Contribution of alloantigens to hepatic ischemia/reperfusion injury: Roles of natural killer cells and innate immune recognition of nonself.

Hepatic ischemia/reperfusion injury (IRI) remains a major clinical problem and involves the innate immune system's recognition of "nonself." Considering the efficient nonself recognition by natural killer (NK) cells, we hypothesize in this study that hepatic IRI associated with liver transplantation (LT) could be augmented in allogeneic rather than in syngeneic (Syn) grafts due to alloantigen recognition by innate immune cells, especially by NK cells. Using green fluorescent protein (GFP)/Sprague-Dawley rats, we tested our hypothesis in a rat LT model with 18 hours of cold storage in University of Wisconsin solution. Hepatic IRI was significantly augmented in allografts with higher alanine transaminase levels, increased necrosis, and vigorous proinflammatory mediator up-regulation compared to Syn grafts. Injury increased in allografts associated with augmented GFP+ host leukocyte infiltration due to significantly increased host CD11b/c+ and RP-1(+) neutrophil recruitment. A large number of liver-resident (donor) mature CD11b/c+ NK cells quickly diminished from allografts, but not from Syn grafts. Depletion of mature NK cells from liver grafts with anti-asialo monosialotetrahexosylganglioside significantly improved hepatic IRI and reduced neutrophil infiltration and proinflammatory mediators. In conclusion, early innate immune responses were more significantly enhanced in allografts than in Syn grafts during hepatic IRI, in part through NK cell recognition of "missing self."

Laparoscopic distal gastrectomy for pyloric stenosis caused by heterotopic glands in a young female: report of a case.

A 17-year-old female was referred to our hospital with worsening dietary intake and abdominal bloating. She had epigastric fullness, but no abdominal pain. Gastrointestinal endoscopy revealed food residue and pyloric stenosis. A contrast-enhanced radiograph also showed pyloric stenosis, and gastrografin was not passed well through her pylorus. Computed tomography revealed similar findings. The biopsy results indicated hyperplasia of the gastric glands. The patient was diagnosed with a benign lesion, and underwent endoscopic balloon dilation several times. However, her stenosis worsened and we decided to perform surgery. In consideration of the cosmetic outcome, we performed laparoscopic distal gastrectomy. The postoperative course was good, and the patient was discharged on postoperative day 10. The final diagnosis was pyloric stenosis caused by heterotopic glands. No malignant lesions were found. Since gastric stenosis caused by heterotopic glands has not been reported previously, we consider this to be a very rare case.

Roles of dendritic cells in murine hepatic warm and liver transplantation-induced cold ischemia/reperfusion injury.

UNLABELLED: Dendritic cells (DCs) induce and regulate both innate and adaptive immune responses; however, their in vivo functional importance in hepatic ischemia/reperfusion (IR) injury is perplexing. We hypothesized that liver-resident DC and locally recruited blood-borne DC might have distinctive roles in hepatic IR injury. We tested this hypothesis by using DC-deficient, fms-like tyrosine kinase 3 ligand (Flt3L) knockout (KO) mice in hepatic warm (70% partial clamping for 60 minutes) and cold IR injury (liver transplant [LTx] with 24-hour cold storage). Flt3L KO liver and lymphoid organs contained virtually no CD11c+ F4/80- DC. Hepatic warm IR injury was significantly lower in Flt3L KO than in wildtype (WT) mice with lower alanine aminotransferase (ALT) levels, reduced hepatic necrosis, and lower neutrophil infiltration. Hepatic messenger RNA (mRNA) and protein levels for inflammatory cytokines (tumor necrosis factor alpha [TNFα], interleukin [IL]-6) and chemokines (CCL2, CXCL2) were also significantly lower in Flt3L KO than in WT mice, indicating that lack of both liver-resident and blood-borne DC ameliorated hepatic warm IR injury. Adoptive transfer of splenic or hepatic WT DC into Flt3L KO or WT mice increased hepatic warm IR injury, suggesting injurious roles of DC infusion. When Flt3L KO liver was transplanted into WT mice, ALT levels were significantly higher than in WT to WT LTx, with enhanced hepatic necrosis and neutrophil infiltration, indicating a protective role of liver-resident DC. CONCLUSION: Using both warm and cold hepatic IR models, this study suggests differential roles of liver-resident versus blood-borne DC, and points to the importance of the local microenvironment in determining DC function during hepatic IR injury.

