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Nanotechnology is associated with the development of particles in the nano-size range that can be used in a wide range of applications in the medical field. It has gained more importance in the pharmaceutical research field particularly in drug delivery, as it results in enhanced therapeutic drug performance, improved drug solubility, targeted drug delivery to the specific sites, minimized side effects, and prolonged drug retention time in the targeted site. To date, the application of nanotechnology continues to offer several benefits in the treatment of various chronic diseases and results in remarkable improvements in treatment outcomes. The use of nano-based delivery systems such as liposomes, micelles, and nanoparticles in pulmonary drug delivery have shown to be a promising strategy in achieving drug deposition and maintained controlled drug release in the lungs. They have been widely used to minimize the risks of drug toxicity in vivo. In this review, recent advances in the application of nano- and micro-based delivery systems in pulmonary drug delivery for the treatment of various pulmonary diseases, such as lung cancer, asthma, and chronic obstructive pulmonary disease, are highlighted. Limitations in the application of these drug delivery systems and some key strategies in improving their formulation properties to overcome challenges encountered in drug delivery are also discussed.

Background A major discrepancy between clinical and most experimental settings of liver ischemia-reperfusion injury (IRI) is the allogenicity. Methods In the current study, we first established a murine model of allogeneic orthotopic liver transplantation with extended cold ischemia time (18 h). Roles of CD4 T cells in the pathogenesis of IRI in liver allografts were determined using a depleting anti-CD4 antibody. The clinical relevance of CD4 as a marker of liver IRI was analyzed retrospectively in 55 liver transplant patients. Results CD4 depletion in both donors and recipients resulted in the most effective protection of liver allografts from IRI, as measured by serum transaminase levels and liver histology. CD4 depletion inhibited IR-induced intragraft neutrophil/macrophage infiltration and proinflammatory gene expressions. Quantitative reverse-transcriptase polymerase chain reaction analysis of human liver biopsies (2 h postreperfusion) revealed that posttransplant, rather than pretransplant, CD4 transcript levels correlated positively with proinflammatory gene expression profile. When we divided patients into subgroups according to intragraft CD4 levels, the high CD4 cohort developed a more severe hepatocellular damage than that with low CD4 levels. Conclusions CD4 T cells play a key pathogenic role in IRI of allogeneic liver transplants, and intragraft CD4 levels in the early postreperfusion phase may serve as a potential biomarker and therapeutic target to ameliorate liver IRI and improve orthotopic liver transplantation outcomes.

BACKGROUND Organ-associated pseudosarcomatous myofibroblastic proliferation (PMP) is a very rare disorder. In the urogenital tract, PMP preferentially involves the urinary bladder; kidney involvement is rare. Here, we report a rare case of PMP with ossification in the lower pole of the kidney, which mimics urothelial carcinoma or an osteogenic tumor. CASE SUMMARY A Chinese man was admitted to our hospital due to intermittent hematuria for more than 1 mo. Enhanced renal computed tomography revealed a mass in the left renal pelvis and upper ureter. The preoperative clinical diagnosis was renal pelvic carcinoma, determined by imaging examination and biopsy. After a standard preparation for surgery, the patient underwent retroperitoneoscopic radical nephroureterectomy. The operative findings were an extensive renal tumor (6 cm × 4.5 cm × 4.5 cm) invading the lower pole of the kidney and upper ureter. The final pathological diagnosis was organ-associated PMP with ossification. After 6-mo follow-up, no recurrence or metastasis was found. CONCLUSION This case of PMP was unusual for its mimicking renal pelvic carcinoma in imaging examinations, making biopsy necessary.

The fast and reliable connection between modules plays an essential role in modular steel buildings to ensure the structure's integrity. Using complete bolt assembly can better improve the efficiency of on-site construction. To study the seismic ductility reduction of connections and obtain the measures suitable for protecting the beam-column weld in connections, full-scale experiments on the connections were carried out under monotonic and cyclic loading. The failure modes, the static and hysteretic performance of the connections were manifested. The experimental results indicated the satisfactory flexural performance and the excellent energy dissipation capacity of the bolted-cover plate connections. Earthquake action would significantly reduce the ductility of connections. Both diagonal braces and flange reinforcement could dramatically improve the connection's stiffness and bearing capacity, while the flange reinforcement has better hysteretic performance than the diagonal braces reinforcement. The yield energy dissipation of the beam flange contributes most to that of the connection. These experimental studies can provide a reference in the practical design of bolted connections for modular steel buildings.

Splice connection consisting of slip-critical high strength bolt and side plate is a promising connection type for future modular construction. However, recognitions on basic performance for this connection are still limited. To fill this gap, this paper conducted a comprehensive research on basic bending behavior of a simple splice connection proposed by the authors for corner-supported steel modular building. The basic bending behavior of the proposed splice connection was analyzed in details by combined experiments and numerical simulations. It is found that the proposed splice connection can achieve a favorable ductile behavior in bending. Bilinear profile with two stages is clearly observed in the relationship between moment and connection rotation. These two stages have different stiffness but the applied load can keep increasing in the second stage with excessive deformation of the specimen. Such phenomenon reveals adequate reserve of bending resistance for the proposed splice connection under large deformation scenario. Based on method of farthest point, a transition point is defined and determined in the relationship between moment and connection rotation. This transition point represents an important change in the bending behavior, which is valuable for practical design. Numerical simulations are also introduced to supplement the tests. The analyses on stress states of components and variation in bolt pre-tension force properly explains anisotropy in resistance at the transition point for different loading directions. A parametric study is further conducted. Results indicate that the proposed splice connection possesses satisfactory tolerance for loss of bolt pre-tension force in the practical construction.

The world is fulfilled with low-quality, irregular, and widely distributed energy, but highly efficient harvesting of such high-entropy energy is hardly possible due to the limitation of currently existing technologies. Recently, triboelectric nanogenerators (TENGs) were invented to harvest random and ambient mechanical energy, and have shown wide applications because of their high efficiency and low-cost. However, the output of the TENG is dictated by the irregularity of the input mechanical agitation. This paper reports a mechanical regulator using the principle of auto-winding mechanical watch for driving the triboelectric nanogenerator with constant AC output (Constant Output TENG, CO-TENG). The device which comprised energy harvest and storage module, energy controllable release module, and energy conversion module can achieve energy harvesting-storage-release not only steadily but also continually under even low-frequency (0.5 Hz) and random working stimulation. The CO-TENG has been successfully applied to harvest the energy of random waves, wind, and water flows, through the adjustment of the input gear train and energy collection parts, such as water turbine, wind scoop, and oscillating buoy. The CO-TENG can steadily and continually produce an open-circuit voltage of 550 V, a short-circuit current of 6 μA, and transferred charges of 190 nC under low-frequency and random mechanical stimulation. With the rectifier filter, a commercial thermometer and 450 LEDs in serial connection were separately powered by the CO-TENG. It demonstrates that the CO-TENG is a potential solution for effective usage of high entropy energy that has a random amplitude and irregular low-frequency.

