Prognostic factors influencing outcome in adult liver transplantation using hypernatremic organ donation after brain death.

BACKGROUND: Hypernatremic donors was regarded as the expanded criteria donors in liver transplantation. The study was to investigate the effects of donor hypernatremia on the outcomes of liver transplantation and identify the prognostic factors possibly contributing to the poor outcomes. METHODS: Donor serum sodium levels before procurement were categorized as normal sodium (< 155 mmol/L), moderate high sodium (155-170 mmol/L), and severe high sodium (≥ 170 mmol/L). Furthermore, we subdivided the 142 hypernatremic donors (≥ 155 mmol/L) into two subgroups: subgroup A, the exposure time of liver grafts from hypernatremia to reperfusion was < 36 h; and subgroup B, the exposure time was ≥ 36 h. The outcomes included initial graft function, survival rates of grafts and recipients, graft loss and early events within the first year following liver transplantation. RESULTS: There were no significant differences in the 1-year survival rates of grafts and recipients, 1-year graft loss rates and early events among the normal, moderate high and severe high sodium groups. However, the overall survival rates of grafts and recipients in subgroup A were significantly higher than those in subgroup B. Cox model showed that the exposure time (HR = 1.117; 95% CI: 1.053-1.186; P < 0.001), cold ischemia time (HR = 1.015; 95% CI: 1.006-1.024; P = 0.001) and MELD (HR = 1.061; 95% CI: 1.003-1.121; P = 0.037) were the important prognostic factors contributing to the poor outcomes of recipients with hypernatremic donors. CONCLUSIONS: The level of donor sodium immediately before organ procurement does not have negative effects on the early outcomes following adult liver transplantation. For hypernatremia liver donors, minimization of the exposure time from hypernatremia to reperfusion is critical to prevent graft loss.

MicroRNA-146a-5p enhances radiosensitivity in hepatocellular carcinoma through replication protein A3-induced activation of the DNA repair pathway.

Hepatocellular carcinoma (HCC) is known for its high mortality rate worldwide. Based on intensive studies, microRNA (miRNA) expression functions in tumor suppression. Therefore, we aimed to evaluate the contribution of miR-146a-5p to radiosensitivity in HCC through the activation of the DNA damage repair pathway by binding to replication protein A3 (RPA3). First, the limma package of R was performed to differentially analyze HCC expression chip, and regulative miRNA of RPA3 was predicted. Expression of miR-146a-5p, RPA3, and DNA damage repair pathway-related factors in tissues and cells was determined. The effects of radiotherapy on the expression of miR-146a-5p and RPA3 as well as on cell radiosensitivity, proliferation, cell cycle, and apoptosis were also assessed. The results showed that there exists a close correlation between miR-146a and the radiotherapy effect on HCC progression through regulation of RPA3 and the DNA repair pathway. The positive rate of ATM, pCHK2, and Rad51 in HCC tissues was higher when compared with that of the paracancerous tissues. SMMC-7721 and HepG2 cell proliferation were significantly inhibited following 8 Gy 6Mv dose. MiR-146a-5p restrained the expression of RPA3 and promoted the expression of relative genes associated with the DNA repair pathway. In addition, miR-146a-5p overexpression suppresses cell proliferation and enhances radiosensitivity and cell apoptosis in HCC cells. In conclusion, the present study revealed that miR-146a-5p could lead to the restriction of proliferation and the promotion of radiosensitivity and apoptosis in HCC cells through activation of DNA repair pathway and inhibition of RPA3.

The impact of serum incubation time on IgM/IgG binding to porcine aortic endothelial cells.

