes) and graft quality (standard liver graft [SLG], Donor Risk Index < 2.3 vs marginal liver graft [MLG], Donor Risk Index ≥ 2.3). RESULTS: Revascularization time of 44 minutes or longer resulted in significantly greater incidence of early allograft dysfunction (EAD) (29% vs 47%, P < 0.001), posttransplant acute kidney injury (AKI) (39% vs 60%, P < 0.001), and new-onset AKI (37% vs 56%, P < 0.001), along with poor long-term outcome (3-year graft survival 92% vs 83%, P = 0.001; 3-year patient survival 87% vs 79%, P = 0.004). On multivariable analysis, RT ≥ 44 was a significant independent predictor of EAD, renal dysfunction, and overall graft survival, but not patient survival. The cumulative effect of prolonged revascularization in marginal grafts (MLG) resulted in the worst transplant outcome compared with all other groups, which could be mitigated by rapid revascularization (SLG, SLG, MLG vs MLG; EAD 24%, 39%, 39% vs 69%; AKI 32%, 46%, 51% vs 70%; 3-year graft survival 94%, 87%, 88% vs 70%, respectively; each P < 0.001). Factors associated with lack of abdominal space, larger grafts, and surgical skills were predictive of RT ≥ 44. CONCLUSIONS: Shorter graft revascularization is a protective factor in LT, particularly in the setting of graft marginality. Careful graft-recipient matching and emphasis on surgical expertise may aid in achieving better outcomes in LT.

Ileocecal Anastomosis Type Significantly Influences Long-Term Functional Status, Quality of Life, and Healthcare Utilization in Postoperative Crohn's Disease Patients Independent of Inflammation Recurrence.

OBJECTIVES: Anastomotic reconstruction following intestinal resection in Crohn's disease (CD) may employ side-to-side anastomosis (STSA; anti-peristaltic orientation) or end-to-end anastomosis (ETEA). Our aim was to determine the impact of these two anastomotic techniques on long-term clinical status in postoperative CD patients. METHODS: We performed a comparative effectiveness study of prospectively collected observational data from consented CD patients undergoing their first or second ileocolonic bowel resection and re-anastomosis between 2008 and 2012, in order to assess the association between anastomosis type and 2-year postoperative quality of life (QoL), healthcare utilization, disease clinical or endoscopic recurrence, use of medications, and need for repeat resection. RESULTS: One hundred and twenty eight postoperative CD patients (60 STSA and 68 ETEA) were evaluated. At 2 years postoperatively, STSA patients had higher rates of emergency department visits (33.3% vs. 14.7%; P=0.01), hospitalizations (30% vs. 11.8%; P=0.01), and abdominal computed tomography scans (50% vs. 13.2%; P<0.001) with lower QoL (mean short inflammatory bowel disease questionnaire 47.9 vs. 53.4; P=0.007). There was no difference among the two groups in the 30 day surgical complications and 2-year patterns of disease activity, CD medication requirement, endoscopic recurrence, and need for new surgical management (all P > 0.05). CONCLUSIONS: At 2 years postoperatively, CD patients with ETEA demonstrated better QoL and less healthcare utilization compared with STSA, despite having similar patterns of disease recurrence and CD treatment. These findings suggest that surgical reconstruction of the bowel as an intact tube (ETEA) contribute to improved functional and clinical status in patients with CD.

Retransplantation in Late Hepatic Artery Thrombosis: Graft Access and Transplant Outcome.

BACKGROUND: Definitive treatment for late hepatic artery thrombosis (L-HAT) is retransplantation (re-LT); however, the L-HAT-associated disease burden is poorly represented in allocation models. METHODS: Graft access and transplant outcome of the re-LT experience between 2005 and 2016 was reviewed with specific focus on the L-HAT cohort in this single-center retrospective study. RESULTS: Ninety-nine (5.7%) of 1725 liver transplantations were re-LT with HAT as the main indication (n = 43; 43%) distributed into early (n = 25) and late (n = 18) episodes. Model for end-stage liver disease as well as United Kingdom model for end-stage liver disease did not accurately reflect high disease burden of graft failure associated infections such as hepatic abscesses and biliary sepsis in L-HAT. Hence, re-LT candidates with L-HAT received low prioritization and waited longest until the allocation of an acceptable graft (median, 103 days; interquartile range, 28-291 days), allowing for progression of biliary sepsis. Balance of risk score and 3-month mortality score prognosticated good transplant outcome in L-HAT but, contrary to the prediction, the factual 1-year patient survival after re-LT was significantly inferior in L-HAT compared to early HAT, early non-HAT and late non-HAT (65% vs 82%, 92% and 95%) which was mainly caused by sepsis and multiorgan failure driving 3-month mortality (28% vs 11%, 16% and 0%). Access to a second graft after a median waitlist time of 6 weeks achieved the best short- and long-term outcome in re-LT for L-HAT (3-month mortality, 13%; 1-year survival, 77%). CONCLUSIONS: Inequity in graft access and peritransplant sepsis are fundamental obstacles for successful re-LT in L-HAT. Offering a graft for those in need at the best window of opportunity could facilitate earlier engrafting with improved outcomes.

