A phase 1 study to evaluate the effect of dolutegravir on renal function via measurement of iohexol and para-aminohippurate clearance in healthy subjects.

AIM: Dolutegravir (DTG; S/GSK1349572) is under clinical development as a once daily, unboosted integrase inhibitor for the treatment of HIV infection. The effect of DTG on glomerular filtration rate (GFR), effective renal plasma flow (ERPF), and creatinine clearance (CLcr ) was evaluated in 34 healthy volunteers. METHODS: Subjects received DTG 50 mg (once daily or twice daily) or placebo for 14 days. GFR was measured by iohexol plasma clearance, ERPF was assessed by para-aminohippurate plasma clearance and CLcr was measured by 24 h urine collection. RESULTS: All treatments were generally well tolerated. A modest decrease (10-14%) in CLcr was observed, consistent with clinical study observations. DTG 50 mg once daily and twice daily had no significant effect on GFR or ERPF compared with placebo over 14 days in healthy subjects. CONCLUSIONS: These findings support in vitro data that DTG increases serum creatinine by the benign inhibition of the organic cation transporter 2, which is responsible for tubular secretion of creatinine.

[Pharmacokinetics and safety of dolutegravir in healthy Japanese subjects].

Dolutegravir (DTG) is a once-daily, unboosted integrase inhibitor that has been shown to be effective by once daily dosing. The impact of race on dolutegravir pharmacokinetics has not yet been fully characterized. This study was a Phase 1, open label, single dose study to determine pharmacokinetics (PK), safety and tolerability of DTG following 50mg single oral administration in the fasted state in healthy adult Japanese subjects. Subjects had a screening visit within 30 days prior to the administration of study drug, and a follow-up visit within 7 to 14 days after the administration of study drug. Safety evaluation and serial PK samplings were performed pre-dose and for 72 hours after dosing. Non-compartmental analysis was performed for PK parameter calculation. Data were compared to historical values from a similar Phase 1 study conducted in non-Asian, healthy adult subjects (n = 18, 17 Caucasian and 1 Arabic/North African). Ten subjects were enrolled and completed the study. DTG was well tolerated with no adverse events reported throughout the study period. Geometric means (CV%) of single dose DTG PK parameters in Japanese subjects for C(max), AUC(inf), t1/2 and C24 were 2.14microg/mL (47%), 43.4 microg x hr/mL (46%), 14.6 hours (10%) and 0.67 microg/mL (45%). The pharmacokinetics and safety profiles of DTG following single dose oral administration to Japanese healthy adult subjects were consistent with findings previously observed in non-Japanese healthy adult subjects. DTG may be given to Japanese subjects without dose-adjustment.

Factors affecting operational tolerance after pediatric living-donor liver transplantation: impact of early post-transplant events and HLA match.

Pediatric recipients of living-donor liver transplants (LDLT) can often discontinue immunosuppression (IS). We examined factors affecting development of operational tolerance (OT), defined as off IS for >1 year, in this population. A historic cohort analysis was conducted in 134 pediatric primary semi-allogeneic LDLT. Multivariate logistic regression analysis was used. The frequency of peripheral regulatory T cells (Tregs) was determined at >10 years post-Tx by FACS analysis. IS was successfully discontinued in 84 tolerant patients (Gr-tol), but not in 50 intolerant patients (Gr-intol). The Gr-intol consisted of 24 patients with rejection (Gr-rej) and 26 with fibrosis of grafts (Gr-fib). The absence of early rejection [odds ratio (OR) 2.79, 95% CI 1.11-7.02, P = 0.03], was a positive independent predictor, whereas HLA-A mismatch (0.18, 0.03-0.91, P = 0.04) was a negative predictor. HLA-DR mismatches did not affect OT. The Treg frequency was significantly decreased in Gr-intol (4.9%) compared with Gr-tol (7.6%) (P = 0.003). There were increased levels of tacrolimus in the first week in Gr-Tol (P = 0.02). Although HLA-B mismatch (8.73, 1.09-70.0, P = 0.04) was a positive independent predictor of OT, its clinical significance remains doubtful. In this large cohort of pediatric LDLT recipients, absence of early rejection, HLA-A match and the later predominance of Tregs are factors associated with OT.

