JAK3 inhibitor-based immunosuppression in allogeneic islet transplantation in cynomolgus monkeys.

Islet transplantation is efficacious to prevent severe hypoglycemia and glycemic liability of selected patients of type 1 diabetes. However, since calcineurin inhibitor (CNI) causes ß-cell and nephrotoxicity, alternative drug(s) with similar potency and safety profile to CNI will be highly desirable. Here we tested whether JAK3 inhibitor, tofacitinib could be used instead of tacrolimus in CIT07 immunosuppression regimen in cynomolgus nonhuman primate (NHP) model. Five independent streptozotocin (STZ)-induced diabetic monkeys were transplanted with MHC-mismatched allogeneic islets and three animals were further re-transplanted upon insufficient glycemic control or early islet graft rejection. After islet transplantation, blood glucose levels were quickly stabilized and maximal islet graft survival as measured by serum C-peptide concentration was >330, 98, >134, 31, or 22 days, respectively, after transplantation (median survival day; 98 days). Cellular and humoral immune responses were efficiently suppressed by JAK3 inhibitor-based immunosuppression during the follow-up periods. Although intermittent increases of the genome copy number of cynomolgus cytomegalovirus (CMV) were detected by quantitative real-time PCR analyses, serious infections or posttransplant lymphoproliferative disease (PTLD) was not found in all animals. Taken together, we have shown that JAK3 inhibitor could be used in replacement of tacrolimus in a highly translatable NHP islet transplantation model and these results suggest that JAK3 inhibitor will be potentially incorporated in human allogeneic islet transplantation.

Ginsenoside Rg1 protects mouse liver against ischemia-reperfusion injury through anti-inflammatory and anti-apoptosis properties.

BACKGROUND: Ginsenoside Rg1, the major effective component of ginseng, possesses a variety of pharmacologic activities. The objective of this study was to investigate the effects of Rg1 on liver ischemia-reperfusion (IR) injury and explore its potential mechanisms. MATERIALS AND METHODS: Liver warm IR injury was achieved by occluding the portal vein and hepatic artery for 1 h followed by 6-h reperfusion. Eighteen mice were equally randomized into three groups: sham group, IR group, and IR plus Rg1 group (n = 6 mice per group). Mice received an intravenous dose of 20 mg/kg Rg1 or an equivalent volume of saline before ischemic insult. Liver samples and serum were collected for analyses. Serum aminotransferase, histopathology, and apoptosis were determined. Cytokines were measured by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). The phosphorylation of nuclear factor kappa B (NF-κB) p65 was assessed by Western blotting. In addition, the effect of Rg1 in a simulated IR model in vitro was also investigated. Rg1 (100 ug/mL and 500 ug/mL) was administered 1 h before hypoxia insult, and then apoptosis was measured after 12-h reperfusion. RESULTS: Liver IR injury led to a dramatic increase in aminopherase activity, apoptosis and necrosis of hepatocytes, and production of proinflammatory cytokines. Pretreatment with Rg1 protected mice from IR-induced liver injury. Treatment with a high-dose Rg1 (500 ug/mL) significantly suppressed apoptosis compared with a lower dose or control (both P < 0.001). Phosphorylation of NF-κB p65 was increased significantly in IR group, and administration with Rg1 suppressed the level of phosphorylation. CONCLUSIONS: Pretreatment of mice with Rg1 reduced hepatocellular apoptosis and inhibited inflammatory response, which was in part through the NF-κB signaling pathway. Rg1 may provide a novel therapeutic strategy for the treatment of IR-induced liver injury.

Tetrandrine attenuates dendritic cell-mediated alloimmune responses and prolongs graft survival in mice.

Tetrandrine, a bisbenzylisoquinoline alkaloid, has significant immunosuppressive effects; however, the effects of tetrandrine on dendritic cells (DCs) and the associated immune reactions are unclear. In this study, we investigated the effects of tetrandrine on DCs and the effects of the tetrandrine-treated DCs on alloimmune reactions in vitro and graft survival in vivo. Tetrandrine significantly downregulated the expression of CD80 and CD86 of DCs and increased their secretion of IL-10 (p = 0.0001). Mixed leukocyte reaction showed that tetrandrine inhibited dendritic-cell allo-stimulatory activity, which was reversed by the anti-IL-10 treatment. An in vivo study demonstrated that tetrandrine-treated DCs prolonged the survival time of skin grafts in mice compared to control (p = 0.005) and decreased cellular infiltration of the graft in the histopathological study. The data suggest that tetrandrine-treated DCs cause immunosuppression and protect skin grafts from rejection. The tetrandrine-induced immunosuppression seems to be partially due to increased IL-10 secretion.

