Intrahepatic Transplantation Research Articles

Conjugating homogenized liver-extracellular matrix into decellularized hepatic scaffold for liver tissue engineering.

The generation of a transplantable liver scaffold is crucial for the treatment of end-stage liver failure. Unfortunately, decellularized liver scaffolds suffer from lack of bioactive molecules and functionality. In this study, we conjugated homogenized liver-extracellular matrix (ECM) into a decellularized liver in a rat model to improve its structural and functional properties. The homogenized ECM was prepared, characterized, and subsequently perfused into ethyl carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) activated liver scaffolds. Various techniques were performed to confirm the improvements that were accomplished through the conjugation process; these included micro/ultra-structural analyses, biochemical analysis of ECM components, DNA quantification, swelling ratio, structural stability, calcification properties, platelet activation study, static and dynamic seeding with EAhy926 endothelial cells and HepG2 hepatocarcinoma cells, subcutaneous implantation and intrahepatic transplantation. The results showed that the conjugated scaffolds have superior micro- and ultrastructural and biochemical characteristics. In addition, DNA contents, swelling ratios, calcification properties, platelet reactions, and host inflammatory reactions were not altered with the conjugation process. The conjugated scaffolds revealed better cellular spreading and popularity compared to the non-conjugated scaffolds. Intrahepatic transplantation showed that the conjugated scaffold had higher popularity of hepatic regenerative cells with better angiogenesis. The conjugation of the decellularized liver scaffold with homogenized liver-ECM is a promising tool to improve the quality of the generated scaffold for further transplantation.

359-OR: a- or ß-Adrenergic Blockade Does Not Affect Transplanted Islet Responses to Hypoglycemia in Type 1 Diabetes (T1D)

Recipients of intrahepatic islet transplantation for T1D exhibit appropriate suppression of endogenous insulin and activation of glucagon secretion in response to insulin-induced hypoglycemia with restored glucose counterregulation and protection against clinically significant hypoglycemia. Whether sympathetic activation of adrenergic receptors on transplanted islets is required for these responses is not known. To evaluate the adrenergic contribution to post-transplant glucose counterregulation, we performed a randomized, double-blind crossover study of responses during a hyperinsulinemic (1 mU·kg-1·min-1) euglycemic (EU, ∼90 mg/dL)-hypoglycemic (HYPO, ∼50 mg/dL) clamp conducted under phentolamine (0.95 µg·kg-1·min-1; α-adrenergic blockage), propranolol (0.48 µg·kg-1·min-1; β-adrenergic blockage), or placebo infusion. Subjects were 5F/4M with median (range) age 53 (34 - 63) years, T1D duration 29 (18 - 56) years, post-transplant 7 (2 - 8) years, HbA1c 5.8 (4.5 - 6.8) %, insulin in-/dependent 5/4, on tacrolimus-based immunosuppression, and spent 97 (76 - 99) % time in range 70 - 180 mg/dL and 1 (0 - 3) % time with hypoglycemia < 70 mg/dL by CGM. During the EU-HYPO clamp, blood pressure was lower with phentolamine and heart rate lower with propranolol vs. placebo (P < 0.05). There was no difference in suppression of C-peptide or activation of glucagon with phentolamine or propranolol vs. placebo. Pancreatic polypeptide was greater with phentolamine vs. placebo during EU (P < 0.05), and free fatty acids were lower and the glucose infusion rate higher with propranolol vs. placebo during HYPO (P < 0.05) with no differences seen for endogenous glucose production. These results indicate physiologic α- and β-adrenergic blockage had no effect on transplanted islet responses to hypoglycemia. Sympathetic re-innervation, as occurs with re-vascularization of intrahepatic islets, may not be necessary for post-transplant recovery of glucose counterregulation in T1D. Disclosure M.R. Rickels: Consultant; Self; Semma Therapeutics, Inc. Research Support; Self; Xeris Pharmaceuticals, Inc. M. Bellin: Research Support; Self; Dexcom, Inc., Viacyte, Inc. Other Relationship; Self; Insulet Corporation. D. Stefanovski: None. A.J. Peleckis: None. C.V. Dalton-Bakes: None. E. Markmann: None. H.T. Nguyen: None. A. Naji: None. Funding National Institutes of Health (R01DK091331, UL1TR001878, P30DK19525)