ET-Kyoto solution plus dibutyryl cyclic adenosine monophosphate is superior to University of Wisconsin solution in rat liver preservation.

ET-Kyoto solution (ET-K) is an extracellular-type organ preservation solution containing the cytoprotective disaccharide, trehalose. A previous study reported the supplement of dibutyryl cyclic adenosine monophosphate (db-cAMP) in conventional ET-K to attenuate lung ischemia-reperfusion injury. In this study, the efficacy of this modified ET-K for liver preservation was investigated by comparison with University of Wisconsin solution (UW). ET-K was supplemented with db-cAMP (2 mmol/L). Lewis rats were randomly assigned to two groups, and liver grafts were flushed and stored at 40C for 24 h with ET-K or UW before syngeneic liver transplantation. The graft function and histological changes at 4 h posttransplant as well as 7-day survival were evaluated. Recipient rat survival rate was significantly higher in the ET-K group than in the UW group. Preservation in ET-K resulted in a significant reduction in serum parenchymal transaminase level and promotion of bile production in comparison with UW. The serum hyaluronic acid level, an indicator of sinusoidal endothelial cell injury, was significantly lower after ET-K preservation than that in UW. Histologically, at 4 h after transplantation, the liver grafts preserved in UW solution demonstrated a greater degree of injury than those in ET-K, which appeared to be apoptosis, rather than necrosis. The continuity of the sinusoidal lining was better preserved in ET-K than in UW. In conclusion, ET-K supplemented with db-cAMP is superior to UW in rat liver preservation. This modified ET-K might therefore be a novel candidate for the procurement and preservation of multiple organs.

Clinical, immunological, and pathological aspects of operational tolerance after pediatric living-donor liver transplantation.

In the setting of our pediatric living-donor liver transplantation (LDLT), 87 patients (15.0% of all the patients: significantly higher proportion, compared with those of other transplant centers) achieved complete withdrawal of immunosuppression, which is referred to as "operational tolerance". Immunosuppressants were completely discontinued for 54 patients as scheduled, and for 33 because of EBV infection or other complications. Immunological analyses of the peripheral blood derived from operationally tolerant patients demonstrated that non-deletional tolerance takes place in which potentially reactive T cells to donor-antigens remain physically in the immune repertoire, but specifically suppressed by certain mechanisms. Not only CD4(+)CD25(high+) T cells were increased in the proportion in the tolerant patients' peripheral lymphocytes and suppressed MLR specifically to the donor antigen, but also FOXP3 expressing cells were present within the tolerant liver. Thus, among several mechanisms accounting for non-deletional tolerance, Tregs are likely to involve at least in part in our tolerant patients. Vdelta1gammadeltaT cells, a subset of gammadeltaT cells, which otherwise reside mainly in the intestine, emerge into the peripheral blood during successful pregnancy but not abortive pregnancy. Since Vdelta1gammadeltaT cells produce massive IL-10, it is proposed that Vdelta1gammadeltaT cells induce fetomaternal tolerance by promoting Th2 immune deviation. Consistent with pregnancy, IL-10 producing Vdelta1gammadeltaT cells emerge into the blood of our tolerant patients. This may reflect a common feature between fetomaternal tolerance and transplant tolerance. We began protocol biopsy in post-LDLT patients who exhibit normal liver function from January 2003. Operationally tolerant patients, albeit showing normal liver function, exhibited decrease in size and increase in number of the bile duct and the fibrosis to a greater extent, compared with patients on maintenance immunosuppression. This warrants serial protocol biopsy before and after complete cessation of immunosuppression even in the presence of normal liver function.

Gene expression profile analysis of the peripheral blood mononuclear cells from tolerant living-donor liver transplant recipients.