[A case of an elderly patient with recurrent GIST, which was effectively treated with low-dose imatinib mesylate].

The recommended dose of imatinib for recurrent gastrointestinal stromal tumors (GIST) is 400mg/day. However, adverse effects limit the use of the standard dose in elderly patients. We report a case of an elderly patient with recurrent GIST, where long-term control of the disease was achieved with low-dose imatinib therapy. An 86-year-old man presenting with tarry stool was admitted to the hospital; upper GI endoscopy revealed a gastric submucosal tumor of the stomach at the posterior wall of the cardia. Partial gastrectomy was performed laparoscopically. The submucosal lesion was histopathologically diagnosed as malignant GIST. Administration of imatinib was initiated 17 months after surgery because of recurrence of GIST. The initial dose of imatinib was 400mg/day, which was later adjusted to 200mg or 300 mg/day because of adverse effects. Though imatinib was withdrawn several times due to strong side effects, the disease was well controlled for 6 years after surgery.

Conversion surgery after lenvatinib treatment for multiple lung metastases from hepatocellular carcinoma.

Although systemic treatment for hepatocellular carcinoma has advanced after the development of tyrosine kinase inhibitors such as sorafenib and lenvatinib, the effectiveness of a single tyrosine kinase inhibitor in survival extension of unresectable hepatocellular carcinoma is limited to a few months. Therefore, novel treatment options are required for unresectable hepatocellular carcinomas, including those with multiple lung metastases. This case report describes a hepatocellular carcinoma patient with a recurrence of multiple lung metastases, which was successfully treated with conversion pneumonectomy after treatment with tyrosine kinase inhibitors. A 79-year-old man underwent right hepatectomy for hepatocellular carcinoma, along with removal of the tumor thrombus in the inferior vena cava. Multiple lung metastases were detected 4 months after hepatectomy. Treatment with tyrosine kinase inhibitors, mainly lenvatinib, resulted in complete remission of the lung metastases, except for one lesion in segment 3 of the right lung which gradually enlarged. Twenty-three months after hepatectomy, partial resection of the right lung was performed using video-assisted thoracic surgery for this residual lesion in the right lung. The patient remained disease-free for 11 months after conversion pneumonectomy, without any adjuvant therapies. This is the first case report of multiple lung metastases originating from hepatocellular carcinoma which were successfully treated with conversion pneumonectomy after treatment with tyrosine kinase inhibitors. Conversion pneumonectomy after systemic therapy with tyrosine kinase inhibitors should be considered as a treatment strategy for patients with unresectable multiple lung metastases from hepatocellular carcinomas.

The loss of renal dendritic cells and activation of host adaptive immunity are long-term effects of ischemia/reperfusion injury following syngeneic kidney transplantation.

Ischemia/reperfusion injury associated with kidney transplantation induces profound acute injury, influences early graft function, and affects long-term graft outcomes. To determine whether renal dendritic cells play any role during initial innate ischemia/reperfusion injury and the subsequent development of adaptive immune responses, we studied the behavior and function of renal graft and host infiltrating dendritic cells during early and late phases of renal ischemia/reperfusion injury. Wild type to green fluorescent protein (GFP) transgenic rat kidney transplantation was performed with and without 24-h cold storage. Ischemia/reperfusion injury in cold-stored grafts resulted in histopathological changes of interstitial fibrosis and tubular atrophy by 10 weeks, accompanied by upregulation of mRNAs of mediators of interstitial fibrosis and inflammation. In normal rat kidneys, we identified two populations of renal dendritic cells, predominant CD103(-)CD11b/c(+) and minor CD103(+)CD11b/c(+) cells. After transplantation without cold storage, grafts maintained CD103(-) but not CD103(+) GFP-negative renal dendritic cells for 10 weeks. In contrast, both cell subsets disappeared from cold-stored grafts, which associated with a significant GFP-expressing host CD11b/c(+) cell infiltration that included CD103(+) dendritic cells with a TNF-α-producing phenotype. These changes in graft/host dendritic cell populations were associated with progressive infiltration of host CD4(+) T cells with effector/effector-memory phenotypes and IFN-γ secretion. Thus, renal graft ischemia/reperfusion injury caused graft dendritic cell loss and was associated with progressive host dendritic cell and T-cell recruitment. Renal-resident dendritic cells might function as a protective regulatory network.