Donor-Specific Regulatory T Cell-Mediated Immune Tolerance in an Intrahepatic Murine Allogeneic Islet Transplantation Model with Short-Term Anti-CD154 mAb Single Treatment

Anti-CD154 blockade-based regimens remain unequaled in prolonging graft survival in various organ transplantation models. Several studies have focused on transplantation tolerance with the anti-CD154 blockade, but none of these studies has investigated the mechanisms associated with its use as the sole treatment in animal models, delaying our understanding of anti-CD154 blockade-mediated immune tolerance. The purpose of this study was to investigate the mechanism underlying the anti-CD154 monoclonal antibody (mAb) blockade in inducing immune tolerance using an intrahepatic murine allogeneic islet transplantation model. Allogeneic BALB/c AnHsd (BALB/c) islets were infused into the liver of diabetic C57BL/6 (B6) mice via the cecal vein. Anti-CD154 mAb (MR1) was administered on −1, 0, 1, 3, 5, and 7 d posttransplantation at 0.5 mg per mouse. We showed that short-term MR1 monotherapy could prolong the allogeneic islet grafts to more than 250 d in the murine intrahepatic islet transplantation model. The second islet grafts transplanted under the kidney capsule of the recipients were protected from rejection. We also found that rejection of same-donor skin grafts transplanted to the tolerant mice was modestly delayed. Using a DEREG mouse model, FoxP3+ regulatory T (Treg) cells were shown to play important roles in transplantation tolerance. In mixed lymphocyte reactions, Treg cells from the tolerant mice showed more potency in suppressing BALB/c splenocyte-stimulated Teff cell proliferation than those from naïve mice. In this study, we demonstrated for the first time that a short-term anti-CD154 mAb single treatment could induce FoxP3+ Treg cell-mediated immune tolerance in the intrahepatic murine allogeneic islet transplantation model.

The Role of Vitamin D and Omega-3 PUFAs in Islet Transplantation.

Recurrence of autoimmunity and allograft rejection represent major challenges that impact the success of islet transplantation. Despite the remarkable improvements achieved in immunosuppression strategies after the publication of the Edmonton protocol, long-term data of intra-hepatic islet transplantation show a gradual decline in beta-cell function. Therefore, there is a growing interest in the investigation of novel, safe and effective anti-inflammatory and immunomodulatory strategies able to promote long-term islet graft survival and notable improvements in clinical outcomes of islet transplant recipients. Vitamin D has been shown to exert anti-inflammatory and immunomodulatory effects. Pre-clinical studies investigating the use of vitamin D and its analogs (alone or in combination with immunosuppressive agents and/or other anti-inflammatory agents, such as omega-3 polyunsaturated fatty acids) showed beneficial results in terms of islet graft survival and prevention of recurrence of autoimmunity/allograft rejection in animal models of syngeneic and allogeneic islet transplantation. Moreover, epidemiologic studies demonstrated that vitamin D deficiency is highly prevalent after solid organ transplantation (e.g., heart, liver or kidney transplantation). However, studies that critically assess the prevalence of vitamin D deficiency among islet transplant recipients have yet to be conducted. In addition, prospective studies aimed to address the safety and efficacy of vitamin D supplementation as an adjuvant immunomodulatory strategy in islet transplant recipients are lacking and are therefore awaited in the future.

Therapeutic potential of spheroids of stem cells from human exfoliated deciduous teeth for chronic liver fibrosis and hemophilia A.