Induction of transplant tolerance is a clinically desirable goal. To provide unbiased insight into transplant tolerance, we analyzed gene expression profiling in peripheral blood mononuclear cells from recipients of living-donor liver transplants (LDLTs) who had retained an immune tolerance with a well-functioning graft for several years using cDNA microarray. The comparative analyses with nontransplanted normal healthy volunteers showed that the majority of reliable detected genes were similar, and 5.6% of the genes in the tested genome (of which 627 up-regulated and 90 down-regulated) were significantly regulated and specific to tolerant LDLT recipients, indicating a significant genetic feature for inducing and maintaining immune tolerance. Moreover, the expression of several selected genes was confirmed by semiquantitative reverse transcriptase-polymerase chain reaction, which correlated to microarray data. Our data indicated that cDNA microarray technology was useful for this application and provided many informative insights into transplant tolerance mechanism.

[Regulatory T cell based transplant tolerance--freedom from immunosuppression].

Tolerance after clinical transplantation (Tx) is still extremely rare. However, Kyoto elective protocol enabled a substantial number of patients to weaned off immunosuppression after liver Tx. This is referred to as an immunoprivilege. Nevertheless, the operating mechanisms for liver Tx tolerance remain elusive. The authors demonstrated that regulatory T cells (Tregs) are likely to play an important role in liver Tx tolerance. In addition, we found that precursor like Tregs exist in the human peripheral blood. This can propagate upon stimulation with allo-antigen, in contrast to anergic property of Tregs. Thus, the exploitation of precursor like Tregs as a cellular source of ex vivo and in vivo expansion may lead to the widespread clinical use of Tregs for Tx.

Evaluating the Imbalance Between Increasing Hemodialysis Patients and Medical Staff Shortage After the Great East Japan Earthquake: Report From a Hemodialysis Center Near the Fukushima Nuclear Power Plants.

The Great East Japan Earthquake in 2011 caused an unprecedented imbalance between an increasing number of hemodialysis patients and medical staff shortage in the Sousou area, the site of the Fukushima nuclear power plants. In 2014, capacity of our hemodialysis center reached a critical limit due to such an imbalance. We attempted to evaluate the effort of medical staff to clarify to what extent their burden had increased post-disaster. The ratio of total dialysis sessions over total working days of medical staff was determined as an approximate indicator of effort per month. The mean value of each year was compared. Despite fluctuations of the ratio, the mean value did not differ from 2010 to 2013. However, the ratio steadily increased in 2014, and there was a significant increase in the mean value. This proposed indicator of the effort of medical staff appears to reflect what we experienced, although its validity must be carefully examined in future studies.

Utility of CD127 combined with FOXP3 for identification of operational tolerance after liver transplantation.

Loss of cell surface expression of CD127 on CD4(+)CD25(++) regulatory T-cells (Tregs) may be a useful marker to efficiently isolate Tregs. As FOXP3 was specifically used to identify Tregs, combining these two markers could give better identification for patient with operational tolerance (OT) after liver transplantation. To testify this mixed lymphocyte reaction (MLR), the function of circulating CD4(+)CD25(++)CD127(dim) cells (CD127(dim) cells) was examined in immunosuppression (IS)-free pediatric recipients after liver transplantation (LTx) (group operational tolerance: OT) (Gr-tol n=25) compared to recipients who could not stop IS due to clinically overt rejection (group intolerance) (Gr-intol n=18), recipients who were weaning IS (Gr-weaning n=11) and age-matched healthy volunteers (Gr-vol n=11). In addition, the frequencies of CD127(dim) cells vs CD4(+)CD25(++)CD127(dim)FOXP3(+) (CD127(dim)FOXP3(+)) cells were compared in these four groups by FACS analyses. Our results showed that The proliferation of CD4 cells to donor antigens was reduced compared to third-party antigens only in Gr-tol (P=0.022) but not in other groups (P=NS). Depletion of CD127(dim) cells resulted in a donor antigen-specific abrogation of this MLR hyporesponsiveness in Gr-tol (P<0.001) but not other groups (P=NS). This implied that CD127 efficiently isolated donor antigen-specific Tregs. The frequencies of CD127(dim) cells were significantly lower in Gr-intol (5.2%±1.9%) compared to those in Gr-tol (7.8%±1.8%) (P<0.001) as were the frequencies of CD127(dim) FOXP3(+) cells (Gr-tol: 5.4%±1.7% vs Gr-intol: 2.9%±1.0%, P<0.001). Of interest, there were fewer CD127(dim)FOXP3(+) cells in Gr-intol (2.9%±1%) than in Gr-weaning (5.1%±1.8%) (P=0.002), but no difference in CD127(dim) cells (Gr-intol: 5.2%±1.9% vs Gr-weaning: 6.7%±2.0%) (NS). Thus, combining FOXP3 with CD127 for phenotype analysis demonstrated an unequivocal difference between Gr-intol and Gr-weaning that was not detected by CD127 alone. In conclusion CD127 was a useful surface marker to isolate donor-antigen-specific-Tregs in OT after LTx. The additive effect of its combination with FOXP3 is important in phenotypical Treg analyses of OT patients.

