A new bicornuate model of rat uterus transplantation.

INTRODUCTION: Uterus transplantation has revolutionized reproductive medicine for women with absolute uterine factor infertility, resulting in more than 40 reported successful live births worldwide to date. Small animal models are pivotal to refine this surgical and immunological challenging procedure aiming to enhance safety for both the mother and the child. MATERIAL AND METHODS: We established a syngeneic bicornuate uterus transplantation model in young female Lewis rats. All surgical procedures were conducted by an experienced and skilled microsurgeon who organized the learning process into multiple structured steps. Animals underwent meticulous preoperative preparation and postoperative care. Transplant success was monitored by sequential biopsies, monitoring graft viability and documenting histological changes long-term. RESULTS: Bicornuate uterus transplantation were successfully established achieving an over 70% graft survival rate with the passage of time. The bicornuate model demonstrated safety and feasibility, yielding outcomes comparable to the unicornuate model in terms of ischemia times and complications. Longitudinal biopsies were well-tolerated, enabling comprehensive monitoring throughout the study. CONCLUSIONS: Our novel bicornuate rat uterus transplantation model provides a distinctive opportunity for sequential biopsies at various intervals after transplantation and, therefore, comprehensive monitoring of graft health, viability, and identification of potential signs of rejection. Furthermore, this model allows for different interventions in each horn for comparative studies without interobserver differences contrary to the established unicornuate model. By closely replicating the clinical setting, this model stands as a valuable tool for ongoing research in the field of uterus transplantation, promoting further innovation and deeper insights into the intricacies of the uterus transplant procedure.

Depletion of donor dendritic cells ameliorates immunogenicity of both skin and hind limb transplants.

Acute cellular rejection remains a significant obstacle affecting successful outcomes of organ transplantation including vascularized composite tissue allografts (VCA). Donor antigen presenting cells (APCs), particularly dendritic cells (DCs), orchestrate early alloimmune responses by activating recipient effector T cells. Employing a targeted approach, we investigated the impact of donor-derived conventional DCs (cDCs) and APCs on the immunogenicity of skin and skin-containing VCA grafts, using mouse models of skin and hind limb transplantation. By post-transplantation day 6, skin grafts demonstrated severe rejections, characterized by predominance of recipient CD4 T cells. In contrast, hind limb grafts showed moderate rejection, primarily infiltrated by CD8 T cells. Notably, the skin component exhibited heightened immunogenicity when compared to the entire VCA, evidenced by increased frequencies of pan (CD11b-CD11c+), mature (CD11b-CD11c+MHCII+) and active (CD11b-CD11c+CD40+) DCs and cDC2 subset (CD11b+CD11c+ MHCII+) in the lymphoid tissues and the blood of skin transplant recipients. While donor depletion of cDC and APC reduced frequencies, maturation and activation of DCs in all analyzed tissues of skin transplant recipients, reduction in DC activities was only observed in the spleen of hind limb recipients. Donor cDC and APC depletion did not impact all lymphocyte compartments but significantly affected CD8 T cells and activated CD4 T in lymph nodes of skin recipients. Moreover, both donor APC and cDC depletion attenuated the Th17 immune response, evident by significantly reduced Th17 (CD4+IL-17+) cells in the spleen of skin recipients and reduced levels of IL-17E and lymphotoxin-α in the serum samples of both skin and hind limb recipients. In conclusion, our findings underscore the highly immunogenic nature of skin component in VCA. The depletion of donor APCs and cDCs mitigates the immunogenicity of skin grafts while exerting minimal impact on VCA.

Tissue-based miRNA mapping in alcoholic liver cirrhosis: different profiles in cirrhosis with or without hepatocellular carcinoma.

Context: Alcoholic liver cirrhosis is a significant risk factor for the development of hepatocellular carcinoma (HCC). The importance of tumour-associated cirrhosis in the development or progression of HCC is not understood. MiRNAs are important regulators for HCC development, but their role in HCC due to alcoholic liver cirrhosis is unclear.Objective: The aim of this study is the detection of miRNA expression in alcoholic liver cirrhosis, tumour-associated cirrhosis, and HCC.Materials and methods: We analysed the differences in the miRNA profiles of HCC, tumour-associated cirrhosis, and cirrhosis without HCC samples from 30 patients who underwent liver transplantation because of alcoholic liver disease.Results: Microarray analyses revealed 40 significantly differentially expressed miRNAs between HCC tissue and tumour-associated cirrhosis tissue. Furthermore, the microarray analysis discovered 56 differentially expressed miRNAs in tumour-associated cirrhosis and cirrhosis without HCC.Discussion: The differences of miRNA profile in alcoholic liver cirrhosis with and without HCC could improve understanding of HCC development, as well as lead to a new diagnostic tool in HCC screening.Conclusion: We were able to show for the first time, the differences of miRNA profile as promising biomarker in HCC, tumour-associated cirrhosis, and cirrhosis without HCC in context of alcoholic liver disease.

