Chao Long Chen

Mesenchymal stem cells prolong composite tissue allotransplant survival in a swine model.

Background. This study investigated whether mesenchymal stem cells (MSCs) combined with bone marrow transplantation (BMT), irradiation, or short-term immunosuppressant therapy could prolong composite tissue allotransplant survival in a swine hind-limb model. Methods. Heterotopic hind-limb transplantation was performed in outbred miniature swine. Group I (n=5) was the untreated control. Group II (n=3) received MSCs alone (given on days −1, +3, +7, +14, +21). Group III (n=6) received cyclosporine A (CsA days 0 to +28). Group IV (n=4) received preconditioning irradiation (day −1), BMT (day +1), and CsA (days 0 to +28). Group V (n=5) received irradiation (day −1), BMT (day +1), CsA (days 0 to +28), and MSCs (days +1, +7,+14). The expression and localization of CD4+/CD25+ T cells and MSCs were assessed using flow cytometry and immunohistochemistry. Results. The allografts survival with MSCs alone revealed a significant prolongation, when compared with the controls (P=0.02). Allografts with CsA treatment exhibited delayed rejection. Irradiation and BMT-CsA treatment revealed no significant allograft survival benefit when compared with the CsA treatment group, but graft-versus-host disease (GVHD) was evident. However, combination of MSCs-BMT-CsA treatment demonstrated significant prolongation of allograft survival (>200 days, P<0.001) and no signs of GVHD with the lowest degree of rejection in the allo-skin and interstitial muscle layers. The CD4+/CD25+ regulatory-like T-cell expression in the circulating blood and allo-skin significantly increased in the MSC-BMT-CsA group. Examination of bromodeoxyuridine-labeled MSCs revealed donor MSC engraftment into the recipient and donor skin and the recipient liver parenchymal tissue. Conclusion. These results suggested that the regulatory activity of MSCs on T cells and GVHD might contribute to significant prolongation of composite tissue allotransplant survival in the MSC-BMT-CsA treatment.

Assessment of donor fatty livers for liver transplantation

Aim. The effect of fatty liver on graft survival, especially with reference to macrovesicular and microvesicular steatosis, is still uncertain. This preliminarily study was designed to create a noninvasive method for the quantification of the hepatic fat content in vivo and to establish provisional criteria for the assessment of fatty donor livers before liver transplantation among transplant surgeons, radiologists, and pathologists. Methods and Materials. Different degrees of rat fatty liver model were established by feeding rats a diet deficient in choline and methionine for different periods of time. Computed tomography (CT) with test tubes containing variable percentages of fat equivalent substance were used to assess the severity of fatty change of the rat liver. This was then correlated with the histological classification, level of hepatic enzymes, and graft survival. Results. Linear correlation between the fat volume fraction added to the test tubes and CT density were found. The process of producing a fatty liver via diet alteration peaked at week 3. At this time hepatic enzymes, radiological fat content, and posttransplantation survival were worse (P =0.013), compared with other time points. Radiological assessment of fatty liver correlated well with survival and serum glutamic oxaloacetic transaminase and glutamic pyruvate transaminase levels. Conclusion. Severe microvesicular steatosis does not influence recipient survival, however, macrovesicular steatosis affects graft survival. Caliber CT is a practical and simple method that allows an accurate noninvasive quantitative assessment of hepatic fatty infiltration. It has potential to be a useful parameter for the assessment of donor livers for clinical liver transplantation.

Risk factors for intraoperative portal vein thrombosis in pediatric living donor liver transplantation.

Abstract:  Pathologic changes of the recipient native portal venous system may cause thrombosis of the portal vein, especially in pediatric living donor liver transplantation (LDLT). This study assessed the utility of Doppler ultrasound (US) for the detection of intraoperative portal vein occlusion and identification of predisposing risk factors in the recipients. Seventy‐three pediatric recipients who underwent LDLT at Chang Gung Memorial Hospital, Taiwan, from 1994 to 2002 were included. Preoperative and intraoperative Doppler US evaluation of the portal vein was performed. Age, body weight, native liver disease, type of graft, graft recipient weight ratio (GRWR), type of portal anastomosis, portal velocity, portal venous size and presence of portosystemic shunt were analyzed for statistical significance of predisposing risk factors. Eight episodes of intraoperative portal vein thrombosis, with typical findings of absent Doppler flow in portal vein and prominent hepatic artery with a resistant index lower than 0.5 (p < 0.001), were detected during transplantation, which was then corrected by thrombectomy and re‐anastomosis. Children age ≤1 yr (p = 0.025), weight ≤10 kg (p = 0.024), low portal flow ≤7 cm/s (p = 0.021), portal venous size ≤4 mm (p = 0.001), and GRWR >3 (p < 0.017) were all risk factors for intraoperative portal vein thrombosis. Doppler US is essential in the preoperative evaluation, early detection and monitoring of outcome of the portal vein in liver transplant.

