LRG-1 promotes fat graft survival through the RAB31-mediated inhibition of hypoxia-induced apoptosis.

Autologous adipose tissue is an ideal soft tissue filling material, and its biocompatibility is better than that of artificial tissue substitutes, foreign bodies and heterogeneous materials. Although autologous fat transplantation has many advantages, the low retention rate of adipose tissue limits its clinical application. Here, we identified a secretory glycoprotein, leucine-rich-alpha-2-glycoprotein 1 (LRG-1), that could promote fat graft survival through RAB31-mediated inhibition of hypoxia-induced apoptosis. We showed that LRG-1 injection significantly increased the maintenance of fat volume and weight compared with the control. In addition, higher fat integrity, more viable adipocytes and fewer apoptotic cells were observed in the LRG-1-treated groups. Furthermore, we discovered that LRG-1 could reduce the ADSC apoptosis induced by hypoxic conditions. The mechanism underlying the LRG-1-mediated suppression of the ADSC apoptosis induced by hypoxia was mediated by the upregulation of RAB31 expression. Using LRG-1 for fat grafts may prove to be clinically successful for increasing the retention rate of transplanted fat.

Evaluation of an alternative heterotopic transplantation model for ovarian tissue to test pharmaceuticals improvements for fertility restoration.

BACKGROUND: Ovarian tissue cryopreservation and transplantation (OTCTP) is currently the main option available to preserve fertility in prepubertal patients undergoing aggressive cancer therapy treatments. However, a major limitation of OTCTP is follicle loss after transplantation. The mouse is a model of choice for studying ovarian function and follicle development after ovarian tissue grafting in vivo. In these mouse models, ovarian tissue or ovaries can be transplanted to different sites. Our aim was to evaluate a new alternative to heterotopic transplantation models that could be useful to test pharmaceutical improvement for ovarian grafts after OTCTP. METHODS: Slow frozen murine whole ovaries were transplanted into the mouse ears (between the external ear skin layer and the cartilage). Ovarian transplants were recovered after 3, 14 or 21 days. Grafts were analyzed by immunohistochemistry and follicle density analyses were performed. RESULTS: An increase of ovarian vascularization (CD31 and Dextran-FITC positive staining), as well as cellular proliferation (Ki67 staining) were observed 3 weeks after transplantation in comparison to 3 days. Fibrosis density, evaluated after Van Gieson staining, decreased 3 weeks after transplantation. Furthermore, transplantation of cryopreserved ovaries into ovariectomized mice favored follicle activation compared to transplantation into non-ovariectomized mice. CONCLUSION: The present study indicates that surgical tissue insertion in the highly vascularized murine ear is an effective model for ovarian grafting. This model could be helpful in research to test pharmaceutical strategies to improve the function and survival of cryopreserved and transplanted ovarian tissue.

Rocaglamide Prolonged Allograft Survival by Inhibiting Differentiation of Th1/Th17 Cells in Cardiac Transplantation.