Regulatory cells, TH1/TH2 unbalance, and antibody-induced chronic rejection in operational tolerance induced by donor-specific blood transfusion.

We developed a rodent model in which donor-specific blood transfusion (DSBT) promotes hyporesponsiveness and graft acceptance. In this model, signs of immune activation are present early posttransplant, with preserved proliferative responses against the donor and a dense cellular infiltrate in tolerant grafts. Intriguingly, an early accumulation of IFN-gamma is seen in grafts destined to become tolerized, supporting recent evidence that Th1 cytokines play a role in tolerance induction. Specific regulatory cells capable of propagating tolerance into naive recipients are operating. These mechanisms of immune activation and the generation of regulatory cells are influenced by immunosuppression (steroids and calcineurin inhibitors). In this model, in a second phase, a Th2 immune deviation occurs and is associated with the development of chronic rejection (vascular obliteration, endothelial IgG deposition, and complement binding). It remains unclear whether chronic rejection in this model is caused by Th2 type regulatory cells or whether chronic rejection is the consequence of an insufficient number of regulatory cells. In the clinic, the current strategy of profoundly inhibiting immune activation (in particular Th1 cytokines/responses) by using high dose calcineurin inhibitors and steroids may prove antagonistic with the development of tolerance, particularly when immunomodulatory strategies (such as DSBT) are applied. Development of chronic rejection in a regulation-based tolerance model suggests that deletion-based tolerogenic strategies may offer a more robust protection against chronic rejection.

FTY720, an immunosuppressant that alters lymphocyte trafficking, abrogates chronic rejection in combination with cyclosporine A.

BACKGROUND: Chronic rejection remains the leading cause of failure after transplantation (Tx). FTY720, a new immunosuppressant altering lymphocyte trafficking, is effective against acute rejection, but its activity against chronic rejection is not known. METHODS: A valid model of chronic rejection was produced. Heart transplantation (HTx) was performed using fully mismatched RA (RT1p) and PVG (RT1c) rats. Administration of donor-specific blood transfusion 12 days before HTx prolongs graft survival, but features of chronic rejection including intimal hyperplasia and vascular obliteration (VO) develop with time only in allogeneic Tx. This is therefore a valid model of chronic rejection. VO was assessed on post-Tx day 90 in six groups differing according to the maintenance immunosuppressive regimen administered. group 1, donor-specific blood transfusion only and no other treatment; group 2, FTY720 (0.3 mg/kg/day orally) for 90 days; group 3, cyclosporine A (CsA) (1 mg/kg/day orally) for 90 days; group 4, combined administration of FTY720 and CsA for 90 days; group 5, transient administration of combined FTY720 and CsA for 7 days; and group 6, syngeneic HTx (RA to RA). Graft infiltrate, endothelial immunoglobulin (Ig) G deposition, and complement binding were also examined on post-Tx day 90. RESULTS: In control group 1, severe VO was observed, compared with syngeneic HTx (group 6). Monotherapy with FTY720 (group 2) or with CsA (group 3) significantly but partially reduced VO. On the contrary, combined administration of FTY720 and CsA (group 4) abrogated VO. A 1-week treatment with combined FTY720 and CsA (group 5) reduced VO but only partially. In group 1, arteriosclerosis was accompanied by graft infiltrate, endothelial IgG deposition, and complement binding. In groups 2, 3, and 5, graft infiltrating scores were partially decreased compared with group 1 but remained higher than in syngeneic controls; endothelial IgG deposition and complement binding were still present. In group 4, continuous administration of combined FTY720 and CsA reduced graft infiltrate to the level of syngeneic control and abrogated both endothelial IgG deposition and complement binding. CONCLUSIONS: Maintenance treatment with either FTY720 or CsA monotherapy partially prevents chronic rejection; short-term treatment with combined FTY720 and CsA reduces chronic rejection only partially; and continuous treatment with combined FTY720 and CsA abrogates chronic rejection, and this is accompanied by dramatic reduction of graft infiltrating cells, endothelial IgG deposition, and complement binding. Prevention of chronic rejection by maintenance treatment with FTY720 and CsA represents indirect evidence that normal lymphocyte trafficking and function are mandatory for development of chronic rejection.