Interferon regulatory factor-2 is protective against hepatic ischemia-reperfusion injury.

Interferon regulatory factor (IRF)-1 is a nuclear transcription factor that induces inflammatory cytokine mediators and contributes to hepatic ischemia-reperfusion (I/R) injury. No strategies to mitigate IRF1-mediated liver damage exist. IRF2 is a structurally similar endogenous protein that competes with IRF1 for DNA binding sites in IRF-responsive target genes and acts as a competitive inhibitor. However, the role of IRF2 in hepatic injury during hypoxic or inflammatory conditions is unknown. We hypothesize that IRF2 overexpression may mitigate IRF1-mediated I/R damage. Endogenous IRF2 is basally expressed in normal livers and is mildly increased by ischemia alone. Overexpression of IRF2 protects against hepatic warm I/R injury. Furthermore, we demonstrate that IRF2 overexpression limits production of IRF1-dependent proinflammatory genes, such as IL-12, IFNß, and inducible nitric oxide synthase, even in the presence of IRF1 induction. Additionally, isograft liver transplantation with IRF2 heterozygote knockout (IRF2(+/-)) donor grafts that have reduced endogenous IRF2 levels results in worse injury following cold I/R during murine orthotopic liver transplantation. These findings indicate that endogenous intrahepatic IRF2 protein is protective, because the IRF2-deficient liver donor grafts exhibited increased liver damage compared with the wild-type donor grafts. In summary, IRF2 overexpression protects against I/R injury by decreasing IRF1-dependent injury and may represent a novel therapeutic strategy.

Hepatic B7 homolog 1 expression is essential for controlling cold ischemia/reperfusion injury after mouse liver transplantation.

UNLABELLED: Ischemia/reperfusion (I/R) injury remains a key risk factor significantly affecting morbidity and mortality after liver transplantation (LT). B7 homolog 1 (B7-H1), a recently identified member of the B7 family, is known to play important roles in regulating local immune responses. We hypothesized that B7-H1 plays crucial roles during innate immune responses induced by hepatic I/R injury, and using B7-H1 knockout (KO) liver grafts, we tested this hypothesis in the mouse LT model with 24 hours of cold storage. Cold I/R injury in wild type (WT)-to-WT LT enhanced constitutive B7-H1 expression on dendritic cells and sinusoidal endothelial cells and promptly induced B7-H1 on hepatocytes. When B7-H1 KO liver grafts were transplanted into WT recipients, serum alanine aminotransferase (ALT) and graft necrosis levels were significantly higher than those after WT-to-WT LT. Augmented tissue injury in B7-H1 KO grafts was associated with increased frequencies and absolute numbers of graft CD3(+) T cells (particularly CD8(+) T cells). B7-H1 KO grafts had significantly fewer annexin V(+) CD8(+) T cells, and this indicated a failure to delete infiltrating CD8(+) T cells. To evaluate the relative contributions of parenchymal cell and bone marrow-derived cell (BMDC) B7-H1 expression, we generated and transplanted into WT recipients chimeric liver grafts lacking B7-H1 on parenchymal cells or BMDCs. A selective B7-H1 deficiency on parenchymal cells or BMDCs resulted in similar levels of ALT and liver injury, and this suggested that parenchymal cell and BMDC B7-H1 expression was involved in liver damage control. Human livers up-regulated B7-H1 expression after LT. CONCLUSION: The study demonstrates that graft tissue expression of B7-H1 plays a critical role in regulating inflammatory responses during LT-induced hepatic I/R injury, and negative coregulatory signals may have an important function in hepatic innate immune responses.

Carbon monoxide inhibits apoptosis during cold storage and protects kidney grafts donated after cardiac death.