BACKGROUND: Aglaia (Meliaceae) species are used for treating autoimmune disorders and allergic diseases in Asian countries. Rocaglamide, an extract obtained from Aglaia species, exhibits suppressive effect by regulating the T cell subset balance and cytokine network in cancer. However, whether it can be used in organ transplantation is unknown. In this study, we investigated the antirejection effect and mechanism of action of rocaglamide in a mouse cardiac allograft model. METHODS: Survival studies were performed by administering mice with phosphate-buffered saline (PBS) (n = 6) and rocaglamide (n = 8). Heart grafts were monitored until they stopped beating. After grafting, the mice were sacrificed on day 7 for histological, mixed lymphocyte reaction (MLR), enzyme-linked immunosorbent assay (ELISA), and flow cytometric analyses. RESULTS: Rocaglamide administration significantly prolonged the median survival of the grafts from 7 to 25 days compared with PBS treatment (P < 0.001). On posttransplantation day 7, the rocaglamide-treated group showed a significant decrease in the percentage of Th1 cells (7.9 ± 0.9% vs. 1.58 ± 0.5%, P < 0.001) in the lymph nodes and spleen (8.0 ± 2.5% vs. 2.4 ± 1.3%, P < 0.05). Rocaglamide treatment also significantly inhibited the production of Th17 cells (6.4 ± 1.0% vs. 1.8 ± 0.4%, P < 0.01) in the lymph nodes and spleen (5.9 ± 0.3% vs. 2.9 ± 0.8%, P < 0.01). Furthermore, the prolonged survival of the grafts was associated with a significant decrease in IFN-γ and IL-17 levels. Our results also showed that NF-AT activation was inhibited by rocaglamide, which also induced p38 and Jun N-terminal kinase (JNK) phosphorylation in Jurkat T cells. Furthermore, by using inhibitors that suppressed p38 and JNK phosphorylation, rocaglamide-mediated reduction in NF-AT protein levels was prevented. CONCLUSION: We identified a new immunoregulatory property of rocaglamide, wherein it was found to regulate oxidative stress response and reduce inflammatory cell infiltration and organ injury, which have been associated with the inhibition of NF-AT activation in T cells.

Tolerogenic Delivery of a Hybrid Insulin Peptide Markedly Prolongs Islet Graft Survival in the NOD Mouse.

The induction of antigen (Ag)-specific tolerance and replacement of islet ß-cells are major ongoing goals for the treatment of type 1 diabetes (T1D). Our group previously showed that a hybrid insulin peptide (2.5HIP) is a critical autoantigen for diabetogenic CD4+ T cells in the NOD mouse model. In this study, we investigated whether induction of Ag-specific tolerance using 2.5HIP-coupled tolerogenic nanoparticles (NPs) could protect diabetic NOD mice from disease recurrence upon syngeneic islet transplantation. Islet graft survival was significantly prolonged in mice treated with 2.5HIP NPs, but not NPs containing the insulin B chain peptide 9-23. Protection in 2.5HIP NP-treated mice was attributed both to the simultaneous induction of anergy in 2.5HIP-specific effector T cells and the expansion of Foxp3+ regulatory T cells specific for the same Ag. Notably, our results indicate that effector function of graft-infiltrating CD4+ and CD8+ T cells specific for other ß-cell epitopes was significantly impaired, suggesting a novel mechanism of therapeutically induced linked suppression. This work establishes that tolerance induction with an HIP can delay recurrent autoimmunity in NOD mice, which could inform the development of an Ag-specific therapy for T1D.

The Influence of Topical Vasodilator-Induced Pharmacologic Delay on Cutaneous Flap Viability and Vascular Remodeling.

BACKGROUND: Surgical delay is a well-described technique to improve survival of random and pedicled cutaneous flaps. The aim of this study was to test the topical agents minoxidil and iloprost as agents of pharmacologic delay to induce vascular remodeling and decrease overall flap necrosis as an alternative to surgical delay. METHODS: Seven groups were studied (n = 8 in each group), including the following: vehicle, iloprost, or minoxidil before treatment only; vehicle, iloprost, or minoxidil before and after treatment; and a standard surgical delay group as a positive control. Surgical flaps (caudally based modified McFarlane myocutaneous skin flaps) were elevated after 14 days of pretreatment, reinset isotopically, and observed at various time points until postoperative day 7. Gross viability, histology, Doppler blood flow, perfusion imaging, tissue oxygenation measurement, and vascular casting were performed for analysis. RESULTS: Pharmacologic delay with preoperative application of topical minoxidil or iloprost was found to have comparable flap viability when compared to surgical delay. Significantly increased viability in all treatment groups was observed when compared with vehicle. Continued postoperative treatment with topical agents had no effect on flap viability. The mechanism of improved flap viability was inducible increases in flap blood volume and perfusion rather than the acute vasodilatory effects of the topical agents or decreased flap hypoxia. CONCLUSIONS: Preoperative topical application of the vasodilators minoxidil or iloprost improved flap viability comparably to surgical delay. Noninvasive pharmacologic delay may reduce postoperative complications without the need for an additional operation. CLINICAL RELEVANCE STATEMENT: Preoperative use of topical vasodilators may lead to improved flap viability without the need for a surgical delay procedure. This study may inform future clinical trials examining utility of preoperative topical vasodilators in flap surgery.