Hepatocyte transplantation for glycogen storage disease type Ib.

Glycogen storage disease type I (GSD-I) is a group of autosomal recessive disorders with an incidence of 1 in 100,000. The two major subtypes are GSD-Ia, caused by a deficiency of glucose-6-phosphatase (G6Pase), and GSD-Ib, caused by a deficiency of glucose-6-phosphate transporter (G6PT). We report that a substantial improvement was achieved following several infusions of hepatocytes in a patient with GSD-Ib. Hepatocytes were isolated from the unused cadaveric whole livers of two donors. At the first transplantation, approximately 2 x 10(9) cells (2% of the estimated recipient's total hepatocytes) were infused. Seven days later 1 x 10(9) (1% of liver mass) cryopreserved hepatocytes from the same donor were infused, and an additional 3 x 10(9) (3% of liver mass) cells from the second donor were infused 1 month after the second transplantation. After the hepatocyte transplantation, the patient showed no hypoglycemic symptoms despite the discontinuation of cornstarch meals. Liver biopsies on posttransplantation days 20 and 250 showed a normal level of glucose-6-phosphatase activity in presolubilization assay that was very low before transplantation. This was the first and successful clinical hepatocyte transplantation in Korea. In this study, hepatocyte transplantation allowed a normal diet in a patient with GSD-Ib, with substantial improvement in their quality of life. Hepatocyte transplantation might be an alternative to liver transplantation and dietary therapy in GSD-Ib.

Potential mechanisms of photopheresis in hematopoietic stem cell transplantation.

Immune tolerance describes specific unresponsiveness to antigens. In clinical situations such as graft-versus-host disease it may be useful to capitalize on these pre-existing tolerance mechanisms to treat patients. Extracorporeal photopheresis is a pheresis treatment whereby the approximately 5 x 10(9) leukocytes are treated with a photoactivatable compound (8-methoxypsoralen) and UVA light, and immediately returned to the patient in a closed-loop, patient-connected system. This therapy induces apoptosis of virtually all the treated leukocytes. There is growing evidence that infusion of apoptotic cells may trigger certain tolerance mechanisms and, thus, be of therapeutic use in graft-versus-host disease. These apoptotic cells are taken up by phagocytes (antigen-presenting cells) in the body of the patient. Apoptotic cell engagement has been reported to induce several changes and functional activities in the engulfing antigen-presenting cell. These antigen-presenting cells: (1) decrease production of proinflammatory cytokines; (2) increase production of anti-inflammatory cytokines; (3) lower ability to stimulate T-cell responses; (4) delete CD8 T effector cells; and (5) induce regulatory T cells. Any and all of these mechanisms could explain the noted effect in graft-versus-host disease. It is still unclear which one or ones are truly responsible. Ongoing studies in animals and human trials will ultimately unravel these details.

Effect of oral vitamin E and C therapy on calcineurin inhibitor levels in heart transplant recipients.

BACKGROUND: A recent prospective trial demonstrated that oral vitamins C and E retard the early progression of transplant-associated coronary arteriosclerosis; as a result, a number of centers have added these agents to their maintenance regimens. This study reviewed the impact of vitamin E and C supplementation on calcineurin inhibitor trough concentrations. METHODS: A retrospective chart review of the first 29 heart transplant patients prescribed anti-oxidant agents was performed. Twenty-two patients taking cyclosporin A (CsA) and 7 patients taking tacrolimus were prescribed vitamin C (500 mg twice a day) and vitamin E (400 IU twice a day). Serum chemistries and drug levels were measured before and after vitamin therapy was initiated. RESULTS: The baseline CsA trough concentration (mean +/- SD) was 137 +/- 39 ng/ml and it declined to 99 +/- 54 ng/ml (p = 0.007) after anti-oxidant therapy was initiated. The average percentage decrease in the CsA trough concentration was 30%. No significant changes were seen in the patients taking tacrolimus. CONCLUSIONS: These data demonstrate that supplementation with the anti-oxidant agents vitamin C and vitamin E decreases CsA concentrations but does not appear to effect tacrolimus concentrations. Although more detailed pharmacokinetic analysis is necessary to clarify the exact mechanism of this interaction, physicians who take care of transplant recipients should be aware that more frequent CsA concentration monitoring is warranted after initiating these anti-oxidant agents.