3DCT angiography for detection of vascular complications in pediatric liver transplantation

Catheter angiography for early diagnosis of vascular complications in pediatric liver transplant yields excellent results but remains an extremely invasive examination for younger children, precluding its routine use. We assessed the efficacy of three‐dimensional multislice computed tomographic angiography (3DCTA) as an alternative option in these patients. Methods Twenty children suspected of vascular complications on clinical grounds, laboratory findings, or Doppler ultrasound underwent 3DCTA between April 2000 and April 2003. Interventional procedures via conventional angiography were subsequently performed in 5 cases, thrombolytic therapy in 4, surgical in 1, and conservative treatment in 10. Results Two hepatic artery stenosis,1 hepatic artery thrombosis, 5 hepatic vein stenosis, 4 portal vein occlusion, 1 portal vein stenosis, and 7 non‐vascular lesions were detected, all of which paralleled the findings of catheter angiography, Doppler ultrasound, and operations. The diagnostic accuracy for vascular complication was 90%. The sensitivity and specificity were 86.7% and 100%, respectively. The positive and negative predictive values were 100% and 71.4%, respectively. To date 19 patients are alive, with a median follow‐up period of 24.8 months. In conclusion, 3DCTA is accurate and efficient in the identification of pathological vascular insults and offers essential information for major decision on further management of the vascular complications in pediatric recipients of liver transplant. (Liver Transpl 2004;10:248–252.)

Magnetic resonance of the hepatic veins with angular reconstruction: application in living-related liver transplantation.

BACKGROUND Preoperative mapping of the hepatic venous system of the partial liver graft is indispensable to the success of living-related liver transplantation. We assessed the accuracy of magnetic resonance (MR) venography with angular reconstruction in depicting the tributaries of the middle hepatic vein and left hepatic vein in the donors, which was essential in graft retrieval and venoplasty. METHODS Nineteen living-related liver transplantation donors underwent a pretransplantation survey, including sonography and MRI for hepatic venous evaluation. T1-weighted images were reconstructed manually, using the inferior vena cava as a fixed point for tilting to produce an oblique plane image where both the middle hepatic vein and left hepatic vein could be demonstrated draining into the inferior vena cava. The reconstructed images of the hepatic veins were compared with preoperative sonography, intraoperative sonography, and operative findings. RESULTS Preoperative sonography and MR findings correlated well with the operative findings in the major hepatic veins. The MR venography of the ramification of the hepatic veins has an accuracy of 93%, the sonography, 84%. Sonography is slightly inferior in the evaluation of the hepatic vein in segment 4 and the left superior hepatic vein, with an accuracy of 73% and 67%, respectively. CONCLUSION MR venography with angular reconstruction is accurate in depicting the complex distribution of the hepatic veins of the left liver, providing important information for decision making as to the cutting plane during graft retrieval and the method of venoplasty and anastomosis. Thus, unnecessary blood loss could be avoided and vascular complications could be prevented, as these conditions would be unacceptable for a healthy living donor. We propose that MR venography, a rapid and reliable technique, is an appropriate alternative examination or complementary modality to sonography in the pretransplantation evaluation of the living donor.

