Successful Unilateral Dual-Kidney Transplant of Discarded Kidneys From Two Expanded Criteria Donors.

In recent years, dual-kidney transplant has become an established method to overcome the inferior quality of donor organs and to allow the recovery of discarded human kidneys. However, in some cases, 1 of the 2 donor kidneys is unsuitable for transplant because of severe pathological changes, and the remaining marginal kidney is often discarded regardless of whether it meets criteria for dual-kidney transplant. Here, we report the use of marginal kidneys from 2 different donors, both of whom had missed kidney donation as a result of the serious pathological changes in their contralateral kidney. We combined the 2 donors' marginal kidneys for dual-kidney transplant, which were implanted into the right iliac fossa of the recipient after cold ischemia times of 13 hours 40 minutes and 30 hours 30 minutes, respectively. The recipient had fully recovered and showed favorable renal function without complications at discharge and at the 1.5-year follow-up. To the best of our knowledge, this is the first case report of successful unilateral dual-kidney transplant of discarded kidneys from 2 expanded criteria donors.

Romidepsin (FK228) improves the survival of allogeneic skin grafts through downregulating the production of donor-specific antibody via suppressing the IRE1α-XBP1 pathway.

Antibody-mediated rejection (AMR) is one of the major causes of graft loss after transplantation. Recently, the regulation of B cell differentiation and the prevention of donor-specific antibody (DSA) production have gained increased attention in transplant research. Herein, we established a secondary allogeneic in vivo skin transplant model to study the effects of romidepsin (FK228) on DSA. The survival of grafted skins was monitored daily. The serum levels of DSA and the number of relevant immunocytes in the recipient spleens were evaluated by flow cytometry. Then, we isolated and purified B cells from B6 mouse spleens in vitro by magnetic bead sorting. The B cells were cultured with interleukin-4 (IL-4) and anti-clusters of differentiation 40 (CD40) antibody with or without FK228 treatment. The immunoglobulin G1 (IgG1) and IgM levels in the supernatant were evaluated by enzyme-linked immunosorbent assay (ELISA). Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and western blotting were conducted to determine the corresponding levels of messenger RNA (mRNA) and protein expression in cultured cells and the recipient spleens. The results showed that FK228 significantly improved the survival of allogeneic skin grafts. Moreover, FK228 inhibited DSA production in the serum along with the suppression of histone deacetylase 1 (HADC1) and HDAC2 and the upregulation of the acetylation of histones H2A and H3. It also inhibited the differentiation of B cells to plasma cells, decreased the transcription of positive regulatory domain-containing 1 (Prdm1) and X-box-binding protein 1 (Xbp1), and decreased the expression of phosphorylated inositol-requiring enzyme 1 α (p-IRE1α), XBP1, and B lymphocyte-induced maturation protein-1 (Blimp-1). In conclusion, FK228 could decrease the production of antibodies by B cells via inhibition of the IRE1α-XBP1 signaling pathway. Thus, FK228 is considered as a promising therapeutic agent for the clinical treatment of AMR.

Progress in Liver Transplant Tolerance and Tolerance-Inducing Cellular Therapies.

Liver transplantation is currently the most effective method for treating end-stage liver disease. However, recipients still need long-term immunosuppressive drug treatment to control allogeneic immune rejection, which may cause various complications and affect the long-term survival of the recipient. Many liver transplant researchers constantly pursue the induction of immune tolerance in liver transplant recipients, immunosuppression withdrawal, and the maintenance of good and stable graft function. Although allogeneic liver transplantation is more tolerated than transplantation of other solid organs, and it shows a certain incidence of spontaneous tolerance, there is still great risk for general recipients. With the gradual progress in our understanding of immune regulatory mechanisms, a variety of immune regulatory cells have been discovered, and good results have been obtained in rodent and non-human primate transplant models. As immune cell therapies can induce long-term stable tolerance, they provide a good prospect for the induction of tolerance in clinical liver transplantation. At present, many transplant centers have carried out tolerance-inducing clinical trials in liver transplant recipients, and some have achieved gratifying results. This article will review the current status of liver transplant tolerance and the research progress of different cellular immunotherapies to induce this tolerance, which can provide more support for future clinical applications.