Mesenchymal stem cell (MSC)-based cell therapies have emerged as a promising treatment option for various diseases. Due to the superior survival and higher differentiation efficiency, three-dimensional spheroid culture systems have been an important topic of MSC research. Stem cells from human exfoliated deciduous teeth (SHED) have been considered an ideal source of MSCs for regenerative medicine. Thus, in the present study, we introduce our newly developed method for fabricating SHED-based micro-hepatic tissues, and demonstrate the therapeutic effects of SHED-based micro-hepatic tissues in mouse disease models. SHED-converted hepatocyte-like cells (SHED-HLCs) were used for fabricating spherical micro-hepatic tissues. The SHED-HLC-based spheroids were then transplanted both into the liver of mice with CCl4-induced chronic liver fibrosis and the kidney of factor VIII (F8)-knock-out mice. At 4weeks after transplantation, the therapeutic efficacy was investigated. Intrahepatic transplantation of SHED-HLC-spheroids improved the liver dysfunction in association with anti-fibrosis effects in CCl4-treated mice. Transplanted SHED-converted cells were successfully engrafted in the recipient liver. Meanwhile, renal capsular transplantation of the SHED-HLC-spheroids significantly extended the bleeding time in F8-knock-out mice. These findings suggest that SHED-HLC-based micro-hepatic tissues might be a promising source for treating pediatric refractory diseases, including chronic liver fibrosis and hemophilia A.

Multisite Injection of Bioengineered Hepatic Units from Collagen Hydrogel and Neonatal Liver Cells in Parenchyma Improves Liver Cirrhosis.

Intrahepatic transplantation of hepatocytes/progenitor cells remodels or repopulates livers resulting in improved hepatic function, which represents a promising treatment option for liver diseases, but poor engraftment of transplanted liver cells in livers has been the major obstacle for exerting therapeutic efficacy on liver diseases. We herein demonstrated great potential of multisite injection of bioengineered hepatic units generating from collagen hydrogel and neonatal liver cells (MSI) in promoting engraftment, survival of liver cells, and the therapeutic potential of liver diseases in livers. The MSI allowed transplantation of large number of liver cells into parenchyma in one time. Superiorly, the MSI promoted the engraftment and survival of liver cells, and generated large, dispersively distributed tissue masses whose sizes were positively correlated with the number of injection sites in livers. Consistently, the MSI improved liver cirrhosis as evidenced by the reduction of collagen matrix deposit, improved liver architecture, and hepatic function in the MSI group compared to that of the nontreated control. These data indicate that the MSI represents a novel, improved bioengineered strategy that can exert therapeutic efficacy on liver diseases.

CCL2/MCP-1 and CXCL12/SDF-1 blockade by L-aptamers improve pancreatic islet engraftment and survival in mouse.

The blockade of pro-inflammatory mediators is a successful approach to improve the engraftment after islet transplantation. L-aptamers are chemically synthesized, nonimmunogenic bio-stable oligonucleotides that bind and inhibit target molecules conceptually similar to antibodies. We aimed to evaluate if blockade-aptamer-based inhibitors of C-C Motif Chemokine Ligand 2/monocyte chemoattractant protein-1 (CCL2/MCP-1) and C-X-C Motif Chemokine Ligand 12/stromal cell-derived factor-1 (CXCL12/SDF-1) are able to favor islet survival in mouse models for islet transplantation and for type 1 diabetes. We evaluated the efficacy of the CCL2-specific mNOX-E36 and the CXCL12-specific NOX-A12 on islet survival in a syngeneic mouse model of intraportal islet transplantation and in a multiple low doses of streptozotocin (MLD-STZ) diabetes induction model. Moreover, we characterized intrahepatic infiltrated leukocytes by flow cytometry before and 3 days after islet infusion in presence or absence of these inhibitors. The administration for 14days of mNOX-E36 and NOX-A12 significantly improved islet engraftment, either compound alone or in combination. Intrahepatic islet transplantation recruited CD45+ leucocytes and more specifically CD45+/CD11b+ mono/macrophages; mNOX-E36 and NOX-A12 treatments significantly decreased the recruitment of inflammatory monocytes, CD11b+ /Ly6Chigh /CCR2+ and CD11b+ /Ly6Chigh /CXCR4+ cells, respectively. Additionally, both L-aptamers significantly attenuated diabetes progression in the MLD-STZ model. In conclusion, CCL2/MCP-1 and CXCL12/SDF-1 blockade by L-aptamers is an efficient strategy to improve islet engraftment and survival.

