Th17 Responses to Collagen Type V, kα1-Tubulin, and Vimentin Are Present Early in Human Development and Persist Throughout Life.

T helper 17 (Th17)-dependent autoimmune responses can develop after heart or lung transplantation and are associated with fibro-obliterative forms of chronic rejection; however, the specific self-antigens involved are typically different from those associated with autoimmune disease. To investigate the basis of these responses, we investigated whether removal of regulatory T cells or blockade of function reveals a similar autoantigen bias. We found that Th17 cells specific for collagen type V (Col V), kα1-tubulin, and vimentin were present in healthy adult peripheral blood mononuclear cells, cord blood, and fetal thymus. Using synthetic peptides and recombinant fragments of the Col V triple helical region (α1[V]), we compared Th17 cells from healthy donors with Th17 cells from Col V-reactive heart and lung patients. Although the latter responded well to α1(V) fragments and peptides in an HLA-DR-restricted fashion, Th17 cells from healthy persons responded in an HLA-DR-restricted fashion to fragments but not to peptides. Col V, kα1-tubulin, and vimentin are preferred targets of a highly conserved, hitherto unknown, preexisting Th17 response that is MHC class II restricted. These data suggest that autoimmunity after heart and lung transplantation may result from dysregulation of an intrinsic mechanism controlling airway and vascular homeostasis.

Clinical repigmentation patterns in paediatric vitiligo.

BACKGROUND: Repigmentation is an essential outcome measure in vitiligo. However, clinical studies describing vitiligo repigmentation patterns are lacking. OBJECTIVES: To assess and clearly define the repigmentation patterns in a series of patients with vitiligo, correlating these with clinicoepidemiological characteristics. METHODS: Patients with vitiligo seen at least at twice (initial consultation and follow-up visit) in the Department of Paediatric Dermatology, Hôpital Pellegrin des Enfants, Bordeaux University Hospital from 2006 to 2014 were included. Clinical photographs and case records were reviewed. RESULTS: There were 109 patients (64 female, 45 male) mostly with Fitzpatrick skin type III (n = 67, 61%). The majority had nonsegmental (n = 71, 65%) or segmental vitiligo (n = 29, 27%). In total 172 representative vitiligo lesions were analysed. Overall, a combined pattern of repigmentation was most commonly seen (n = 106, 62%). The combined pattern occurred more frequently in patients with segmental vs. nonsegmental vitiligo (P = 0·009), whereas the diffuse pattern was more frequent in the latter (P = 0·007). Diffuse repigmentation was the predominant pattern on the eyelids (P < 0·001). We observed a new pattern in sites with few to absent hair follicles, which we propose to call 'medium spotted repigmentation'. This begins as circular macules of repigmentation, wider than 5 mm in diameter, which, from the outset, are larger than the initial macules of perifollicular repigmentation. This study is limited by its retrospective nature and small sample size for subgroup assessment. CONCLUSIONS: The combined pattern of repigmentation was most frequently observed. Medium spotted repigmentation is a new pattern, which will benefit from larger studies for a better understanding.

Pilot study of an automated method to determine Melasma Area and Severity Index.

BACKGROUND: Objective outcome measures for melasma severity are essential for the evaluation of severity as well as results of treatment. The modified Melasma Area and Severity Index (mMASI) score is a validated tool for assessing melasma severity but is often subject to inter-observer variability. OBJECTIVES: To develop and validate a novel image analysis software designed to automatically calculate the area and degree of hyperpigmentation in melasma from computer image analysis of whole-face digital photographs, thereby deriving an automated mMASI score (aMASI). METHODS: The algorithm was developed in collaboration between dermatologists and image analysis experts. Firstly, using an adaptive threshold method, the algorithm identifies, segments and calculates the areas involved. It then calculates the darkness. Finally, the derived area and darkness are then used to calculate mMASI. The scores derived from the algorithm are validated prospectively. Twenty-nine patients with melasma using depigmenting agents were recruited for validation. Three dermatologists scored mMASI at baseline and post-treatment using standardized photographs. These scores were compared with aMASI scores derived from computer analysis. RESULTS: aMASI scores correlated well with clinical mMASI pre-treatment (r = 0·735, P < 0·001) and post-treatment (r = 0·608, P < 0·001). aMASI was reliable in detecting changes with treatment. These changes in aMASI scores correlated well with changes in clinician-assessed mMASI (r = 0·622, P < 0·001). CONCLUSIONS: This study proposes a novel approach in melasma scoring using digital image analysis. It holds promise as a tool that would enable clinicians worldwide to standardize melasma severity scoring and outcome measures in an easy and reproducible manner, enabling different treatment options to be compared accurately.