Ischemia/reperfusion (I/R) injury remains as a serious deleterious factor in kidney transplantation (KTx). We hypothesized that carbon monoxide (CO), an endogenous potent cytoprotective molecule, inhibits hypothermia-induced apoptosis of kidney grafts. Using the rat KTx model mimicking the conditions of donation after cardiac death (DCD) as well as nontransplantable human kidney grafts, this study examined effects of CO in preservation solution in improving the quality of marginal kidney grafts. After cardiac cessation, rat kidneys underwent 40 min warm ischemia (WI) and 24 h cold storage (CS) in control UW or UW containing CO (CO-UW). At the end of CS, kidney grafts in control UW markedly increased mitochondrial porin release into the cytosol and resulted in increased cleaved caspase-3 and PARP expression. In contrast, grafts in CO-UW had significantly reduced mitochondrial breakdown and caspase pathway activation. After KTx, recipient survival significantly improved with CO-UW with less TUNEL(+) cells and reduced mRNA upregulation for proinflammatory mediators (IL-6, TNF-α, iNOS). Furthermore, when nontransplantable human kidney grafts were stored in CO-UW for 24 h, graft PARP expression, TUNEL(+) cells, and proinflammatory mediators were less than those in control UW. CO in UW inhibited hypothermia-induced apoptosis and significantly improved kidney graft function and outcomes of KTx.

Critical role of interferon regulatory factor-1 in murine liver transplant ischemia reperfusion injury.

Interferon regulatory factor-1 (IRF-1) is a transcription factor that regulates gene expression during immunity. We hypothesized that IRF-1 plays a pivotal role in liver transplant (LTx) ischemia/reperfusion (I/R) injury. Mouse orthotopic LTx was conducted after 24 hours cold storage in University of Wisconsin (UW) solution in wildtype (WT) C57BL/6 and IRF-1 knockout (KO) mice. IRF-1 deficiency in liver grafts, but not in recipients, resulted in significant reduction of hepatocyte apoptosis and liver injury, as well as improved survival. IRF-1 mRNA up-regulation was typically seen in graft hepatocytes in WT-->WT LTx. Deficiency of IRF-1 signaling in graft resulted in significantly reduced messenger RNA (mRNA) levels for death ligands and death receptors in hepatocytes, as well as decreased caspase-8 activities, indicating that IRF-1 mediates death ligand-induced hepatocyte death. Further, a smaller but significant IRF-1 mRNA up-regulation was seen in WT graft nonparenchymal cells (NPC) and associated with interferon gamma (IFN-gamma) mRNA up-regulation exclusively in NPC. IFN-gamma mRNA was significantly reduced in IRF-1 KO graft. Thus, IRF-1 in graft hepatocytes and NPC has distinct effects in hepatic I/R injury. However, LTx with chimeric liver grafts showed that grafts lacking hepatocellular IRF-1 had better protection compared with those lacking IRF-1 in NPC. The study identifies a critical role for IRF-1 in liver transplant I/R injury.

Liver graft exposure to carbon monoxide during cold storage protects sinusoidal endothelial cells and ameliorates reperfusion injury in rats.

Hepatic ischemia/reperfusion (I/R) injury significantly influences short-term and long-term outcomes after liver transplantation (LTx). The critical step initiating the injury is known to include sinusoidal endothelial cell (SEC) alteration during the cold preservation period. As carbon monoxide (CO) has potent cytoprotective functions on vascular endothelial cells, this study examined if CO treatment of excised liver grafts during cold storage could protect SECs and ameliorate hepatic I/R injury. Rat liver grafts were preserved in University of Wisconsin (UW) solution containing 5% CO (CO-UW solution) for 18 to 24 hours and were transplanted into syngeneic Lewis rats. After 18 hours of cold preservation, SEC damage was evident with propidium iodide (PI) nuclear staining on SECs, and the frequency of PI(+) SECs was significantly lower in grafts stored in CO-UW solution versus those stored in control UW solution. SEC protection with CO was associated with decreased intercellular cell adhesion molecule translocation and less matrix metalloproteinase release during cold preservation. After LTx with 18 hours of cold preservation, serum alanine aminotransferase levels and hepatic necrosis were significantly less in the CO-UW group than in the control UW group. With 24 hours of cold storage, 35% (7/20) survived with control UW solution, whereas the survival with CO-UW solution improved to 80% (8/10). These beneficial effects of CO-UW solution were associated with a significant reduction of neutrophil extravasation, down-regulation of hepatic messenger RNA for tumor necrosis factor alpha and intercellular cell adhesion molecule 1, and less hepatic extracellular signal-regulated kinase activation. Liver grafts from Kupffer cell-depleted donors or pseudogerm-free donors showed less SEC death during cold preservation, and CO-UW solution further reduced SEC death. In conclusion, CO delivery to excised liver grafts during cold preservation efficiently ameliorates SEC damage and hepatic I/R injury.