Operational immune tolerance towards transplanted allogeneic pancreatic islets in mice and a non-human primate.

Patients with autoimmune type 1 diabetes transplanted with pancreatic islets to their liver experience significant improvement in quality of life through better control of blood sugar and enhanced awareness of hypoglycaemia. However, long-term survival and efficacy of the intrahepatic islet transplant are limited owing to liver-specific complications, such as immediate blood-mediated immune reaction, hypoxia, a highly enzymatic and inflammatory environment and locally elevated levels of drugs including immunosuppressive agents, all of which are injurious to islets. This has spurred a search for new islet transplant sites and for innovative ways to achieve long-term graft survival and efficacy without life-long systemic immunosuppression and its complications. We used our previously established approach of islet transplant in the anterior chamber of the eye in allogeneic recipient mouse models and a baboon model of diabetes, which were treated transiently with anti-CD154/CD40L blocking antibody in the peri-transplant period. Survival of the intraocular islet allografts was assessed by direct visualisation in the eye and metabolic variables (blood glucose and C-peptide measurements). We evaluated longitudinally the cytokine profile in the local microenvironment of the intraocular islet allografts, represented in aqueous humour, under conditions of immune rejection vs tolerance. We also evaluated the recall response in the periphery of the baboon recipient using delayed-type hypersensitivity (DTH) assay, and in mice after repeat transplant in the kidney following initial transplant with allogeneic islets in the eye or kidney. Results in mice showed >300days immunosuppression-free survival of allogeneic islets transplanted in the eye or kidney. Notably, >70% of tolerant mice, initially transplanted in the eye, exhibited >400days of graft survival after re-transplant in the kidney without immunosuppression compared with ~30% in mice that were initially transplanted in the kidney. Cytokine and DTH data provided evidence of T helper 2-driven local and peripheral immune regulatory mechanisms in support of operational immune tolerance towards the islet allografts in both models. We are currently evaluating the safety and efficacy of intraocular islet transplantation in a phase 1 clinical trial. In this study, we demonstrate immunosuppression-free long-term survival of intraocular islet allografts in mice and in a baboon using transient peri-transplant immune intervention. These results highlight the potential for inducing islet transplant immune tolerance through the intraocular route. Therefore, the current findings are conceptually significant and may impact markedly on clinical islet transplantation in the treatment of diabetes.

Pancreatic Islet Transplantation in Humans: Recent Progress and Future Directions.

Pancreatic islet transplantation has become an established approach to β-cell replacement therapy for the treatment of insulin-deficient diabetes. Recent progress in techniques for islet isolation, islet culture, and peritransplant management of the islet transplant recipient has resulted in substantial improvements in metabolic and safety outcomes for patients. For patients requiring total or subtotal pancreatectomy for benign disease of the pancreas, isolation of islets from the diseased pancreas with intrahepatic transplantation of autologous islets can prevent or ameliorate postsurgical diabetes, and for patients previously experiencing painful recurrent acute or chronic pancreatitis, quality of life is substantially improved. For patients with type 1 diabetes or insulin-deficient forms of pancreatogenic (type 3c) diabetes, isolation of islets from a deceased donor pancreas with intrahepatic transplantation of allogeneic islets can ameliorate problematic hypoglycemia, stabilize glycemic lability, and maintain on-target glycemic control, consequently with improved quality of life, and often without the requirement for insulin therapy. Because the metabolic benefits are dependent on the numbers of islets transplanted that survive engraftment, recipients of autoislets are limited to receive the number of islets isolated from their own pancreas, whereas recipients of alloislets may receive islets isolated from more than one donor pancreas. The development of alternative sources of islet cells for transplantation, whether from autologous, allogeneic, or xenogeneic tissues, is an active area of investigation that promises to expand access and indications for islet transplantation in the future treatment of diabetes.