The results of the assay for measuring anti-non-Gal antibodies (which affect pig xenograft survival) in recipients are important. Serum incubation time and concentration may be important factors in the extent of antibody binding to the graft. The aim of this in vitro study was to determine the optimal incubation time and serum concentration for measuring anti-non-Gal antibody binding to porcine aortic endothelial cells (pAECs). Pooled human, naive, and sensitized baboon sera were incubated with wild-type, α1,3-galactosyltransferase gene-knockout (GTKO), and GTKO/human CD55 pAECs. IgM/IgG binding to pAECs after varying serum incubation times (0.5, 1, 2, and 3 hour) and concentrations (5, 10, 20, and 40 µL) was determined by flow cytometry. An increase in incubation time from 30 minutes to 2 hour was associated with increases in anti-non-Gal IgM/IgG binding to GTKO and GTKO/hCD55 pAECs of pooled human, naive and sensitized baboon sera (P<.05). Pooled human serum showed a significant increase in anti-non-Gal IgM (1.5 times) and a minimal increase in anti-non-Gal IgG antibody binding. IgM/IgG binding of sensitized baboon serum to GTKO pAECs after 2-hour incubation was 1.5 times and 2 times greater than after 30-minutes incubation, respectively, whereas naïve baboon sera showed minimal (non-significant) increase in anti-non-Gal IgM/IgG antibody binding. With 2-hour incubation, increasing the serum concentration from 5 µL to 20 µL significantly increased antibody binding to non-Gal antigens in pooled human and sensitized baboon serum. With naïve baboon serum, only IgG was significantly increased. Increasing the serum incubation time contributed to improve the sensitivity of detecting anti-non-Gal antibodies, without affecting cell viability in vitro.

Foxp3-modified bone marrow mesenchymal stem cells promotes liver allograft tolerance through the generation of regulatory T cells in rats.

BACKGROUND: The transcription factor forkhead box P3 (Foxp3) is a master regulatory gene necessary for the development and function of CD4(+)CD25(+) regulatory T cells (Tregs). Mesenchymal stem cells (MSC) have recently emerged as promising candidates for cell-based immunosuppression/tolerance induction protocols. Thus, we hypothesized that MSC-based Foxp3 gene therapy would improve immunosuppressive capacity of MSC and induce donor-specific allograft tolerance in rat's liver allograft model. METHODS: The present study utilized a lentivirus vector to overexpress the therapeutic gene Foxp3 on MSC. In vivo, Injections of 2 × 10(6) MSC, FUGW-MSC or Foxp3-MSC into the portal vein were carried out immediately after liver transplantation. RESULTS: Successful gene transfer of Foxp3 in MSC was achieved by lentivirus carrying Foxp3 and Foxp3-MSC engraftment in liver allograft was confirmed by fluorescence microscopy. Foxp3-MSC treatment significantly inhibited the proliferation of allogeneic ACI CD4(+) T cells to splenocytes (SC) from the same donor strain or third-party BN rat compared with MSC. Foxp3-MSC suppressive effect on the proliferation of CD4(+) T cells is contact dependent and associated with Programmed death ligand 1(PD-L1) upregulation in MSC. Co-culture of CD4(+) T cells with Foxp3-MSC results in a shift towards a Tregs phenotype. More importantly, Foxp3-MSC monotherapy achieved donor-specific liver allograft tolerance and generated a state of CD4(+)CD25(+)Foxp3(+) Tregs-dependent tolerance. CONCLUSION: Foxp3-engineered MSC therapy seems to be a promising and attractive cell therapy approach for inducing immunosuppression or transplant tolerance.

Splenic artery trunk embolization reduces the surgical risk of liver transplantation.

BACKGROUND: Portal hypertension is one of the most important clinical conditions that cause intraoperative intensive hemorrhage in cirrhotic patients undergoing liver transplantation. Pre-transplant portal decompression may reduce the intraoperative bleeding during liver transplantation. METHODS: Splenic artery trunk embolization (SATE) was performed one month prior to liver transplantation. Platelet count, prealbumin, international normalized ratio, and blood flow in the portal vein and hepatic artery were monitored before and one month after SATE. The measurements above were collected on admission and before surgery in the non-SATE patients, who served as controls. We also recorded the intraoperative blood loss, operating time, required transfusion, post-transplant ascites, and complications within three months after operation in all patients. RESULTS: SATE significantly reduced portal venous blood flow, increased hepatic arterial blood flow, normalized platelet count, and improved prealbumin and international normalized ratio in the patients before liver transplantation. Compared to the non-SATE patients, the pre-transplant SATE significantly decreased the operating time, intraoperative bleeding, post-transplant ascites and severe surgical complications. CONCLUSION: Pre-transplant SATE decreases portal pressure, improves liver function reserve, and reduces the surgical risk of liver transplantation effectively in patients with severe portal hypertension.

[Protective effect of minocycline on hepatic ischemia-reperfusion injury in rats].