Differential requirement for P2X7R function in IL-17 dependent vs. IL-17 independent cellular immune responses.

IL17-dependent autoimmunity to collagen type V (Col V) has been associated with lung transplant obliterative bronchiolitis. Unlike the T helper 1 (Th1)-dependent immune responses to Tetanus Toxoid (TT), the Th17 response to Col V in lung transplant patients and its Th1/17 variant observed in coronary artery disease patients requires IL-1ß, tumor necrosis factor α and CD14(+) cells. Given the involvement of the P2X7 receptor (P2X7R) in monocyte IL-1ß responses, we investigated its role in Th17-, Th1/17- and Th1-mediated proinflammatory responses. Transfer of antigen-pulsed peripheral blood mononucleated cells (PBMCs) from Col V-reactive patients into SCID mouse footpads along with P2X7R antagonists revealed a selective inhibition of Col V-, but not TT-specific swelling responses. P2X7R inhibitors blocked IL-1ß induction from monocytes, including both Col V-α1 peptide-induced (T-dependent), as well as native Col V-induced (T-independent) responses. Significantly higher P2X7R expression was found on CXCR3(neg) CCR4(+)/6(+) CD4(+) [Th17] versus CXCR3(+)CCR4/6(neg) CD4(+) [Th1] subsets in PBMCs, suggesting that the paradigm of selective dependence on P2X7R might extend beyond Col V autoimmunity. Indeed, P2X7R inhibitors suppressed not only anti-Col V, but also Th1/17-mediated alloimmunity, in a heart transplant patient without affecting anti-viral Epstein-Barr virus responses. These results suggest that agents targeting the P2X7R might effectively treat Th17-related transplant pathologies, while maintaining Th1-immunity to infection.

Consensus statement on the diagnosis, treatment and follow-up of patients with primary adrenal insufficiency.

Primary adrenal insufficiency (PAI), or Addison's disease, is a rare, potentially deadly, but treatable disease. Most cases of PAI are caused by autoimmune destruction of the adrenal cortex. Consequently, patients with PAI are at higher risk of developing other autoimmune diseases. The diagnosis of PAI is often delayed by many months, and most patients present with symptoms of acute adrenal insufficiency. Because PAI is rare, even medical specialists in this therapeutic area rarely manage more than a few patients. Currently, the procedures for diagnosis, treatment and follow-up of this rare disease vary greatly within Europe. The common autoimmune form of PAI is characterized by the presence of 21-hydroxylase autoantibodies; other causes should be sought if no autoantibodies are detected. Acute adrenal crisis is a life-threatening condition that requires immediate treatment. Standard replacement therapy consists of multiple daily doses of hydrocortisone or cortisone acetate combined with fludrocortisone. Annual follow-up by an endocrinologist is recommended with the focus on optimization of replacement therapy and detection of new autoimmune diseases. Patient education to enable self-adjustment of dosages of replacement therapy and crisis prevention is particularly important in this disease. The authors of this document have collaborated within an EU project (Euadrenal) to study the pathogenesis, describe the natural course and improve the treatment for Addison's disease. Based on a synthesis of this research, the available literature, and the views and experiences of the consortium's investigators and key experts, we now attempt to provide a European Expert Consensus Statement for diagnosis, treatment and follow-up.

A lightweight hybrid vision transformer network for radar-based human activity recognition.

Radar-based human activity recognition (HAR) offers a non-contact technique with privacy protection and lighting robustness for many advanced applications. Complex deep neural networks demonstrate significant performance advantages when classifying the radar micro-Doppler signals that have unique correspondences with human behavior. However, in embedded applications, the demand for lightweight and low latency poses challenges to the radar-based HAR network construction. In this paper, an efficient network based on a lightweight hybrid Vision Transformer (LH-ViT) is proposed to address the HAR accuracy and network lightweight simultaneously. This network combines the efficient convolution operations with the strength of the self-attention mechanism in ViT. Feature Pyramid architecture is applied for the multi-scale feature extraction for the micro-Doppler map. Feature enhancement is executed by the stacked Radar-ViT subsequently, in which the fold and unfold operations are added to lower the computational load of the attention mechanism. The convolution operator in the LH-ViT is replaced by the RES-SE block, an efficient structure that combines the residual learning framework with the Squeeze-and-Excitation network. Experiments based on two human activity datasets indicate our method's advantages in terms of expressiveness and computing efficiency over traditional methods.

Donor-specific indirect pathway analysis reveals a B-cell-independent signature which reflects outcomes in kidney transplant recipients.