Inhibition of class I HDACs attenuates renal interstitial fibrosis in a murine model.

Renal interstitial fibrosis is the most common of all the forms of chronic kidney disease (CKD). Research has shown that histone deacetylases (HDACs) participate in the process leading to renal fibrosis. However, the effects of class I HDAC inhibitors on the mechanisms of onset and progression of renal interstitial fibrosis are still unclear. Here, we present the effects and mechanisms of action of FK228 (a selective inhibitor of class I HDACs) in the murine model of unilateral ureteral obstruction (UUO) and in vitro models. We investigated the antifibrotic role of FK228 in a murine model of UUO. We used two key effector cell populations, rat renal interstitial fibroblasts and renal tubular epithelial cells exposed to recombinant transforming growth factor-beta 1 (TGF-ß1), to explore the mechanistic pathways among in vitro models. The results indicated that FK228 significantly suppressed the production of extracellular matrix (ECM) in both in vivo and in vitro models. FK228 inhibited renal fibroblast activation and proliferation and increased the acetylation of histone H3. We found that FK228 also inhibited the small mothers against decapentaplegic (Smad) and non-Smad signaling pathways. So FK228 could significantly suppress renal interstitial fibrosis via Smad and non-Smad pathways. FK228 may be the basis for a new and effective medicine for alleviating renal fibrosis in the future.

A novel MyD88 inhibitor attenuates allograft rejection after heterotopic tracheal transplantation in mice.

BACKGROUND: After lung transplantation, the major complication limiting the long-term survival of allografts is obliterative bronchiolitis (OB), characterized by chronic rejection. Innate immune responses contribute to the development of OB. In this study, we used a murine heterotopic tracheal transplantation mouse model to examine the effects of a newtype of innate immune inhibitor, TJ-M2010-5. METHODS: Syngeneic tracheal grafts were transplanted heterotopically from C57BL/6 mice to C57BL/6 mice. Allografts from BALB/c mice were transplanted to C57BL/6 mice. The allograft recipients were treated with TJ-M2010-5, and anti-mouse CD154 (MR-1). The grafts were harvested at 7, 14, and 28 days and evaluated by histological and real-time RT-PCR analyses. RESULTS: In untreated allografts, almost all epithelial cells fell off at 7 days and tracheal occlusion reached a peak at 28 days. However, the loss of the epithelium and airway obstruction were significantly improved in mice treated with TJ-M2010-5 combined with MR-1. The relative mRNA expression levels of pro-inflammatory cytokines were upregulated in allogeneic tracheal grafts, and treatment with the two drugs reduced the production of pro-inflammatory cytokines and infiltration of inflammatory cells. CONCLUSIONS: In heterotopic tracheal transplantation models, TJ-M2010-5 combined with MR-1 could ameliorate the development of OB.

Galectin-7 promotes proliferation and Th1/2 cells polarization toward Th1 in activated CD4+ T cells by inhibiting The TGFß/Smad3 pathway.

Galectin-7 (Gal-7) has been associated with cell proliferation and apoptosis. It is known that Gal-7 antagonises TGFß-mediated effects in hepatocytes by interacting with Smad3. Previously, we have demonstrated that Gal-7 is related to CD4+ T cells responses; nevertheless, its effect and functional mechanism on CD4+ T cells responses remain unclear. The murine CD4+ T cells were respectively cultured with Gal-7, anti-CD3/CD28 mAbs, or with anti-CD3/CD28 mAbs & Gal-7. The effects of Gal-7 on proliferation and the phenotypic changes in CD4+ T cells were assessed by flow cytometry. The cytokines from CD4+ T cells were analysed by quantitative real-time PCR. Subcellular localisation and expression of Smad3 were determined by immunofluorescence staining and Western blot, respectively. Gal-7 enhanced the proliferation of activated CD4+ T cells in a dose- and ß-galactoside-dependent manner. Additionally, Gal-7 treatment did not change the ratio of Th2 cells in activated CD4+ T cells, while it increased the ratio of Th1 cells. Gal-7 also induced activated CD4+ T cells to produce a higher level of IFN-γ and TNF-α and a lower level of IL-10. Moreover, Gal-7 treatment significantly accelerated nuclear export of Smad3 in activated CD4+ T cells. These results revealed a novel role of Gal-7 in promoting proliferation and Th1/2 cells polarization toward Th1 in activated CD4+ T cells by inhibiting the TGFß/Smad3 pathway.