OBJECTIVE: To explore the protective eff ect of minocycline on hepatic ischemia-reperfusion injury (IRI) in rats and the underlying mechanisms. METHODS: A total of 54 male Sprague-Dawley rats were randomly divided into 3 groups: the sham-operated group (control group), the ischemic-reperfusion (IR group), and the minocycline preconditioning group (n=18 per group). The rats in the minocycline preconditioning group were given minocycline (45 mg/kg) by gastric irrigation at 36 h before operation and then were subsequently administered with minocycline (22.5 mg/kg) at every 12 h. Th e rats were sacrifi ced at 2, 6, 24 h after reperfusion. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were measured. HE staining of liver tissues was performed to detect the histological changes, and the degree of liver IRI according to Suzuki score were calculated. The levels of malondialdehyde (MDA) and myeloperoxidase (MPO) were determined by spectrophotometer; the mRNA expression of tumor necrosis factor-α (TNF-α) and interleukin-1 beta (IL-1ß) in the liver were measured by real-time PCR; Dickkopf-1 (DKK-1) and beta-catenin (ß-catenin) protein expression in the liver were detected by Western blot. RESULTS: After 2, 6, 24 h reperfusion, compared with the IR group, the liver function (ALT, AST and LDH) in the minocycline group was significantly improved (all P<0.05); the Suzuki's scores and the levels of hepatic TNF-α and IL-1ß mRNA were significantly decreased (all P<0. 05); the MDA and MPO levels the liver were decreased (both P<0.05); the protein expression of hepatic DKK-1 was decreased (P<0.05), while the protein expression of ß-catenin was increased (P<0.05). CONCLUSION: Minocycline can alleviate the ischemic-reperfusion injury mainly through reducing oxidative stress and inhibiting the release of pro-inflammatory cytokines depends on the activation of the Wnt/ß-catenin signaling pathway in the liver.

Influence of sex on outcomes of liver transplantation for hepatocellular carcinoma: a multicenter cohort study in China.

OBJECTIVE: Sex-specific differences are observed in various liver diseases, but the influence of sex on the outcomes of hepatocellular carcinoma (HCC) after liver transplantation (LT) remains to be determined. This study is the first Chinese nationwide investigation of the role of sex in post-LT outcomes in patients with HCC. METHODS: Data for recipients with HCC registered in the China Liver Transplant Registry between January 2015 and December 2020 were analyzed. The associations between donor, recipient, or donor-recipient transplant patterns by sex and the post-LT outcomes were studied with propensity score matching (PSM). The survival associated with different sex-based donor-recipient transplant patterns was further studied. RESULTS: Among 3,769 patients enrolled in this study, the 1-, 3-, and 5-year overall survival (OS) rates of patients with HCC after LT were 96.1%, 86.4%, and 78.5%, respectively, in female recipients, and 95.8%, 79.0%, and 70.7%, respectively, in male recipients after PSM (P = 0.009). However, the OS was comparable between recipients with female donors and male donors. Multivariate analysis indicated that male recipient sex was a risk factor for post-LT survival (HR = 1.381, P = 0.046). Among the donor-recipient transplant patterns, the male-male donor-recipient transplant pattern was associated with the poorest post-LT survival (P < 0.05). CONCLUSIONS: Our findings highlighted that the post-LT outcomes of female recipients were significantly superior to those of male recipients, and the male-male donor-recipient transplant pattern was associated with the poorest post-LT survival. Livers from male donors may provide the most benefit to female recipients. Our results indicate that sex should be considered as a critical factor in organ allocation.

Alterations of axis inhibition protein 1 (AXIN1) in hepatitis B virus-related hepatocellular carcinoma and overexpression of AXIN1 induces apoptosis in hepatocellular cancer cells.