Inhibition of Kupffer cell-mediated early proinflammatory response with carbon monoxide in transplant-induced hepatic ischemia/reperfusion injury in rats.

UNLABELLED: Proinflammatory responses play critical roles in hepatic ischemia/reperfusion (I/R) injury associating with liver transplantation (LTx), and carbon monoxide (CO) can effectively down-regulate them. Using wild-type (WT) to enhanced green fluorescent protein (EGFP)-transgenic rat LTx with 18-hour cold preservation in University of Wisconsin solution, this study analyzed the relative contribution of donor and host cells during early posttransplantation period and elucidated the mechanism of hepatic protection by CO. CO inhibited hepatic I/R injury and reduced peak alanine aminotransferase levels at 24 hours and hepatic necrosis at 48 hours. Abundant EGFP(+) host cells were found in untreated WT liver grafts at 1 hour and included nucleated CD45(+) leukocytes (myeloid, T, B, and natural killer cells) and EGFP(+) platelet-like depositions in the sinusoids. However, reverse transcription polymerase chain reaction (RT-PCR) analysis of isolated graft nonparenchymal cells (NPCs) revealed that I/R injury-induced proinflammatory mediators [for example, tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS)] were not up-regulated in purified CD45(+) cells of donor or host origin. Instead, TNF-alpha and IL-6 messenger RNA (mRNA) elevation was exclusively seen in isolated CD68(+) cells, whereas iNOS mRNA up-regulation was seen in hepatocytes. Nearly all CD68(+) cells at 1 hour after LTx were EGFP(-) donor Kupffer cells, and CO efficiently inhibited TNF-alpha and IL-6 up-regulation in the CD68(+) Kupffer cell fraction. When graft Kupffer cells were inactivated with gadolinium chloride, activation of inflammatory mediators in liver grafts was significantly inhibited. Furthermore, in vitro rat primary Kupffer cell culture also showed significant down-regulation of lipopolysaccharide (LPS)-induced inflammatory responses by CO. CONCLUSION: These results indicate that CO ameliorates hepatic I/R injury by down-regulating graft Kupffer cells in early postreperfusion period. The study also suggests that different cell populations play diverse roles by up-regulating distinctive sets of mediators in the acute phase of hepatic I/R injury.

The novel NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin, prevents local and remote organ injury following intestinal ischemia/reperfusion in rats.

BACKGROUND: Nuclear factor-kappaB regulates the expression of several genes involved in inflammation, the immune response, apoptosis, cell survival, and proliferation. Many of these same genes are activated during ischemia/reperfusion (I/R) injury. Here, we examined the anti-inflammatory efficacy of a newly developed nuclear factor-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), in the intestinal I/R injury model of rats. MATERIALS AND METHODS: Intestinal ischemia was induced by occluding the superior mesenteric artery for 60 min. The experimental animals were divided into two groups: untreated group, control; treated group, DHMEQ-treated (20 mg/kg). DHMEQ were administered intraperitoneally at 60 min prior to clamping and 5 min prior to reperfusion. Animal survival rates, intestinal tissue blood flow, serum levels of tumor necrosis factor-alpha, and interleukin-6, and the histopathology of both the intestine and the lung were analyzed. RESULTS: The DHMEQ-treated animals exhibited higher values of intestinal tissue blood flow and suppression of tumor necrosis factor-alpha and interleukin-6 production, resulting in marked prolongation of their survival times. Histopathological findings obtained by examining tissues from control animals revealed severe intestinal mucosal damage and disruption of the lung alveolar architecture accompanied by hemorrhage and marked neutrophilic infiltration. These findings were significantly ameliorated in DHMEQ-treated animals. CONCLUSION: DHMEQ effectively prevented both intestine and lung injuries in rat intestinal I/R models. This agent may possess a good potency for clinical application in various pathological settings including intestinal I/R and/or inflammatory acute lung injury.