Graft-versus-host-disease after kidney transplantation: A case report and literature review.

INTRODUCTION: Acute graft-versus-host-disease (GVHD) in kidney recipients is extremely rare. Knowledge about its clinical manifestations, diagnosis, treatment, and prognosis is limited and needs to be increased. CLINICAL FINDINGS: One male kidney transplant recipient developed diarrhea and suffered kidney function damage. Primarily diagnosed with acute rejection, he was given methylprednisolone (MP) bolus treatment. Meanwhile, intravenous immunoglobulin (IVIG) and decreased immunosuppressive agents were applied for the corresponding infection. During the treatment, skin rashes occurred over his whole body. Biopsies were then taken. The pathology of the kidney graft showed no rejection, while the skin pathology revealed typical GVHD. Furthermore, fluorescence in situ hybridization proved the presence of donor-derived cells in the skin lesions, and infiltrating cytotoxic T cells and NK cells were identified in the rash. OUTCOME: Based on the clinical presentations, pathological findings, and chimerism detection, GVHD after kidney transplantation was confirmed as the final diagnosis. The recipient responded well to treatment. His kidney function recovered, and the skin lesions were completely resolved. He has been followed for 1 year without any further episodes. CONCLUSION: GVHD after kidney transplantation has its own characteristics. In the presence of a highly immunocompromised state, diarrhea and rashes, a diagnosis of GVHD needs to be considered. Kidney function impairment may be involved. Pathological changes and detection of chimerism and immunocyte infiltration are required for diagnosis. MP bolus, IVIG, and decreased immunosuppression could be beneficial to the clinical outcome. Kidney recipients have a prognosis superior to recipients of organs bearing large numbers of lymphocytes.

Short-term MyD88 inhibition ameliorates cardiac graft rejection and promotes donor-specific hyporesponsiveness of skin grafts in mice.

Recognition of evolutionarily conserved ligands by Toll-like receptors (TLRs) triggers signaling cascades in innate immune cells to amplify adaptive immune responses. Nearly all TLRs require MyD88 to transduce downstream signaling. MyD88 deficiency has been shown to promote the allograft acceptance in mice. However, direct evidence for therapeutic potential of MyD88 inhibitors remains lacking. Herein, we used a MyD88 inhibitor, namely ST2825, to explore its therapeutic potential and mechanisms in fully allogeneic skin and heart transplant models. Phenotypic maturation of dendritic cells stimulated by TLR ligands was alleviated by ST2825 in parallel with reduced T-cell proliferation in vitro. A short-course treatment with ST2825 significantly prolonged cardiac graft survival (mean survival time = 18.5 ± 0.92 days vs. 7.25 ± 0.46 days). ST2825-treated group had significantly reduced proinflammatory cytokines in allografts compared with control group. ST2825 combined with anti-CD154 induced long-term skin allograft acceptance in about one-third of recipients (>100 days). 'Skin-tolerant' recipients showed attenuated donor-specific IFN-γ responses, intact IL-4 responses, and compromised alloantibody responses. We conclude that MyD88 inhibitor ST2825 attenuates acute cardiac rejection and promotes donor-specific hyporesponsiveness in stringent skin transplant models. The direct evidence suggests that pharmacological inhibition of MyD88 hold promising potential for transplant rejection.

Donor pretreatment with adenosine monophosphate-activated protein kinase activator protects cardiac grafts from cold ischaemia/reperfusion injury.

