A prospective, randomized, non-blinded, non-inferiority pilot study to assess the effect of low-dose anti-thymocyte globulin with low-dose tacrolimus and early steroid withdrawal on clinical outcomes in non-sensitized living-donor kidney recipients.

BACKGROUND: The optimal dose of anti-thymocyte globulin (ATG) as an induction regimen in Asian living-donor kidney recipients is unclear. METHODS: This is a pilot study in which 36 consecutive patients undergoing living-donor kidney transplantation were randomly assigned to receive either 4.5 mg/kg (n = 19) or 6.0 mg/kg (n = 17) of ATG; all patients had corticosteroid withdrawal within 7 days. The primary end point was a composite of biopsy-proven acute rejection, de novo donor-specific antibody formation, and graft failure. RESULTS: At 12 months post-transplant, biopsy-proven acute rejection was more common in the ATG4.5 group (21.1%) than in the ATG6.0 group (0%)(P = .048). Importantly, the rate of the composite end point was significantly higher in the ATG4.5 group (36.8% vs 0%)(P = .006). There were significant differences in neither the renal function nor adverse events between the two groups. One case of death-censored graft failure occurred in the ATG4.5 group and no mortality was observed overall. Compared with pre-transplantation, T cells, natural killer (NK) cells, and natural killer T (NKT) cells were significantly decreased in the first week post-transplantation except for B cells. Although T and NKT cells in both groups and NK cells in the ATG4.5 group had recovered to the pre-transplant levels, NK cells in the ATG6.0 group remained suppressed until six months post-transplant. CONCLUSIONS: Compared with ATG 6.0 mg/kg, ATG 4.5 mg/kg with early corticosteroid withdrawal and low dose maintenance regimen was associated with higher rates of acute rejection in non-sensitized Asian living-donor kidney recipients. TRIAL REGISTRATION: ClinicalTrials.gov: NCT02447822.

Islet isograft transplantation improves insulin sensitivity in a murine model of type 2 diabetes.

PURPOSE: Type 2 diabetes develops in the presence of chronic overnutrition and genetic susceptibility, and causes insulin resistance and relative insulin deficiency. We hypothesized that islet transplantation can improve insulin sensitivity by modifying the mediators of insulin sensitivity in the pancreas, liver, muscle, and adipose tissues. METHODS: Eight-week-old male mice were used as both recipients and donors in this study. To induce type 2 diabetes with partial ß-cell failure, the mice were fed a high-fat diet for 4 weeks and then injected with low-dose streptozotocin. Approximately 400 islet cells from a donor mouse were injected into the renal capsule of a recipient mouse for islet transplantation. After 6 weeks following transplantation, the mediators of insulin sensitivity in the pancreas, liver, muscle, and adipose tissues were quantitatively compared between islet-transplanted and non-transplanted groups. RESULTS: Intravenous glucose tolerance test showed that whereas the non-transplanted mice failed to show notable reductions in the glucose level, the islet-transplanted mice showed significant reductions in the serum glucose level to ~200 mg/dL at 6 weeks after islet transplantation. The islet-transplanted mice showed significantly higher Matsuda index and significantly lower HOMA-IR than did the non-transplanted mice, thus signifying improved insulin sensitivity. CONCLUSIONS: Islet transplantation resulted in improvements in multiple indices of insulin sensitivity in a murine model of type 2 diabetes. Islet transplantation may be utilized to improve insulin sensitivity in patients with type 2 diabetes.

CD56+CD57+ infiltrates as the most predominant subset of intragraft natural killer cells in renal transplant biopsies with antibody-mediated rejection.

Little is known about the characteristics and clinical implications of specific subsets of intragraft natural killer (NK) cells in kidney transplant recipients. We analyzed 39 for-cause renal transplant biopsies performed at our center from May 2015 to July 2017. According to histopathologic reports, 8 patients (20.5%) had no rejection (NR), 11 (28.2%) had T cell-mediated rejections (TCMR) only, and 20 (51.3%) had antibody-mediated rejection (ABMR). NK cells were defined as CD3-CD56+ lymphocytes that are positive for CD57, CD49b, NKG2A, or KIR. The density of NK cells was significantly higher in the ABMR group (2.57 ± 2.58/mm2) than in the NR (0.12 ± 0.22/mm2) or the TCMR (0.25 ± 0.34/mm2) group (P = 0.002). Notably, CD56+CD57+ infiltrates (2.16 ± 1.89) were the most frequently observed compared with CD56+CD49b+ (0.05 ± 0.13), CD56+NKG2A+ (0.21 ± 0.69), and CD56+KIR+ (0.15 ± 0.42) cells in the ABMR group (P < 0.001). Death-censored graft failure was significantly higher in patients with NK cell infiltration than those without (Log-rank test, P = 0.025). In conclusion, CD56+CD57+ infiltrates are a major subset of NK cells in kidney transplant recipients with ABMR and NK cell infiltration is significantly associated with graft failure post-transplant.

