Human iPSC-MSC-Derived Xenografts Modulate Immune Responses by Inhibiting the Cleavage of Caspases.

Mesenchymal stem cells (MSCs) negatively modulate immune properties. Induced pluripotent stem cells (iPSCs)-derived MSCs are alternative source of MSCs. However, the effects of iPSC-MSCs on T cells phenotypes in vivo remain unclear. We established an iPSC-MSC-transplanted host versus graft reaction mouse model using subcapsular kidney injection. Th1, Th2, regulatory T cells (Treg), and Th17 phenotypes and their cytokines were investigated in vivo and in vitro. The role of caspases and the soluble factors involved in the effects of MSCs were examined. We found that iPSC-MSC grafts led to more cell survival and less infiltration of inflammatory cells in mice. iPSC-MSC transplantation inhibited T cell proliferation, decreased Th1 and Th2 phenotypes and cytokines, upregulated Th17 and Treg subsets. Moreover, iPSC-MSCs inhibited the cleavage of caspases 3 and 8 and inhibition of caspases downregulated Th1, Th2 responses and upregulated Th17, Treg responses. Soluble factors were determined using protein array and TGF-ß1/2/3, IL-10, and MCP-1 were found to be highly expressed in iPSC-MSCs. The administration of the soluble factors decreased Th1/2 response, upregulated Treg response and inhibited the cleavage of caspases. Our results demonstrate that iPSC-MSCs regulate T cell responses as a result of a combined action of the above soluble factors secreted by iPSC-MSCs. These factors suppress T cell responses by inhibiting the cleavage of caspases. These data provide a novel immunomodulatory mechanism for the underlying iPSC-MSC-based immunomodulatory effects on T cell responses. Stem Cells 2017;35:1719-1732.

Preparation and characterization of small-diameter decellularized scaffolds for vascular tissue engineering in an animal model.

BACKGROUND: The development of a suitable extracellular matrix (ECM) scaffold is the first step in vascular tissue engineering (VTE). Synthetic vascular grafts are available as an alternative to autologous vessels in large-diameter arteries (>8 mm) and medium-diameter arteries (6-8 mm). In small-diameter vessels (<6 mm), synthetic vascular grafts are of limited use due to poor patency rates. Compared with a vascular prosthesis, natural tissue ECM has valuable advantages. Despite considerable progress in recent years, identifying an optimal protocol to create a scaffold for use in small-diameter (<6 mm) fully natural tissue-engineered vascular grafts (TEVG), remains elusive. Although reports on different decellularization techniques have been numerous, combination of and comparison between these methods are scarce; therefore, we have compared five different decellularization protocols for making small-diameter (<6 mm) ECM scaffolds and evaluated their characteristics relative to those of fresh vascular controls. RESULTS: The protocols differed in the choice of enzymatic digestion solvent, the use of non-ionic detergent, the durations of the individual steps, and UV crosslinking. Due to their small diameter and ready availability, rabbit arteria carotis were used as the source of the ECM scaffolds. The scaffolds were subcutaneously implanted in rats and the results were evaluated using various microscopy and immunostaining techniques. CONCLUSIONS: Our findings showed that a 2 h digestion time with 1× EDTA, replacing non-ionic detergent with double-distilled water for rinsing and the application of UV crosslinking gave rise to an ECM scaffold with the highest biocompatibility, lowest cytotoxicity and best mechanical properties for use in vivo or in situ pre-clinical research in VTE in comparison.

Overexpression of Jagged-1 combined with blockade of CD40 pathway prolongs allograft survival.