Regulatory cell-mediated tolerance does not protect against chronic rejection.

BACKGROUND: Regulatory cells prevent graft loss to acute rejection and induce tolerance, possibly by promoting Th2 deviation. Th2 cytokines stimulate B cells, which cause alloantibody-mediated chronic rejection. We searched to determine whether regulatory cell-mediated tolerance protects or not against chronic rejection. METHODS: Heart transplantation (Htx) was performed using RA (RT1P) and PVG (RT1c) rats as donor and recipients. Donor-specific blood transfusion (DSBT) was given on preTx day 12. Secondary grafts were implanted at day 100. Splenocytes were transferred from tolerant rats (and controls) into lightly irradiated (450 rad) naive PVG, which received RA Htx. Primary Htx were investigated for the development of vascular occlusion (VO), the production of Th1/Th2 intragraft cytokines, and for the nature of graft infiltrate as well as for endothelial deposition of immunoglobulin (Ig)G isotypes and complement (C3) binding. Results were compared with rejecting controls (no DSBT) and syngeneic Htx. RESULTS: RA Htx were rejected within 10 days (8, 9, 10x4). PreTx DSBT prolonged primary Htx survival indefinitely (>140 days) with acceptance of secondary donor-specific (but not third-party) grafts (P<0.001). Naive irradiated PVG rats given splenocytes from tolerant rats but not from controls accepted RA Htx, showing the existence of regulatory cells in allograft acceptors. Despite being tolerant, DSBT-treated rats displayed typical features of chronic rejection at day 90 (VO=77%; P<0.001 vs. VO=4% in syngeneic rats). An overt Th2 deviation, particularly intragraft production of interleukin (IL)-4, was observed at day 30. Simultaneously to this Th2 deviation, B cells emerged in the grafts and endothelial deposition of IgG1 (Th2 dependent) and C3 binding were observed. CONCLUSIONS: Regulatory cells that prevent graft loss to acute rejection in primary and secondary grafts do not protect against the development of chronic rejection.

Early accumulation of interferon-gamma in grafts tolerized by donor-specific blood transfusion: friend or enemy?