Does Local Zoledronate Applied to Pasteurized Bone Autografts Improve the Likelihood of Union of Graft-Host Junctions after Limb-sparing Surgery?

BACKGROUND: Pasteurized bone autograft is a recycling biological reconstruction method for limb-sparing surgery when an allograft or other reconstruction technique is unavailable. Since the application of a local bisphosphonate to morselized allografts can reduce graft resorption and enhance bone formation without systemic complications, adding the local bisphosphonate to pasteurized bone autografts should reduce the graft resorption and improve the graft incorporation to host bone. However, no study that we know of has described the outcomes of local bisphosphonate application to massive allografts or pasteurized bone autografts. Thus, this study compared the outcomes of pasteurized bone autografts with and without local zoledronate. QUESTIONS/PURPOSES: (1) What is the survival of pasteurized bone autografts and what complications lead to graft removal? (2) Does treatment of pasteurized bone autografts with zoledronate alter the survival of pasteurized bone autografts compared with grafts without treatment? (3) Does the local application of zoledronate reduce the proportion of patients with fractures because of metaphyseal graft resorption? (4) Does local application of zoledronate improve union at the graft-host bone junction compared with untreated grafts? METHODS: Between July 2011 and December 2019, we performed 538 musculoskeletal bone tumor resections. Of these, 101 patients underwent reconstruction with pasteurized bone autografts. Other reconstructions included tumor prostheses (150 patients), allografts (70 patients), reconstruction using a bone cement-plate construct (62 patients), and resection only (155 patients). We generally used pasteurized bone autograft when tumors showed an osteoblastic pattern, had less than one-third cortical destruction, and less than half of metaphyseal bone destruction. Six percent (6 of 101) were lost to follow-up, 6% (6 of 101) had incomplete clinical data, and 16% (16 of 101) had a follow-up period less than 2 years without an event, leaving 73 patients for evaluation. The median (interquartile range) age of the patients was 18 years (15 to 26). Ninety-seven percent (71 of 73) had a diagnosis of bone sarcoma. The median follow-up time was 46 months (33 to 75). From 2011 to 2014, 21 pasteurized bone autografts were prepared without local zoledronate, and from 2014 to 2019, 52 pasteurized bone autografts were prepared with local zoledronate because we thought it might improve union and reduce resorption of the graft. From our tumor registry database, we obtained age, sex, use of chemotherapy, graft length and location, pasteurized bone graft type, fixation methods, the use of local zoledronate, osteotomy gap, complications, proportion of grafts that united by 2 years, and local recurrences. Curves for graft survival were determined using the Kaplan-Meier method with the endpoint of autograft removal and metaphyseal fracture from graft resorption. The probabilities of graft removal were estimated by cumulative incidences using the competing risk analysis, where death was considered as the competing event. Intergroup differences in survival and multivariable analyses were performed using the log-rank test and a Cox regression analysis. A logistic regression model was used to evaluate the association between graft-host osseous union by 2 years and other baseline factors. Union was defined when a callus was seen to bridge the osteotomy line for at least three cortices in both the AP and mediolateral planes. RESULTS: The 5-year survival rate of all 73 pasteurized grafts was 85% (95% confidence interval 74% to 92%). With the numbers available, we found no difference in the 5-year survival rates between grafts with and without local zoledronate (90% [95% CI 78% to 96%] versus 74% [95% CI 48% to 89%]; p = 0.30). Eleven percent (8 of 73) of patients had metaphyseal fractures because of graft resorption, primarily associated with osteoarticular grafts (5-year fracture-free survival 56% [95% CI 20 to 80]) rather than pasteurized graft-prosthesis composites (94% [95% CI 78% to 98%]) and intercalary grafts (91% [95% CI 50 to 99]; p = 0.001); there was no association with the use of local zoledronate (13%; 7 of 52) compared with those without local zoledronate (5%; 1 of 21) (odds ratio 3.1 [95% CI 0.4 to 27]; p = 0.43). Of the 84 graft-host bone junctions, 85% (71) of the grafts unified within 2 years, 7% (6) unified after 2 years, and 8% (7) of grafts showed nonunion. Union within 2 years was associated with fixation using plate compared with those with stem and with both stem and plate (odds ratio 6.6 [95% CI 1.4 to 31]; p = 0.02) and grafts treated with local zoledronate compared with those without treatment (OR 5.9 [95% CI 1.3 to 28]; p = 0.02). CONCLUSION: The application of local zoledronate to pasteurized bone autografts for limb-sparing surgery improved the likelihood of graft union compared with untreated grafts, especially when the osteotomy junctions were fixed using plate osteosynthesis, but it did not appear to alter the proportion of patients who experience metaphyseal fracture of the grafts because of graft resorption. Although this is a small study, it suggests that the treatment of pasteurized bone autografts and perhaps bone allografts should be studied further to determine whether bisphosphonates or other adjuncts can improve the union time and return to function in patients undergoing bone tumor resections using these reconstruction types. LEVEL OF EVIDENCE: Level III, therapeutic study.