Dendritic cells (DCs) have the tolerogenic potential to regulate adaptive immunity and induce allografts acceptance. Here we investigated whether blockade of the CD40 pathway could enhance the immune tolerance induced by DC2.4 cells modified to express Jagged-1 (JAG1-DC) in heart transplantation. Results showed that JAG1-DC treatment combined with anti-CD40L monoclonal antibody (mAb) administration significantly prolonged cardiac allograft survival in mice, with long-term survival (>110 days) of 50% of the allografts in the recipients. The therapy specifically inhibited the immune response, induced alloantigen-specific T-cell hyporesponsiveness, upregulated transforming growth factor-ß synthesis and increased the population of regulatory T cells (Tregs) driven by Jagged-1-Notch activation. These results highlight the potential application of gene therapy to induce alloantigen-specific Tregs effectively by providing the Jagged-1 stimulation.

Ectopic drainage from the inferior vena cava to the left atrium together with a partial anomalous pulmonary venous connection.

We report a case of a 44-year-old male patient with ectopic drainage from the inferior vena cava to the left atrium accompanied by partial anomalous pulmonary venous drainage. After the patient was hospitalized, his diagnosis was confirmed by Doppler echocardiography and angiography. A pericardial patch was used to divert the blood to the atrium. The surgical procedure was successful, and the patient began a rehabilitation program 8 days later. This type of ectopic drainage pattern is an unusual and infrequent clinical finding. The definitive diagnosis should be made by Doppler ultrasound combined with angiography.

Inhibition of accelerated rejection mediated by alloreactive CD4⁺ memory T cells and prolonged allograft survival by arsenic trioxide.

The aim of this study was to evaluate and determine the potential mechanisms of As2O3 in accelerated rejection mediated by alloreactive CD4⁺ memory T cells. Vascularized heterotopic cardiac transplantation from C57BL/6 mice to nude mice (pre-transferred CD4⁺ memory T cells) was performed on Day 0, and As2O3 was administered to recipient mice from Day 0 to 10. As a result, As2O3 could reduce the proliferation of allo-primed CD4⁺ memory T cells in vitro in MLR and the baseline rate of proliferation was restored by the addition of exogenous IL-2. In vivo, compared with the control[+] group, the mean survival time of cardiac allografts in the As2O3 group was prolonged from 5.8 ± 0.7 to 14.2 ± 2.5 days. Five days after transplantation, the relative gene expression of IL-2, IFN-γ and Foxp3 was reduced in the grafts by As2O3 treatment, but the expression of IL-10 and TGF-ß was increased. Correspondingly, the proportions of CD4⁺ T cells, CD4⁺ memory T cells and regulatory T cells (Tregs), both in recipient spleens and lymph nodes, were lowered. These results indicate the potential of As2O3 as a novel immunosuppressant targeting CD4⁺ memory T cells.

Effect of dorsal nerve fascial island flap on repairing distal soft tissue defects at the proximal segment of the index, middle, ring, and little fingers.

BACKGROUND: To investigate the effect of the dorsal nerve fascial island (DNFI) flap on repairing finger soft tissue defects at the distal segments. METHODS: Fifty patients with distal soft tissue defects at the index, middle, ring, or little fingers were treated with a DNFI flap at the proximal phalanx between February 2008 and May 2018. The nutrient vascular chain around the dorsal branch of the proper palmar digital nerves served as the flap axis. The dorsal branch of the proper palmar digital arteries provided blood supply. The fascia pedicle served as the venous system. All patients were followed for 6 months. RESULTS: All 50 flaps survived. The appearance, color, and texture of the skin returned to normal. The sensory function was partially restored. The two-point discrimination of the finger flap was 7-10 mm. CONCLUSIONS: The DNFI flap at the proximal phalanges of the index, middle, ring, and little fingers is an effective surgical option. The technique has a high flap survival rate and long pedicle, which can repair different parts of the finger. The flap also restores the sensory function of the finger without damaging the main nerves or blood vessels. The flap treatment is an optimal option for finger soft tissue defects at the distal segments.

Ghrelin enhances cisplatin sensitivity in HO-8910 PM human ovarian cancer cells.