BACKGROUND: We previously documented an early (day-2) interferon (IFN)-gamma accumulation in cardiac allografts of rats made tolerant by donor-specific blood transfusion (DSBT) but not in rejecting controls. This contrasted with the IFN-gamma peak seen later (day 5) in rejecting but not in tolerant rats. METHODS: To further examine the role of early intragraft IFN-gamma in DSBT-induced tolerance, we studied whether IFN-gamma up-regulation correlates with the magnitude of the DSBT effect and how IFN-gamma is influenced by interventions abrogating tolerance. RESULTS: The protective effect of DSBT depended upon the timing of administration: day-12 DSBT induced indefinite graft survival; day-6 DSBT gave a moderate, and day-0 DSBT, no graft prolongation. IFN-gamma up-regulation correlated with the DSBT effect: it was maximal after day-12 DSBT, intermediate after day-6 DSBT, and absent after day-0 DSBT. Tolerant splenocytes transferred tolerance into naive rats in a donor-specific manner, indicating that alloantigen-specific regulatory cells operate. Thymectomy prevented regulatory cells development, caused further amplification of intragraft IFN-gamma, and led to rejection, although graft survival was still prolonged. CONCLUSIONS: Day 2 intragraft IFN-gamma correlates with the DSBT protective effect. Thymectomy abrogates DSBT-induced tolerance, prevents regulatory cell development, and paradoxically causes further accumulation of intragraft IFN-gamma. These data indicate that DSBT has a stimulatory and a (thymus-dependent) inhibitory effect on early intragraft IFN-gamma. Intragraft IFN-gamma is beneficial, providing it occurs early and remains moderate. The role of intragraft IFN-gamma in tolerance and rejection depends upon the timing and the degree of production and perhaps the type of IFN-gamma producing cells (regulatory or effector).

Complete freedom from rejection after intestinal transplantation using a new tolerogenic protocol combined with low immunosuppression.

BACKGROUND: Intestinal transplantation (Itx) remains the most difficult form of transplantation. This is due to the high immunogenicity of the bowel that currently obligates Itx patients to heavy immunosuppression, which causes infection, posttransplant lymphoproliferative disease (PTLD), and drug toxicity. Wider application of Itx depends on the development of tolerogenic strategies to promote engraftment while reducing the need for immunosuppression. We applied a strategy to clinical Itx that combines intraportal donor-specific blood transfusion with a deliberately low immunosuppression protocol (no high-dose steroids; lower tacrolimus level). METHODS: A 55-year-old patient received a combined liver/Itx. Donor-specific whole blood was taken from the donor during procurement and transfused in the recipient portal vein after graft reperfusion. For induction immunosuppression, no intravenous bolus of steroids was given; only two doses of anti-interleukin 2 receptor antibody were administered. The patient received posttransplantation maintenance immunosuppression with lower tacrolimus levels than average (15 ng/ml first month; 5-10 ng/ml thereafter), low-dose azathioprine (1 mg/kg first to third months; 0.5 mg/kg thereafter), and low-dose steroids (Medrol 8 mg twice daily first and second months; 4 mg twice thereafter). The patient was monitored for rejection, graft-versus-host disease, infection, and PTLD. Protocol biopsy specimens were taken from the distal ileum (2 per week). RESULTS: Clinical, endoscopic, and histologic signs of rejection did not develop. Chimerism was identified at day 28. Graft-versus-host disease was absent clinically. Chimerism was self-limiting and disappeared without modifying baseline immunosuppression and without observing a change in graft function. The patient remained free of systemic opportunistic infections, PTLD, and drug toxicity. Total parenteral nutrition was stopped at 7 weeks after transplantation. The patient remains free of total parenteral nutrition and free of rejection at 14 months after transplantation. CONCLUSIONS: We describe an Itx patient who remained rejection free despite receiving significantly lower immunosuppression than average. We hypothesize that intraoperative immunomodulation via intraportal donor-specific blood transfusion in the absence of nonspecific overimmunosuppression promoted Itx acceptance.

Intragraft Vδ1 γδ T cells with a unique T-cell receptor are closely associated with pediatric semiallogeneic liver transplant tolerance.