Hepatic Hydrothorax Complicated by Spontaneous Bacterial Empyema: An Under-Recognized Clinical Entity

Spontaneous bacterial empyema (SBE) is an under-recognized clinical entity and significant complication of cirrhosis that has a high mortality if left untreated. This case report reviews a presentation of recurrent hepatic hydrothorax with thoracentesis revealing this diagnosis and highlights the importance of treatment. A 62-year-old female with a history of cirrhosis secondary to autoimmune liver disease presented with acute abdominal pain, fevers, chills, and dyspnea. The patient had discontinued prophylactic antibiotics and her home diuretics were stopped one week prior for increasing serum creatinine. She was afebrile with a pulse of 92, blood pressure of 102/56, respiratory rate of 16 and stable oxygen saturation. Her abdomen was tender and she had decreased breath sounds extending up to the right mid-lung. Laboratory data were significant for hemoglobin of 8.4 g/dl, platelets of 67,000/ml, sodium of 130 mEq/L, albumin of 2.5 g/dl, creatinine of 1.7 mg/dl, bilirubin of 1.5 mg/dl, and INR of 1.5 (MELD-Na of 18). Computed tomography of the abdomen revealed a large right pleural effusion with small volume abdominal ascites. Thoracentesis showed a transudative effusion with 3,581 WBC / mm3, 60% neutrophils, pH of 7.53, protein 1.0 g/dL, albumin 0.5 g/dL, and LDH 91 U/L significant for SBE. The patient received a cephalosporin antibiotic and albumin. She had recurrent hepatic hydrothorax requiring repeat thoracentesis on day 6 with cell count of 185 WBC / mm3 and 12% neutrophils. Home diuretics were cautiously restarted and she was discharged in stable condition. This case demonstrates hepatic hydrothorax complicated by SBE after holding home diuretics. SBE is a rare finding defined as hepatic hydrothorax with a pleural fluid neutrophil count greater than 500 / mm3 or greater than 250 / mm3 with positive fluid cultures. One must exclude parapneumonic effusion before making a diagnosis of SBE; treatment regimens and clinical implications differ. SBE will affect up to 16% of patients with hepatic hydrothorax with a high mortality of 20%. It often occurs in the absence of spontaneous bacterial peritonitis or appreciable ascites. A low threshold to perform diagnostic thoracentesis should be applied to patients with suspected hepatic hydrothorax and constitutional symptoms. The risks and benefits of diuresis in addition to evaluation for transjugular intrahepatic portosystemic shunt and transplant should be considered in these patients to prevent recurrence.2370_A Figure 1. Computed tomography of chest showing large right hepatic hydrothorax.2370_B Figure 2. Chest radiography showing large right hepatic hydrothorax.2370_C Figure 3. Chest radiography following thoracentesis showing resolution of right hepatic hydrothorax.

Alpha-1 Antitrypsin (AAT) Promotes Islets Graft Survival by Modulating Macrophage Activation