Acute pancreatitis (AP) is a sudden inflammatory reaction, caused by the activation of pancreatic enzymes in the pancreas, and in severe cases can lead to systemic inflammation and multiple organ failure. Oxidative stress contributed to the further deterioration of inflammation and played an important role in AP development. Bilirubin has been found to exert antioxidative, anti-inflammatory, and anti-apoptotic effects in a series of diseases accompanied by a high level of oxidative stress. However, the therapeutic effects of bilirubin for AP management have not yet been demonstrated. Additionally, the poor solubility and potential toxicity of bilirubin also limit its application. Thus, we developed bilirubin encapsulated silk fibrin nanoparticles (BRSNPs) to study the protective effects and mechanisms of bilirubin nanomedicine for the treatment of AP. BRSNPs could selectively delivery to the inflammatory lesion of the pancreas and release bilirubin in an enzyme-responsive manner. In the model of AP caused by L-Arginine hyperstimulation, BRSNPs exerted strong therapeutic effects against AP by the reduction of oxidative stress, decreased expression of pro-inflammatory cytokines, and impaired recruitment of macrophages and neutrophils. The mechanism study indicated that BRSNPs protected acinar cells against extensive oxidative damage and inflammation through inhibiting NF-κB pathway and activating the Nrf2/HO-1 pathway. Collectively, for the first time, this study demonstrated that bilirubin nanomedicine, BRSNPs, are effective in alleviating experimental acute pancreatitis, and the mechanisms are associated with its inhibition of NF-κB regulated pro-inflammatory signaling and activation of Nrf2-regulated cytoprotective protein expression.

High-power solid-state lasers with good beam quality are attracting great attention on account of their important applications in industry and military. However, the thermal effects generated in the laser host materials seriously limit power scaling and degrade the beam quality. Thermal lensing and thermally induced wavefront deformation are the main causes of the beam quality deterioration. Here we investigate the performance of a zero thermal expansion (ZTE) solid-state laser gain material. In a proof-of-principle experiment, an a -cut rod N d : Y A l O 3 (Nd:YAP) perovskite crystal is chosen to be the gain medium for ZTE around 180 K. The laser performance spanning the temperature range from 80 to 290 K is studied. The maximum output power and minimum threshold pump power were obtained at a temperature of 180 K. Moreover, the measured thermal focal power and peak-to-valley value of the wavefront distortion also reach a minimum at this temperature, an additional benefit from the crystal’s ZTE coefficient. We envisage that these results will open a new route towards the development of high-power and high-beam-quality lasers through the use of ZTE gain materials.

Liver ischemia reperfusion activates innate immune system to drive the full development of inflammatory hepatocellular injury. Damage-associated molecular patterns (DAMPs) stimulate myeloid and dendritic cells via pattern recognition receptors (PRRs) to initiate the immune response. Complex intracellular signaling network transduces inflammatory signaling to regulate both innate immune cell activation and parenchymal cell death. Recent studies have revealed that DAMPs may trigger not only proinflammatory but also immune regulatory responses by activating different PRRs or distinctive intracellular signaling pathways or in special cell populations. Additionally, tissue injury milieu activates PRR-independent receptors which also regulate inflammatory disease processes. Thus, the innate immune mechanism of liver ischemia-reperfusion injury involves diverse molecular and cellular interactions, subjected to both endogenous and exogenous regulation in different cells. A better understanding of these complicated regulatory pathways/network is imperative for us in designing safe and effective therapeutic strategy to ameliorate liver ischemia-reperfusion injury in patients.

Objective To explore the relationships of the expression of miR-145 to the clinicopathological characteristics and prognosis of patients with breast cancer complicated by type 2 diabetes mellitus (T2DM). Methods A total of 257 female patients with breast cancer were enrolled for our experiment, including 140 patients with simple breast cancer (control group) and 117 patients with breast cancer complicated by T2DM (observation group). Patients were treated with modified radical mastectomy supplemented with radiotherapy, chemotherapy and endocrine therapy. qRT-PCR was used for the detection of miR-145 expression in patients of both groups. Follow-up lasted 13-60 months. Results The relative expression of miR-145 in the observation group was significantly lower than that in the control group (pConclusions Loss of miR-145 expression is related to the development of breast cancer complicated by T2DM, and low miR-145 expression might be an adverse prognostic factor in patients with this disease.

An adjustable slab-aberration compensator (ASAC) with the ability to compensate the large magnitude inherent wavefront aberrations in the slab width direction is proposed and experimentally demonstrated. The ASAC has a size of 130mm×45mm (effective aperture of 75mm×28mm) and 11 actuators along the length with a contact spacing of 8 mm. The design is optimized by simulations in terms of the mirror’s coupling coefficient with the contact areas, mechanical properties of the driving units, and the mirror thickness. The initial surface figure of the ASAC has PV and RMS values of 55 nm and 10 nm, and the dynamic range is 30 µm. In our experiments, a 20 kW Nd: YAG quasi-continuous wave (QCW) slab laser is further compensated by the ASAC system. The beam quality increases from 15× to 3.5× diffraction limit at 20 kW output after correction. Besides, the proposed ASAC can maintain the surface shape after power shutdown and have good thermal stability. The temperature rise of the ASAC is less than 7 °C in the 20 kW laser correction experiment.

Although pretransplant diabetes is a risk factor for mortality post-liver transplant, the underlying mechanism has not been fully defined. In a murine liver partial warm ischemia model, we addressed the question of how diabetes/hyperglycemia impacted tissue inflammatory injuries against ischemia reperfusion (IR), focusing on the advanced glycation endproduct (AGE) and its receptor (RAGE) pathway. Our results showed that hepatocellular injury was exacerbated in streptozotocin-induced diabetic mice against IR, in association with hyper-inflammatory immune activation in livers. Serum levels of AGEs, but not HMGB1, were increased in diabetic mice in response to liver IR. Both RAGE antagonist peptides and small interfering RNA alleviated liver injuries and inhibited inflammatory immune activation against IR in diabetic, but not normal, mice. Kupffer cells (KCs)/macrophages, but not hepatocytes, from diabetic mice expressed significantly higher levels of RAGE, leading to their hyper-inflammatory responsiveness to both TLR ligands and AGEs. In vitro, hyperglycemia increased macrophage RAGE expression and enhanced their TLR responses. Our results demonstrated that activation of the AGE-RAGE signaling pathway in KCs was responsible for hyper-inflammatory immune responses and exacerbated hepatocellular injuries in diabetic/hyperglycemic hosts against liver IR.