BACKGROUND: T-cell receptor Vδ2 γδ T cells (Vδ2 cells) participate in host defense, whereas Vδ1 γδ T cells (Vδ1 cells) may regulate immune responses. Vδ1 cells appear to play a role in fetomaternal tolerance and our aim was to examine their role in liver transplant tolerance. METHODS: To determine whether Vδ1 cells increase within accepted grafts after semiallogeneic pediatric liver transplantation, the Vδ1/Vδ2 ratio was assessed at the transcriptional level and the complementarity-determining region 3 loop of the δ chain of Vδ1 cells was sequenced in biopsies from immunosuppression-free (n=6) or almost free (n=3) liver transplant recipients, referred to as group tolerance (Gr-Tol; n=9). The results were compared with biopsies from grafts of recipients on maintenance immunosuppression due to concern of rejection (Gr-IS; n=11). Chronically rejected grafts (Gr-CR; n=6) and normal livers (Gr-NL; n=8) were also examined. RESULTS: The Vδ1/Vδ2 ratio was the highest in Gr-Tol (0.07±0.06) compared with Gr-IS (0.03±0.02; P=0.04), Gr-CR (0.01±0.02; P=0.008), and Gr-NL (0.02±0.04; P=0.01). There was an identical complementarity-determining region 3 sequence (100% homologous) among all recipients in Gr-Tol, which was dominant in six of nine recipients. This sequence was not seen in Gr-IS or Gr-CR, although it was observed in five of six normal livers. CONCLUSIONS: A unique Vδ1-bearing T-cell clone accumulates within accepted human liver grafts. It might be useful as a biomarker of tolerance and the identification of its ligand might aid in the development of a novel strategy for tolerance induction.

The generation of donor-specific CD4+CD25++CD45RA+ naive regulatory T cells in operationally tolerant patients after pediatric living-donor liver transplantation.

BACKGROUND: CD4+CD25++CD45RA+ cells (naïve regulatory T cells [naïve-Tregs]) have been identified as a functionally premature form of CD4+CD25+++CD45RA(-) cells (conventional-Tregs). However, their contribution to transplant tolerance remains to be elucidated. METHOD: We examined the frequency and the function of conventional and naive-Tregs in the peripheral blood derived from operationally tolerant patients after pediatric living-donor liver transplant (Gr-tol). The data were compared with those of patients who were unable to be weaned off immunosuppression due to rejection (group-intolerance [Gr-intol]), patients in the process of weaning immunosuppression (Gr-weaning) and healthy volunteers (group-healthy volunteers [Gr-vol]). RESULTS: In Gr-tol, the frequency of conventional-Tregs was significantly higher than that in Gr-vol and tended to be higher than that in Gr-intol. The frequency of naive-Tregs was significantly decreased in Gr-intol versus those in Gr-tol, -weaning, and -vol. In mixed lymphocyte reactions, donor-specific hyporesponsiveness of CD4+ cells was observed only in Gr-tol but not in the other groups. Depletion of conventional or naive-Tregs from CD4+ cells demonstrated that the suppressive properties of donor antigen-reactive conventional and naïve-Tregs were upregulated compared with those of third-party antigen-reactive conventional and naïve-Tregs in Gr-tol only. CONCLUSIONS: This is the first report providing detailed evidence that donor-specific naïve-Tregs were generated and their suppressive properties were upregulated in the peripheral blood of tolerant patients, whereas their frequency was downregulated in intolerant patients. Therefore, we speculate that not only conventional-Tregs play a role in Tx tolerance but also the role of naïve-Tregs is critical.

Intestinal ischemia reperfusion and lipopolysaccharide transform a tolerogenic signal into a sensitizing signal and trigger rejection.

BACKGROUND: After intestinal transplantation (ITx), rejection is vigorous and tolerance is difficult to achieve. A possible reason for this phenomenon is that ITx and ischemia reperfusion injury are accompanied by translocation of lipopolysaccharide (LPS), a "danger" signal capable of activating adaptive alloimmunity against Tx antigens. METHODS: To study that, we used our previously described rat model where donor-specific blood transfusion (DSBT) induces tolerance to fully mismatched heart allografts. RESULTS: In this model, control grafts are rejected within 9 days, whereas 100% of DSBT pretreated grafts survive long term. In contrast with heart Tx, an identical DSBT protocol failed to induce tolerance in 57% of recipients after ITx (P<0.05). We hypothesized that DSBT promotes tolerance to heart but not intestinal allografts (partly) because of ischemia/reperfusion injury and LPS translocation associated with ITx. We tested this hypothesis by adding to DSBT an inflammatory signal in the form of a remote intestinal/ischemia reperfusion injury or by administrating 0.1 mg of LPS. These maneuvers (partially) abrogated tolerance induction (P<0.05). CONCLUSIONS: Addition of a danger signal (remote intestinal ischemia/reperfusion) transforms a tolerogenic signal (DSBT) into a sensitizing signal and may lead to graft rejection. This effect is mediated in part by LPS. This may, partly, account for the high rejection rate and the difficulty to induce graft acceptance after ITx.