Axis inhibition protein 1 (AXIN1) is a negative regulator of Wnt/beta-catenin signaling via regulating the level of beta-catenin. However, the role of AXIN1 in the tumorigenesis and progression of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) is less clear. PCR sequence analysis, immunohistochemistry, and Western blot were performed on 22 HBV-related HCC samples and corresponding nontumor liver tissues to detect variants in AXIN1 gene and the expression level of AXIN1. Human hepatoma cell lines SNU475 and SNU423 were transfected with pCDNA3.1-AXIN1-myc or AXIN1 G425S-myc mutant. The growth curve and apoptosis rate of cell lines, phosphorylation of beta-catenin, and cell cycle regulatory proteins depending on beta-catenin transcriptional activity were detected. We identified four mutations of AXIN1 in 22 primary HBV-related HCCs and demonstrated a lower expression of AXIN1 in HBV-related HCC tissues than that in paired adjacent nontumor tissues. Overexpression of AXIN1 wild-type but not AXIN1 mutant inhibited the growth of HCC cell lines, accelerated their apoptosis, and negatively regulated beta-catenin-dependent transcriptional activity. Our study revealed that alterations of AXIN1 were involved in HBV-related HCC. Overexpression of AXIN1 but not AXIN1 mutant negatively regulated beta-catenin-dependent transcriptional activity and downregulated the level of cell cycle regulatory proteins, suggesting that AXIN1 may be a potential target for gene therapy of primary HCC.

Angiotensin II induces endothelin-1 expression in human hepatic stellate cells.

BACKGROUND: Both angiotensin (Ang)-II and endothelin-1 (ET-1) are involved in the pathogenesis of liver fibrosis. Activated hepatic stellate cells (HSCs) are considered a key effector of liver fibrosis. AIMS: To explore the effect of Ang-II on ET-1 expression in cultured human HSCs and the underlying mechanisms. METHODS: Human HSCs were treated with Ang-II in different concentrations (0.1, 0.5, 1, 5, or 10 nM) for different lengths of time (0.5, 1, 2, 4, or 6 h) with or without transcription inhibitor actinomycin D, Ang-II type 1 (AT1) receptor blocker losartan, AT2 receptor blocker PD123177, or different kinase inhibitors. RESULTS: Ang-II increased the ET-1 mRNA level in a statistically significant dose- and time-dependent manner within 4 h, which led to dose-dependent up-regulation of the ET-1 protein level. Actinomycin D (1 mg/ml), losartan (50 µM), and phosphatidylinositol-3 kinase inhibitor LY294002 (50 µM) abolished the promoting effect of Ang-II on ET-1 expression. Ang-II (10 nM) significantly increased the expression of α-smooth muscle actin and type I collagen in HSCs, which was abolished by losartan, LY294002, ET A receptor blocker BQ123, and ET-1 siRNA, but not PD123177 and ET B receptor blocker BQ788. CONCLUSIONS: Ang-II induces ET-1 expression in human HSCs via the AT1 receptor by the PI3 K/Akt signaling pathway. The ET-1/ET A receptor axis could mediate the promoting effects of Ang-II on HSCs' transdifferentiation into myofibroblast-like cells. This is the first evidence of crosstalk between the Ang-II/AT1 axis and the ET-1 system in regard to the pathogenesis of liver fibrosis.

An updated meta-analysis of the p53 codon 72 polymorphism and gastric cancer risk.

To investigate the association between p53 codon 72 polymorphisms and gastric cancer risk, a meta-analysis published in 2007 was updated with new data. Relevant literature was retrieved by searching PubMed and statistical analysis conducted using Review Manager software. Twenty-eight case-control studies were included in this meta-analysis, with 6,859 cases and 9,277 controls. The pooled results for all included studies showed that patients with gastric cancer had a borderline lower frequency of the Arg/Arg phenotype (odds ratio (OR) = 0.91, 95 % CI = 0.83-1.00, p = 0.04). When stratified for race, the difference in Arg/Arg frequency was significant among Asians (OR = 0.87, 95 % CI = 0.78-0.97, p = 0.01). On stratifying the various studies we found that, among Asians: (i) patients with cardial gastric cancer had a significantly higher frequency of the Pro/Pro genotype (OR = 1.35, 95 % CI = 1.03-1.77, p = 0.04) than those with non-cardial gastric cancer; (ii) patients with advanced (stage III/IV) gastric cancer had a significantly higher frequency of Arg/Arg (OR = 1.30, 95 % CI = 1.06-1.61, p = 0.01) than those with early (stage I/II) cancer; and (iii) patients with metastasis had a significantly higher frequency of Pro/Pro (OR = 3.31, 95 % CI = 1.31-8.41) than those without metastasis. Our study suggests that, among Asians, the p53 codon 72 Arg/Arg genotype is associated with a modestly decreased risk of gastric cancer, and that this difference in genotype distribution may be associated with cancer stage, location, differentiation and metastasis.