The effect of reduction characteristics of microbe dissimilating of Fe(III) under different activated sludge, carbon source and anoxic/anaerobic condition was studied by activated sludge as inoculum under laboratory condition. The results showed that the maximum accumulation of Fe(Ⅱ) and the reduction rate of Fe(III) inoculated by biological iron mud were higher than that inoculated by conventional activated sludge. The reduction characteristics of Fe(III) was better in nutrient solution as carbon source than that in sewage as carbon source when inoculated biological iron mud. But the reduction characteristics of Fe(III) was better in sewage as carbon source than that in nutrient solution as carbon source when inoculated conventional activated sludge. From the whole test, the reduction characteristics of dissimilating Fe(III) microbe under anaerobic condition were better than that under anoxic condition.

BACKGROUND. Orthotopic liver transplant (OLT) is the primary therapy for end-stage liver disease and acute liver failure. However, ischemia/reperfusion injury (IRI) can severely compromise allograft survival. To understand the evolution of immune responses underlying OLT-IRI, we evaluated longitudinal cytokine expression profiles from adult OLT recipients before transplant through 1 month after transplant. METHODS. We measured the expression of 38 cytokines, chemokines, and growth factors in preoperative and postoperative recipient circulating systemic blood (before transplant and 1 day, 1 week, and 1 month after transplant) and intraoperative portal blood (before and after reperfusion) of 53 OLT patients and analyzed this expression in relation to biopsy-proven IRI (n = 26 IRI+; 27 IRI-), clinical liver function tests early (days 1-7) after transplant, and expression of genes encoding cytokine receptors in biopsies of donor allograft taken before and after reperfusion. RESULTS. Bilirubin and arginine transaminase levels early after transplant correlated with IRI. Fourteen cytokines were significantly increased in the systemic and/or portal blood of IRI+ recipients that shifted from innate to adaptive-immune responses over time. Additionally, expression of cognate receptors for 10 of these cytokines was detected in donor organ biopsies by RNAseq. CONCLUSION. These results provide a mechanistic roadmap of the early immunological events both before and after IRI and suggest several candidates for patient stratification, monitoring, and treatment. FUNDING. Ruth L. Kirschstein National Research Service Award T32CA009120, Keck Foundation award 986722, and a Quantitative & Computational Biosciences Collaboratory Postdoctoral Fellowship.

OBJECTIVE To compare the numbers of positive and total lymph nodes and prognosis in gastric cancer patients whose perigastric lymph node retrieval was performed by surgeons and pathologists. METHODS We conducted a retrospective analysis of clinical and follow-up data from 1, 056 patients who underwent gastric cancer D2 radical lymph node resection between January 2008 and December 2010 in the Gastrointestinal Surgery Department of Yantai Yuhuangding Hospital. The follow-up ended in December 2015. Patients were divided into two groups according to the specialty of physicians who performed the postoperative perigastric lymph node retrieval: the surgeon group (475 cases) and the pathologist group (581 cases). The numbers of positive and total perigastric lymph nodes and the 3- and 5-year survival were compared between gastric cancer patients in the two groups overall and stratified by TNM stage (the 7th Edition of the American Joint Committee on Cancer). RESULTS Overall, the numbers of positive and total lymph nodes were significantly higher in the surgeon group than in the pathologist group (6.53±4.07 vs. 4.09±3.70, P=0.021; 29.64±11.50 vs. 20.71±8.56, P

Ischaemia–reperfusion injury (IRI) in the liver, a major complication of haemorrhagic shock, resection and transplantation, is a dynamic process that involves the two interrelated phases of local ischaemic insult and inflammation-mediated reperfusion injury. This Review highlights the latest mechanistic insights into innate–adaptive immune crosstalk and cell activation cascades that lead to inflammation-mediated injury in livers stressed by ischaemia–reperfusion, discusses progress in large animal experiments and examines efforts to minimize liver IRI in patients who have received a liver transplant. The interlinked signalling pathways in multiple hepatic cell types, the IRI kinetics and positive versus negative regulatory loops at the innate–adaptive immune interface are discussed. The current gaps in our knowledge and the pathophysiology aspects of IRI in which basic and translational research is still required are stressed. An improved appreciation of cellular immune events that trigger and sustain local inflammatory responses, which are ultimately responsible for organ injury, is fundamental to developing innovative strategies for treating patients who have received a liver transplant and developed ischaemia–reperfusion inflammation and organ dysfunction.

Ischemia/reperfusion injury (IRI) remains unresolved problem in clinical organ transplantation. We analyzed the role of Type-I interferon (IFN) pathway in a clinically relevant murine model of extended hepatic cold preservation followed by orthotopic liver transplantation (OLT). Livers from Type-I IFN receptor (IFNAR) knockout (KO) or wild-type (WT) mice (C57/BL6) were harvested, preserved at 4°C in UW solution for 20 h and transplanted to groups of syngeneic IFNAR KO or WT recipients. Liver graft but not recipient IFNAR deficiency was required to consistently ameliorate IRI in OLTs. Indeed, disruption of Type-I IFN signaling decreased serum alanine aminotransferase (sALT) levels (p < 0.001), diminished Suzuki's score of histological OLT damage (p < 0.01) and improved 14-day survival (from 42%[5/12] in WT to 92%[11/12] in IFNAR KO; p < 0.05). Unlike in WT group, IFNAR deficiency attenuated OLT expression of TNF-α, IL-1β, IL-6, MCP-1, CXCL-10, ICAM-1; diminished infiltration by macrophages/PMNs; and enhanced expression of antioxidant HO-1/Nrf2. The frequency of TUNEL+ apoptotic cells and caspase-3 activity/expression selectively decreased in IFNAR KO group. Small interfering (si)RNA-directed targeting of HO-1 restored cardinal features of liver IRI in otherwise resistant IFNAR-deficient OLTs. Thus, intact Type-I IFN signaling is required for hepatic IRI, whereas HO-1 is needed for cytoprotection against innate immunity-dominated organ preservation damage in IFNAR-deficient liver transplants.

Nozzle flow model for high pressure variable-rate spraying is indispensable when orchard sprayer is controlling liquid flow based on Pulse Width Modulation (PWM) technology. Three flow models for Teejet AITXA 8002, 8003 and 8004 nozzles are obtained by using nozzle flow model test system which is established in this paper. The results from equation hypothesis test and test for lack of fit of flow model shows that those three flow models work well. Nozzle flow model validation trials show that the relative errors of model flow and actual flow are small, while the maximum relative error is 6.50%; the flows characteristics of different nozzles with the same type are almost the same.