Swine Hemi-Facial Composite Tissue Allotransplantation: A Model to Study Immune Rejection

OBJECTIVE Partial face composite tissue allotransplantation was recently achieved in a human subject. However, the side effects of long-term immunosuppression and chronic rejection area still need concerning. This preliminary study investigated the reproducibility of swine hemi-facial transplantation for preclinical studies. MATERIALS AND METHODS Eleven out-bred miniature swine underwent hemi-facial transplant. The hemi-facial orthotopic transplant consisted of ear cartilage, auricular nerve, parotid gland and lymphoid tissue, muscle with surrounding hemi-facial skin paddle supplied by the carotid artery, and external jugular vein transplanted to recipient swine. Three different experimental designs were studied, as follows: group I (n = 4): autologous hemi-facial transplantation as a normal control; group II (n = 4): hemi-facial allotransplantation without treatment; group III (n = 3): hemi-facial allotransplantation with cyclosporine-A treatment for 4 wk. The transplanted face was observed daily for signs of rejection. Biopsy of donor skin, gland lymphoid tissue, and cartilage were obtained at specified predetermined time (d 7, 14, 28), or at the time of clinically evident rejection. RESULTS The results indicated the survival of group I following autologous hemi-facial transplant was 100% and indefinite until sacrifice. Group II without treatment as the controls revealed allograft rejection by d 7 to 28. The allograft with short-term cyclosporine-A treatment revealed delayed rejection by d 38 to 49 postoperatively. The histological examination in group I revealed abundant lymphocyte infiltration, especially in lymphoid gland and alloskin at 1 wk and sacrifice. In contrast, the cyclosporine treatment group showed no significant rejection signs in 4 wk posttransplants. These results demonstrated that lymphoid tissue and alloskin are both susceptible to early rejection. CONCLUSION The experimental results revealed this model is suitable to investigate the new strategies for preclinical facial allotransplantation studies. Monitoring and modulation of early rejection in alloskin and gland lymphoid tissue is a useful strategy to evaluate composite tissue allotransplantation survival.

Alloantigen-Pulsed Host Dendritic Cells Induce T-Cell Regulation and Prolong Allograft Survival in a Rat Model of Hindlimb Allotransplantation

BACKGROUND Composite tissue allotransplantation is restricted due to the risks presented by long-term therapeutic immunosuppression. This study is conducted to investigate whether treatment with recipient immature dendritic cells (DCs) pulsed with donor alloantigens can prolong allograft survival and induce T-cell regulation in a rodent model. MATERIALS AND METHODS Orthotopic hindlimb transplants from Brown-Norway (RT1(n)) to Lewis (RT1(1)) rats were performed (day 0). DCs were propagated from the recipient bone marrow and pulsed with the donor alloantigen lysate. Group 1 (control group) did not receive any treatment. Groups 2 and 3 received cyclosporine A (CsA) at a concentration of 10 and 16 mg.kg(-1).day(-1), respectively, on days 0-20 following composite tissue allotransplantation. Group 4 received antilymphocyte serum (i.p. administered 4 d before and 1 d after transplantation) therapy. Group 5 received combined treatment with CsA (10 mg.kg(-1).day(-1), days 0-20) and donor alloantigen-pulsed recipient DCs (i.v. administered on days 7, 14, and 21). Group 6 received combined treatment with CsA (10 mg.kg(-1).day(-1) on days 0-20), antilymphocyte serum (administered i.p. 4 d before and 1 d after transplantation), and DCs (administered i.v. on days 7, 14, and 21). Graft rejection was defined as epidermolysis/desquamation of the donor skin. The mixed lymphocyte reaction was performed to determine the donor T-cell reactivity. Tissue samples were biopsied to analyze the histological changes, and flow cytometry was performed to quantify the donor T-cells. RESULTS Allograft survival was significantly prolonged (>200 d) in Group 6 when compared with the other groups (P < 0.001). The mixed lymphocyte reaction performed for Group 6 revealed hyporesponsiveness of the T-cells to donor alloantigens. Flow cytometric analysis in Group 6 revealed a significant increase in the percentage of CD4(+)/CD25(+) and CD4(+)/foxP3(+) T-cells expression, and significant increase in the percentage of donor cells (RT1(n)) in the recipient peripheral blood. Immunohistochemical staining of allo-skin revealed a significant increase in the proportion of CD25(+) cells in the subcutaneous and dermis layers in Group 6, as compared to other groups. CONCLUSION Treatment with donor alloantigen-pulsed recipient immature DCs in combination with transient immunosuppression prolongs allograft survival and induced tolerance by inducing T-cell hyporesponsiveness to donor alloantigens and increasing the CD4(+)/CD25(+) T-cell population.