The Predictive Value of the Maximal Liver Function Capacity Test for the Isolation of Primary Human Hepatocytes.

The need for primary human hepatocytes is constantly growing for basic research, as well as for therapeutic applications. However, the isolation outcome strongly depends on the quality of liver tissue, and we are still lacking a preoperative test that allows the prediction of the hepatocyte isolation outcome. In this study, we evaluated the "maximal liver function capacity test" (LiMAx) as predictive test for the quantitative and qualitative outcome of hepatocyte isolation. This test is already used in clinical routine to measure preoperative and to predict postoperative liver function. The patient's preoperative mean LiMAx was obtained from the patient records, and preoperative computed tomography and magnetic resonance images were used to calculate the whole liver volume to adjust the mean LiMAx. The outcome parameters of the hepatocyte isolation procedures were analyzed in correlation with the adjusted mean LiMAx. Primary human hepatocytes were isolated from partial hepatectomies (n = 64). From these 64 hepatectomies we included 48 to our study and correlated their isolation outcome parameters with volume corrected LiMAx values. From a total of 11 hepatocyte isolation procedures, metabolic parameters (albumin, urea, and aspartate aminotransferase or AST) were assessed during the hepatocyte cultivation period of 5 days. The volume adjusted mean LiMAx showed a significant positive correlation with the total cell yield (p = 0.049; r = 0.242; n = 48). The correlations of volume adjusted LiMAx values with viable cell yield and cell viability did not reach statistical significance. To create a more homogenous study group regarding tumor entities, subgroup analyses were performed. A subgroup analysis of isolations from patients with colorectal metastasis revealed a significant correlation between volume adjusted mean LiMAx and total cell yield (p = 0.012; r = 0.488; n = 21) and viable cell yield (p = 0.034; r = 0.405; n = 21), whereas a subgroup analysis of isolations of patients with carcinoma of the biliary tree showed significant correlations of volume adjusted mean LiMAx with cell viability (r = 0.387; p = 0.046; n = 20) and lacked significant correlations with total cell yield (r = -0.060; p = 0.401; n = 20) and viable cell yield (r = 0.012; p = 0.480; n = 20). The volume-adjusted mean LiMAx did not show a significant correlation with any of the metabolic parameters. In conclusion, the LiMAx test might be a useful tool to predict the quantitative outcome of hepatocyte isolation, as long as underlying liver disease is taken into consideration.

The Magnetic Field of Magnetic Resonance Imaging Systems Does Not Affect Cells Labeled with Micrometer-Sized Iron Oxide Particles.

INTRODUCTION: Labeling using iron oxide particles enables cell tracking through magnetic resonance imaging (MRI). However, the magnetic field can affect the particle-labeled cells. Here, we investigated the effects of a clinical MRI system on primary human hepatocytes labeled using micrometer-sized iron oxide particles (MPIOs). METHODS: HuH7 tumor cells were incubated with increasing concentrations of biocompatible, silica-based, micrometer-sized iron oxide-containing particles (sMPIOs; 40-160 particles/cell). Primary human hepatocytes were incubated with 100 sMPIOs/cell. The particle-labeled cells and the native cells were imaged using a clinical 3.0 T MRI system, whereas the control groups of the labeled and unlabeled cells were kept at room temperature without exposure to a magnetic field. Viability, formation of reactive oxygen species (ROS), aspartate aminotransferase leakage, and urea and albumin synthesis were assessed for a culture period of 5 days. RESULTS: The dose finding study showed no adverse effects of the sMPIOs labeling on HuH7 cells. MRI had no adverse effects on the morphology of the sMPIO-labeled primary human hepatocytes. Imaging using the T1- and T2-weighted sequences did not affect the viability, transaminase leakage, formation of ROS, or metabolic activity of the sMPIO-labeled cells or the unlabeled, primary human hepatocytes. CONCLUSION: sMPIOs did not induce adverse effects on the labeled cells under the conditions of the magnetic field of a clinical MRI system.

Bile: miRNA pattern and protein-based biomarkers may predict acute cellular rejection after liver transplantation.