PIAS1 Alleviates Hepatic Ischemia-Reperfusion Injury in Mice through a Mechanism Involving NFATc1 SUMOylation.

Recently, attentions have come to the alleviatory effect of protein inhibitor of activated STAT1 (PIAS1) in hepatic ischemia-reperfusion injury (HIRI), but the underlying molecular mechanistic actions remain largely unknown, which were illustrated in the present study. Microarray-based analysis predicted a possible regulatory mechanism involving the PIAS1/NFATc1/HDAC1/IRF-1/p38 MAPK signaling axis in HIRI. Then, growth dynamics of hypoxia/reoxygenation- (H/R-) exposed hepatocytes and liver injury of HIRI-like mice were delineated after the alteration of the PIAS1 expression. We validated that PIAS1 downregulation occurred in H/R-exposed hepatocytes and HIRI-like mice, while the expression of NFATc1, HDAC1, and IRF-1 and phosphorylation levels of p38 were increased. PIAS1 inactivated p38 MAPK signaling by inhibiting HDAC1-mediated IRF-1 through NFATc1 SUMOylation, thereby repressing the inflammatory response and apoptosis of hepatocytes in vitro, and alleviated liver injury in vivo. Collectively, the NFATc1/HDAC1/IRF-1/p38 MAPK signaling axis is highlighted as a promising therapeutic target for potentiating hepatoprotective effects of PIAS1 against HIRI.

IL-17 in the early diagnosis of acute renal allograft rejection in mice.

OBJECTIVE: To investigate the expression of T helper (Th) 17 cells and the related interleukin 17 (IL-17) in acute renal allograft rejection in mice and its significance. METHODS: We established a mouse renal allograft model, in which mice were randomly divided into a renal isograft group and an acute renal allograft rejection group. Three and 7 d after the transplantation, the serum interferon (IFN)-γ and IL-17 levels in the mice were determined by enzyme-linked immunosorbent assay, the percentage of Th1 and Th17 cells in the total kidney-infiltrating lymphocytes was investigated by flow cytometry, and the transplanted kidney species were given routine pathological examination after fixation with 10% formalin. RESULTS: Compared with the isograft group, the allograft mice showed a significantly higher content of IL-17 (P<0.05) but not IFN-γ in the serum 3 d after transplantation, and showed significantly higher serum IL-17 and IFN-γ contents 7 d after transplantation (P<0.05). Also, compared with the isograft group, the allograft mice exhibited significantly higher percentage of Th1 and Th17 cells on both day 3 and day 7 (P<0.05). In the allograft group, the contents of serum IFN-γ and IL-17 and the percentage of Th1 and Th17 cells were significantly higher on day 7 than on day 3 (P<0.05). Routine pathological examination indicated that, as time passed, the allograft mice showed gradually stronger rejection responses. CONCLUSION: Th17 cells might play an important role in the development of acute renal allograft rejection, and IL-17 can be used as an early indicator of acute rejection.

Mouse model of orthotopic small bowel transplantation.

OBJECTIVE: To investigate the surgical procedures of orthotopic small bowel transplantation (SBT) model in mice to study the function and rejection of SBT. METHODS: We established a mouse SBT allograft model as follows: the donor portal vein was anastomosed end by side with the recipient inferior vena cava; the donor superior mesenteric artery with aorta patch was anastomosed end by side with recipient abdominal aorta. After an appropriate length of the recipient's small bowel was removed, the donor's small bowel and the recipient's small bowel were end-to-end anastomosed discontinuously. The mice were fasted for 4 d after the operation, free access to water and subcutaneously injection of 2 mL of 5% glucose saline twice daily. Operation success was regarded as survival for more than 5 d. There is no antibiotic and immunosuppressor. RESULTS: A total of 30 transplantations were done, the 5 d survival rate was 60% (18/30), and 12 died within 5 d. Among the dead recipients, 5 died of arterial anastomotic stenosis and anastomotic thrombosis, 2 of hemorrhagic shock caused by anastomotic bleeding, and the other 5 of intra-abdominal infection caused by postoperative intestinal fistula. The donors' operative time was (40 ± 4.5) min, warm ischemia time was about 0.5 min, donor preparation time was about 3 min, and cold preservation time was (30 ± 7.5) min. The recipients' operative time was (95 ± 8.0) min, among which, the abdominal aorta and inferior vena cava clamping time was (38 ± 3.5) min, the venous anastomotic time was (10 ± 2.0) min and the arterial anastomotic time was (15 ± 3.0) min. The mean intra-operative blood loss of the surviving recipient mice was about 0.2 mL. CONCLUSION: High quality vascular anastomosis, and rehydration of donors and recipients are crucial factors for improving the success rate of SBT.