Infiltrating macrophages can induce inflammation by secretion of proinflammatory cytokines and contribute to islet graft loss shortly after islet transplantation. In this study, we tested the mechanistic effects of AAT in mitigating macrophage mediated inflammation in an intrahepatic islet transplantation model in which human islets (2500 islet equivalent numbers) were transplanted into NOD-SCID mice that had been rendered diabetic (blood glucose >350mg/dl) by streptozotocin treatment. AAT (Prolastin-C, Grifols) was given to recipients intraperitoneally at 4 mg/mouse, every two days for 14 days, beginning before transplantation. In those recipients receiving AAT (n=29), 69.0% reached normoglycemia, compared to 35.7% in controls (n=28, p=0.002, logrank test) at 30 days post transplantation. Compared to mice treated with vehicle, AAT-treated mice had lower serum levels of interleukin 1 β (IL-1β), interferon- γ (IFN-γ) and interleukin 6 (IL-6), at day 1 post transplantation. To explore potential cell target(s) of AAT action, we analyzed activation of macrophages infiltrated into the liver after transplantation. Fewer M1 macrophages were observed in AAT-treated mice than in controls. To measure the effects of AAT on macrophage activation, we added AAT to Raw264.7 cells stimulated with IFN-γ in vitro. Expressions of M1 markers (iNOS, CD11c, TNFα, and IL-6) were dramatically decreased in AAT-treated cells compared to vehicle-treated controls. Co-culture of IFN-γ activated macrophages with islets led to islet cell apoptosis. Addition of AAT in the co-culture system reduced macrophage activation and protected islet cells from apoptosis. In summary, AAT treatment significantly enhances human islet graft survival and function after transplantation into the liver of NOD-SCID mice, in part by suppression of macrophage activation and pro-inflammatory cytokine production. Disclosure W. Gou: None. J. Wang: None. D. Kim: None. L. Song: None. C. Strange: Research Support; Self; Grifols, CSL Behring, Shire. Consultant; Self; Shire. Stock/Shareholder; Self; Abeona. Consultant; Self; CSL Behring. Research Support; Self; Adverum. Consultant; Self; Adverum. Research Support; Self; MatRx. D. Adams: None. K. Morgan: None. H. Wang: None.

MONITORING OF MORPHOLOGICAL EFFECTS AUTOLOGICAL MESENCHIMAL STEM CELLS, TRANSPLANTED IN LIVER WITH VIRUS CYRROSIS (clinical observation)

Introduction. The importance of the HCV-infection is determined by the wide spread, progressive course, the formation of liver cirrhosis (LC) and hepatocellular carcinoma. The mechanisms of the effect of the virus on hepatic cells, the processes of fibrogenesis and fibrolysis, mechanisms of the reverse development of the LC remain unexplored. There is no effective pathogenetic therapy. The aim of the study — determination of the effectiveness and safety of intrahepatic transplantation of mesenchymal stem cells (MSC) in chronic HCV-infection at the stage оf LC. Methods. A patient with HCV-LC who has a secondary hemorrhagic vaculities who underwent autologous MSC transplantation into the liver tissue. The liver biopsy specimens were studied in dynamics by light and electronic microscopy and by immunohistochemistry. Results. The transplantation and posttransplantation period proceeded without complications. After the introduction of MSC the features of the formed micronodular LC remained. In some parts of the samples, the septa looked thin, sometimes perforated, indicating a resorption in this place of fibrous tissue. There was a decrease in the degree of transdifferentiation of stellate cells into myofibroblasts, a decrease in the number of fibroblasts and fibroblasts, there were no immune reactions in the form of deposition of amorphous and fibrous masses of moderate electron density along the sinusoidal capillaries that were significantly expressed in the primary biopsy. These changes were combined with the appearance of hepatocyte heterogeneity in the density of the cytoplasmic matrix, the state and quantity of organelles and inclusions, and the structural improvement of intracellular organelles. Conclusion. Autologous transplantation of mesenchymal bone marrow stem cells reduces the degree of destructive changes in hepatocytes, the severity of fibrosis and contributes to the improvement of the morpho-functional state of the liver, and therefore, it can be recommended as an important component of medical interventions.

Local, Controlled Release In Vivo of Vascular Endothelial Growth Factor Within a Subcutaneous Scaffolded Islet Implant Reduces Early Islet Necrosis and Improves Performance of the Graft