OBJECTIVES: Adenosine monophosphate-activated protein kinase (AMPK) is a master regulator of energy metabolism and has been shown to be protective in ischaemia/reperfusion injury (IRI). We hypothesized that preactivation of AMPK with an activator before donor heart procurement could protect heart grafts from cold IRI. METHODS: Donor Sprague-Dawley rats were injected intravenously with AMPK activator 5-amino-imidazole-4-carboxamide ribonucleotide (AICAR) or vehicle 30 min before heart procurement. Heart grafts were then preserved in histidine-tryptophan-ketoglutarate (HTK) solution at 4°C for 8 h. After preservation, grafts were immediately mounted on the Langendorff perfusion system and perfused with Krebs-Henseleit buffer at 37°C for 1 h. Adenosine triphosphate (ATP) and malondialdehyde (MDA) content in graft tissue were quantified post-preservation and post-reperfusion. After reperfusion, isolated heart function was assessed using a pressure transducer; cumulative release of creatine kinase (CK) and lactate dehydrogenase (LDH) into the perfusate was measured to assess cardiomyocyte necrosis; ultrastructural changes in the mitochondria of the grafts were examined using transmission electron microscopy (TEM). RESULTS: After preservation, myocardial ATP content in the pretreated hearts was significantly higher than in the control hearts (3.247 ± 0.3034 vs 1.817 ± 0.2533 µmol/g protein; P < 0.05). AICAR-pretreated heart grafts exhibited significantly higher coronary flow (9.667 ± 0.3159 vs 8.033 ± 0.2459 ml/min; P < 0.05) and left ventricular developing pressure (58.67 ± 2.894 vs 42.67 ± 3.333 mmHg; P < 0.05) than the vehicle treated after reperfusion. Cumulative release of CK (300.0 ± 25.30 vs 431.7 ± 42.39 U/l; P < 0.05) and LDH (228.0 ± 16.68 vs 366.8 ± 57.41 U/l; P < 0.05) in the perfusate was significantly lower in the AICAR-pretreated group than that in the control group. Myocardial MDA content was also reduced in the pretreated group (0.5167 ± 0.1046 vs 0.9333 ± 0.1333 nmol/mg protein; P < 0.05). TEM suggested that the mitochondrial structure of AICAR-pretreated hearts was much better preserved. Moreover, AICAR-pretreated hearts significantly diminished cytosolic cytochrome c release after reperfusion. CONCLUSIONS: This study demonstrates that pretreatment with AMPK activator AICAR significantly protects heart grafts from extended cold IRI. This novel protocol may be useful and feasible in clinical heart transplantation.

MicroRNA-19a and CD22 Comprise a Feedback Loop for B Cell Response in Sepsis.

BACKGROUND: MicroRNA-19a (miR-19a), an oncogenic microRNA, has been recently reported to target CD22 in B cell lymphoma cell lines, but its role in inflammatory response is unclear. CD22 is a negative regulator for BCR signaling, and we hypothesize that miR-19a regulates B cell response by targeting CD22 in sepsis. MATERIAL AND METHODS: In order to determine whether miR-19a-CD22 pathway was involved in sepsis, and what role it played in the regulatory mechanisms, we detected the levels of miR-19a in B cells obtained from patients with sepsis, and measured the levels of miR-19a and CD22 expression in B cells activated by LPS in vitro. Additionally, we investigated the correlation between miR-19a and CD22, as well as the influence of this pathway on BCR signaling, in transfected B cells. RESULTS: We found that septic patients displayed up-regulated miR-19a in B cells. In vitro, miR-19a was increased in activated B cells, with CD22 expression initially enhanced but subsequently decreased. Moreover, overexpression of miR-19a resulted in an amplified BCR signaling, while overexpression of CD22 attenuated the effect of miR-19a and increased its expression. CONCLUSIONS: Our study demonstrated that miR-19a and CD22 comprised a feedback loop for B cell response in sepsis, providing a potential therapeutic target to recover the immune homeostasis.

Galectin-9 in combination with rapamycin induces cardiac allograft tolerance in mice.