BACKGROUND: Resistance to platinum-based chemotherapy is one of the crucial problems in ovarian cancer treatment. Ghrelin, a widely distributed peptide hormone, participates in a series of cancer progression. The aim of this study is to determine whether ghrelin influences the sensitivity of ovarian cancer to cisplatin, and to demonstrate the underlying mechanism. METHODS: The anti-tumor effects of ghrelin and cisplatin were evaluated with human ovarian cancer cells HO-8910 PM in vitro or in vivo. Cell apoptosis and cell cycle were analyzed via flow cytometry assay. The signaling pathway and the expression of cell cycle protein were analyzed with Western Blot. RESULTS: Our results showed that treatment with ghrelin specifically inhibited cell proliferation of HO-8910 PM and sensitized these cells to cisplatin via S phase cell cycle arrest, and enhanced the inhibitory effect of cisplatin on tumor growth of HO-8910 PM derived xenografts in vivo. Treatment with ghrelin inhibited the expression of p-Erk1/2 and p-p38, which was opposite the effect of cisplatin. However, under the treatment of ghrelin, cisplatin treatment exhibited a stronger effect on inhibiting P21 expression, upregulating p-CDK1 and cyclin B1 expression, and blocking cell cycle progression. Mechanistically, ghrelin promoted S phase cell cycle arrest and upregulated p-CDK1 and cyclin B1 expression induced by cisplatin via inhibition of p38. CONCLUSION: This study revealed a specifically inhibitory effect of ghrelin on platinum-resistance via suppressing p-P38 and subsequently promoting p-CDK1 mediated cell cycle arrest in HO-8910 PM.

Vitamin D3 combined with antibody agents suppresses alloreactive memory T-cell responses to induce heart allograft long-term survival.

BACKGROUND: The pre-stored memory T cells in organ transplant patient carry a high risk of allograft rejection. The current study aimed to determine whether the allogenic response of adoptively transferred memory T cells in mice was suppressed by vitamin D3 monotherapy alone or in combination with monoclonal antibody treatment. METHODS: Prior to vascularized heterotopic heart transplantation, naïve C57BL/6 mice were primed with memory T cells. Recipient mice were administered vitamin D3 alone or in combination with monoclonal antibodies (anti-CD40L/ anti-LFA-1). Memory T cells and CD4+ forkhead box P3+ T cells in recipient spleens were measured using flow cytometry. Additionally, the expression of cytokines was measured by ELISA and quantitative PCR. Inflammatory factors in the grafts were identified by hematoxylin and eosin staining. RESULTS: Vitamin D3 in conjunction with anti-CD40L/ anti-LFA-1 antibodies were administered according to the median survival time from 6.5 to 80 days. The results revealed that grafts were protected through the prevention of inflammatory cell infiltration. Combined treatment decreased the mRNA levels of IL-2, IFN-γ and IL-10 and increased the mRNA levels of IL-4, Foxp3 and TGF-ß in the allograft. Rejection was suppressed by a reduction of CD4+CD44high CD62L+ and CD8+ CD44high CD62L+ memory T cells, the induction of regulatory T cells in the recipient spleen and a reduction of serum IL-2, IFN-γ and IL-10 levels. CONCLUSION: Vitamin D3 efficiently protected allografts from memory T-cell allo-responses when combined with anti-CD40L/anti-LFA-1 antibodies therapy.

Combination of mesenchymal stem cells and FK506 prolongs heart allograft survival by inhibiting TBK1/IRF3-regulated-IFN-γ production.