CXCR4 blockade reduces the severity of murine heart allograft rejection by plasmacytoid dendritic cell-mediated immune regulation.

Allograft-specific regulatory T cells (Treg cells) are crucial for long-term graft acceptance after transplantation. Although adoptive Treg cell transfer has been proposed, major challenges include graft-specificity and stability. Thus, there is an unmet need for the direct induction of graft-specific Treg cells. We hypothesized a synergism of the immunotolerogenic effects of rapamycin (mTOR inhibition) and plerixafor (CXCR4 antagonist) for Treg cell induction. Thus, we performed fully-mismatched heart transplantations and found combination treatment to result in prolonged allograft survival. Moreover, fibrosis and myocyte lesions were reduced. Although less CD3+ T cell infiltrated, higher Treg cell numbers were observed. Noteworthy, this was accompanied by a plerixafor-dependent plasmacytoid dendritic cells-(pDCs)-mobilization. Furthermore, in vivo pDC-depletion abrogated the plerixafor-mediated Treg cell number increase and reduced allograft survival. Our pharmacological approach allowed to increase Treg cell numbers due to pDC-mediated immune regulation. Therefore pDCs can be an attractive immunotherapeutic target in addition to plerixafor treatment.

Characterization of Novel P-Selectin Targeted Complement Inhibitors in Murine Models of Hindlimb Injury and Transplantation.

The complement system has long been recognized as a potential druggable target for a variety of inflammatory conditions. Very few complement inhibitors have been approved for clinical use, but a great number are in clinical development, nearly all of which systemically inhibit complement. There are benefits of targeting complement inhibition to sites of activation/disease in terms of efficacy and safety, and here we describe P-selectin targeted complement inhibitors, with and without a dual function of directly blocking P-selectin-mediated cell-adhesion. The constructs are characterized in vitro and in murine models of hindlimb ischemia/reperfusion injury and hindlimb transplantation. Both constructs specifically targeted to reperfused hindlimb and provided protection in the hindlimb ischemia/reperfusion injury model. The P-selectin blocking construct was the more efficacious, which correlated with less myeloid cell infiltration, but with similarly reduced levels of complement deposition. The blocking construct also improved tissue perfusion and, unlike the nonblocking construct, inhibited coagulation, raising the possibility of differential application of each construct, such as in thrombotic vs. hemorrhagic conditions. Similar outcomes were obtained with the blocking construct following vascularized composite graft transplantation, and treatment also significantly increased graft survival. This is outcome may be particularly pertinent in the context of vascularized composite allograft transplantation, since reduced ischemia reperfusion injury is linked to a less rigorous alloimmune response that may translate to the requirement of a less aggressive immunosuppressive regime for this normally nonlife-threatening procedure. In summary, we describe a new generation of targeted complement inhibitor with multi-functionality that includes targeting to vascular injury, P-selectin blockade, complement inhibition and anti-thrombotic activity. The constructs described also bound to both mouse and human P-selectin which may facilitate potential translation.