BACKGROUND: Galectin-9 serves opposing roles in the innate and adaptive immune systems. Galectin-9 triggers T-cell immunoglobulin mucin-3 (Tim-3) on T helper type 1 (Th1) cells, thereby terminating Th1 immunity and protecting allografts from host immune attacks. Meanwhile, galectin-9 promotes the maturation of dendritic cells (DCs) that deliver proinflammatory signals. We previously showed that galectin-9 significantly prolongs cardiac allograft survival in mice but failed to induce tolerance. This study aimed at improving the administration protocol to induce allograft tolerance. We examined whether rapamycin can reverse the proinflammatory effects of galectin-9 on DCs and whether rapamycin synergizes with galectin-9 to induce cardiac allograft tolerance. METHODS: Monocytes/DCs from cardiac allografts were assessed for Tim-3 expression by flow cytometry. Costimulatory molecules CD80/CD86 were measured on galectin-9/rapamycin-treated bone marrow-derived DCs by flow cytometry. We performed heterotopic cervical cardiac transplantation using BALB/c donors and C57BL/6 recipients and assessed graft survival time. T cells of long-term surviving recipients were immunoassayed for interferon-γ and interleukin-4 secretion. RESULTS: Allograft-infiltrating monocytes/DCs expressed high Tim-3 levels (47.3%±5.6%). Expression of CD80/CD86 was up-regulated on galectin-9-treated bone marrow-derived DCs, which was reversed by rapamycin. Combined treatment with galectin-9 and rapamycin promoted the permanent acceptance of fully mismatched grafts (survival time >180 days; n=6). However, treatment with galectin-9 or rapamycin alone was not sufficient to induce tolerance. Galectin-9/rapamycin-induced tolerance was associated with low donor-specific interferon-γ and interleukin-4 secretion. CONCLUSIONS: Rapamycin inhibits proinflammatory effects of galectin-9 on DCs. Combined treatment of galectin-9 and rapamycin promotes allografts tolerance, which is associated with reduced Th1 and Th2 responses.

Tim-3 ligand galectin-9 reduces IL-17 level and accelerates Klebsiella pneumoniae infection.

T cell immunoglobulin and mucin domain (Tim)-3 is expressed on activated CD4(+) and CD8(+) T cells. Identification of galectin-9 as a ligand for Tim-3 has now firmly established the Tim-3/galectin-9 pathway, which results in apoptosis of effector CD4(+) and CD8(+) T cells. Moreover, Th17 cells are a recently discovered CD4(+) effector T cell, which are important in antimicrobial immunity. Whether the Tim-3/galectin-9 pathway affects Th17 immunity has not been elucidated. Here, we demonstrated expression of Tim-3 on Th17 cells by flow cytometry. Th17-skewed cells were sensitive to galectin-9-induced apoptosis. In vitro administration of galectin-9 decreased stimulated Th17 cells and inhibited production of IL-17. Interestingly, Klebsiella pneumoniae (K. pneumoniae) infection led to enhanced IL-17 levels. Recombinant galectin-9 significantly decreased IL-17 in vivo, which resulted in reduced bacterial clearance and high mortality. These observations suggest that the Tim-3/galectin-9 pathway plays an important role in termination of Th17-immune responses, and could be a therapeutic target for inflammatory diseases.

The critical pathway for deceased donation: reportable uniformity in the approach to deceased donation.

The critical pathway of deceased donation provides a systematic approach to the organ donation process, considering both donation after cardiac death than donation after brain death. The pathway provides a tool for assessing the potential of deceased donation and for the prospective identification and referral of possible deceased donors.

Galectin-9 significantly prolongs the survival of fully mismatched cardiac allografts in mice.

