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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.

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 + , CDB + , 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 ofFoxp3 mRNA in Gr-Tol was higher than that in Gr-IS (P=0.07) and Gr-NL (P

Outcome for highly immunogenic lung transplantation remains unsatisfactory despite the development of potent immunosuppressants. The poor outcome may be the result of a lack of minimally invasive methods to detect early rejection. There is emerging clinical evidence that, paradoxically, expression of forkhead box P3 (FOXP3, a specific marker for the regulatory T cells) is upregulated within rejecting grafts.Orthotopic lung transplantation was performed using miniature swine without immunosuppression. Rejection was monitored by chest radiography and open lung biopsy. Expressions levels of FOXP3, perforin, Fas-L and IP-10 mRNA were quantified in the peripheral blood. In addition, rescue immunosuppressive therapy (steroid plus tacrolimus) was administered on post-operative day (POD) 4 or 6.Early rejection was detected by open lung biopsy, but misdiagnosed by chest radiography on POD 4. Expression of FOXP3 in the peripheral blood reached its highest value as early as POD 4, followed by a decline. Such an increase of FOXP3 was not observed in recipients given high-dose tacrolimus. Neither perforin, Fas-L or IP-10 in the peripheral blood exhibited significant fluctuations in the early phase of rejection. Rescue immunosuppressive therapy from POD 4, when peak FOXP3 was seen, prolonged graft survival (27.2 days, versus 9.1 days without immunosuppression, p0.001), in contrast to POD 6, when rejection was suspected by chest radiography (11.5 days, p = not statistically significant [NS]).In a miniature swine lung transplantation model, the FOXP3 mRNA level in the peripheral blood was upregulated at an early phase of rejection. The clinical implication of this finding remains to be elucidated.

Operational tolerance (graft acceptance in an immunosuppression (IS)-free environment) after living-donor liver transplantation (LDLT) could occur by our elective protocol in some patients. There is, nevertheless, no reliable parameter to monitor patients who may discontinue IS without a risk of rejection. To identify such parameters, we systemically phenotyped peripheral blood mononuclear cells from operationally tolerant patients. An increase was observed in the frequency of CD4+CD25high+ cells, B cells and Vdelta1/Vdelta2 gammadeltaT-cells ratio in operationally tolerant patients (Gr-tol; n = 12), compared with those from age-matched volunteers (Gr-vol; n = 24) or patients on IS (Gr-IS; n = 19). The frequency of NK cells was decreased in Gr-tol, compared with those in Gr-IS or Gr-vol. The frequency of NKT cells was decreased after LDLT, compared with that in Gr-vol. Although the contribution of those subsets to the tolerant state remains elusive, the results may provide important clues for reliable indicators of tolerance after LDLT.

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 (RT1 P ) and PVG (RT1 c ) 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

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.

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.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.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.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.

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 for1 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 at10 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.

Objective Kidney transplantation in rats is an important research model. Various methods have been reported, but there is no “standard operation.” We investigated a 1-stage versus a 2-stage native nephrectomy and the type of ureteral anastomosis seeking to establish a standard, reproducible and successful method. Materials and Methods We used PVG (RT1c-RT1Ac: B/Dc) male rats, weighing approximately 200 to 250 g, that underwent transplantation after right recipient nephrectomy. Left recipient nephrectomy was performed either 10 days later or simultaneously. The ureteric anastomosis was fashioned 2 ways: using a ureteral stent or by bladder insertion. Results Urinary complications (obstruction or reflux) were observed in 77.8% when a ureteral stent was used for the ureteric anastomosis versus 28.6% when using the bladder insertion technique (P = .0211). Transplanted rats with nephrectomy of both native kidneys at the time of grafting showed a perioperative mortality of 70%, whereas those hosts with a 2-stage nephrectomy displayed a mortality rate of 22% (P = .0025). Conclusions The bladder insertion technique reduced the incidence of urological complications in rats. In addition, unilateral native nephrectomy at the time of operation with delayed contralateral nephrectomy was better tolerated than simultaneous bilateral nephrectomy. These 2 surgical variants allowed us to perform kidney transplantation with a high degree of success.

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.

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.

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 4°C 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.

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.

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).

Donor-specific blood transfusion (DSBT) promotes graft acceptance in certain experimental and clinical situations. However, the exact nature of the operating mechanisms, and in particular the role of Th1/Th2 cytokines, remains controversial. We recently described a fully mismatched rat combination [RA rat (RT1p) to PVG rat (RT1c)] in which a single pre-transplant (Tx) DSBT induces donor-specific tolerance following heart transplantation (Htx). In order to delineate better the role of Th1 and Th2 cytokines in the development of tolerance versus rejection, we studied the intragraft mRNA for Th1 and Th2 cytokines at different time points post-Tx in rejecting and DSBT-tolerized heart grafts. We performed reverse transcriptase-polymerase chain reaction (RT-PCR) to examine intragraft Th1/Th2 cytokines profile. In contrast to findings from previous data, we detected an early (post-Tx day 2) Th1 cytokine upregulation. Th1 downregulation/Th2 immune deviation was observed only at a later period (post-Tx day 30). Consistent with this Th1 early upregulation, a dense mononuclear cellular infiltrate was seen in tolerized grafts, which was equivalent to that seen in rejecting grafts. Despite in vivo unresponsiveness, peripheral lymphocyte derived from tolerant animals proliferated against donor-matched antigens to the same degree as against those of a third party until post-Tx day 30. Altogether, these observations (dense graft cellular infiltrate, early post-Tx Th1 cytokine production, early preserved cellular proliferative responses), suggest that the development of tolerance is preceded by an active Th1 cytokine-mediated immune response. The mechanisms by which an early Th1 response promotes tolerance need to be further studied.