Urinary transglutaminase 2 as a potent biomarker to predict interstitial fibrosis and tubular atrophy of kidney allograft during early posttransplant period in deceased donor kidney transplantation.

PURPOSE: Transglutaminase type 2 (TG2) is an extracellular matrix crosslinking enzyme with a pivotal role in kidney fibrosis. We tested whether quantification of urinary TG2 may represent a noninvasive method to estimate the severity of kidney allograft fibrosis. METHODS: We prospectively collected urine specimens from 18 deceased donor kidney transplant recipients at 1-day, 7-day, 1-month, 3-month, and 6-month posttransplant. In addition, kidney allograft tissue specimens at 0-day and 6-month posttransplant were sampled to analyze the correlation of urinary TG2 and kidney allograft fibrosis. RESULTS: Thirteen recipients had increased interstitial fibrosis and tubular atrophy (IFTA) scores at the 6-month protocol biopsy (IFTA group). The mean level of urinary TG2 in the IFTA group was higher compared to that of 5 other recipients without IFTA (no IFTA group). Conversely, the mean level of urinary syndecan-4 in the IFTA group was lower than levels in patients without IFTA. In the IFTA group, double immunofluorescent staining revealed that TG2 intensity was significantly upregulated and colocalizations of TG2/heparin sulfate proteoglycan and nuclear syndecan-4 were prominent, usually around tubular structures. CONCLUSION: Urinary TG2 in early posttransplant periods is a potent biomarker for kidney allograft inflammation or fibrosis.

Tissue-resident natural killer cells exacerbate tubulointerstitial fibrosis by activating transglutaminase 2 and syndecan-4 in a model of aristolochic acid-induced nephropathy.

Despite reports suggesting that tissue-resident natural killer (trNK) cells cause ischemic kidney injury, their contribution to the development of tubulointerstitial fibrosis has not been determined. This study hypothesized that the depletion of trNK cells may ameliorate renal fibrosis by affecting transglutaminase 2/syndecan-4 interactions. Aristolochic acid nephropathy (AAN) was induced in C57BL/6 mice as an experimental model of kidney fibrosis. The mice were treated with anti-asialo GM1 (ASGM1) or anti-NK1.1 antibodies to deplete NK cells. Although both ASGM1 and NK1.1 antibodies suppressed renal NKp46＋DX5＋ NK cells, renal NKp46＋DX5- cells were resistant to suppression by ASGM1 or NK1.1 antibodies during the development of tubulointerstitial fibrosis in the AAN-induced mouse model. Western blot analysis showed that both antibodies increased the expression of fibronectin, transglutaminase 2, and syndecan-4. These findings indicate that trNK cells played an exacerbating role in tubulointerstitial fibrosis by activating transglutaminase 2 and syndecan-4 in the AAN-induced mouse model. [BMB Reports 2019; 52(9): 554-559].

Novel colchicine derivatives enhance graft survival after transplantation via suppression of T-cell differentiation and activity.

Immunosuppressants are crucial in organ transplantation but their side effects are a trade-off for long-term use. Colchicine has been shown to be effective in various diseases, albeit with many side effects. To enhance the immunosuppressive effects of colchicine, in addition to minimizing its side effects, we attempted to synthesize new colchicine derivatives (KR compounds). In rat cardiac and pancreatic islet allograft, long-term graft survival was identified in KR compound-treated groups. The use of cyclosporine A (CsA) or colchicine inhibited the CD3+ and CD4+ T-cell proliferation, whereas KR compounds inhibited the CD8+ T cells as well as CD4+ T cells. KR compounds reduced the apoptosis, interleukin-2 receptor expression, and signal transducer and activator of transcription 3 phosphorylation more than CsA. These results indicate that KR compounds have a potential therapeutic value as novel agents for prevention of graft deterioration by allograft of rejection.

A composite of urinary biomarkers for differentiating between tubulointerstitial inflammation and interstitial fibrosis/tubular atrophy in kidney allografts.