Sequencing of Cell-Free DNA to Monitor Cytomegalovirus Infection After Liver Transplant.

OBJECTIVES: In the present study, we investigated donor-derived cell-free DNA dynamics and assessed the diagnostic efficacy of 2 tests: the sequencing of cytomegalovirus-derived cell-free DNA and the quantitative nucleic acid amplification test in cytomegalovirus infection following liver transplant. MATERIALS AND METHODS: We first examined 6 patients who were identified with active cytomegalovirus DNAemia by both quantitative nucleic acid amp-lification test and next-generation sequencing of cytomegalovirus-derived cell-free DNA and then performed a receiver operating characteristic analysis to evaluate the efficacy of cell-free DNA sequencing and establish a cutoff for this assay. Further validation of the next-generation sequencing method was also performed in 84 liver transplant recipients. The study protocol conformed to the ethical guidelines of the Declaration of Helsinki and the Declaration of Istanbul. RESULTS: In the first 6 patients, there was no significant correlation between the cytomegalovirus infection and donor-derived cell-free DNA. We determined that the levels of cytomegalovirus-derived cell-free DNA sequencing directly correlate with the results of the quantitative nucleic acid amplification test (area under the curve 0.982) and obtained a value of 0.015% as a cutoff for the cell-free DNA sequencing assay. In the validation cohort composed of 84 liver transplant recipients, next-generation sequencing of cell-free DNA revealed the occurrence of cytomegalovirus infection that remains otherwise undetected by the quantitative nucleic acid amplification test. CONCLUSIONS: Cytomegalovirus infections that do not cause direct graft injury (cytomegalovirus-related hepatitis) did not result in elevations of donor-derived cell-free DNA. Next-generation sequencing of cytomegalovirus-derived cell-free DNA provides a potential tool for detection of cytomegalovirus infection that remains undetected by the quantitative nucleic acid amplification test.

Induction of lymphocyte apoptosis in rat liver allograft by adenoviral gene transfection of human interleukin-10.

BACKGROUND/AIMS: Gene therapy can provide a possible avenue in organ transplantation to treat acute allograft rejection. This study was designed to investigate the effect of adenovirus-mediated human IL-10 (hIL-10) gene transfer on the apoptosis of infiltrating lymphocytes and examine the efficacy of hIL-10 gene transfer in combination with subtherapeutic doses of cyclosporine A (CsA) in a rat liver transplantation model. METHODS: Inbred male DA and LEW rats were used for liver donors and recipients, respectively. The rats were divided into saline, Ad-lacZ, CsA, Ad-hIL-10 and Ad-hIL-10 + CsA groups. Graft survival, histopathological, enzyme-linked immunosorbent assay, reverse transcriptase-polymerase chain reaction and flow cytometry were performed in liver specimens obtained from different time points after transplantation in the 5 groups. RESULTS: Ad-hIL-10 pretreatment inhibited allograft rejection, prolonged the survival of hepatic allografts, and downregulated the expression of IFN-gamma and IL-2 mRNA, with simultaneous upregulation of IL-4 mRNA. In addition, Ad-hIL-10 pretreatment upregulated the expression of Fas mRNA in the isolated graft-infiltrating lymphocytes and induced graft-infiltrating lymphocyte apoptosis. A single subtherapeutic dose of CsA acted synergistically with it. CONCLUSION: hIL-10 gene therapy induced alloreactive lymphocyte apoptosis via Fas/FasL pathway. hIL-10 gene transfection in combination with subtherapeutic doses of CsA facilitates the long-term survival of liver grafts.

Recombinant adenovirus vector Ad-hIL-10 protects grafts from cold ischemia-reperfusion injury following orthotopic liver transplantation in rats.