Conception, Pregnancy, and Lactation Despite Chronic Intestinal Failure Requiring Home Parenteral Nutrition.

BACKGROUND: Short-term parenteral nutrition is commonly accepted to be safe in pregnancy, but knowledge about the management of pregnancy during long-term home parenteral nutrition (HPN) is sparse. METHODS AND RESULTS: A systematic literature review revealed that the published experience is limited to 15 pregnancies with parenteral nutrition from preconception to delivery and beyond. Maternal morbidity was surprisingly low, and fetal outcome was good; however, micronutrient deficiencies may have contributed to fetal anomalies. Herein, we additionally report the case of a 26-year-old Caucasian woman with long-term HPN dependence secondary to short bowel syndrome caused by recurrent thromboembolic mesenteric infarctions who delivered a healthy fetus at 37 weeks of gestation. Individual macronutrient support and adequate micronutrient supplementation ensured normal maternal weight gain and fetal development. Based on the individual maternal risk of recurrent thrombosis, anticoagulant treatment was carefully titrated throughout pregnancy. Furthermore, loss of abdominal domain with a rigid maternal abdominal wall secondary to short bowel syndrome and multiple laparotomies resulted in food intolerance during the third trimester. Still, with multidisciplinary efforts, both mother and the breast-fed infant were in good health at 12 months after delivery. CONCLUSIONS: Taking the reported literature into consideration, we conclude that under the premise of optimal medical care, the risk:benefit ratio for pregnancy of HPN-dependent women seems to be justifiable. To minimize the risks, we recommend preconception counseling and early referral to a tertiary center offering both a high-risk pregnancy unit and a nutrition service. In particular, maternal micronutrient levels should be monitored.

Myocyte TLR4 enhances enteric and systemic inflammation driving late murine endotoxic ileus.

Myocytes are nonhemopoietic in origin and functionally essential in generating gastrointestinal motility. In endotoxemia, a rapid-onset nonhemopoietic mechanism potently triggers early ileus in a Toll-like receptor 4 (TLR4)/myeloid differentiation primary response gene 88 (MyD88)-dependent manner. Moreover, synergistically with hemopoietic cells, nonhemopoietic cells escalate late ileus via an IL-6 receptor-dependent inflammation-driven pathway. We therefore specifically investigated the role of myocytes in TLR4-triggered inflammation and ileus. TLR4(+/+), TLR4(-/-), bmTLR4(+/+)/TLR4(-/-) chimera, SM22-Cre(-/-)TLR4(flox/flox), and selective myocyte TLR4-deficient (SM22-Cre(+/-)TLR4(flox/flox)) mice were injected intraperitoneally with purified lipopolysaccharide. SM22-driven Cre recombinase activity was selectively detected in cardiac, gastrointestinal, skeletal, and vascular myocytes, of small-sized vessels in a two-color fluorescent Cre reporter mouse. In contrast to nonhemopoietic TLR4 deficiency, deletion of myocyte TLR4 signaling prevented neither endotoxin-induced suppression of spontaneous jejunal contractility in vitro nor early ileus in vivo at 6 h. Circulating plasma colony-stimulating factor 3 was greatly elevated during endotoxemia, independent of myocyte TLR4 signaling or time. TLR4 activation of myocytes contributed significantly to an early enteric IL-6 mRNA induction and systemic IL-6 release, as well as to a late increase in circulating chemokine (C-X-C motif) ligand 1 (CXCL1) and IL-17. Consequently, inhibition of myocyte TLR4 signaling allowed functional recovery of motility by preventing inflammation-driven late ileus at 24 h. Direct TLR4 activation of myocytes is not responsible for nonhemopoietic-mediated early ileus. However, myocytes are proinflammatory cells that potently drive enteric and systemic inflammation, subsequently fueling late mediator-triggered ileus. Specifically, the myocyte TLR4-dependent inflammatory signature of elevated plasma IL-6, CXCL1, and IL-17 is strongly associated with late rodent ileus.

Differential molecular and cellular immune mechanisms of postoperative and LPS-induced ileus in mice and rats.