Islet transplantation remains the only alternative to daily insulin therapy for control of type 1 diabetes (T1D) in humans. To avoid the drawbacks of intrahepatic islet transplantation, we are developing a scaffolded islet implant to transplant islets into nonhepatic sites. The implant test bed, sized for mice, consists of a limited (2-mm) thickness, large-pore polymeric sponge scaffold perforated with peripheral cavities that contain islets suspended in a collagen hydrogel. A central cavity in the scaffold holds a 2-mm diameter alginate sphere for controlled release of the angiogenic cytokine vascular endothelial growth factor (VEGF). Host microvessels readily penetrate the scaffold and collagen gel to vascularize the islets. Here, we evaluate the performance of the implant in a subcutaneous (SC) graft site. Implants incorporating 500 syngeneic islets reversed streptozotocin-induced diabetes in mice approximately 30 d after SC placement. Controlled release of a modest quantity (20 ng) of VEGF within the implant significantly reduced the time to normoglycemia compared to control implants lacking VEGF. Investigation of underlying causes for this effect revealed that inclusion of 20 ng of VEGF in the implants significantly reduced central necrosis of islets 24 h after grafting and increased implant vascularization (measured 12 d after grafting). Collectively, our results demonstrate (1) that the scaffolded islet implant design can reverse diabetes in SC sites in the absence of prevascularization of the graft site and (2) that relatively low quantities of VEGF, delivered by controlled release within the implant, can be a useful approach to limit islet stress after grafting.

Maximum intensity projection of an intrahepatic transplant 14 days after Tx

Maximum intensity projection of an intrahepatic transplant 14 days after Tx

Maximum intensity projection of an intrahepatic transplant 1 day after Tx

Maximum intensity projection of an intrahepatic transplant 1 day after Tx

Anti-Inflammatory Strategies in Intrahepatic Islet Transplantation

The identification of pathway(s) playing a pivotal role in peritransplant detrimental inflammatory events represents the crucial step toward a better management and outcome of pancreatic islet transplanted patients. Recently, we selected the CXCR1/2 inhibition as a relevant strategy in enhancing pancreatic islet survival after transplantation. Here, the most clinically used anti-inflammatory compounds (IL1-receptor antagonist, steroids, and TNF-α inhibitor) alone or in combination with a CXCR1/2 inhibitor were evaluated in their ability to improve engraftment or delay graft rejection. To rule out bias related to transplantation site, we used well-established preclinical syngeneic (250 C57BL/6 equivalent islets in C57BL/6) and allogeneic (400 Balb/c equivalent islets in C57BL6) intrahepatic islet transplantation platforms. In mice, we confirmed that targeting the CXCR1/2 pathway is crucial in preserving islet function and improving engraftment. In the allogeneic setting, CXCR1/2 inhibitor alone could reduce the overall recruitment of transplant-induced leukocytes and significantly prolong the time to graft rejection both as a single agent and in combination with immunosuppression. No other anti-inflammatory compounds tested (IL1-receptor antagonist, steroids, and TNF-α inhibitor) alone or in combination with CXCR1/2 inhibitor improve islet engraftment and significantly delay graft rejection in the presence of MMF + FK-506 immunosuppressive treatment. These findings indicate that only the CXCR1/2-mediated axis plays a crucial role in controlling the islet damage and should be a target for intervention to improve the efficiency of islet transplantation.

Posttransplant oxygen inhalation improves the outcome of subcutaneous islet transplantation: A promising clinical alternative to the conventional intrahepatic site.

Subcutaneous tissue is a promising site for islet transplantation, due to its large area and accessibility, which allows minimally invasive procedures for transplantation, graft monitoring, and removal of malignancies as needed. However, relative to the conventional intrahepatic transplantation site, the subcutaneous site requires a large number of islets to achieve engraftment success and diabetes reversal, due to hypoxia and low vascularity. We report that the efficiency of subcutaneous islet transplantation in a Lewis rat model is significantly improved by treating recipients with inhaled 50% oxygen, in conjunction with prevascularization of the graft bed by agarose-basic fibroblast growth factor. Administration of 50% oxygen increased oxygen tension in the subcutaneous site to 140mmHg, compared to 45mmHg under ambient air. In vitro, islets cultured under 140mmHg oxygen showed reduced central necrosis and increased insulin release, compared to those maintained in 45mmHg oxygen. Six hundred syngeneic islets subcutaneously transplanted into the prevascularized graft bed reversed diabetes when combined with postoperative 50% oxygen inhalation for 3days, a number comparable to that required for intrahepatic transplantation; in the absence of oxygen treatment, diabetes was not reversed. Thus, we show oxygen inhalation to be a simple and promising approach to successfully establishing subcutaneous islet transplantation.