Angioplasty treatment of hepatic vein stenosis in pediatric liver transplants: long-term results

We reviewed long‐term results of percutaneous venoplasty in children with hepatic vein stenosis after partial liver transplants, of which excellent early results were shown. Percutaneous transjugular hepatic venoplasty using balloon dilatation or stent implantation was performed in six cases with hepatic vein stenosis identified on routine post‐transplant Doppler sonography and confirmed by transjugular hepatic venography from 1994 to 2003. Repeated procedure was carried out if necessary. Six of 105 patients with partial liver graft developed hepatic stenosis characterized by low hepatic venous velocity with monophasic waveform with significant pressure gradient (>5 mmHg). The incidence was 4.46% for all 112 pediatric liver transplants. Successful balloon venoplasty was achieved in four cases. Self‐expanding stent was used in two cases with absent waisting or angulated balloon catheter during dilatation and persisted pressure gradient (>5 mmHg). Repeated procedure was required in two initially successful cases with additional stent used in one case. Three cases had transient hyperdynamic hepatic venous flow with markedly increased central venous pressure after stent implantation. Nonprocedural‐related mortality rate was 16.7%. Patent hepatic vein was maintained in five patients after a mean follow‐up of 3.67 years (0.75–9.5). Higher incidence of hepatic vein stenosis was noted in pediatric partial liver transplant. However, encouraging long‐term results showed that hepatic venoplasty or stent implantation could be a preferable alterative to surgical revision or retransplantation, which has been the procedure of choice in our hospital.

Multislice computed tomography angiography in pediatric liver transplantation.

Background. Preoperative delineation of any vascular anomalies offers planning for possible alteration of surgical procedures, especially in pediatric recipients undergoing living-related liver transplantation. Purpose. We assess the efficacy of three-dimensional (3D) multislice computed tomography (CT) angiography in the hope of replacing conventional angiography as the pretransplant evaluation of the hepatic vascular system for potential recipients of liver transplantation. Methods. 3D CT angiography was performed in 38 children with biliary atresia. Conventional angiography was also performed in the first 15 patients. Twelve patients underwent living-related liver transplantation. The findings on 3D CT angiography were compared with conventional angiography and operative findings. Results. 3D CT angiography was successfully performed in 37 pediatric patients. All findings of 3D CT angiography on hepatic artery, portal vein, and inferior vena cava paralleled those of catheter angiography and operative findings. Four patients were unsuitable to receive living grafts because of pathologic insults of the hepatic artery (one patient) and the portal vein (three patients). Three patients were advised to undergo a venous graft for portal anastomoses. Eight patients demonstrated portosystemic shunts that may require closure. Conclusion. 3D CT angiography proves to be a better tool in the demonstration of the vascular system and identification of pathologic insults in pediatric patients. It is superior to conventional angiography because it is less invasive, more convenient, and more efficient in providing thorough preoperative information that would have a major impact on patient selection and surgical planning.

The effect of exogenous histone H1 on rat adipose-derived stem cell proliferation, migration, and osteogenic differentiation in vitro

Adipose‐derived stem cells (ASCs) are of great interest for the development of novel cell therapies due to their ease of isolation and expansion, immunosuppressive activity, and multilineage differentiation potential. However, the mechanisms underlying the therapeutic potential of ASCs remain to be elucidated. Others and we have shown that nuclear proteins such as histone H1 and high mobility group box 1 (HMGB1) play important roles in the maturation of dendritic cells (DCs). Furthermore, we previously demonstrated translocation of histone H1 from the nucleus to the cytoplasm and activation of mitogen‐activated protein kinases (MAPKs) in DCs. In the present study, we confirmed that histone H1 does not alter the immunophenotype and immunosuppression potential of ASCs, but that histone H1 enhanced wound healing and increased interleukin (IL)‐6 expression. Moreover, histone H1 treated‐ASCs showed up‐regulation of MAPKs extracellular‐regulated kinase 1/2 (ERK1/2) and sequential NF‐κB translocation. Finally, we found that culture in differentiation media supplemented with histone H1 enhanced ASC osteogenesis. In contrast, inhibition of histone H1 by small interfering RNA (siRNA) reduced osteogenic differentiation markers including ALP. These results suggest that histone H1 may be useful for induction of mesenchymal stem cells in tissue engineering and future potential ASC therapies. J. Cell. Physiol. 227: 3417–3425, 2012.