Laparoscopic gastrectomy for early gastric cancer targeting as a less invasive procedure.

BACKGROUND: Since only a few extensive reports are available on the less invasive nature of laparoscopic gastrectomy, we compared postoperative changes over time in vital signs and hematological parameters between this surgery and laparotomic gastrectomy. METHODS: Of 188 patients who underwent distal gastrectomy for preoperatively diagnosed early gastric cancer between January 2004 and September 2006, 87 underwent laparoscopy-assisted distal gastrectomy (LADG) and 101 underwent laparotomic distal gastrectomy (DG). The invasiveness of the two procedures was evaluated in 164 patients with no postoperative complications (82 cases of LADG and 82 cases of DG by measuing vital signs daily and performing hematological examination on postoperative days (POD) 1, 4, 7, and 10. RESULTS: For body temperature, heart rate, and blood pressure, significantly lower values were obtained with LADG on 3 and 4 POD, 4 POD, and 3 and 4 POD, respectively. For white blood cell counts (WBC) and C-reactive protein (CRP), significantly lower values were obtained with LADG on 7 and 10 POD, and 10 POD, respectively. For serum protein levels and lymphocyte counts, significantly higher values were obtained with LADG on 1, 4, 7, and 10 POD, and 4 and 10 POD, respectively. Body temperature, WBC, and CRP showed no significant difference immediately after surgery but earlier recovery occurred with LADG. For protein levels and lymphocyte counts, higher values were obtained immediately after surgery. There seemed to be two patterns of less invasiveness in the parameters: the early recovery found for body temperature, WBC and CRP, and the smaller shift immediately after surgery in protein level and lymphocyte count, and probably, heart rate and blood pressure. The complication rate was 18.8% for DG and 5.7% for LADG. CONCLUSIONS: LADG is a less-invasive surgical procedure as it produces early normalization or smaller shifts in various parameters and exhibits a low prevalence of complications.

A novel fully human anti-CD40 monoclonal antibody, 4D11, for kidney transplantation in cynomolgus monkeys.

BACKGROUND: CD40-CD154 pathway blockade by anti-CD154 monoclonal antibodies (mAbs) significantly prolongs allograft survival in nonhuman primates. However, thromboembolic complications have prevented clinical application. Thus, blockade of the counter molecule by a novel fully human anti-CD40 mAb, 4D11, is an attractive alternative. METHODS: Kidney transplantations were performed between outbred cynomolgus monkeys (stimulation index >3 in a mixed lymphocyte reaction). The animals were divided into five groups: nontreatment control (Group 1, n=3), 10-week treatment with either 10 mg/kg (Group 2, n=3), 20 mg/kg (Group 3, n=3), or 40 mg/kg (Group 4, n=1), and 4-week treatment (Group 5, n=1 each) with 10 mg/kg, 20 mg/kg, or 40 mg/kg followed by monthly administration. Graft survival, biochemistry, complete blood counts, lymphocyte phenotypes, blood drug levels, antidonor and antidrug antibodies, and renal histology were examined. RESULTS: Survival (days) was as follows: Group 1 (5, 6, 7), Group 2 (150, 108, 108), Group 3 (84, 108, 379), Group 4 (147), and Group 5 (147, 102, 112). Two animals in Group 3 with normal graft function were killed upon development of hydronephrosis and cerebral infarction. B lymphocytes fell to one-third of the preoperative value at 4 weeks after transplantation in all animals. Antidonor antibodies developed in most of the animals after stopping drug treatment or at the time of death. No animals except for one formed anti-4D11 antibody. CONCLUSION: 4D11 appears to be a promising agent for antirejection treatment in clinical organ transplantation.

Control of allograft rejection by applying a novel nuclear factor-kappaB inhibitor, dehydroxymethylepoxyquinomicin.