BACKGROUND: The involvement of T-cell immunoglobulin mucin-3 (Tim-3) in the regulation of solid organ transplantation rejection is largely unknown. We used galectin-9 (Tim-3 ligand) to evaluate the effect and mechanisms of Tim-3/galectin-9 pathway in an allogeneic heart transplant model. METHODS: The murine cardiac transplant model from BALB/c (H-2d) to C57BL/6 (H-2b) was built. The recipients were administered with galectin-9 for 7 days from day 1 or day 3 posttransplant. The complete cessation of cardiac contractility was defined as the observation endpoint. The effect of galectin-9 on cell proliferation was assessed by mixed lymphocyte reaction. Histology and immunohistochemistry were performed to estimate the severity of rejection. The phenotype and cytokine profile of lymphocytes were analyzed by flow cytometry. RESULTS: In vitro, galectin-9 suppressed the proliferation of lymphocytes mixed lymphocyte reactions in a dose- and beta-galactoside-dependent manner. In vivo, galectin-9 treatment from day 1 to day 3 posttransplant achieved similar survival time of grafts (median survival time, 22.7+/-1.2 vs. 23.0+/-1.0 days). The prolonged survival time was associated with reduced infiltration of CD4 and CD8 lymphocytes in allografts. Furthermore, the intragraft transcriptional profiling in galectin-9-treated group showed reduction of IFN-gamma and IL-17 mRNA but elevation of Ebi-3 and galectin-9 mRNA. Flow cytometry analysis indicated that galectin-9 treatment reduced the ratio of T helper (Th) 1 and Th17 cells in the draining lymph nodes and peripheral blood. CONCLUSIONS: A short-course administration of galectin-9 significantly prolonged the survival of fully allogeneic cardiac allografts, which was associated with the suppression of Th1 and Th17 immune responses.

Tim-3-Galectin-9 pathway involves the suppression induced by CD4+CD25+ regulatory T cells.

CD4(+)CD25(+) regulatory T cells (Tregs) are considered to play a key role as suppressors of immune-mediated reactions. The mechanisms of this suppression in animals and patients with autoimmune, allergic or oncogenic diseases have been investigated under various conditions. However, the precise mode of suppression by CD4(+)CD25(+) Tregs is still not clear. In this report, Tim-3-Galectin-9 pathway was explored as one of the mechanisms for the suppression and cytotoxicity induced by Tregs. Here, we demonstrated that Galectin-9 was expressed on CD4(+)CD25(+) Tregs by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Interestingly, blocking Tim-3-Galectin-9 pathway resulted in an obvious decreased suppression activity of Tregs and enhanced Th1 cytokine level in vitro. Furthermore, blocking Tim-3-Galectin-9 pathway negated prolonged survival of allogeneic skin grafts induced by CD4(+)CD25(+) Tregs in vivo. Our results suggest that Tim-3-Galectin-9 pathway involves the function of CD4(+)CD25(+) Tregs.

Activation of Tim-3-Galectin-9 pathway improves survival of fully allogeneic skin grafts.

T cell immunoglobulin and mucin domain (Tim)-3 is a molecule expressed on terminally differentiated murine Th1 cells but not on Th2 cells. Identification of Galectin-9 as a ligand for Tim-3 has now firmly established the Tim-3-Galectin-9 pathway as an important regulator of Th1 immunity, which results in apoptosis of Th1 cells. Here, we demonstrate that engagement of Tim-3 by mouse recombinant Galectin-9 remarkably suppresses allograft rejection and improves survival of allogeneic skin grafts. Furthermore, administration of recombinant Galectin-9 decreases Tim-3 positive cells in draining lymph node and selectively inhibits production of IFN-gamma after skin transplantation. At last, even low dose of Galectin-9 (1 microg/ml) can obviously inhibit TCR crosslinking-induced primary T cell proliferation in vitro. These observations suggest that Tim-3-Galectin-9 pathway plays an important role in the termination of productive Th1-immune response and could lead to developing novel therapies in transplant medicine.

The Tim-3 ligand galectin-9 negatively regulates CD8+ alloreactive T cell and prolongs survival of skin graft.