BACKGROUNDS/AIMS: Compared with a single urinary biomarker, a composite of multiple urinary biomarkers may be more helpful for differentiating tubulointerstitial inflammation from interstitial fibrosis/tubular atrophy (IFTA) in kidney allografts. METHODS: In this cross-sectional cohort study, we collected urine samples from 115 patients with for-cause biopsy, 53 patients with stable allografts, and 50 living kidney donors. We measured the urinary levels of transglutaminase 2 (TG2), syndecan-4 (SDC4), alpha 1 microglobulin (A1M), interferon-inducible protein 10 (IP-10), interleukin 6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1). RESULTS: The for-cause biopsy group showed significantly higher levels of logeTG2/Cr, logeA1M/Cr, logeIL-6/Cr, and logeMCP-1/Cr compared with other groups. In the for-cause biopsy group, logeTG2/Cr level was positively correlated with the severity of IFTA. After adjusting for age, sex, body mass index, diabetes, hypertension, cardiovascular disease, and the interval between kidney transplant and biopsy, TG2 and the interval between transplant and biopsy were significantly correlated variables for the severity of IFTA. Regarding tubulointerstitial inflammation, Body mass index, TG2, SDC4, and IP-10 were positively-correlated variables, and MCP-1 and the interval between transplant and biopsy were negatively-correlated variables. CONCLUSIONS: Our results show that post-transplant urinary levels of TG2, SDC4, MCP-1 and IP-10 may be a useful biomarker for tubulointerstitial inflammation and IFTA.

Involvement of indirectly allostimulated CD4+CD43highCD45RO+ T cell proliferation in the development of chronic allograft nephropathy.

The goal of this study was to identify immunological markers for use in antigen-specific assays that predict long-term survival after renal allograft and distinguish stable-functioning (SP) patients from poorly functioning (PP) patients. For this prospective study, 20 patients were enrolled. Eight SP and six PP patients were enrolled in this study. Serum cytokine/chemokine levels were analyzed by the Luminex multiplex assay. To detect indirect alloreactive T cells, we performed indirect mixed lymphocyte reaction using donor-antigen-pulsed autologous dendritic cells as stimulators. Serum induced protein-10 levels were significantly higher in the serum of PP patients, whereas sCD40L levels were higher in SP patients. The PP patients had significantly higher numbers of donor-specific CD4(+)CD43(high)CD45RO(+) T cells after indirect allostimulation, whereas this cell population was unchanged in SP patients. The donor-specific CD4(+)CD43(high)CD45RO(+) T cells had the effector memory T cell phenotype. Prospectively, we studied whether these cells influence graft outcome and found that their strong proliferation in pre-transplant patients is related to a poorly functioning graft. Indirectly allostimulated CD4(+)CD43(high)CD45RO(+) T cells may not only contribute to chronic allograft nephropathy development but may also have a role in the progression of acute rejection. Thus, these cells may have potential use as immune-monitoring markers in a noninvasive in vitro assay that predicts graft outcome.

Interleukin (IL)-10 induced by CD11b(+) cells and IL-10-activated regulatory T cells play a role in immune modulation of mesenchymal stem cells in rat islet allografts.

Mesenchymal stem cells (MSCs) are suggested to be immune modulators because of their therapeutic potential in transplantation. In the present study, we evaluated the therapeutic potential of autologous MSCs for preventing graft rejection after allogeneic rat islet transplantation. We assessed the ability of MSCs to elicit an antiproliferative response in alloreactive lymphocytes and tested the immunosuppressive effect of MSCs in allogeneic islet transplantation. In islet allotransplantation, injection of autologous MSCs or a subtherapeutic dose of cyclosporine A (CsA; 5 mg/kg) alone did not prolong allograft survival. However, graft survival was attained for >100 d in 33% of autologous MSC-plus-CsA-treated recipients, indicating that graft acceptance was achieved in a subgroup of allograft recipients. Splenocytes from autologous MSC-plus-CsA-treated rats exhibited a reduced mixed lymphocyte reaction (MLR)-proliferative response to donor stimulators and increased interleukin (IL)-10 release. Interestingly, after excluding host CD11b(+) cells, splenic T cells from autologous MSC-plus-CsA-treated rats did not produce IL-10 or did not inhibit proliferative responses under the same conditions. The use of autologous MSC-plus-CsA downregulated immune responses, inducing donor-specific T-cell hyporesponsiveness by reducing the production of proinflammatory cytokines and inducing antiinflammatory cytokine production, especially that of IL-10, during the early posttransplantation period. T-regulatory cells made a contribution at a later phase. In conclusion, the combined use of autologous MSCs and low-dose CsA exerted a synergistic immunosuppressive effect in an islet allograft model, suggesting a role for autologous MSCs as an immune modulator.