BACKGROUND: Nuclear factor (NF)-kappaB plays a crucial role in lymphocyte activation, proliferation, and survival. We examined the immunosuppressive effect of a newly developed NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ) in allotransplantation. METHODS: Purified C57BL/6 (H-2b) T cells were used for in vitro studies examining activation, proliferation, cytokine production and nuclear NF-kappaB and nuclear factor of activated T cells (NFAT) protein levels. A fully major histocompatibility complex incompatible BALB/c (H-2d)-to-C57BL/6 mice cardiac transplantation model was utilized for in vivo studies. DHMEQ was given intraperitoneally to transplant recipients at a various dose starting from day 0. In some, DHMEQ was administered concomitantly with tacrolimus. RESULTS: DHMEQ significantly suppressed alphaCD3 + alphaCD28 monoclonal antibody-triggered T-cell proliferation, CD25/CD69 expressions, and both interleukin-2 and interferon (IFN)-gamma production in a dose-dependent fashion. DHMEQ blocked nuclear translocation of NF-kappaB but not NFAT in activated T cells. Combined treatment with DHMEQ and tacrolimus significantly suppressed T cell activation as compared to that of mono-therapy with either agent alone. Single DHMEQ treatment moderately prolonged cardiac allograft survival. Further, combination of DHMEQ plus tacrolimus markedly prolonged graft mean survival time (MST) to 59.5 days when compared to either DHMEQ (MST: 10 days) or tacrolimus (MST: 13 days) treatment alone. Such effect was associated with inhibition of mixed lymphocyte reaction against donor antigen, IFN-gamma producing splenocytes and graft cellular infiltration as examined at 5 and 12 days posttransplantation. CONCLUSION: DHMEQ inhibits nuclear translocation of NF-kappaB but not NFAT in activated T cells, and prolongs allograft survival. Blocking both NF-kappaB and NFAT by DHMEQ and tacrolimus induces potent immunosuppression, which may become a new modality in controlling allograft rejection.

Orthotopic mouse liver transplantation to study liver biology and allograft tolerance.

Orthotopic liver transplantation in the mouse is a powerful research tool that has led to important mechanistic insights into the regulation of hepatic injury, liver immunopathology, and transplant tolerance. However, it is a technically demanding surgical procedure. Setup of the orthotopic liver transplantation model comprises three main stages: surgery on the donor mouse; back-table preparation of the liver graft; and transplant of the liver into the recipient mouse. In this protocol, we describe our procedure in stepwise detail to allow efficient completion of both the donor and recipient operations. The protocol can result in consistently high technical success rates when performed by personnel experienced in the protocol. The technique can be completed in ∼2-3 h when performed by an individual who is well practiced in performing mouse transplantation in accordance with this protocol. We have achieved a perioperative survival rate close to 100%.

Immunomodulatory effect of nuclear factor-κB inhibition by dehydroxymethylepoxyquinomicin in combination with donor-specific blood transfusion.

BACKGROUND: Nuclear factor-κB (NF-κB) is a key molecule in alloimmune responses, however, its role in tolerance induction is not clear. We have previously reported that dehydroxymethylepoxyquinomycin (DHMEQ), a novel NF-κB inhibitor, prolongs cardiac allograft survival. In this study, we evaluated the immunomodulatory effects of DHMEQ when combined with a donor-specific blood transfusion (DST), and assessed whether the treatment induces tolerance in a mouse heart transplantation model. METHODS: DST (20×10 splenocytes) was given intravenously at day -7. DHMEQ (30 mg/kg/day) was administered intraperitoneally for 14 days after DST. Graft survival and histology were evaluated. The underlying mechanisms of immunomodulation by DST and DHMEQ treatments were investigated by assessing alloimmune responses after transplantation. RESULTS: In fully mismatched H2-to-H2 heart transplants, DST alone prolonged allograft median survival time to 15 days, whereas when DST was combined with DHMEQ treatment, the graft median survival time was prolonged to 39.5 days. When the donor-recipient strain combination was reversed, that is, H2-to-H2, heart transplants were accepted (>150 days survival) in more than 60% of recipients treated with a DST and DHMEQ, whereas control allografts were all rejected within 8 days. The combined therapy markedly inhibited immune responses by both the direct and indirect allorecognition pathways mainly attributed to promotion of activation-induced cell death and Treg generation. CONCLUSIONS: Our results demonstrate the distinctive ability of NF-κB inhibition in combination with donor alloantigen to promote transplantation tolerance through multiple cellular mechanisms.