The Simplified model of both calculating leakage magnetic field and mechanical force of windings at short circuit condition for a 120MVA/220kV power transformer is set up. The leakage magnetic field ‚ short-circuit force and stress in transformer windings are analyzed and their distributing characteristics are acquired respectively by using FEM. The calculation method and results are validated by withstanding short circuit strength test of the product. A number of useful suggestions are given for design of power transformers.

CD8(+) memory T cells endanger allograft survival by causing acute and chronic rejection and prevent tolerance induction. We explored the role of CD27:CD70 T-cell costimulatory pathway in alloreactive CD8(+)/CD4(+) T-cell activation. CD27-deficient (CD27(-/-)) and wild-type (WT) B6 mice rejected BALB/c cardiac allografts at similar tempo, with or without depletion of CD4(+) or CD8(+) T cells, suggesting that CD27 is not essential during primary T-cell alloimmune responses. To dissect the role of CD27 in primed effector and memory alloreactive T cells, CD27(-/-) or WT mice were challenged with BALB/c hearts either 10 or 40 days after sensitization with donor-type skin grafts. Compared to WT controls, allograft survival was prolonged in day 40- but not day 10-sensitized CD27(-/-) recipients. Improved allograft survival was accompanied by diminished secondary responsiveness of memory CD8(+) T cells, which resulted from deficiency in memory formation rather than their lack of secondary expansion. Chronic allograft vasculopathy and fibrosis were diminished in CD27(-/-) recipients of class I- but not class II-mismatched hearts as compared to WT controls. These data establish a novel role for CD27 as an important costimulatory molecule for alloreactive CD8(+) memory T cells in acute and chronic allograft rejection.

Based on the results of similar material simulation, the laws of slope movement due to mining under a gully were analyzed. Selected a slope rock as objective, the mechanisms of slope movement influence upon underground mining were proposed, and respective structural models were built by means of numerical modeling and physical simulation. It holds the point that the influence of slope movement on underground mining could be controlled to some extent by appropriate measures. The results indicate that, for gully-ward mining, which mines toward a gully, the slope rock slides horizontally and rotates in layers; for gully-away mining, which mines away from the gully, the slope rock rotates in a reversed polygon. The slope movement associated with mining under a gully is attributed to pre-existing free faces in the ground gully and underground mining-induced free faces.

Allograft rejection in sensitized recipients remains the major problem in clinical organ transplantation. We have developed a donor-type skin-sensitized mouse cardiac allograft model (BALB/c→C57BL/6) in which both rejection (30 days), paralleled by selective target gene depression at the graft site. These results provide the rationale to probe potential synergy of adjunctive therapy targeting CD4 and CD154 to overcome graft rejection in sensitized recipients.

Background Although toll-like receptor 4 (TLR4) activation has been demonstrated to play a key role in the induction of intrahepatic inflammation, leading to hepatocellular damage in liver ischemia/reperfusion injury (IRI), the nature of TLR4 ligands generated during tissue injury remains to be elucidated. We hypothesized that endogenous TLR4 ligands, rather than endotoxin (lipopolysaccharide [LPS]), are instrumental in the activation of liver TLR4 leading to local inflammation response that culminates in ultimate organ IRI. Methods and results By using the LPS-neutralizing agent, recombinant bactericidal/permeability-increasing protein, we showed that the endotoxin blockade failed to protect mouse livers from warm IRI, as assessed by serum alanine aminotransferase levels, intrahepatic inflammatory gene induction profile, and liver pathology. The recombinant bactericidal/permeability-increasing protein did not cause any hepatocytoxicity by itself if injected into normal naive mice. Furthermore, we demonstrated that liver perfusates, generated by isolated liver perfusion system, contained LPS-independent, heat-sensitive protein molecules that activated macrophages to produce tumor necrosis factor (TNF)-alpha through TLR4 but not TLR2 pathway. Conclusion This study provides a definitive evidence that endogenous TLR4 ligands are critical in the pathogenesis of liver IRI.

Ischemia/reperfusion injury (IRI) remains an important problem in clinical transplantation. Following ischemia, phosphatidylserine (PS) translocates to surfaces of endothelial cells (ECs) and promotes the early attachment of leukocytes/platelets, impairing microvascular blood flow. Diannexin, a 73 KD homodimer of human annexin V, binds to PS, prevents attachment of leukocytes/platelets to EC, and maintains sinusoidal blood flow. This study analyzes whether Diannexin treatment can prevent cold IRI in liver transplantation. Rat livers were stored at 4 degrees C in UW solution for 24 h, and then transplanted orthotopically (OLT) into syngeneic recipients. Diannexin (200 microg/kg) was infused into: (i) donor livers after recovering and before reperfusion, (ii) OLT recipients at reperfusion and day +2. Controls consisted of untreated OLTs. Both Diannexin regimens increased OLT survival from 40% to 100%, depressed sALT levels, and decreased hepatic histological injury. Diannexin treatment decreased TNF-alpha, IL-1beta, IP-10 expression, diminished expression of P-selectin, endothelial ICAM-1, and attenuated OLT infiltration by macrophages, CD4 cells and PMNs. Diannexin increased expression of HO-1/Bcl-2/Bcl-xl, and reduced Caspase-3/TUNEL+ apoptotic cells. Thus, by modulating leukocyte/platelet trafficking and EC activation in OLTs, Diannexin suppressed vascular inflammatory responses and decreased apoptosis. Diannexin deserves further exploration as a novel agent to attenuate IRI, and thereby improve OLT function/increase organ donor pool.

Background & AimsLiver graft injury and tumor recurrence are the major challenges of liver transplantation for the patients with hepatocellular carcinoma (HCC). Here, we aimed to explore the role and mechanism of liver graft injury mobilizing regulatory T cells (Tregs), which lead to late phase tumor recurrence after liver transplantation.MethodsThe correlation among tumor recurrence, liver graft injury and Tregs mobilization were studied in 257 liver transplant recipients with HCC and orthotopic rat liver transplantation models. The direct roles of CXCL10/CXCR3 signaling on Tregs mobilization and tumor recurrence were investigated in CXCL10−/− and CXCR3−/− mice models with hepatic IR injury.ResultsClinically, patients received the graft with graft weight ratio (GWR)

Nitric oxide (NO), produced via inducible NO synthase (iNOS), is implicated in the pathophysiology of liver ischemia/reperfusion injury (IRI). We examined the effects of a novel iNOS inhibitor, FK330 (FR260330), in well-defined rat liver IRI models. In a model of liver cold ischemia followed by ex vivo reperfusion, treatment with FK330 improved portal venous flow, increased bile production and decreased hepatocellular damage. FK330 prevented IRI in rat model of 40-h cold ischemia followed by syngeneic orthotopic liver transplantation (OLT), as evidenced by: (1) increased OLT survival (from 20% to 80%); (2) decreased hepatocellular damage (serum glutamic oxaloacetic transaminase/glutamic pyruvic transaminase levels); (3) improved histological features of IRI; (4) reduced intrahepatic leukocyte infiltration, as evidenced by decreased expression of P-selectin/intracellular adhesion molecule 1, ED-1/CD3 cells and neutrophils; (5) depressed lymphocyte activation, as evidenced by expression of pro-inflammatory cytokine (TNF-alpha, IL-1beta, IL-6) and chemokine (IP-10, MCP-1, MIP-2) programs; (6) prevented hepatic apoptosis and down-regulated Bax/Bcl-2 ratio. Thus, by modulating leukocyte trafficking and cell activation patterns, treatment of rats with FK330, a specific iNOS inhibitor, prevented liver IRI. These results provide the rationale for novel therapeutic approaches to maximize organ donor pool through the safer use of liver grafts despite prolonged periods of cold ischemia.