Impact of St John's wort treatment on the pharmacokinetics of tacrolimus and mycophenolic acid in renal transplant patients.

BACKGROUND: This study investigated the effect of St John's wort (SJW) extract on the pharmacokinetics of the immunosuppressants tacrolimus (TAC) and mycophenolic acid (MPA). METHODS: Ten stable renal transplant patients received 600 mg SJW extract for 14 days in addition to their regular regimen of TAC and mycophenolate mofetil. RESULTS: Dose-corrected AUC((0-12)) of TAC decreased significantly from 180 ng/ml/h at baseline to 75.9 ng/ml/h after 2 weeks of SJW treatment. To maintain therapeutic TAC concentrations, dose adjustments from a median 4.5 mg/day at baseline to 8.0 mg/day under SJW treatment were required. Two weeks after discontinuation of SJW, TAC doses were reduced to a median of 6.5 mg/day. MPA pharmacokinetics remained unaffected by comedication with hypericum extract. CONCLUSIONS: Administration of SJW extract to patients receiving TAC treatment can result in a serious drug interaction leading to markedly reduced TAC blood concentrations associated with the risk of organ rejection.

Leucyl-leucine methyl ester-treated haploidentical donor lymphocyte infusions can mediate graft-versus-leukemia activity with minimal graft-versus-host disease risk.

L-leucyl-L-leucine methyl ester (LLME) prevents GVHD in several animal models by depleting dipeptidyl peptidase I (DPPI)-expressing cytotoxic cellular subsets. However, clinical application has been hampered by difficulties in stem cell engraftment following treatment of donor bone marrow inocula with LLME at the concentrations necessary to purge DPPI-expressing T-cells. Noting that T-cells can mediate graft-versus-leukemia (GVL) responses via both perforin (usually co-expressed in cytotoxic granules with DPPI) and Fas ligand pathways in a murine model, we hypothesized that LLME might be useful for treatment of delayed DLIs for potential GVL activity with a decreased risk of GVHD induction. In regard to the clinical setting, the ex vivo use of LLME for this purpose would circumvent any toxicity issues for donor stem cells, because by that time patients would have already achieved successful engraftment. For our preclinical studies, we used the haploidentical C57BL/6 (B6) (H2b) --> ((B6 x DBA/2)F1 (H(2b/d)) murine model with lethally irradiated hosts that had received transplants of T-cell-depleted bone marrow cells and were challenged with the MMD2-8 myeloid leukemia line (H2d) of DBA/2 origin. A DLI of LLME-treated donor splenocytes, from B6 mice presensitized to recipient alloantigens, was administered in varying doses 14 days post-marrow transplantation, and the potential for both GVHD and GVL activity was assessed. All mice that received any dose of LLME-treated DLI survived indefinitely, without evidence of cachexia nor B-cell hypoplasia, in contrast to the severe and lethal GVHD induced by mock-treated DLI. Histological analysis largely correlated with the symptomatic findings and revealed no GVHD-like lesions in the spleens of LLME-treated DLI recipients, although some mice displayed various degrees of hepatic mononuclear infiltration. Most notably, mice given LLME-treated DLI also experienced DLI dose-dependent increases in survival against the challenge with the MMD2-8 leukemia. LLME-treated splenocytes remained immunocompetent, as these cells could proliferate in response to mitogens and to restimulation with ovalbumin when used as a recall antigen. In conclusion, LLME-treated DLI possesses immune potential and, in particular, GVL activity without inducing clinically evident GVHD.

Recombinant human erythropoietin treatment of anemia in renal transplant patients.