CONTEXT: Bile rather than blood depicts the local inflammation in the liver and may improve prediction and diagnosis of acute cellular rejection (ACR) after liver transplantation (OLT). METHODS: Secretome and miRNAs were analyzed during the first two weeks and on clinical suspicion of ACR in the bile of 45 OLT recipients. RESULTS: Levels of CD44, CXCL9, miR-122, miR-133a, miR-148a and miR-194 were significantly higher in bile of patients who developed ACR within the first 6 months after OLT and during ACR. CONCLUSION: Analysis of secretome and miRNA in bile could improve our understanding of the local inflammatory process during rejection.

Improved rat liver decellularization by arterial perfusion under oscillating pressure conditions.

One approach of regenerative medicine to generate functional hepatic tissue in vitro is decellularization and recellularization, and several protocols for the decellularization of livers of different species have been published. This appears to be the first report on rat liver decellularization by perfusion under oscillating pressure conditions, intending to optimize microperfusion and minimize damage to the ECM. Four decellularization protocols were compared: perfusion via the portal vein (PV) or the hepatic artery (HA), with (+P) or without (-P) oscillating pressure conditions. All rat livers (n = 24) were perfused with 1% Triton X-100 and 1% sodium dodecyl sulphate, each for 90 min with a perfusion rate of 5 ml/min. Perfusion decellularization was observed macroscopically and the decellularized liver matrices were analysed by histology and biochemical analyses (e.g. levels of DNA, glycosaminoglycans and hepatocyte growth factor). Livers decellularized via the hepatic artery and under oscillating pressure showed a more homogeneous decellularization and less remaining DNA, compared with the livers of the other experimental groups. The novel decellularization method described is effective, quick (3 h) and gentle to the extracellular matrix and thus represents an improvement of existing methodology. Copyright © 2014 John Wiley & Sons, Ltd.

microRNA signatures in peripheral blood fail to detect acute cellular rejection after liver transplantation.

OBJECTIVE: We investigated whether microRNA signatures in whole blood samples are associated with acute cellular rejection (ACR) after liver transplantation. MATERIALS AND METHODS: Blood samples were collected using Paxgene technology and analyzed by microarrays and quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: microRNA signatures failed to distinguish between 19 patients with ACR and 16 controls. Let-7b-5p and let-7c were upregulated in a subgroup of patients with ACR during the 6th and 7th postoperative days but failed in an independent validation of 20 patients. CONCLUSION: microRNA signatures in whole blood processed by Paxgene technology are not suited for the detection of ACR after liver transplantation.

Human Hepatocyte Isolation: Does Portal Vein Embolization Affect the Outcome?

Primary human hepatocytes are widely used for basic research, pharmaceutical testing, and therapeutic concepts in regenerative medicine. Human hepatocytes can be isolated from resected liver tissue. Preoperative portal vein embolization (PVE) is increasingly used to decrease the risk of delayed postoperative liver regeneration by induction of selective hypertrophy of the future remnant liver tissue. The aim of this study was to investigate the effect of PVE on the outcome of hepatocyte isolation. Primary human hepatocytes were isolated from liver tissue obtained from partial hepatectomies (n = 190) using the two-step collagenase perfusion technique followed by Percoll purification. Of these hepatectomies, 27 isolations (14.2%) were performed using liver tissue obtained from patients undergoing PVE before surgery. All isolations were characterized using parameters that had been described in the literature as relevant for the outcome of hepatocyte isolation. The isolation outcomes of the PVE and the non-PVE groups were then compared before and after Percoll purification. Metabolic parameters (transaminases, urea, albumin, and vascular endothelial growth factor secretion) were measured in the supernatant of cultured hepatocytes for more than 6 days (PVE: n = 4 and non-PVE: n = 3). The PVE and non-PVE groups were similar in regard to donor parameters (sex, age, and indication for surgery), isolation parameters (liver weight and cold ischemia time), and the quality of the liver tissue. The mean initial viable cell yield did not differ between the PVE and non-PVE groups (10.16 ± 2.03 × 10(6) cells/g vs. 9.70 ± 0.73 × 10(6) cells/g, p = 0.499). The initial viability was slightly better in the PVE group (77.8% ± 2.03% vs. 74.4% ± 1.06%). The mean viable cell yield (p = 0.819) and the mean viability (p = 0.141) after Percoll purification did not differ between the groups. PVE had no effect on enzyme leakage and metabolic activity of cultured hepatocytes. Although PVE leads to drastic metabolic alterations and changes in hepatic blood flow, embolized liver tissue is a suitable source for the isolation of primary human hepatocytes and is equivalent to untreated liver tissue in regard to cell yield and viability.

Independent effects of sham laparotomy and anesthesia on hepatic microRNA expression in rats.