Ileus is caused by the initiation of a complex cascade of molecular and cellular inflammatory responses within the intestinal muscularis, which might be species specific. Our objective was to investigate a possible immunological divergence in the mechanisms of postoperative- and endotoxin-induced ileus in C57BL/6 mice and Sprague-Dawley rats. Gastrointestinal transit (GIT) was measured at 24 h after the injurious stimulus. MPO-staining and F4/80 immunohistochemistry were used to quantify polymorphonuclear and monocyte infiltration of jejunal muscularis whole-mounts, and intestinal muscularis MCP-1, ICAM-1 and iNOS gene expression was assessed by RT-PCR. Intestinal muscularis subjected to in vivo surgical manipulation (SM) or LPS treatment was cultured for 24 h, and the liberation of nitric oxide and chemokines/cytokines into the culture medium was analyzed by Griess reaction and Luminex multiplex assay. Intestinal SM and lipopolysaccharide (LPS) (15 mg/kg) caused a significant delay in gastrointestinal transit, which was more severe in mice compared to rats in both injury models. Both SM- and LPS-triggered neutrophil and monocytic extravasation into the rat jejunal muscularis exceeded the cellular infiltration seen in mice. These results correlated with significantly greater increases in rat muscularis MCP-1 (syn. CCL2), ICAM-1 and iNOS message with more subsequent NO production after SM or LPS compared to mouse. The cultured muscularis obtained from SM mice released significantly more inflammatory proteins such as TNF-α, IL-1-α, IL-4 and GM-CSF compared to the manipulated rat muscularis. In contrast, LPS initiated the secretion of significantly more IL-1ß by the inflamed rat muscularis compared to the mouse, but GM-CSF (syn. CSF2) liberation from mouse muscularis was markedly higher compared to LPS-treated rat muscularis. The data indicate that mechanistically the development of ileus in rat is mediated predominately through a leukocytic pathway consisting of chemotaxis, cellular extravasation and NO liberation. Whereas, the more intense mouse ileus evolves via a potent but injury-specific local cytokine response.

Hydrogen-enriched preservation protects the isogeneic intestinal graft and amends recipient gastric function during transplantation.

BACKGROUND: Inhaled hydrogen gas exerts antioxidant and anti-inflammatory effects in rat intestinal transplantation. Here, we investigated whether ex vivo donor organ treatment with dissolved hydrogen would prevent intestinal graft injury. METHODS: Isogeneic intestinal transplantation was performed in Lewis rats with vascular flush, luminal preservation, and cold graft storage in nitrogen-bubbled (SITxN2) or hydrogen-bubbled (SITxH2) preservation solution. Lactated Ringer's solution and 3-hr cold ischemia time were used for mechanistic investigations, whereas survival experiments were performed with University of Wisconsin solution and 6-hr cold ischemia time. RESULTS: During the early phase of ischemia-reperfusion injury, hydrogen-enriched solution significantly preserved mucosal graft morphology, diminished graft malondialdehyde levels demonstrating substantial reduction potential and blunted proinflammatory molecular responses (early growth response gene [EGR-1], interleukin [IL]-6, IL-1ß, and inducible nitric oxide synthase) within the reperfused intestinal graft muscularis. During the late phase of ischemia-reperfusion injury, circulating IL-6 protein and lactate dehydrogenase levels were significantly ameliorated in SITxH2 animals, which were associated with a favorable functional outcome in in vivo liquid gastrointestinal transit and recipient solid gastric emptying of chrome steel balls, and marked prevention of the posttransplant associated suppression of in vitro muscarinic jejunal contractility. Reflecting improved graft preservation, hydrogen preloading of grafts increased recipient survival rates from 41% to 80%. Anti-inflammatory and antiapoptotic heme oxygenase-1 was significantly upregulated in the hydrogen-treated graft muscularis but not mucosa before reperfusion. CONCLUSIONS: Graft preloading with hydrogen demonstrated superior morphologic and functional graft protection in rodent intestinal transplantation, ultimately facilitating recipient survival. Antioxidant capacity and muscularis heme oxygenase-1 upregulation are possible protective mechanisms.

Hydrogen inhalation ameliorates ventilator-induced lung injury.