CD8+ alloreactive T cells are the key mediators of accelerated rejection. Vigorous CD8+ alloreactive T cells responses against alloantigens, which is the main effector mechanism in acute allograft rejection, has been well described. But the molecular mechanisms to dampen activated CD8+ T cells are largely unknown. On the other hand, Tim-3 is a molecule expressed on terminally differentiated CD4+ Th1 cells. Engaging Tim-3 with its ligand galectin-9 causes an inhibitory signal, resulting in apoptosis of Th1 cells and negatively regulates Th1 type immunity. However, the question whether CD8+ T cells express surface molecular Tim-3 has not been fully elucidated. In this study, we have investigated which CD8+ subset express molecular Tim-3 by flow cytometric assay. In addition, cytotoxic assay was applied to analyze whether CD8+ alloreactive T cells were sensitive to galectin-9 induced apoptosis. Here, our results demonstrated that Tim-3 was expressed on activated CD8+ alloreactive T cells (CD8+CD44highCD62Llow), but not expressed on naïve CD8+ T cells. Furthermore, alloreactive CD8+ cytotoxic T cells were sensitive to galectin-9 induced apoptosis both in vitro and vivo, resulting in attenuation of CD8+ alloreactive T cells mediated cytotoxicity and prolonged survival of skin graft.

Negative costimulatory molecules: the proximal of regulatory T cells?

Regulatory T cells (Tregs) are a central mechanism of immune regulation. It is well known that their regulatory effect is antigen-specific and depends on cell-cell contact. In addition, some immunological phenomenon such as linked suppression and iDC-induced tolerance are related with Tregs. But the surface markers, which reliably distinguish Treg from other T cell populations, and the regulatory mechanism still remain to be further revealed. Negative costimulatory molecule (NCM) family is one natural intrinsic mechanism that delivers the negative signal into cytoplasma to modulate immunoresponse and its expression can be induced not only on the immune cells but also on the parenchymal cells. Based on the present knowledge, we hypothesize NCMs are the specific surface markers to define Tregs. Tregs are one kind of activated T cells with high expression of NCM receptor and have the capability to induce NCM ligands expression on the membrane of APCs and the target cells of the activated cells. The NCM receptor-ligand complexes deliver negative signal into lymphocytes to regulate the immune response. This hypothesis remains to be fully elucidated.

Inhibition of xenogeneic response in porcine endothelium using RNA interference.

BACKGROUND: Rejection mediated by antibody recognition of the alpha-Gal epitope (Galalpha1-3Galbeta1-4GlcNAc-R) is a major barrier in porcine-to-human xenotransplantation. Because the synthesis of alpha-Gal is dependent on alpha1,3 galactosyltransferase (alpha1,3GT), methods of blocking this enzyme are needed. RNA interference induced by small interfering RNA (siRNA) is a powerful technique for allowing the silencing of mammalian genes with great specificity and potency. In this study, we use siRNA for silencing of alpha1,3GT with the purpose of reducing expression of the alpha-Gal epitope and subsequently decreasing immunogenicity of porcine endothelial cells. METHODS: alpha1,3GT-specific and control siRNAs were transfected into the porcine aortic endothelial cell line, PED. alpha-Gal expression was assessed by Western blotting, flow cytometry, and immunofluorescence. Protection from human-complement and natural killer (NK)-cell-mediated cytotoxicity was evaluated by Cr-release assays after incubation of PED with normal human serum (NHS) and NK92 cell, respectively. RESULTS: RNA interference was successfully achieved in PED as witnessed by the specific knock-down of alpha1,3GT mRNA levels. Flow cytometric analysis using the Griffonia simplicifolia isolectin B4 lectin confirmed the suppression of alpha1,3GT activity as evidenced by decreased alpha-Gal. Functional relevance of the knock-down phenotype was illustrated by the finding that silenced PED were protected from cytotoxicity of NHS. Protection from NK-mediated cytotoxicity was not observed. CONCLUSIONS: Our data are the first to demonstrate that RNA interference is a potent tool to down modulate alpha-Gal expression and to protect endothelial cells from complement-mediated cytotoxicity. Gene silencing by siRNA may represent a new approach for overcoming hyperacute and acute vascular rejection.