BACKGROUND: Studies on liver regeneration following partial hepatectomy (PH) have identified several microRNAs (miRNAs) that show a regulated expression pattern. These studies involve major surgery to access the liver, which is known to have intrinsic effects on hepatic gene expression and may also affect miRNA screening results. We performed two-third PH or sham laparotomy (SL) in Wistar rats to investigate the effect of both procedures on miRNA expression in liver tissue and corresponding plasma samples by microarray and qRT-PCR analyses. As control groups, non-treated rats and rats undergoing anesthesia only were used. RESULTS: We found that 49 out of 323 miRNAs (15%) were significantly deregulated after PH in liver tissue 12 to 48 hours postoperatively (>20% change), while 45 miRNAs (14%) were deregulated following SL. Out of these miRNAs, 10 miRNAs were similarly deregulated after PH and SL, while one miRNA showed opposite regulation. In plasma, miRNA upregulation was observed for miR-133a and miR-133b following PH and SL, whereas miR-100 and miR-466c were similarly downregulated following anesthesia and surgery. CONCLUSIONS: We show that miRNAs are indeed regulated by sham laparotomy and anesthesia in rats. These findings illustrate the critical need for finding appropriate control groups in experimental surgery.

Hedgehog pathway as a potential treatment target in human cholangiocarcinoma.

BACKGROUND: Innovative treatment concepts targeting essential signaling pathways may offer new chances for patients suffering from cholangiocarcinoma (CCC). For that, we performed a systematic molecular genetic analysis concerning the Hedgehog activity in human CCC samples and analyzed the effect of Hh inhibition on CCC cells in vitro and in vivo. METHODS: Activation of the Hh pathway was analyzed in 50 human CCC samples using quantitative polymerase chain reaction (qPCR). The efficacy of Hh inhibition using cyclopamine and BMS-833923 was evaluated in vitro. In addition, the effect of BMS-833923, alone or in combination with gemcitabine, was analyzed in vivo in a murine subcutaneous xenograft model. RESULTS: Expression analysis revealed a significant activation of the Hh-signaling pathway in nearly 50% of CCCs. Hh inhibition resulted in a significant decrease in cell proliferation of CCC cells. Moreover, a distinct inhibition of tumor growth could be seen as a result of a combined therapy with BMS-833923 and gemcitabine in CCC xenografts. CONCLUSION: The results of our study suggest that the Hh pathway plays a relevant role at least in a subset of human CCC. Inhibition of this pathway may represent a possible treatment option for CCC patients in which the Hh pathway is activated.

Combination of hedgehog signaling blockage and chemotherapy leads to tumor reduction in pancreatic adenocarcinomas.

OBJECTIVES: Activation of the hedgehog signal transduction pathway, triggered by hedgehog binding to the transmembrane receptor patched 1 (PTCH1) or by mutations in the PTCH1 gene, plays an important role in the development of various tumors. METHODS: To investigate whether the Hedgehog signaling pathway is also active in human pancreatic adenocarcinomas, we determined the expression levels of the known Hedgehog target genes PTCH1 and GLI-1 in pancreatic tumors. To determine whether alterations in the PTCH1 gene are responsible for this pathway activation, we screened pancreatic carcinomas for mutations in PTCH. To investigate the contribution of hedgehog signaling to the tumorigenicity of pancreatic tumor cells, we blocked the Hedgehog pathway in cultured tumor cells and xenografts using the steroidal alkaloid cyclopamine and the small-molecule Hedgehog inhibitor Hh-Antag. RESULTS: We identified single nucleotide polymorphisms (SNPs) within the PTCH1 gene but no somatic PTCH1 mutations. Pathway-blockage resulted in a significant dose-dependent reduction of tumor cell growth in vitro and in vivo. Moreover, combined treatment with cyclopamine and the conventional antimetabolite gemcitabine revealed a synergistic effect on the reduction of tumor growth in pancreatic adenocarcinoma xenografts. CONCLUSIONS: Inhibition of Hedgehog signaling could be a promising approach for the treatment of pancreatic adenocarcinomas.

Hopping on the methadone bus.

This paper investigates the impact of a 'free drug program' on the market equilibrium of drugs. We introduce a screening model of the hard drug market in which dealers use payment and punishment options to screen between high and low risk users. We show that, if a free drug program selects sufficiently many high-risk drug users, the pure-strategy separating market equilibrium ceases to exist and a symmetric mixed-strategy equilibrium results, in which drug users derive a higher expected utility. This encourages new drug users to enter the market. The novelty of the paper is the transmission mechanism for this effect, which is via the influence on market price.

Erythropoietin reduces ischemia-reperfusion injury after liver transplantation in rats.