Bortezomib alleviates antibody-mediated rejection in kidney transplantation by facilitating Atg5 expression.

Antibody-mediated rejection (AMR) is one of the most dominant mechanisms responsible for the loss of kidney grafts. Previous researches have shown that donor-specific antibodies (DSAs) are the major mediators of AMR. In order to prolong the survival time of grafts, it is vital to reduce the incidence of AMR and inhibit the generation of DSAs. We established an animal model of AMR by performing kidney transplantation in pre-sensitized rats. Then, we investigated the effect of bortezomib (BTZ) on AMR. We found that BTZ could reduce the serum level of DSAs and alleviate post-transplantation inflammation in peritubular capillaries (PTCs) and glomeruli, which was demonstrated by the reduction of C4d and IgG deposition in PTCs, and the reduced number of B cell and plasma cell in peripheral blood and the transplanted kidney (p < 0.05). Our results also suggested that BTZ increased the number of regulatory T cell (Treg) and significantly reduced the proportion of T helper (Th17) cell (p < 0.05). Besides, BTZ induced the significant upregulation of anti-inflammatory cytokines but downregulated pro-inflammatory cytokines (p < 0.05). After dealing with Atg5 siRNA-lentivirus, the effect of BTZ alleviating AMR was reversed and Th17/Treg proportions were also significantly modulated. Collectively, these findings show that BTZ slows down the process of AMR and Atg5 may be the key mechanism. Furthermore, Atg5 silencing results may be demonstrated that Atg5 alleviated AMR by modulating the ratio of Th17/Treg.

Blockade of IL-6/IL-6R Signaling Attenuates Acute Antibody-Mediated Rejection in a Mouse Cardiac Transplantation Model.

Acute antibody-mediated rejection (AAMR) is an important cause of cardiac allograft dysfunction, and more effective strategies need to be explored to improve allograft prognosis. Interleukin (IL)-6/IL-6R signaling plays a key role in the activation of immune cells including B cells, T cells and macrophages, which participate in the progression of AAMR. In this study, we investigated the effect of IL-6/IL-6R signaling blockade on the prevention of AAMR in a mouse model. We established a mouse model of AAMR for cardiac transplantation via presensitization of skin grafts and addition of cyclosporin A, and sequentially analyzed its features. Tocilizumab, anti-IL-6R antibody, and recipient IL-6 knockout were used to block IL-6/IL-6R signaling. We demonstrated that blockade of IL-6/IL-6R signaling significantly attenuated allograft injury and improved survival. Further mechanistic research revealed that signaling blockade decreased B cells in circulation, spleens, and allografts, thus inhibiting donor-specific antibody production and complement activation. Moreover, macrophage, T cell, and pro-inflammatory cytokine infiltration in allografts was also reduced. Collectively, we provided a highly practical mouse model of AAMR and demonstrated that blockade of IL-6/IL-6R signaling markedly alleviated AAMR, which is expected to provide a superior option for the treatment of AAMR in clinic.

Celastrol-based nanomedicine promotes corneal allograft survival.