INTRODUCTION: Mechanical ventilation (MV) can provoke oxidative stress and an inflammatory response, and subsequently cause ventilator-induced lung injury (VILI), a major cause of mortality and morbidity of patients in the intensive care unit. Inhaled hydrogen can act as an antioxidant and may be useful as a novel therapeutic gas. We hypothesized that, owing to its antioxidant and anti-inflammatory properties, inhaled hydrogen therapy could ameliorate VILI. METHODS: VILI was generated in male C57BL6 mice by performing a tracheostomy and placing the mice on a mechanical ventilator (tidal volume of 30 ml/kg without positive end-expiratory pressure, FiO(2) 0.21). The mice were randomly assigned to treatment groups and subjected to VILI with delivery of either 2% nitrogen or 2% hydrogen in air. Sham animals were given same gas treatments for two hours (n = 8 for each group). The effects of VILI induced by less invasive and longer exposure to MV (tidal volume of 10 ml/kg, 5 hours, FiO(2) 0.21) were also investigated (n = 6 for each group). Lung injury score, wet/dry ratio, arterial oxygen tension, oxidative injury, and expression of pro-inflammatory mediators and apoptotic genes were assessed at the endpoint of two hours using the high-tidal volume protocol. Gas exchange and apoptosis were assessed at the endpoint of five hours using the low-tidal volume protocol. RESULTS: Ventilation (30 ml/kg) with 2% nitrogen in air for 2 hours resulted in deterioration of lung function, increased lung edema, and infiltration of inflammatory cells. In contrast, ventilation with 2% hydrogen in air significantly ameliorated these acute lung injuries. Hydrogen treatment significantly inhibited upregulation of the mRNAs for pro-inflammatory mediators and induced antiapoptotic genes. In the lungs treated with hydrogen, there was less malondialdehyde compared with lungs treated with nitrogen. Similarly, longer exposure to mechanical ventilation within lower tidal volume (10 mg/kg, five hours) caused lung injury including bronchial epithelial apoptosis. Hydrogen improved gas exchange and reduced VILI-induced apoptosis. CONCLUSIONS: Inhaled hydrogen gas effectively reduced VILI-associated inflammatory responses, at both a local and systemic level, via its antioxidant, anti-inflammatory and antiapoptotic effects.

Mechanical strain and TLR4 synergistically induce cell-specific inflammatory gene expression in intestinal smooth muscle cells and peritoneal macrophages.

Mechanical trauma of the gut is an unavoidable event in abdominal surgery. Former studies demonstrated that intestinal manipulation induces a strong inflammation within the tunica muscularis. We hypothesized that mechanical strain initiates or aggravates proinflammatory responses in intestinal smooth muscle cells (iSMC) or macrophages. First, an appropriate isolation and culture method for neonatal rat iSMC was established. Purified iSMC and primary peritoneal macrophages (pMacs) were subjected to static or cyclic strain, and gene expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), IL-6, and IL-1ß was analyzed by quantitative PCR. Supernatants from stretched iSMC were transferred to untreated pMacs or contrariwise, and medium transfer-triggered inflammatory gene expression was measured in unstretched cells. Finally, we investigated the synergistic effect of static strain on LPS-induced proinflammatory gene expression. Although cyclic strain failed, static strain significantly induced iNOS, COX-2, and IL-1ß mRNA in iSMC. pMacs showed an increase in all inflammatory genes investigated as well as macrophage inflammatory protein (MIP)-1α and MIP-2 mRNA after static strain. Both cell entities liberated unknown mediators in response to stretch that mutually stimulated iNOS gene expression. Finally, mechanostimulation amplified LPS-induced iNOS and IL-1ß gene expression in iSMC as well as COX-2 and IL-6 mRNA in pMacs. In conclusion, static strain initiates proinflammatory gene expression in iSMC and pMacs and triggers a bidirectional paracrine communication between both cultured cell entities via the liberation of unknown mediators. Furthermore, static strain synergistically operates with Toll-like receptor 4 ligation in a cell-specific manner. Hence, this study demonstrates that mechanical strain functions as an immunomodulatory stimulus in abdominal cells.

Intraesophageal manganese superoxide dismutase-plasmid liposomes ameliorates novel total-body and thoracic radiation sensitivity of NOS1-/- mice.

The effect of deletion of the nitric oxide synthase 1 gene (NOS1(-/-)) on radiosensitivity was determined. In vitro, long-term cultures of bone marrow stromal cells derived from NOS1(-/-) were more radioresistant than cells from C57BL/6NHsd (wild-type), NOS2(-/-) or NOS3(-/-) mice. Mice from each strain received 20 Gy thoracic irradiation or 9.5 Gy total-body irradiation (TBI), and NOS1(-/-) mice were more sensitive to both. To determine the etiology of radiosensitivity, studies of histopathology, lower esophageal contractility, gastrointestinal transit, blood counts, electrolytes and inflammatory markers were performed; no significant differences between irradiated NOS1(-/-) and control mice were found. Video camera surveillance revealed the cause of death in NOS1(-/-) mice to be grand mal seizures; control mice died with fatigue and listlessness associated with low blood counts after TBI. NOS1(-/-) mice were not sensitive to brain-only irradiation. MnSOD-PL therapy delivered to the esophagus of wild-type and NOS1(-/-) mice resulted in equivalent biochemical levels in both; however, in NOS1(-/-) mice, MnSOD-PL significantly increased survival after both thoracic and total-body irradiation. The mechanism of radiosensitivity of NOS1(-/-) mice and its reversal by MnSOD-PL may be related to the developmental esophageal enteric neuronal innervation abnormalities described in these mice.