Human recombinant Erythropoietin (rHuEpo) has recently been shown to be a potent protector of ischemia- reperfusion injury in warm-liver ischemia. Significant enhancement of hepatic regeneration and survival after large volume partial hepatic resection has also been demonstrated. It was the aim of this study to evaluate the capacities of rHuEpo in the setting of rat liver transplantation. One-hundred-and-twenty Wistar rats were used: 60 recipients received liver transplantation following donor organ treatment (60 donors) with either 1000 IU rHuEpo or saline injection (controls) into portal veins (cold ischemia 18 h, University of Wisconsin (UW) solution). Recipients were allocated to two groups, which either received 1000 IU rHuEpo at reperfusion or an equal amount of saline (control). Animals were sacrificed at defined time-points (2, 4.5, 24, 48 h and 7 days postoperatively) for analysis of liver enzymes, histology [hematoxylin-eosin (HE) staining, periodic acid Schiff staining (PAS)], immunostaining [terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), Hypoxyprobe] and real-time polymerase chain reaction (RT-PCR) of cytokine mRNA (IL-1, IL-6). Lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) values were significantly reduced among the epo-treated animals 24 and 48 h after liver transplantation (LT). The TUNEL and Hypoxyprobe analyses as well as necrotic index evaluation displayed significant reduction of apoptosis and necrosis in rHuEpo-treated graft livers. Erythropoietin reduces ischemia-reperfusion injury after orthotopic liver transplantation in rats.

Erythropoietin promotes hepatic regeneration after extended liver resection in rats.

BACKGROUND AND AIM: It has been proven in various animal studies that recombinant human erythropoietin (rHuEPO) protects renal, cardiac and neuronal, as well as hepatic, tissue from ischemia, and promotes regeneration of adult central nervous system neurons. To date, no data are available as to whether rHuEPO has the ability to stimulate liver regeneration after liver resection. METHODS: Rats undergoing 70% or 90% hepatectomy received an intraportalvenous administration (i.p.) of rHuEPO prior to resection or a subcutaneous injection (s.c.) for 3 days postoperatively, control animals were treated with surgery and saline injection only. Regeneration capacity of remnant livers was studied over 7 days by histology and immunohistochemistry (Ki-67, proliferating cell nuclear antigen [PCNA]). Polymerase chain reaction was carried out to measure transforming growth factor beta (TGF-beta), hypoxia induced factor (HIF), signal transducing activator 3 and vascular endothelial growth factor. RESULTS: Ten-day survival in rats undergoing 90% hepatectomy significantly increased in i.p.-pretreated animals. After 70% hepatectomy the mitotic index was significantly increased in both rHuEPO-treated groups. These data were confirmed by PCNA and Ki-67 expression, which was significantly increased in the treated groups 24 h and 2 days after liver resection. TGF-beta and HIF mRNA both were upregulated in control animals 3 h after surgery. CONCLUSION: rHuEPO effectively increased liver regeneration in rats after 70% liver resection and enhanced survival after 90% hepatectomy. Thus, rHuEPO may increase the regenerative capacity after major hepatectomy.

Combination therapy of poly (ADP-ribose) polymerase inhibitor 3-aminobenzamide and gemcitabine shows strong antitumor activity in pancreatic cancer cells.

BACKGROUND AND AIM: Poly (ADP-ribose) polymerase (PARP) inhibitors such as 3-aminobenzamide (3-ABA) enhance the in vitro cytotoxicity of DNA mono-functional alkylating agents such as radiation or chemotherapeutic agents. The aim of this study was to test an approach combining the PARP inhibitor 3-ABA with standard gemcitabine therapy in human pancreatic cancer cells. METHODS: Cell viability was determined by proliferation assay (XTT). Cell-cycle analysis (FACS), ELISA (M30 Apoptosense), Western blot for caspase 8 and PARP, and electron microscopy were used to identify apoptosis. Tumor growth and survival was assessed in nude mice by subcutaneously injected Capan-1 cells. In addition, Ki67 staining was performed on tumors for cell proliferation and in vivo apoptosis induction was measured by TUNEL assay and ELISA. RESULTS: Combination therapy of gemcitabine and 3-ABA suppressed tumor cell growth more than gemcitabine alone in XTT, FACS and ELISA analysis. CONCLUSION: This in vivo study demonstrated a significantly reduced tumor weight and increased survival up to 40 days after cell inoculation with combination therapy compared to animals treated with PBS, gemcitabine or 3-ABA alone. Furthermore, TUNEL assay revealed a significant apoptosis induction and reduced proliferation in the combination group.