Donor graft interferon regulatory factor-1 gene transfer worsens liver transplant ischemia/reperfusion injury.

BACKGROUND: Liver ischemia and reperfusion (IR) injury is a phenomenon that leads to graft dysfunction after liver transplantation. Understanding the molecular mechanisms behind this process is crucial to developing strategies to prevent short- and long-term graft dysfunction. The purpose of this study was to explore the role of the transcription factor interferon regulatory factor-1 (IRF-1) in a model of orthotopic rat liver transplantation. METHODS: Orthotopic syngeneic LEW rat liver transplantation (OLT) was performed after 18 or 3 hours preservation in cold University of Wisconsin solution. Adenovirus-expressing IRF-1 (AdIRF-1) or control gene vector (Adnull) was delivered to the liver by donor intravenous pretreatment 4 days before graft harvesting. Uninfected grafts also served as controls. Recipients were humanely killed 1-24 hours post-transplantation. RESULTS: Rats that underwent OLT with long-term preserved grafts (18 hours) displayed increased hepatic nuclear expression of IRF-1 protein at 1 and 3 hours. Rats pretreated with AdIRF-1 before transplantation had elevated alanine aminotransferase levels and increased expression of interferon (IFN)-beta, IFN-gamma, interleukin-12, and inducible nitric oxide synthase in the short-term period (3 hours) when compared with donor livers pretreated with Adnull. AdIRF-1 pretreated donor livers also exhibited increased susceptibility to early apoptosis in the transplanted grafts as shown by increased terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) staining and expression of cleaved caspase-3. Additionally, AdIRF-1 pretreated donor livers had increased activation of the MAP kinase Jun N-terminal kinase as compared with Adnull pretreated donor livers. CONCLUSION: IRF-1 is an important regulator of IR injury after OLT in rats. Targeting of IRF-1 may be a potential strategy to ameliorate ischemic liver injury after transplantation to minimize organ dysfunction.

Protection of transplant-induced hepatic ischemia/reperfusion injury with carbon monoxide via MEK/ERK1/2 pathway downregulation.

Carbon monoxide (CO), a product of heme degradation by heme oxygenases (HO), has been shown to provide cytoprotection in various tissue injury models. This study examined the efficacy and molecular mechanisms of exogenously delivered inhaled CO in protecting liver grafts from cold ischemia/reperfusion (I/R) injury associated with liver transplantation. Orthotopic syngenic liver transplantation (OLT) was performed in Lewis rats with 18-h cold preservation in University of Wisconsin solution. Recipients were exposed to air or different concentrations of CO (20-250 ppm) for 1 h before and 24 h after OLT and killed 1-48 h posttransplant. CO inhalation significantly decreased serum alanine transaminase (ALT) levels and suppressed hepatic necrosis and neutrophil accumulation at 24-48 h after OLT in a dose-dependent manner. Reduced hepatic injury with inhaled CO is associated with marked downregulation of early mRNA expression for TNF-alpha and IL-6. Expression in liver grafts of mRNA and protein of the stress-responding enzyme inducible nitric oxide synthase was significantly reduced by CO, while HO-1 was only marginally suppressed. Cold hepatic I/R injury was associated with prompt MAPK phosphorylation in liver grafts at 1 h after OLT, and CO significantly inhibited phosphorylation of ERK1/2 MAPK and its upstream MEK1/2 and downstream transcriptional factor c-Myc. CO also significantly inhibited I/R injury-induced STAT1 and STAT3 activation. In contrast, CO did not inhibit p38 or JNK MAPK pathways during hepatic I/R injury. Results demonstrate that exogenous CO suppresses early proinflammatory and stress-response gene expression and efficiently ameliorates hepatic I/R injury. The possible mechanism may include the downregulation of MEK/ERK1/2 signaling pathway with CO.