BACKGROUND: Interleukin 10 (IL-10), a Th2 type cytokine, modulates inflammatory responses by inhibiting the production of proinflammatory cytokines. This study was designed to investigate the protective effects of adenovirus-mediated human IL-10 (Ad-hIL-10) gene transfer on protecting grafts from cold ischemia-reperfusion injury following orthotopic liver transplantation in rats. METHODS: Adenoviruses encoding hIL-10 or beta-galactosidase (Ad-lacZ) were injected via the superior mesenteric vein into prospective donor animals. The donor liver was harvested 48 hours after transduction, and stored for 12 hours at 4 degree centigrade in lactated Ringer's solution prior to transplantation. The rats were divided into saline, Ad-lacZ, and Ad-hIL-10 groups. Liver function test, histopathological examination, reverse transcriptase-polymerase chain reaction (RT-PCR), and Western blotting were performed at 24 hours after transplantation in the three groups. RESULTS: Liver function (ALT and AST) was significantly improved, and the Suzuki score was significantly decreased in the Ad-hIL-10 group. The levels of hepatic TNF-alpha, MIP-2, ICAM-1 mRNA, and NF-kappaB protein in the Ad-hIL-10 group were significantly decreased. The expression of hIL-10 mRNA was detected by RT-PCR in Ad-hIL-10-treated grafts but not in controls treated with saline or Ad-lacZ. CONCLUSIONS: Donor pretreatment with Ad-hIL-10 down-regulates the expression of proinflammatory cytokines TNF-alpha, MIP-2, and ICAM-1 mRNA. hIL-10 protects against hepatic cold ischemia-reperfusion injury, at least in part, by suppressing NF-kappaB activation and subsequent expression of proinflammatory mediators.

Cytoprotective effects of human interleukin-10 gene transfer against necrosis and apoptosis induced by hepatic cold ischemia/reperfusion injury.

BACKGROUND: Apoptosis as well as necrosis may play an important role in hepatic ischemia/reperfusion (I/R) injury. Interleukin 10 (IL-10), a Th2 type cytokine, modulates inflammatory responses by inhibiting the production of proinflammatory cytokines. The study focused on cytoprotective and antiapoptotic pathways to assess mechanisms by which gene transduction of human IL-10 (hIL-10) may renders grafts resistant to the cold I/R injury. MATERIALS AND METHODS: Adenoviruses encoding hIL-10 or beta-galactosidase (LacZ) were injected via the superior mesenteric vein into prospective donor animals. The donor liver was harvested 48h after transduction, and stored for 12h at 4 degrees C lactated Ringer's solution prior to being transplanted. Graft survival, liver function, the degree of necrosis and apoptosis, and the molecules of apoptotic networks were assessed. RESULTS: Ad-hIL-10 pretreatment significantly prolonged the survival of liver grafts by improving liver function, preserving hepatocyte integrity and architecture, and depressing intrahepatic apoptosis and necrosis. In addition, Ad-hIL-10 pretreatment diminished the release of cytochrome c from mitochondria into cytoplasm and caspase-3 activity, with simultaneous up-regulated of antioxidant HO-1 and anti-antiapoptotic Bcl-2 molecules. CONCLUSION: Adenoviral gene transfer of hIL-10 ameliorated cold I/R injury by decreasing hepatic necrosis and apoptosis. The underlying mechanism of cytoprotective effects may at least be involved with the inhibition of caspase-3 activity and mitochondrial cytochrome c release, and the up-regulation of antiapoptotic (Bcl-2) and antioxidant (HO-1) molecules.

Immune Responses of HLA Highly Sensitized and Nonsensitized Patients to Genetically Engineered Pig Cells.