The current models of liver ischemia/reperfusion injury (IRI) in mice are largely limited to a warm ischemic component. To investigate the mechanism of hepatic "cold" IRI, we developed and validated a new mouse model of prolonged cold preservation followed by syngeneic orthotopic liver transplantation (OLT). Two hundred and forty-three OLTs with or without rearterialization and preservation in University of Wisconsin solution at 4 degrees C were performed in Balb/c mice. The 14-day survivals in the nonarterialized OLT groups were 92% (11/12), 82% (9/11), and 8% (1/12) after 1-hour, 6-hour and 24-hour preservation, respectively. In contrast, hepatic artery reconstruction after 1-hour, 6-hour, and 24-hour preservation improved the outcome as evidenced by 2-week survival of 100% (12/12), 100% (10/10), and 33% (4/12), respectively, and diminished hepatocellular damage (serum alanine aminotransferase /histology). Moreover, 24-hour (but not 1-h) cold preservation of rearterialized OLTs increased hepatic CD4+ T-cell infiltration and proinflammatory cytokine (tumor necrosis factor-alpha, interleukin 2, interferon-gamma) production, as well as enhanced local apoptosis, and Toll-like receptor 4/caspase 3 expression. These cardinal features of hepatic IRI validate the model. In conclusion, we have developed and validated a new mouse model of IRI in which hepatic artery reconstruction was mandatory for long-term animal survival after prolonged (24-h) OLT preservation. With the availability of genetically manipulated mouse strains, this model should provide important insights into the mechanism of antigen-independent hepatic IRI and help design much needed refined therapeutic means to combat hepatic IRI in the clinics.

The CD154-CD40 costimulation pathway is one of the most extensively studied in organ transplantation. However, despite impressive results reported in a number of allograft models, precise mechanisms behind its in vivo blockade remain unclear. Several issues related to its potential clinical application have been addressed, resulting in some intriguing findings, particularly on interaction with conventional immunosuppressive agents and differential effects on T cells under the influence of infections. This review focuses on recent data from rodent studies to provide new insights and builds a cohesive picture of the interlocked immune mechanisms in CD154-CD40 signaling at work in transplant recipients.

The impact of memory T cells on graft rejection and tolerance induction in transplant recipients is well recognized, particularly in clinical settings. This review discusses how alloreactive memory T cells are generated, either directly by alloantigen sensitization or indirectly by cross-reactive memory T cells against environmental antigens. Examples are provided of memory T-cell resistance to one of the most effective tolerance induction strategies today, ie, costimulation blockade, followed by a literature review on the role of memory T cells, especially of CD8 phenotype, in host immunity. The generation of distinct memory T-cell subsets, their different costimulation requirements for activation, and homeostasis are analyzed in the context of novel therapeutic strategies. Thus, modifications in current immunosuppressive regimens or adjunctive use of new agents is needed to control memory T-cell–mediated transplant rejection successfully. Indeed, comprehensive basic research on alloreactive memory T cells is warranted to develop much-needed specific strategies to overcome this seemingly formidable obstacle in the allograft rejection cascade.

We have shown that CD8+ CTLs are the key mediators of accelerated rejection, and that CD8+ T cells represent the prime targets of CD154 blockade in sensitized mouse recipients of cardiac allografts. However, the current protocols require CD154 blockade at the time of sensitization, whereas delayed treatment fails to affect graft rejection in sensitized recipients. To elucidate the mechanisms of costimulation blockade-resistant rejection and to improve the efficacy of CD154-targeted therapy, we found that alloreactive CD8+ T cells were activated despite the CD154 blockade in sensitized hosts. Comparative CD8 T cell activation study in naive vs primed hosts has shown that although both naive and primed/memory CD8+ T cells relied on the CD28 costimulation for their activation, only naive, not primed/memory, CD8+ T cells depend on CD154 signaling to differentiate into CTL effector cells. Adjunctive therapy was designed accordingly to deplete primed/memory CD8+ T cells before the CD154 blockade. Indeed, unlike anti-CD154 monotherapy, transient depletion of CD8+ T cells around the time of cardiac engraftment significantly improved the efficacy of delayed CD154 blockade in sensitized hosts. Hence, this report provides evidence for 1) differential requirement of CD154 costimulation signals for naive vs primed/memory CD8+ T cells, and 2) successful treatment of clinically relevant sensitized recipients to achieve stable long term graft acceptance.

Background Ischemia/reperfusion (I/R) injury remains an important clinical problem that affects both early and later allograft outcome. This study was designed to analyze the role of T cells and CD154-CD40 T- cell costimulation pathway in a mouse liver I/R model. Methods and results Ninety minutes of warm ischemia followed by 4 h of reperfusion in wild-type (WT) mice resulted in a significant hepatic damage, as assessed by liver function (serum alanine aminotransferase [sALT] levels), local neutrophil accumulation (myeloperoxidase activity), and histology (Suzuki's score). In contrast, T-cell deficiency (in T-cell deficient [nu/nu] mice), disruption of the CD154 signaling (in knockout [KO] mice), or its blockade in WT recipients (after MR1 monoclonal antibody [mAb] treatment), virtually prevented hepatic I/R insult. Unlike CD154-deficient T cells, adoptive transfer of WT spleen cells fully restored hepatic I/R injury in nu/nu mice. Finally, the improved hepatic function in CD154 KO recipients, WT mice treated with CD154 mAb, or nu/nu mice infused with CD154-deficient cells resulted in consistently enhanced expression of heme oxygenase-1 (HO-1), a heat-shock protein with cytoprotective functions. Conclusion This study confirms the importance of T cells, and documents for the first time the role of CD154 costimulation signals in the mechanism of hepatic I/R injury. We also show that CD154 blockade-mediated cytoprotection results and depends on HO-1 overexpression. Our data provide the rationale for human trials to target CD154-CD40 costimulation in hepatic I/R injury, particularly in the transplant patient.