Bone marrow-derived mesenchymal stromal cells support rat pancreatic islet survival and insulin secretory function in vitro.

BACKGROUND AIMS: Recent evidence has suggested that transplanted bone marrow (BM)-derived mesenchymal stromal cells (MSC) are able to engraft and repair non-hematopoietic tissues successfully, including central nervous system, renal, pulmonary and skin tissue, and may possibly contribute to tissue regeneration. We examined the cytoprotective effect of BM MSC on co-cultured, isolated pancreatic islets. METHODS: Pancreatic islets and MSC isolated from Lewis rats were divided into four experimental groups: (a) islets cultured alone (islet control); (b) islets cultured in direct contact with MSC (IM-C); (c) islets co-cultured with MSC in a Transwell system, which allows indirect cell contact through diffusible media components (IM-I); and (d) MSC cultured alone (MSC control). The survival and function of islets were measured morphologically and by analyzing insulin secretion in response to glucose challenge. Cytokine profiles were determined using a cytokine array and enzyme-linked immunosorbent assays. RESULTS: Islets contact-cultured with MSC (IM-C) showed sustained survival and retention of glucose-induced insulin secretory function. In addition, the levels of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) were decreased, and tissue inhibitor of metalloproteinases-1 (TIMP-1) and vascular endothelial growth factor (VEGF) levels were increased at 4 weeks in both the IM-C and IM-I groups. CONCLUSIONS: These results indicate that contact co-culture is a major factor that contributes to islet survival, maintenance of cell morphology and insulin function. There might also be a synergic effect resulting from the regulation of inflammatory cytokine production. We propose that BM MSC are suitable for generating a microenvironment favorable for the repair and longevity of pancreatic islets.

The synergistic effect of Tautomycetin on Cyclosporine A-mediated immunosuppression in a rodent islet allograft model.

Most immunosuppressive drugs that support successful allograft survival act by inhibiting or depleting T lymphocytes. Tautomycetin (TMC) is a specific inhibitor of protein phosphatase 1, which has a role in cell-cycle control and T-cell activation and promotes T-cell-specific apoptosis. In this study, we investigated the effect on rat islet transplantation of TMC alone and in combination with cyclosporine A (CsA). TMC treatment inhibited splenocyte proliferation in mixed lymphocyte reactions (MLR) without affecting cell viability. When used alone in islet allograft recipients, TMC did not significantly increase the survival of grafted islets. However, cotreatment of TMC and subtherapeutic doses of CsA significantly prolonged islet graft survival from 5.1 d to more than 100 d (P<0.05). At 100 d, there was no evidence of specific organ toxicity, and histological analyses of grafted liver tissue revealed the presence of viable islets. CD4+ and CD8+ T-cell infiltration and interleukin (IL)-2 mRNA levels were decreased in TMC/CsA-cotreated rats, whereas IL-10 levels were increased. In addition, the number of FoxP3-expressing cells and FoxP3 mRNA levels were also increased. We suggest that CsA and TMC act synergistically to reduce the function of T-effector cells and enhance regulatory cell function in this islet allotransplantation model.

Viral IL-10 gene transfer prolongs rat islet allograft survival.

Islet transplantation is a potential cure for diabetes. However, allotransplant rejection severely limits its clinical application. In this study, we sought to transfect rat islets with an adenoviral vector containing the viral IL-10 (vIL-10) gene and examine its efficacy in preventing graft rejection. The immunosuppressive effect of vIL-10 is reported but its efficacy is somehow debatable in transplantation model. vIL-10 transfected islets were transplanted into streptozotocin-induced diabetic rats. Blood glucose, serum vIL-10 concentration, graft histology, and graft cytokine expression were used to monitor graft function up to day 21 after transplantation. Transfected islets released a large amount of vIL-10 protein without affecting their viability and functional integrity. When we transplanted the transfected islets into allogeneic hosts, the survival of grafted islets was not significantly increased. However, the combined use of vIL-10 and subtherapeutic doses of CsA (cyclosporine) significantly prolonged graft survival beyond that achieved with either agent alone (p < 0.001). vIL-10 and CsA-treated rats contain high level of vIL-10 in serum, which is evidenced by the inhibition of allogeneic mixed lymphocyte reaction (MLR). Histological analysis additionally revealed the presence of viable islets up to 21 days. IL-10 mRNA expression in grafted liver was higher and IFN-gamma mRNA was lower in vIL-10 and CsA-treated animals, compared with other groups. The synergistic effect of this combination therapy is potentially correlated with the induction of inhibitory cytokine secretion and downregulation of proinflammatory cytokine secretion from host cells.