Dominant role of the MyD88-dependent signaling pathway in mediating early endotoxin-induced murine ileus.

TLR4 ligation by pathogen-associated molecular patterns, such as Gram-negative bacteria-derived LPS, triggers a nonhematopoietic cell-mediated ileus during early endotoxemia. Our objective was to investigate the quantitative contributions of the two downstream signaling pathways of TLR4, namely the adapter proteins myeloid differentiation primary response gene 88 (MyD88) and Toll-IL-1-resistance (TIR) domain-containing adaptor-inducing IFN-beta (TRIF). Six hours after intraperitoneal injection of highly purified LPS (UP-LPS, 5 mg/kg), in vivo gastrointestinal transit and intestinal muscularis gene transcripts of inflammatory mediators chemokine (C-X-C motif) ligand 10, synonymous IP-10 (CXCL10), granulomonocyte colony stimulating factor (GM-CSF, synonymous CSF-2), IL-1beta, IL-6, IL-10, and inducible NO synthase (iNOS) were assessed in mice with transgenic loss-of-function for MyD88 or TRIF. LPS-induced MyD88 and TRIF mRNA upregulation was quantified within the intestinal muscularis of TLR4-competent and TLR4-mutant mice, and MyD88 mRNA levels were additionally measured in TLR4 bone marrow chimeras. MyD88 deficiency completely protected mice from early endotoxin-induced ileus, while TRIF deficiency partially ameliorated ileus severity. LPS induction of the primary downstream signaling element MyD88 was TLR4 dependent and was derived in equal amounts from both the hematopoietic and the nonhematopoietic cells. Conversely, no induction of TRIF mRNA was detectable. Significant gene induction of all inflammatory mediators was dependent on intracellular signal transduction by MyD88, while the TRIF MyD88-independent pathway predominantly regulated the molecular levels of CXCL10. In summary, MyD88 and TRIF are nonredundant signaling pathways in early endotoxin-induced rodent ileus, but MyD88 is the essential adaptor molecule for transduction of early TLR4-induced ileus and inflammatory signaling. The dependency of ileus on individual adaptor protein pathways is also reflected in the manifestation of specific molecular inflammatory events within the intestinal muscularis externa.

Amelioration of rat cardiac cold ischemia/reperfusion injury with inhaled hydrogen or carbon monoxide, or both.

BACKGROUND: Recent advances in novel medical gases, including hydrogen and carbon monoxide (CO), have demonstrated significant opportunities for therapeutic use. This study was designed to evaluate the effects of inhaled hydrogen or CO, or both, on cold ischemia/reperfusion (I/R) injury of the myocardium. METHODS: Syngeneic heterotopic heart transplantation was performed in rats after 6 or 18 hours of cold ischemia in Celsior solution. Survival, morphology, apoptosis and marker gene expression were assessed in the grafts after in vivo inhalation of hydrogen (1% to 3%), CO (50 to 250 ppm), both or neither. Both donors and recipients were treated for 1 hour before and 1 hour after reperfusion. RESULTS: After 6-hour cold ischemia, inhalation of hydrogen (>2%) or CO (250 ppm) alone attenuated myocardial injury. Prolonged cold ischemia for 18 hours resulted in severe myocardial injury, and treatment with hydrogen or CO alone failed to demonstrate significant protection. Dual treatment with hydrogen and CO significantly attenuated I/R graft injury, reducing the infarcted area and decreasing in serum troponin I and creatine phosphokinase (CPK). Hydrogen treatment alone significantly reduced malondialdehyde levels and serum high-mobility group box 1 protein levels as compared with air-treated controls. In contrast, CO only marginally prevented lipid peroxidation, but it suppressed I/R-induced mRNA upregulation for several pro-inflammatory mediators and reduced graft apoptosis. CONCLUSIONS: Combined therapy with hydrogen and CO demonstrated enhanced therapeutic efficacy via both anti-oxidant and anti-inflammatory mechanisms, and may be a clinically feasible approach for preventing cold I/R injury of the myocardium.