A Rare Presentation of Hepatic Hydrothorax in a Patient with Alcohol induced Liver Cirrhosis

Hepatic hydrothorax is defined as significant pleural effusion greater than 500 ml in a patient with liver cirrhosis without any underlying pulmonary, cardiac and pleural disease. This case report describes a 47 year old Indian gentleman who was diagnosed as alcohol induced liver cirrhosis, Child-Turcotte-Pugh score B, with gross ascites. He presented with recurrent right sided pleural effusion. Pleural fluid analysis revealed transudative pleural effusion. A diagnosis of hepatic hydrothorax was made after excluding other causes of pleural effusion. He did not respond to medical therapy and sodium restriction. His recurrent pleural effusion was treated with tube thoracostomy and chemical talc pleurodesis. He was referred to the tertiary hepatology unit for transjugular intrahepatic portosystemic shunt (TIPSS) and liver transplantation. Hepatic hydrothorax should always be suspected in a patient who presents with liver cirrhosis with portal hypertension and transudative pleural effusion.

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice.

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy may incur acute whole blood reactions and result in poor islet engraftment. Here, we report a robust protocol for the transplantation of islets at the extrahepatic transplantation site-the epididymal fat pad (EFP)-in a diabetic mouse model. A protocol to isolate and purify islets at high yields from C57BL/6J mice is described, as well as a transplantation method performed by seeding islets onto a decellularized scaffold (DCS) and implanting them at the EFP site in syngeneic C57BL/6J mice rendered diabetic by streptozotocin. The DCS graft containing 500 islets reversed the hyperglycemic condition within 10 days, while the free islets without DCS required at least 30 days. The normoglycemia was maintained for up to 3 months until the graft was explanted. In conclusion, DCS enhanced the engraftment of islets into the extrahepatic site of the EFP, which could easily be retrieved and might provide a reproducible and useful platform for investigating the scaffold materials, as well as other transplantation parameters required for a successful islet engraftment.

Human liver mesenchymal stem/progenitor cells inhibit hepatic stellate cell activation: in vitro and in vivo evaluation

BackgroundProgressive liver fibrosis leads to cirrhosis and end-stage liver disease. This disease is a consequence of strong interactions between matrix-producing hepatic stellate cells (HSCs) and resident and infiltrating immune cell populations. Accumulated experimental evidence supports the involvement of adult-derived human liver mesenchymal stem/progenitor cells (ADHLSCs) in liver regeneration. The aim of the present study was to evaluate the influence of ADHLSCs on HSCs, both in vitro and in vivo.MethodsActivated human HSCs were co-cultured with ADHLSCs or ADHLSC-conditioned culture medium. The characteristics of the activated human HSCs were assessed by microscopy and biochemical assays, whereas proliferation was analyzed using flow cytometry and immunocytochemistry. The secretion profile of activated HSCs was evaluated by ELISA and Luminex. ADHLSCs were transplanted into a juvenile rat model of fibrosis established after co-administration of phenobarbital and CCl4.ResultsWhen co-cultured with ADHLSCs or conditioned medium, the proliferation of HSCs was inhibited, beginning at 24 h and for up to 7 days. The HSCs were blocked in G0/G1 phase, and showed decreased Ki-67 positivity. Pro-collagen I production was reduced, while secretion of HGF, IL-6, MMP1, and MMP2 was enhanced. Neutralization of HGF partially blocked the inhibitory effect of ADHLSCs on the proliferation and secretion profile of HSCs. Repeated intrahepatic transplantation of cryopreserved/thawed ADHLSCs without immunosuppression inhibited the expression of markers of liver fibrosis in 6 out of 11 rats, as compared to their expression in the vehicle-transplanted group.ConclusionsThese data provide evidence for a direct inhibitory effect of ADHLSCs on activated HSCs, which supports their development for the treatment of liver fibrosis.