The rHuEpo effect on anemia in eight renal transplant patients (group A) with severe anemia (Hb 6.0-7.5 g/dL) and chronic graft failure (CGF) (sCr 281-794 mumol/L) was compared to the rHuEpo effect on anemia in predialysis (20 patients-group B) and hemodialysis patients (17 patients-group C) in order to examine the rHuEpo effect on anemia and graft failure progression, and to find out whether the response to therapy in these three patient groups differed. Although renal function impairment was similar in patients from group A and B, anemia was more severe in patients from group A. Serum immunoreactive erythropoietin levels were within normal limits for nonanemic persons, that is, inadequate for the level of anemia in all patients before therapy. Maintenance immunosuppression given after renal transplantation consisted of cyclosporine, azathioprine, and prednisone in standard doses. The startig rHuEpo dose of 150 U/kg/wk increased by 25 U/kg if the target Hb of 10.0 g/dL was not achieved at the end of a 4-week period. When target Hb was achieved, the rHuEpo dose was regularly adjusted to maintain Hb of 10.0 g/dL. Most patients from group A and group C were polytransfused before rHuEpo therapy and consequently with iron overload so that only some patients from these groups and all predialysis patients needed iron supplementation given orally. Anemia improved in all patients with 2 to 10 weeks of treatment. Mean rHuEpo doses for the first 2 months were similar in three studied groups, but the patients with the lowest initial hemoglobin values responded better to rHuEpo therapy. The rate of Hb increase during the initial phase of therapy was significantly higher in patients from group A and B comparing to patients from group C, indicating the importance of residual renal function for rHuEpo effect on anemia. Progression of CGF expressed by the slope of l/sCr vs. time did not change in either patients from group A or in predialysis patients. It could be concluded that rHuEpo therapy improved anemia in transplant patients as in predialysis and hemodialysis patients. Anemia improvement by rHuEpo did not accelerate the progression of graft function.

Possible immunological effects of psychotropic medication.

Psychoactive drugs provide an essential intervention in the care of organ transplant recipients, yet little is known of their effect on immunological function. Human and animal data on immunological effects of neuroleptic (e.g., haloperidol, phenothiazines) and antidepressant agents (e.g., tricyclic antidepressants, fluoxetine) lead to conflicting hypotheses. Lithium carbonate acts through the phosphoinositide system and has a bipolar effect on neuronal activity. Lithium is known to have immune-enhancing properties and is difficult to administer in the peritransplant period but nonetheless deserves study for possible beneficial effects on allograft function.

[FK-506. A new immunosuppressive drug]. / FK-506. Un nuovo farmaco immunosoppressore.

In this brief review, we outline the properties of the new macrolide immunosuppressant FK-506. This selective anti-T cell agent has a similar mode of action to cyclospirin A (CSA). It is, however, more powerful, has the capacity (unlike CSA) to reverse liver allograft rejection and appears to have a higher therapeutic index than CSA in patients receiving organ transplants. Preliminary data suggest that renal function is better in recipients of liver transplants given FK-506 as primary therapy compared with those given CSA. In addition, evidence has been presented that FK-506-treated patients have shorter hospital stay than those receiving treatment with CSA. Other factors require to be taken into account, but it may be that it will prove less expensive in the future to treat patients with FK-506 than with CSA. The potential of FK-506 for the control of autoimmune diseases has been demonstrated in rodent models of rheumatoid arthritis, type I diabetes, posterior uveitis, allergic encephalomyelitis and glomerulonephritis. In the clinical field, FK-506 has been used in two cases of nephrotic syndrome resulting from glomerulonephritis; there was improvement in proteinuria and no decline in renal function. Studies to date have been restricted to one clinical centre although many hundreds of organ graft recipients have been studied. Both longer term and multi-centre investigations are urgently required, but it appears that FK-506 may offer considerable potential as an immunotherapeutic agent.

Experimental murine and primate models for dissection of the immunosuppressive potential of photochemotherapy in autoimmune disease and transplantation.

This paper reviews the results achieved in murine and primate models of autoimmune disease and transplantation. These studies have attempted to clarify the nature and specificity of the response induced by reinfusion of phototreated immunoactive lymphocytes. Results obtained in murine lupus have demonstrated that some of the disease features related to the abnormal proliferation of inducer T cells can be inhibited both prophylactically and therapeutically by exposure to photoinactivated autoimmune splenocytes. Radiolabeling studies performed in normal syngeneic mice have shown that, if immunoactive cells are phototreated and injected, their recirculation pattern is altered, and increased sequestration in the spleen, bone marrow, and kidney is noted. These studies suggest that reinfused, phototreated, antigen-activated lymphocytes may localize in sites where they are available for induction of immune responses. Primate cardiac xenotransplantation models have demonstrated that reinfusion of phototreated autologous leukocytes, administered with cyclosporine A and steroids, mediates enhanced specific suppression of both the cellular and humoral host response to foreign tissue. Taken as a whole, the experimental models suggest that photopheresis may provide a means of inducing specific suppression of immunoactive T cells. This form of therapy may have a role as an immunosuppressive agent in both autoimmune disease and transplantation.