Effectively promoting corneal allograft survival remains a challenge in corneal transplantation. The emerging therapeutic agents with high pharmacological activities and their appropriate administration routes provide attractive solutions. In the present study, a celastrol-loaded positive nanomedicine (CPNM) was developed to enhance corneal penetration and to promote corneal allograft survival. The in vitro, in vivo and ex vivo results demonstrated the good performance of CPNM prolonging the retention time on ocular surface and opening the tight junction in cornea, which resulted in enhanced corneal permeability of celastrol. Both in vitro and in vivo results demonstrated that celastrol inhibited the recruitment of M1 macrophage and the expression of TLR4 in corneal allografts through the TLR4/MyD88/NF-κB pathway, thereby significantly decreasing secretion of multiple pro-inflammatory cytokines to promote corneal allograft survival. This is the first celastrol-based topical instillation against corneal allograft rejection to provide treatment more potent than conventional eye drops for ocular anterior segment diseases.

Intrapatient Variability in Tacrolimus Trough Levels Over 2 Years Affects Long-Term Allograft Outcomes of Kidney Transplantation.

This study aimed to determine the impact of tacrolimus (TAC) trough level (C0) intrapatient variability (IPV) over a period of 2 years after kidney transplantation (KT) on allograft outcomes. In total, 1,143 patients with low immunologic risk were enrolled. The time-weighted coefficient variability (TWCV) of TAC-C0 was calculated, and patients were divided into tertile groups (T1: < 24.6%, T2: 24.6%-33.7%, T3: ≥ 33.7%) according to TAC-C0-TWCV up to post-transplant 1st year. They were classified into the low/low, low/high, high/low, and high/high groups based on a TAC-C0-TWCV value of 33.7% during post-transplant 0-1st and 1st-2nd years. The allograft outcomes among the three tertile and four TAC-C0-TWCV groups were compared. The T3 group had the highest rate of death-censored allograft loss (DCGL), and T3 was considered an independent risk factor for DCGL. The low/low group had the lowest and the high/high group had the highest risk for DCGL. Moreover, patients with a mean TAC-C0 of ≥5 ng/ml in the high/high group were at the highest risk for DCGL. Thus, TAC-IPV can significantly affect allograft outcomes even with a high mean TAC-C0. Furthermore, to improve allograft outcomes, a low TAC-IPV should be maintained even after the first year of KT.

Oridonin Prolongs the Survival of Mouse Cardiac Allografts by Attenuating the NF-κB/NLRP3 Pathway.

Background: Oridonin (Ori), the main bioactive ingredient of the natural anti-inflammatory herb Rabdosia rubescens, could be a covalent inhibitor of the NLRP3 inflammasome. Solid organ transplantation provides a life-saving optional therapy for patients with end-stage organ dysfunction. The long-term survival of solid organ transplantation remains restricted because of the possibility of rejection and the toxicity, infection, cardiovascular disease, and malignancy related to immunosuppressive (IS) drugs. However, the pathogenic mechanisms involved remain unclear. The ideal IS drugs to prevent allograft rejection have not been identified. Here, we investigated whether Ori could prolong the in vivo survival of completely mismatched cardiac allografts. Methods: The cardiac transplantation models were conducted among three groups of mice from C57BL/6NCrSlc (B6/N) or C3H/HeNSlc (C3H) to C3H: the syngeneic and the allogeneic group, whose recipients were treated with vehicle of Ori, and the Ori treatment group, in which the recipients were transplanted hearts from MHC-I mismatched donors and treated with different dosages of Ori from post-operative day (POD) 0 to 7. Then, we investigated the effect of Ori on bone marrow-derived dendritic cell (BMDC) and allogeneic mixed lymphocyte reaction in vitro. Results: Ori with 3, 10, and 15 mg/kg Ori could prolong the survival (MST = 22.8, 49.2, and 65.3 days, respectively). We found that infiltrating CD8+ T cells and macrophages were decreased, and regulatory T cells (Tregs) were expanded in allografts on POD7. The mRNA level of IL-1ß and IFN-γ of allografts was downregulated. Mechanistically, Ori-treated BMDCs suppressed T-cell proliferation and IFN-γ+CD4+ T-cell differentiation, along with the expansion of Tregs and IL-10+CD4+ T cells. Ori inhibited NOD, LRR-, and pyrin domain-containing protein 3 (NLRP3) expression; attenuated NF-κB and IκBα phosphorylation in LPS-activated BMDCs; downregulated NLRP3, Caspase-1, IL-1ß, IL-18, and IFN-γ; and upregulated IL-10 expression. Conclusions: Our findings highlight the potential of Ori as a novel and natural IS agent to improve transplant tolerance. Ori could exert IS activity through decreasing IL-1ß and IL-18 production and Th1 differentiation and proliferation and expanding Tregs via inhibiting the NF-κB/NLRP3 signaling pathway.