Anti-inflammatory action of alpha-melanocyte stimulating hormone (alpha-MSH) in anti-CD3/CD28-mediated spleen and CD4(+)CD25(-) T cells and a partial participation of IL-10.

alpha-Melanocyte stimulating hormone (alpha-MSH) has been shown to inhibit the production and the effects of pro-inflammatory cytokine by inflammatory cells in innate immunity. We have determined whether alpha-MSH inhibits anti-CD3/CD28-mediated spleen cells and CD4(+)CD25(-) T cells proliferation and its mechanism of action. The proliferation of anti-CD3/CD28-mediated spleen cells and CD4(+)CD25(-) T cells markedly were suppressed by 50-100 nM and 5-100 nM alpha-MSH, respectively. alpha-MSH (100 nM) increased the production of the anti-inflammatory cytokine IL-10 and decreased the production of the pro-inflammatory cytokine IL-2 and IFN-gamma from CD4(+)CD25(-) T cells. Moreover, anti-IL-10 blocking Ab decreased the inhibitory effects of anti-CD3/CD28-mediated spleen cells and CD4(+)CD25(-) T cells proliferation by alpha-MSH, indicating a partial participation of IL-10 in its mechanism of inhibitory action. These results suggest that alpha-MSH may be useful for treatment of autoimmune diseases and transplantation involving innate and adaptive immunity.

Cell surface modification by activated polyethylene glycol prevents allosensitization after islet transplantation.

The necessity to transplant islet tissue without the need for immunosuppressant therapy has led to the development of materials for immune modulation. Pegylation makes islets antigenically silent, protecting them from the adsorption of foreign protein and thus avoiding immune injury. The aim of this study is to determine whether pegylation of islets prolongs islet survival and function both during tissue culture and posttransplantation. We used cyanuric chloride-activated methoxy-polyethylene glycol for cell surface modification. To detect the pegylation effect on splenocytes, we measured antibody binding inhibition and abrogation of lymphocyte proliferation. To detect the pegylation effect on islet grafts, we performed rodent islet transplantation. Islet viability and function were maintained after pegylation. Pegylated islets showed a 90% decrease in antibody binding and decreased lymphocyte proliferation in a mixed lymphocyte culture. However, when pegylated islets were transplanted, no prolongation of graft survival was observed. When a subtherapeutic dose of immunosuppressant was given at the time of transplantation of pegylated islets, islet graft survival was significantly prolonged. In addition, when rats were sensitized with donor splenocytes, graft survival was prolonged by pegylation. We observed that pegylation of islets, combined with a subtherapeutic dose of immunosuppressant, protects the graft from rejection. Prolonged graft survival in sensitized recipients showed that pegylation of islets shifted the pattern of rejection from an acute humoral response to a less aggressive cellular alloresponse.

Effect of immunosuppressants on the expansion and function of naturally occurring regulatory T cells.

The induction of immune tolerance is one of the final therapeutic goals in clinical transplantation. Regulatory T lymphocytes are important for the induction and maintenance of immune tolerance to grafts. If immunosuppressive drugs used clinically to prevent immune rejection also inhibit regulatory T lymphocytes, tolerance would not be achieved. We therefore tested the effect of several immunosuppressants with different mechanisms of action on the proliferation and suppressive activity of CD4(+)CD25(+) regulatory T cells. Highly purified CD4(+)CD25h(+) T cells from C57BL/6 (H-2(b)) mice were stimulated with allogeneic T-depleted splenocytes (BALB/c; H-2(d)) in the presence of various immunosuppressants. After one week in culture, viable T cells were recovered, their regulatory capacity was assessed by their ability to inhibit responder T cell proliferation in MLR, and their cytokine production profile was measured by ELISA. The immunosuppressants rapamycin, cyclosporine A, and methylprednisolone significantly inhibited the expansion of regulatory T cells upon stimulation with alloantigen, whereas mycophenolic acid and the costimulatory blockers, anti-CD40L and CTLA4Ig, did not. None of these immunosuppressants, however, reduced the suppressive capacity of regulatory T cells. Pretreatment with immunosuppressants did not induce significant changes in the cytokine production profile of regulatory T cells. Our results suggest that costimulatory blockers and mycophenolate mofetil can be utilized therapeutically in the induction of immune tolerance. In contrast, the use of rapamycin, cyclosporine A, and methylprednisolone should be reconsidered, due to their deleterious effects on the expansion of naturally occurring regulatory T cells.