Lifelong immunosuppression use presents many serious side effects to transplant recipients. Previous studies have shown that mesenchymal stem cells (MSC) regulate the progress of inflammation and protect allograft function. However, the benefits of MSC combined with low-dose tacrolimus (FK506) has not been investigated in heart transplant recipients, and its mechanism deserves further investigation. SD Rat bone marrow-derived MSC were infused into recipient mouse (C57BL/6, B6) through the tail vein, followed by a BALB/c donor cervical ectopic heart transplantation on the next day of infusion. T-lymphocyte subsets and their functions were determined using flow cytometry, ELISA, and qPCR. Thereafter, in vitro and in vivo experiments were conducted to identify the mechanisms regarding MSC and FK506 combination (MF group) use in regulating IFN-γ signaling. MF group in the allogeneic heart transplantation mouse model inhibited acute rejection and prolonged mean survival time (MST) of grafts from 7 days (d) to 22d. Pathological examination of heart grafts suggested that inflammatory cell infiltration decreased, and tissue damage was significantly reduced in the MF group. IFN-γ mRNA expression levels in the grafts and recipients decreased, while IL-4 and TGF-ß mRNA expression increased in the MF group. Phosphorylation of TBK1/IRF3 in recipient immune cells decreased under donor antigen stimulation. Combination use of MSC and FK506 can prolong graft survival, possibly by down-regulating TBK1/IRF3 phosphorylation, thus reducing IFN-γ production to prevent infiltration of inflammatory cells in the graft and extend graft survival. The findings provide a potential new approach to immunosuppression selection.

Optimization of the Cuff Technique for Murine Heart Transplantation.

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to improve surgical efficiency. In our lab, we have optimized the cuff technique with two major steps. First, we used the inner tube technique to insert a temporary inner tube into the external jugular vein and carotid artery blood vessel to facilitate eversion of the vessel over the cuff. Second, we performed complete heterotopic cardiac transplantation through the collaboration of two experienced surgeons. These modifications effectively reduced the operation time to 25 minutes, with a success rate of 95%. In this report, we describe these procedures in detail and provide a supplemental video. We believe that this report on the improved cuff technique will offer practical guidance for murine heterotopic heart transplantation and will enhance the utility of this mouse model for basic research.

Combined treatment with vitamin D3 and antibody agents suppresses secondary heart transplant rejection in the early postoperative period.

BACKGROUND: Accelerated transplant rejection mediated by donor reactive memory T cells is another barrier to the induction of graft tolerance. The aim of this study was to investigate the immunosuppressing effects of vitamin D (1,25(OH)2D3), administered alone or in combination with a costimulatory blockade treatment, on rejection of secondary heart allografts in a mouse model. METHODS: Circular full-thickness skin grafts from BALB/c mice were cut and grafted onto the lumbar regions of C57BL/6 mice as allo-primed recipients. Four weeks after skin grafting, the vascularized hearts from the BALB/c mice were transplanted heterotopically into the allo-primed recipients using a non-suture cuff technique. The recipients were then randomly divided into four groups and given either intraperitoneal injection of isotype, Ab, 1,25(OH)2D3, or a combination of Ab and 1,25(OH)2D3. Allograft incidence was determined by hematoxylin-eosin staining, and cytokine expression was assessed by the quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assays, and cytometric bead arrays. Spleen cells from the recipient were used to assess mixed lymphocyte reactions. Memory T cells and regulatory T cells (Tregs) in spleen cells were measured by flow cytometry. RESULTS: The median allograft survival time was longer with the combined treatment with Ab and 1,25(OH)2D3 than with no treatment or with treatment with Ab or 1,25(OH)2D3 alone. The grafts were protected from infiltration by inflammatory cells and by inhibition of interleukin 2 and interferon gamma expression. Rejection was initially suppressed in the early postoperative period by a reduction in the number of memory T cells and induction of Foxp3+ Tregs, but this effect disappeared by day 15 after transplantation upon withdrawal of the treatment. CONCLUSION: Vitamin D3 administered as an immunosuppressive agent, when combined with monoclonal antibody treatment, may protect heart grafts from memory T cell responses in a secondary heart transplant model.

The development of an extra-anatomic tissue-engineered artery with collateral arteries for therapeutic angiogenesis in ischemic hind limb.