C3 complement inhibition prevents antibody-mediated rejection and prolongs renal allograft survival in sensitized non-human primates.

Sensitized kidney transplant recipients experience high rates of antibody-mediated rejection due to the presence of donor-specific antibodies and immunologic memory. Here we show that transient peri-transplant treatment with the central complement component C3 inhibitor Cp40 significantly prolongs median allograft survival in a sensitized nonhuman primate model. Despite donor-specific antibody levels remaining high, fifty percent of Cp40-treated primates maintain normal kidney function beyond the last day of treatment. Interestingly, presence of antibodies of the IgM class associates with reduced median graft survival (8 vs. 40 days; p = 0.02). Cp40 does not alter lymphocyte depletion by rhesus-specific anti-thymocyte globulin, but inhibits lymphocyte activation and proliferation, resulting in reduced antibody-mediated injury and complement deposition. In summary, Cp40 prevents acute antibody-mediated rejection and prolongs graft survival in primates, and inhibits T and B cell activation and proliferation, suggesting an immunomodulatory effect beyond its direct impact on antibody-mediated injury.

Effect of memantine on the survival of an ischemic random skin flap and the underlying mechanism.

BACKGROUND: Skin flap transplantation is a common wound repair method in orthopedic surgery, but skin flap necrosis remains problematic. Memantine, an excitatory amino acid receptor antagonist, is currently used in the treatment of moderate to severe Alzheimer's disease, due to its ability to promote angiogenesis and reduce oxidative stress. This study investigated the effect of memantine on the survival of random skin flaps in Sprague-Dawley (SD) rats. MATERIALS AND METHODS: Thirty six male SD rats were divided into control, high-dose (20 mg/kg per day), and low-dose (10 mg/kg per day) groups and underwent a McFarland flap procedure. Seven days later, the survival of the flap was evaluated, The microvascular density and neutrophil density were measured by hematoxylin and eosin staining. Lead angiography was used to detect angiogenesis, and laser Doppler was used to detect blood perfusion. Expression levels of vascular endothelial growth factor (VEGF), interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α, Toll-like receptor (TLR) 4, nuclear factor kappa B(NF-κB) and Mitogen-activated protein kinase（MAPK）were detected by immunohistochemistry. Oxidative stress was evaluated by measuring the levels of malondialdehyde (MDA) and superoxide dismutase (SOD). RESULTS: Compared with the control group, the flap survival area of memantine group, especially the high-dose group, was larger, VEGF expression, microvascular density, angiogenesis, blood perfusion, and superoxide dismutase in the flap were higher in the Memantine-H group than in the Memantine-L and control groups (P < 0.01). In addition, levels of neutrophil density, IL-1ß, IL-6, TNF-α, TLR4, NF-κB, MAPK and malondialdehyde decreased significantly in the Memantine-H group (P < 0.01). CONCLUSIONS: Memantine can promote the survival of skin flap in rats by improving the blood supply, promoting angiogenesis, inhibiting the inflammatory response, and reducing ischemia-reperfusion injury.

Outcomes in Hepatitis C Positive Liver Transplantation: Timing of Direct-Acting Antiviral Treatment and Impact on Graft Fibrosis.