INTRODUCTION Accelerated rejection (AccR) in sensitized recipients (second-set rejection) is considered a classic humorally mediated form of allograft rejection, although additional effector mechanisms may be involved. METHODS We developed a model of AccR in which C57BL6 mice are sensitized by BALB/c skin grafts, followed 10 days later by transplantation of BALB/c hearts. We undertook analysis of various humoral and cellular components in this model using knockout or monoclonal antibody-treated allograft recipients. RESULTS Sensitized mice rejected cardiac allografts in 34+/-7 hr. AccR was accompanied by endothelial deposition of immunoglobulins, complement, and fibrin, but also by dense expression of multiple chemokines and a mixed polymorphonuclear and mononuclear cellular infiltrate. Whereas neutrophil or complement depletion had no significant effect on the tempo of AccR, surprisingly B cell-deficient recipients still underwent AccR (41+/-7 hr) in conjunction with T cell and macrophage recruitment. In contrast, T cell-deficient (nude) mice maintained functioning cardiac allografts for >720 hr despite prior skin engraftment. CONCLUSIONS AccR in sensitized experimental recipients involves multiple effector pathways. Although most previous studies have emphasized the key role of humoral pathways in mediating AccR, our data indicate that T cell-dependent mechanisms can also promote AccR, alone or in conjunction with humoral responses.

Although the roles of the metabolic stress in organ ischemia-reperfusion injury (IRI) have been well recognized, the question of whether and how these stress responses regulate innate immune activation against IR remains unclear. In a murine liver partial warm ischemia mode, we showed that prolonged ischemia triggered endoplasmic reticulum (ER) stress response, particularly, the activating transcription factor 6 (ATF6) branch, in liver Kupffer cells (KCs) and altered their responsiveness against Toll-like receptor (TLR) stimulation. Ischemia-primed cells increased pro-, but decreased anti-, inflammatory cytokine productions. Alleviation of ER stress in vivo by small chemical chaperon 4-phenylbutyrate or ATF6 small interfering RNA (siRNA) diminished the pro-inflammatory priming effect of ischemia in KCs, leading to the inhibition of liver immune response against IR and protection of livers from IRI. In vitro, ATF6 siRNA abrogated the ER stress-mediated pro-inflammatory enhancement of macrophage TLR4 response, by restricting NF-κB and restoring Akt activations. Thus, ischemia primes liver innate immune cells by ATF6-mediated ER stress response. The IR-induced metabolic stress and TLR activation function in synergy to activate tissue inflammatory immune response. This study demonstrates that ischemia promotes pro-inflammatory innate immune activation by activating ATF6 signaling pathways in murine liver ischemia and reperfusion injury. © Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons.

© HUANG ETAL. Macrophages are instrumental in the pathophysiology of liver ischemia/reperfusion injury (IRI). Although Nrf2 regulates macrophage-specific heme oxygenase-1 (HO-1) antioxidant defense, it remains unknown whether HO-1 induction might rescue macrophage Nrf2-dependent antiinflammatory functions. This study explores the mechanisms by which the Nrf2–HO-1 axis regulates sterile hepatic inflammation responses after adoptive transfer of ex vivo modified HO-1 overexpressing bone marrow–derived macrophages (BMMs). Livers in Nrf2-deficient mice preconditioned with Ad-HO-1 BMMs, but not Ad-â-Gal- BMMs, ameliorated liver IRI (at 6 h of reperfusion after 90 min of warm ischemia), evidenced by improved hepatocellular function (serum alanine aminotransferase [sALT] levels) and preserved hepatic architecture (Suzuki histological score). Treatment with Ad-HO-1 BMMs decreased neutrophil accumulation, proinflammatory mediators and hepatocellular necrosis/apoptosis in ischemic livers. Moreover, Ad-HO-1 transfection of Nrf2-deficient BMMs suppressed M1 (Nos2+) while promoting the M2 (Mrc-1/Arg-1+) phenotype. Unlike in controls, Ad-HO-1 BMMs increased the expression of Notch1, Hes1, phosphorylation of Stat3 and Akt in IR-stressed Nrf2-deficient livers as well as in lipopolysaccharide (LPS)-stimulated BMMs. Thus, adoptive transfer of ex vivo generated Ad-HO-1 BMMs rescued Nrf2-dependent antiinflammatory phenotype by promoting Notch1/Hes1/Stat3 signaling and reprogramming macrophages toward the M2 phenotype. These findings provide the rationale for a novel clinically attractive strategy to manage IR liver inflammation/damage.

Activating transcription factor 3 (ATF3) is a stress-induced transcription factor that has been shown to repress inflammatory gene expression in multiple cell types and diseases. However, little is known about the roles and mechanisms of ATF3 in liver ischemia/reperfusion injury (IRI). In warm and cold liver IRI models, we showed that ATF3 deficiency significantly increased ischemia/reperfusion (IR)-stressed liver injury, as evidenced by increased serum alanine aminotransferase levels, histological liver damage, and hepatocellular apoptosis. These may correlate with inhibition of the intrahepatic nuclear factor erythroid-derived 2-related factor 2/heme oxygenase-1 (NRF2/HO-1) signaling pathway leading to enhancing Toll-like receptor 4/nuclear factor kappa beta (TLR4/NF-κB) activation, pro-inflammatory programs and macrophage/neutrophil trafficking, while simultaneously repressing anti-apoptotic molecules in ischemic liver. Interestingly, activation of NRF2/HO-1 signaling using an NRF2 activator, oltipraz (M2), during hepatic IRI-rescued ATF3 anti-inflammatory functions in ATF3-deficient mice. For in vitro studies, ATF3 ablation in lipopolysaccharide (LPS)-stimulated bone marrow-derived macrophages (BMMs) depressed levels of NRF2/HO-1 and PI3K/AKT, resulting in enhanced TLR4/NF-κB activation. Pretreatment of LPS-stimulated BMMs with M2 increased NRF2/HO-1 expression, promoted PI3K/AKT, which in turn suppressed TLR4/NF-κB-mediated proinflammatory mediators. Thus, our results first demonstrate ATF3-mediated NRF2/HO-1 signaling in the regulation of TLR4-driven inflammatory responses in IR-stressed livers. Our findings provide a rationale for a novel therapeutic strategy for managing IR-induced liver injury.