To develop tissue-engineered arteries (TEAs) with collateral arteries(CAs) in ischemic hind limb goat models(IHLMs). The IHLMs created by removing femoral arteries were divided into non-treated control group(NG); non-catheter group (NCG) in which TEA was anastomosed to external iliac artery(EIA), and surrounded with collagen sponge containing autologous MSCs and VEGF-gelatin microspheres, the distal end of TEA was ligated; catheter group(CG) which received the same procedure as NCG, also received heparin infusion through catheter in EIA. TEA patency was assessed weekly by Ultrasound. The TEA and CAs were assessed by angiography, gross examination, histology and electron microscopy. In CG, TEAs remained patent for 1 month, but became partly occluded 1 week after catheter withdrawn. In NCG, TEAs were occluded 1 week after implantation. Angiography demonstrated that communication between CAs arising from the TEAs and the native vessels was established in both groups. NCG had fewer CAs than CG (P < 0.01). At 40 days, TEAs in CG demonstrated of endothelium formation, smooth muscle cells infiltration and collagen regeneration. The CG had more capillaries and mature vessels in adventia of TEAs than NCG (P < 0.01). CG group also had more vessels around TEAs than NCG (P < 0.01) or NG (P < 0.001).

Size discrepancy: not a formidable problem in a mouse heterotopic aortic transplant.

OBJECTIVES: We sought to develop a better approach in establishing a heterotopic aortic transplant model. MATERIALS AND METHODS: We used "sleeve " operation and set up 2 heterotopic aortic transplant experimental groups (groups 1 and 2). In group 1, donors (BALB/c (H-2d) mice; weight, 25-30 g) had the same weight as the recipients (C57BL/6 (H-2d) mice; weight, 25-30 g); in group 2, donors had lower weights (BALB/c (H-2d) mice, weight, 15-20 g). Grafts were examined macroscopically and histologically 60 days after the transplant. RESULTS: The thrombosis incidence was 10% in group 1 (5 of 50) and 2% in group 2 (1 of 50) (P < .05). Intestinal obstructions led to a high mortality rate in both groups: 8% in group 1 and 10% in group 2 (P < .05). CONCLUSIONS: The issue of size discrepancy between donor and recipient aortas is not a problem in a heterotopic aortic transplant model with the use of a sleeve technique, while the high incidence of intestinal obstructions must be considered.

Inhibiting accelerated rejection mediated by alloreactive CD4(+) memory T cells and prolonging allograft survival by 1α,25-dihydroxyvitamin D(3) in nude mice.

Donor-reactive memory T cells are major barriers to long-term survival of transplanted organs due to their capacity to accelerate rejection. In this study we investigated the ability of 1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] to inhibit accelerated rejection mediated by alloreactive CD4(+) memory T cells and to prolong cardiac allograft survival in an adoptive T cell memory/heart transplant model of nude mice. In vitro, the proliferation of CD4(+) memory T cells was significantly inhibited by 1,25(OH)(2)D(3) and was restored following addition of exogenous IL-2. Compared with the control group, the mean survival time of cardiac allografts in the 1,25(OH)(2)D(3) group was prolonged from 6.5±0.3 to 20.2±0.8 days in vivo. Five days after transplantation, the levels of IL-2 and IFN-γ were reduced in the grafts and the recipient sera by 1,25(OH)(2)D(3) treatment, while that of IL-10 increased. The proportions of CD4(+) memory T cells and CD4(+)Foxp3(+) T cells, both in recipient spleen and lymph nodes, were lowered by 1,25(OH)(2)D(3) treatment when compared with the control group. Our data suggests that 1,25(OH)(2)D(3) inhibits expansion of CD4(+) memory T cells, possibly by inducing clonal anergy and/or clonal deletion, resulting in prolongation of cardiac allograft survival in nude mice. These results may provide a rational basis for exploiting 1,25(OH)(2)D(3) as a novel immunosuppressant targeting CD4(+) memory T cells.