To investigate the role of donor-specific indirect pathway T cells in renal transplant tolerance, we analyzed responses in peripheral blood of 45 patients using the trans-vivo delayed-type hypersensitivity assay. Subjects were enrolled into five groups-identical twin, clinically tolerant (TOL), steroid monotherapy (MONO), standard immunosuppression (SI) and chronic rejection (CR)-based on transplant type, posttransplant immunosuppression and graft function. The indirect pathway was active in all groups except twins but distinct intergroup differences were evident, corresponding to clinical status. The antidonor indirect pathway T effector response increased across patient groups (TOL < MONO < SI < CR; p < 0.0001) whereas antidonor indirect pathway T regulatory response decreased (TOL > MONO = SI > CR; p < 0.005). This pattern differed from that seen in circulating naïve B-cell numbers and in a cross-platform biomarker analysis, where patients on monotherapy were not ranked closest to TOL patients, but rather were indistinguishable from chronically rejecting patients. Cross-sectional analysis of the indirect pathway revealed a spectrum in T-regulatory:T-effector balance, ranging from TOL patients having predominantly regulatory responses to CR patients having predominantly effector responses. Therefore, the indirect pathway measurements reflect a distinct aspect of tolerance from the recently reported elevation of circulating naïve B cells, which was apparent only in recipients off immunosuppression.

Repigmentation of leucotrichia in vitiligo with noncultured cellular grafting.

BACKGROUND: Vitiligo in hair-bearing areas is often associated with leucotrichia. Repigmentation of leucotrichia has been reported mainly with tissue and follicular hair grafts. OBJECTIVES: To evaluate the repigmentation response of leucotrichia after noncultured cellular grafting. METHODS: We retrospectively reviewed patients with stable generalized and segmental vitiligo who underwent noncultured cellular grafting from March 2008 to November 2010 in areas with leucotrichia. The percentage repigmentation of white hairs was based on clinical assessment and standardized digital photography, and was graded 'poor', 'fair', 'good' or 'excellent', corresponding to a scale of 0-100% repigmentation with respective intervals of 25%. RESULTS: Eighty-four patients with vitiligo underwent cellular grafting; of these, 13 had grafting in areas with leucotrichia. Twelve patients (92%) had poor repigmentation of leucotrichia 3 months after transplant. One patient defaulted follow-up after this period. At 6months, fair repigmentation was achieved in eight out of the 12 remaining patients (67%). After 9-12months, however, 91% (10 out of 11) of the patients achieved good or excellent repigmentation. One patient did not reach the 9-month follow-up period at the time of data analysis. Leucotrichia of eyebrows yielded excellent repigmentation in eight out of nine patients, whereas hairs on the limbs and scalp showed good repigmentation in two out of two patients at 9-12 months. CONCLUSIONS: Good to excellent repigmentation of leucotrichia can be achieved with noncultured cellular grafting, obviating the need for hair transplantation.

Lymphomatoid papulosis: is a second lymphoma commoner among East Asians?

BACKGROUND: Lymphomatoid papulosis (LyP) is a low-grade cutaneous lymphoma, which lies within the spectrum of primary cutaneous CD30-positive lymphoproliferative disorders. Around 10-20% of LyP cases are associated with a second lymphoma. AIM: To analyse a cohort of Asian patients with LyP, diagnosed from 1987 to 2007 at the National Skin Centre (NSC), Singapore, in terms of epidemiology, treatment and association with a second lymphoma. METHODS: Patients were identified through the NSC clinical and histological databases. RESULTS: During this period, 13 patients were diagnosed with LyP based on clinicopathological criteria. The mean age at diagnosis was 41 years, the male : female ratio was 2.3 : 1, and 92% of the patients were Chinese. Recurrent papulonecrotic lesions were present for a mean of 3 years before diagnosis. Treatment of LyP comprised monotherapy (n = 4) or combination therapy (n = 9) using corticosteroids, oral antibiotics, methotrexate and/or phototherapy. Mean duration of follow-up was 6.4 years. Eight patients (61.5%) were diagnosed with a second lymphoma, either before (n = 2), concurrently with (n = 1) or after (n = 5) the diagnosis of LyP. Mycosis fungoides (MF) was the commonest lymphoma (78%, n = 7), followed by primary cutaneous anaplastic large-cell lymphoma (12%, n = 2). There was one death (mortality rate 7.7%), which occurred in a patient who had developed stage IIA MF after LyP, which subsequently progressed to systemic T-cell lymphoma. CONCLUSIONS: LyP is a chronic, relapsing disease with considerable morbidity, but an overall good prognosis. A strikingly large proportion of our Asian patients (61.5%) had a second lymphoma, compared with previous studies. This emphasizes the importance of regular lifetime surveillance for associated lymphomas in all patients with LyP.