Patients with hepatocellular carcinoma (HCC) receiving living donor liver transplantation appear to possess significantly higher tumor recurrence than the recipients receiving deceased donor liver transplantation. The underlying mechanism for HCC recurrence after transplantation remains unclear. Here, we aim to investigate the impact of small-for-size liver graft injury on HCC recurrence after transplantation.The correlation between tumor recurrence, liver graft injury, CXCL10 expression and endothelial progenitor cell (EPC) mobilization was studied in 115 liver transplant recipients and rat orthotopic liver transplantation (OLT) models. The direct role of CXCL10/CXCR3 signaling on EPC mobilization was investigated in CXCL10(-/-) mice and CXCR3(-/-) mice. The role of EPCs on tumor growth and angiogenesis was further investigated in an orthotopic liver tumor model.Clinically, patients with small-for-size liver grafts (60% of standard liver weight, SLW) had significantly higher HCC recurrence (p=0.04), accompanied by more circulating EPCs and higher early-phase intragraft and plasma CXCL10 levels, than the recipients with large grafts (≥60% of SLW), which were further validated in rat OLT models. Circulatory EPC mobilization was reduced after liver injury both in CXCL10(-/-) mice and CXCR3(-/-) mice in comparison to wild-type controls. CXCL10 recruited EPCs in dose-dependent and CXCR3-dependent manners in vitro. Early-phase EPC/CXCL10 injection enhanced orthotopic liver tumor growth, angiogenesis and metastasis in nude mice.Post-transplant enhanced CXCL10/CXCR3 signaling in small-for-size liver grafts directly induced EPC mobilization, differentiation and neovessel formation, which further promotes tumor growth. Targeting CXCL10/CXCR3 signaling may attenuate early-phase liver graft injury and prevent late-phase tumor recurrence/metastasis after transplantation.

A very simple approach to quantitative analysis is proposed based on the technology of digital image processing using three-dimensional (3D) spectra obtained by high-performance liquid chromatography coupled with a diode array detector (HPLC-DAD). As the region-based shape features of a grayscale image, Zernike moments with inherently invariance property were employed to establish the linear quantitative models. This approach was applied to the quantitative analysis of three compounds in mixed samples using 3D HPLC-DAD spectra, and three linear models were obtained, respectively. The correlation coefficients (R(2)) for training and test sets were more than 0.999, and the statistical parameters and strict validation supported the reliability of established models. The analytical results suggest that the Zernike moment selected by stepwise regression can be used in the quantitative analysis of target compounds. Our study provides a new idea for quantitative analysis using 3D spectra, which can be extended to the analysis of other 3D spectra obtained by different methods or instruments.

Background Inflammatory response plays a pathogenic role in liver ischemia/reperfusion (I/R) injury. All-trans retinoic acid (ATRA) is an active metabolite of vitamin A with anti-inflammatory effects. However, there are few reports on the anti-inflammatory effects of ATRA on liver I/R injury. The purpose of this study was to investigate the effects of ATRA on liver I/R injury and related mechanisms. Methods A total of 54 male Sprague–Dawley rats were randomly divided into three groups (18 rats each), namely, sham, I/R, and I/R + ATRA groups. ATRA was intraperitoneally administered at a dose of 15 mg/kg/d 14 d before ischemia surgery. The segmental (70%) hepatic ischemia model was used by clamping the portal vein, hepatic artery, and bile duct of the left and median for 1 h. The rats were sacrificed 3, 6, and 24 h after reperfusion, and blood and liver tissue samples were obtained. Liver injury was evaluated by biochemical and histopathologic examinations. Myeloperoxidase activity was spectrophotometrically measured. The expression of pro-inflammatory cytokines, such as tumor necrosis factor-α and interleukin-6 was measured by enzyme-linked immunosorbent assay and real-time polymerase chain reaction. Liver nuclear factor kappa B (NF-κB) was detected by immunohistochemistry. The expression of NF-κB p65 and inhibitor NF-κB-α (IκBα) was determined by Western blot analysis. Results The serum alanine aminotransferase level, Suzuki scores of hepatic histology, and hepatic myeloperoxidase activity, as indices of hepatic injury, were increased after reperfusion. The increase was attenuated by preadministration with ATRA. Compared with the I/R group, ATRA treatment increased IκBα expression and suppressed NF-κB p65 expression. Subsequently, the levels of tumor necrosis factor-α and interleukin-6 after liver I/R were effectively downregulated. Conclusions ATRA administration can significantly attenuate I/R injury in rat liver. The protective mechanism is related to its anti-inflammatory function of inhibiting NF-κB activation.

The deleterious sensitization to donor MHC Ags represents one of the most challenging problems in clinical organ transplantation. Although the role of effector/memory T cells in the rejection cascade has been extensively studied, it remains unknown whether and how these 'Ag-specific' cells influence host innate immunity, such as tissue inflammation associated with ischemia and reperfusion injury (IRI). In this study, we analyzed how allogeneic skin transplant (Tx) affected the sequel of host's own liver damage induced by partial warm ischemia and reperfusion. Our data clearly showed that allo-Tx recipients had increased inflammatory response against IR insult in their native livers, as evidenced by significantly more severe hepatocelluar damage, compared with syngeneic Tx recipient controls, and determined by serum ALT levels, liver histology (Suzuki's score) and intrahepatic proinflammatory gene inductions (TNF-alpha, IL-1beta and CXCL10). The CD4 T cells, but neither CD8 nor NK cells, mediated the detrimental effect of allo-Ag sensitization in liver IRI. Furthermore, CD154, but not IFN-gamma, was the key mechanism in allo-Tx recipients to facilitate IR-triggered liver damage. These results provide new evidence that alloreactive CD4 T cells are capable of enhancing innate tissue inflammation and organ injury via an Ag-nonspecific CD154-dependent but IFN-gamma independent mechanism.

The Chinese-Russian Oil Pipeline (CROP), from Mohe to Daqing has a length of about 965 km and traverses the Da Hinggan Mountains and part of Song(hua)-Nen(jiang) Plain in Northeastern China. This paper attempts to estimate some of the important elements concerning the environmental hazards and contingency plans along the pipeline. The first part reviews the potential impacts of the Mohe-Daqing Pipeline on the cold regions environment. It includes 1) the major environmental characteristics such as complicated permafrost, rugged terrain, harsh weather, and the diverse flora and fauna, vegetation the pipeline passes through; 2) the consideration of route selection; 3) potential environmental impacts of the CROP on the environmental factors; 4) countermeasures to alleviate changes to the local environment during the design, construction and operation stages. The second part presents the risk of oil spills, the subsequent environmental challenges after the start of operation of CROP, and contingency plans in response to potential oil spills.