Liver transplantation for hepatitis C virus (HCV)-related disease has the lowest five-year graft survival among all liver transplant recipients. Graft failure due to accelerated fibrosis from unrestrained HCV replication is common. Optimal timing of HCV treatment with direct-acting antiviral agents remains unknown. We compared HCV liver transplant recipients successfully treated for HCV before transplant to those treated within 1 year of transplant to determine if graft fibrosis, measured by Fib-4 scores, differs with timing of treatment. Fib-4 scores less than or equal to 1.45 defined minimal fibrosis and greater than 1.45 defined greater than minimal fibrosis. We identified 117 liver transplant recipients: 52 treated before transplantation and 65 treated within 1 year of transplantation. Overall, 34% of recipients had minimal fibrosis, and the likelihood of having minimal fibrosis following treatment and liver transplantation did not differ by timing of treatment. The odds ratio of having greater than minimal fibrosis was 0.65 (95% CI 0.30, 1.42) among those treated within 1 year after transplantation compared to those treated before transplantation (p-value 0.28). Importantly, nearly 2/3 of these patients had evidence of fibrosis progression one year after sustained virologic response, supporting recommendations for early antiviral-based treatment to prevent accumulation of HCV-related disease.

Oxymatrine protects cardiac allografts by regulating immunotolerant cells.

Organ transplantation is an effective treatment strategy for patients with irreversible organ failure or congenital organ dysfunction. Oxymatrine (OMT) is a quinolizidine alkaloid with protective and anti-inflammatory effects on tissues and organs. The objective of this study was to investigate whether OMT could exert protective effects in cardiac allografts by regulating immune cells. In vitro cell proliferation and co-culture experiments were used to measure the effects of OMT on splenocyte proliferation and differentiation. In the in vivo study, C57BL/6 mice transplanted with BALB/c cardiac grafts were randomly divided into untreated, low-dose OMT treated, middle-dose OMT treated, high-dose OMT treated, and rapamycin-treated groups. Haematoxylin and eosin and immunohistochemical staining were used to assess pathological changes in the grafts, and fluorescence-activated cell sorting analysis was performed to measure the percentages of immune cells. The results showed that, in the in vitro study, OMT inhibited splenocyte proliferation, decreased the percentage of mature dendritic cells (DCs), and increased the percentage of regulatory T cells (Tregs) and regulatory B cells (Bregs). In the in vivo study, OMT exerted allograft protective effects by prolonging survival time, alleviating pathological damages to the cardiac allograft, decreasing intragraft CD3+ cell and increasing intragraft Foxp3+ cell infiltration, decreasing the percentages of mature DCs, increasing the percentages of Tregs and Bregs, and inhibiting the function of DCs. In conclusion, our study demonstrates that OMT exerted a protective effect on cardiac allografts by regulating immunotolerant cells. More in-depth studies of OMT may provide additional insight into the use of immunosuppressive drugs as a post-transplantation treatment strategy.

Effect of hirudin on arterialized venous flap survival in rabbits.

Arterialized venous flap (AVF) is limited in clinical application because its survival remains inconsistent and its exact survival mechanism is still unclear. Hirudin is an effective thrombin specific inhibitor, which is isolated from the salivary gland secretions of the leech. Our study evaluated the impact of hirudin on the viability of AVFs in rabbits. Thirty-six rabbits were randomly divided into three groups: sham group (physiological perfusion), control group (AVF), and hirudin group (AVF + hirudin). In hirudin group, 20 antithrombin units (ATU) hirudin (2.5 ml) were injected into each flap. In sham group and control group, the same amount of normal saline was injected into each flap. Status of flap survival, water content, vascular perfusion, histopathology, expression of CD34, VEGF, eNOS and HIF-1α were analyzed in each group. Analysis of oxidative stress was performed by measuring the activity of superoxide dismutase (SOD) and malondialdehyde (MDA). Compared with flaps in sham group with physiological perfusion mode, results of survival rate, perfusion status, SOD activity, expression of CD34, VEGF, and eNOS of AVFs in control group were significantly lower, while water content, MDA level and expression of HIF-1α were higher. The flap condition of AVFs injected with hirudin in hirudin group was improved significantly, and the results were similar to sham group. Our findings revealed that hirudin can effectively improve survival of AVF.