BACKGROUND: We investigated in vitro whether HLA highly sensitized patients with end-stage renal disease will be disadvantaged immunologically after a genetically engineered pig kidney transplant. METHODS: Blood was drawn from patients with a calculated panel-reactive antibody (cPRA) 99% to 100% (Gp1, n = 10) or cPRA 0% (Gp2, n = 12), and from healthy volunteers (Gp3, n = 10). Serum IgM and IgG binding was measured (i) to galactose-α1-3 galactose and N-glycolylneuraminic acid glycans by enzyme-linked immunosorbent assay, and (ii) to pig red blood cell, pig aortic endothelial cells, and pig peripheral blood mononuclear cell from α1,3-galactosyltransferase gene-knockout (GTKO)/CD46 and GTKO/CD46/cytidine monophosphate-N-acetylneuraminic acid hydroxylase-knockout (CMAHKO) pigs by flow cytometry. (iii) T-cell and B-cell phenotypes were determined by flow cytometry, and (iv) proliferation of T-cell and B-cell carboxyfluorescein diacetate succinimidyl ester-mixed lymphocyte reaction. RESULTS: (i) By enzyme-linked immunosorbent assay, there was no difference in IgM or IgG binding to galactose-α1-3 galactose or N-glycolylneuraminic acid between Gps1 and 2, but binding was significantly reduced in both groups compared to Gp3. (ii) IgM and IgG binding in Gps1 and 2 was also significantly lower to GTKO/CD46 pig cells than in healthy controls, but there were no differences between the 3 groups in binding to GTKO/CD46/CMAHKO cells. (iii and iv) Gp1 patients had more memory T cells than Gp2, but there was no difference in T or B cell proliferation when stimulated by any pig cells. The proliferative responses in all 3 groups were weakest when stimulated by GTKO/CD46/CMAHKO pig peripheral blood mononuclear cell. CONCLUSIONS: (i) End-stage renal disease was associated with low antipig antibody levels. (ii) Xenoreactivity decreased with increased genetic engineering of pig cells. (iii) High cPRA status had no significant effect on antibody binding or T-cell and B-cell response.

[Reconstructive methods for hypopharynx and cervical esophagus].

OBJECTIVE: To review the surgical treatment for reconstructing hypopharynx and cervical esophagus after hypopharyngo-oesophagectomy, and to evalue its efficacy. METHODS: Different methods were adopted to reconstruct the hypopharynx and cervical esophagus among 25 cases, including 14 cases of carcinoma of the hypopharynx and 11 of carcinoma of hypopharynx and cervical esophagus. In accordance with the standard of the International Union Against Cancer in 1997, the 25 cases were divided into different clinic stages, among which 5 were in T(2)N(0), 2 in T(2)N(1), 4 in T(3)N(0), 3 in T(3)N(1), 7 in T(4)N(1) and 3 in T(4)N(2). Treatment protocol was as follow: Pure operation for 5 cases, re-operation after radiotherapy for 2 cases, operation plus radiotherapy for 18 cases, laryngeal conservation operation for 8, and neck dissection for 21 cases. Reconstruction was done by using free jejunal transplantation, gastric pull-up, the laryngotracheal flap, and myocutaneous flap. RESULTS: After the reconstruction, 3 cases of free jejunal graft and gastric pull-up, 4 of laryngotracheal flap recovered oral fleeding within 2 weeks. No serious complications occurred. After 18 cases underwent the myocutaneous flap reconstruction, no complications occurred in 10 patients, but there were different complications in 8 cases, including pharyngocutaneous fistula (6 cases), haryngoesphageal stenosis (7 cases), and pectoralis major myocutaneous flap necrotic (1 case). The 3-year survival rate was 38.9% (7/18). CONCLUSION: Reconstruction with free jejunal graft, gastric pull-up, and laryngotracheal flap constitutes is a safe and reliable method to restore the continuity of the upper digestive tract after pharyngo-laryngo-oesophagectomy. After the reconstruction with myocutaneous flap, there is high incidence of pharyngocutaneous fistula and haryngoesophageal stenosis.

The miR-124-p63 feedback loop modulates colorectal cancer growth.

Among the diverse co-regulatory relationships between transcription factors (TFs) and microRNAs (miRNAs), feedback loops have received the most extensive research attention. The co-regulation of TFs and miRNAs plays an important role in colorectal cancer (CRC) growth. Here, we show that miR-124 can regulate two isoforms of p63, TAp63 and ΔNp63, via iASPP, while p63 modulates signal transducers and activators of transcription 1 (STAT1) expression by targeting miR-155. Moreover, STAT1 acts as a regulator of CRC growth by targeting miR-124. Taken together, these results reveal a feedback loop between miRNAs and TFs. This feedback loop comprises miR-124, iASPP, STAT1, miR-155, TAp63 and ΔNp63, which are essential for CRC growth. Moreover, this feedback loop is perturbed in human colon carcinomas, which suggests that the manipulation of this microRNA-TF feedback loop has therapeutic potential for CRC.