Although the role of CD4 T cells in tissue inflammation and organ injury resulting from ischemia and reperfusion injury (IRI) has been well documented, it remains unclear how CD4 T cells are activated and function in the absence of a specific antigen (Ag). We used a murine liver warm IRI model to determine first whether de novo Ag-specific CD4 T cell activation was required and then what its functional mechanism was. The critical role of CD4 T cells in liver immune activation against ischemia and reperfusion (IR) was confirmed in CD4 knockout mice and CD4 depleted wild-type mice. Interestingly, the inhibition of CD4 T cell activation without target cell depletion failed to protect livers against IRI, and this suggested that T cells function in liver IRI without Ag-specific de novo activation. To dissect the T cell functional mechanism, we found that CD154 blockade, but not interferon (IFN-) neutralization, inhibited local immune activation and protected livers from IRI. Furthermore, agonist anti-CD40 antibodies restored liver IRI in otherwise protected CD4-deficient hosts. Finally, fluorescence-activated cell sorting analysis of liver CD4 T cells revealed the selective infiltration of effector cells, which constitutively expressed a higher level of CD154 in comparison with their peripheral counterparts. IR triggered a significant liver increase in CD40 expression but not CD154 expression, and macrophages responded to toll-like receptor 4 and type I IFN stimulation to up-regulate CD40 expression. Conclusion: These novel findings provide evidence that CD4 T cells function in liver IRI via CD154 without de novo Ag-specific activation, and innate immunity–induced CD40 up-regulation may trigger the engagement of CD154-CD40 to facilitate tissue inflammation and injury. (HEPATOLOGY 2009; 50:1537-1546.)

Background. Although CD 154 costimulation blockade prolongs allograft survival in multiple transplantation models, the underlying immunological mechanisms remain to be elucidated. Methods and Results. We used a murine orthotopic kidney allograft (KTx) model to analyze the impact of CD154 blockade on trafficking and function of alloreactive T effector versus T regulatory cells. A single dose of MR1 Ab treatment at the time of KTx significantly improved the survival of Balb/c KTx in naive C57BL/6 recipients (mean survival time >100 days vs. 52 days in controls; P

CD40L (CD154), a member of TNF-TNFR superfamily, binds to CD40 and several integrins, and plays the key role in host immune responses [1]. Blockade of the CD40-CD40L costimulation pathway has proven to be highly effective in modulating various types of immune responses, including anti-microbial, autoimmune, alloimmune responses, allergy, as well as tissue inflammation of both antigen-specific and nonspecific types. In particular, CD154 blockade has been widely applied in organ transplant models, from rodents to primates [2]. In this review, we focus on recent literature with emphasis on in vivo immunological mechanisms.

We have documented the key role of toll-like receptor 4 (TLR4) activation and its signaling pathway mediated by interferon (IFN) regulatory factor 3, in the induction of inflammation leading to the hepatocellular damage during liver ischemia/reperfusion injury (IRI). Because type I IFN is the major downstream activation product of that pathway, we studied its role in comparison with IFN-γ. Groups of type I (IFNAR), type II (IFNGR) IFN receptor–deficient mice, along with wild-type (WT) controls were subjected to partial liver warm ischemia (90 minutes) followed by reperfusion (1-6 hours). Interestingly, IFNAR knockout (KO) but not IFNGR KO mice were protected from IR-induced liver damage, as evidenced by decreased serum alanine aminotransferase and preservation of tissue architecture. IR-triggered intrahepatic pro-inflammatory response, assessed by tumor necrosis factor (TNF-α), interleukin 6 (IL-6), and chemokine (C-X-C motif) ligand 10 (CXCL-10) expression, was diminished selectively in IFNAR KO mice. Consistent with these findings, our in vitro cell culture studies have shown that: (1) although hepatocytes alone failed to respond to lipopolysaccharide (LPS), when co-cultured with macrophages they did respond to LPS via macrophage-derived IFN-β; (2) macrophages required type I IFN to sustain CXCL10 production in response to LPS. This study documents that type I, but not type II, IFN pathway is required for IR-triggered liver inflammation/damage. Type I IFN mediates potential synergy between nonparenchyma and parenchyma cells in response to TLR4 activation. (HEPATOLOGY 2007.)

This study analyzes how toll-like receptor 4 (TLR4) signaling in the donor organ affects the ischemia and reperfusion injury (IRI) sequel following liver transplantation. Isogenic orthotopic liver transplantations (OLTs) with rearterialization were performed in groups of wild-type (WT) and TLR4 knockout (KO) mice after donor liver preservation in University of Wisconsin solution at 4 degrees C for 24 hours. Unlike WT OLTs, TLR4-deficient OLTs transplanted to either WT or TLR4 KO recipients suffered significantly less hepatocellular damage, as evidenced by serum alanine aminotransferase levels, and histological Suzuki's grading of liver IRI. Disruption of TLR4 signaling in OLTs decreased local neutrophil sequestration, CD4+ T cell infiltration, interferon (IFN)-gamma-inducible protein 10 (CXCL10) and an intercellular adhesion molecule (ICAM-1), as well as tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-2, and IFN-gamma, yet increased IL-4 and IL-10 expression. The well-functioning OLTs from TLR4 KO donors revealed attenuated activity of capase-3, and enhanced heme oygenase-1 (HO-1) expression, along with decreased levels of apoptotic endothelial cells/hepatocytes, as compared with WT OLTs with intact TLR4 signaling. Thus, the functional sentinel TLR4 complex in the donor organ plays a key role in the mechanism of hepatic IRI after OLT. Disruption of TLR4 pathway downregulated the early proinflammatory responses and ameliorated hepatic IRI. These results provide the rationale to locally modify innate TLR4 signaling in the donor organ to more efficiently control the adaptive posttransplantation IRI-dependent responses.

Ischemia/reperfusion injury (IRI) represents the major problem in clinical liver transplantation. We have shown that toll-like receptor 4 (TLR4) signaling is specifically required in initiating antigen-independent IRI leading to liver inflammation, whereas local induction of anti-oxidant heme oxygenase-1 (HO-1) is cytoprotective. This study analyzes in vivo interactions between HO-1 and sentinel TLR system in the pathophysiology of liver IRI. Using a 90-min lobar warm ischemia model, wild type (WT), TLR4 KO/mutant and TLR2 KO mice were first assessed for the severity of hepatocellular damage at 6 h postreperfusion. Unlike in WT or TLR2-deficient mice, disruption/absence of TLR4 pathway reduced IRI, as manifested by liver function (serum alanine aminotransferase levels), histology (Suzuki's scores), neutrophil infiltration (myeloperoxidase activity) and local/systemic TNF-alpha production (mRNA/protein levels). Moreover, defective TLR4 but not TLR2 signaling increased mRNA/protein HO-1 expression. In contrast, tin protoporphyrin-mediated HO-1 inhibition restored hepatic damage in otherwise IRI-resistant TLR4 mutant/KO mice. CoPP-induced HO-1 overexpression ameliorated hepatic damage in IRI-susceptible TLR2 KO mice, comparable with WT controls, and concomitantly diminished TLR4 levels. In conclusion, this study highlights the importance of cross talk between HO-1 and TLR system in the mechanism of hepatic IRI. Hepatic IRI represents a case for innate immunity in which HO-1 modulates proinflammatory responses that are triggered via TLR4 signaling, a putative HO-1 repressor.