Requirement of protocol biopsy before and after complete cessation of immunosuppression after liver transplantation.

BACKGROUND: Operational tolerance is defined as long-term acceptance of a transplanted organ after complete cessation of immunosuppression (IS), but may not always protect against antigen-dependent changes in graft morphology. METHOD: IS free patients after living-donor liver transplantation (LDLT) underwent protocol biopsy (tolerance group [Gr-Tol]) and were evaluated for rejection and fibrosis. The degree of fibrosis was compared with those in the patients on maintenance IS group (Gr-IS) and the base line normal liver group (Gr-BS). When bridging fibrosis or progression of fibrosis was observed, IS was reintroduced or increased in Gr-Tol or in the patients in the weaning process. RESULTS: Neither acute nor chronic rejection was observed. The degree of fibrosis, however, was significantly greater in Gr-Tol than those in Gr-IS and Gr-BS. In Gr-Tol, the number of graft infiltrating FOXP3 cells was significantly increased, the interval between LDLT and biopsy plus the donor age was significantly longer, and recipient age at LDLT was significantly younger, compared with those in Gr-IS. However, none of these three parameters correlated with the degree of fibrosis. In 7 of 11 patients in whom IS was reintroduced or increased, the improvement of fibrosis was observed by the subsequent biopsy. CONCLUSION: Grafts of operationally tolerant patients after LDLT did not exhibit acute or chronic rejection, but they exhibited fibrosis. It remains elusive whether fibrosis observed in tolerant grafts is antigen dependent. The finding that after [corrected] the reintroduction or the increase of IS fibrosis was improved supported the possibility that fibrosis in operationally tolerant patients was antigen dependent.

The presence of Foxp3 expressing T cells within grafts of tolerant human liver transplant recipients.

BACKGROUND: Some experimental transplant-tolerance models have shown that the presence of regulatory T cells within grafts is important for the development of tolerance (Tol). METHODS.: To determine if the presence of regulatory T cells correlates with graft acceptance in living-donor liver-transplantation tolerance, the expression of Foxp3 mRNA and the presence of CD4, CD8, and Foxp3 cells were quantified in biopsies from tolerant recipients by real-time polymerase chain reaction and by immunohistochemistry and immunofluorescent staining (Gr-Tol). The results were compared with biopsies from the recipients on maintenance immunosuppression (Gr-IS), grafts removed because of chronic rejection (Gr-CR), or normal liver (Gr-NL). RESULTS: The expression of Foxp3 mRNA in Gr-Tol was higher than that in Gr-IS (P=0.07) and Gr-NL (P<0.0001), but equivalent to that in Gr-CR. In Gr-Tol, Foxp3 cells were detectable within the clustered CD4 and CD8 cells in the portal areas. Ninety-two percent of those Foxp3 cells were CD4, whereas 8% were CD8. The number of Foxp3 cells was significantly increased in Gr-Tol, compared with that in Gr-IS (P<0.05), although the number of CD4 or CD8 cells did not differ between the two. Foxp3 cells were hardly detectable in Gr-CR or -NL. CONCLUSIONS: This is the first report showing that CD4Foxp3 cells are present within grafts in a subset of tolerant patients after human liver transplantation. A prospective study is needed to elucidate whether the assessment of intragraft expression of Foxp3 protein, but not Foxp3 mRNA, can aid the identification of living-donor liver-transplantation recipients who can successfully withdraw IS.