Computer-assisted quantitative upper airway analysis following modified uvulopalatal flap and lateral pharyngoplasty for obstructive sleep apnoea: a prospective case-controlled study.

OBJECTIVES: The study aims to perform static and dynamic quantitative assessment of the anatomical changes of the upper airway before and after modified uvulopalatal flap and lateral pharyngoplasty and comparison of the improvement in airway dimensions, collapsibility and extent of normalisation to that of control patients. DESIGN: Prospective case-controlled study. SETTING: Computer-assisted quantitative measurement is used to compare upper airway parameters before and after modified uvulopalatal flap and lateral pharyngoplasty in patients with obstructive sleep apnoea (OSA). PARTICIPANTS: Patients with obstructive sleep apnoea diagnosed on sleep study and failed positive airway pressure therapy. MAIN OUTCOME MEASURES: Sleep study results, upper airway parameters and symptom score following surgery and its comparison to normal patients to assess the degree and extent of normalisation. RESULTS: Thirty-five study and 32 control subjects were recruited and completed the study. All the retropalatal airway dimensions like area, transverse diameter, longitudinal diameter and collapsibility showed statistically significant improvement following surgery. The success rate of this surgery is 43% (15 of 35) overall, 58% (14 of 24) for patients with isolated palatal obstruction and only 9% (1 of 11) for patients with multi-level obstruction. Comparing obstructive sleep apnoea to the control subjects, there are obvious and logical differences in their biostatistics, sleep study parameters and airway dimensions. The postoperative obstructive sleep apnoea retropalatal longitudinal diameter has a higher tendency of normalising to be comparable to those of control patients. CONCLUSIONS: Modified uvulopalatal flap and lateral pharyngoplasty is an effective surgical technique for the treatment of obstructive sleep apnoea. The surgery increases the resting retropalatal dimensions and reduces the retropalatal collapsibility.

Molecular typing of Salmonella enterica serovar Sofia in Australia by pulsed-field gel electrophoresis and repetitive element PCR typing.

AIMS: In this study, we used two molecular fingerprinting methods to investigate the genetic and clonal relationship shared by Australian Salmonella Sofia isolates. METHODS AND RESULTS: A total of 84 Australian Salm. Sofia isolates from various states in Australia were typed using pulsed-field gel electrophoresis (PFGE) (XbaI and SpeI) and repetitive element PCR (REP1R-I primer). The previous problem of DNA degradation of Salm. Sofia strains was solved by modifying the lysis solution used to treat the bacterial plugs, allowing Salm. Sofia to be subtyped using PFGE. Molecular typing of isolates resulted in the generation of eight XbaI, six SpeI and five REP1 pattern profiles. Individual typing methods showed low discrimination index values (<0·5), indicating the poor discriminatory ability of the methods. However, the combination of the typing methods was able to improve the discrimination of isolates, further dividing them into 16 subtypes and raising the index value to 0·721. CONCLUSIONS: The combination of typing methods was shown to be the best approach to fingerprint Salm. Sofia. The Australian Salm. Sofia isolates only showed limited genetic diversity and probably share a clonal relationship. A majority of the Salm. Sofia isolates were not geographically restricted with the predominant pattern subtype observed amongst the isolates from various states. SIGNIFICANCE AND IMPACT OF THE STUDY: We have successfully devised a PFGE protocol that counteracts DNase activity of Salm. Sofia, enabling typing of this serovar.

Early and limited use of tacrolimus to avoid rejection in an alemtuzumab and sirolimus regimen for kidney transplantation: clinical results and immune monitoring.

Alemtuzumab induction with 60 days of tacrolimus treatment and continuous sirolimus treatment prevented acute rejection in nine of 10 consecutive renal allograft recipients. All patients are alive with a functioning kidney graft at 27-39 months of follow-up. Extensive immune monitoring was performed in all patients. Alloantibody detection, cytokine kinetics assay (CKA), and trans vivo delayed-type hypersensitivity (DTH) assay were performed every 6 months showing correlation with clinical evolution. Despite alloantibody presence in five patients, eight patients remain without the need for specific treatment and only sirolimus monotherapy in decreasing dosage. Four patients take only 1 mg sirolimus daily with levels of 3-4 ng/mL. One patient showed clinical signs of rejection at month 9 post-transplant, with slow increase in serum creatinine and histological signs of mixed cellular (endarteritis) and humoral rejection (C4d positivity in peritubular capillaries and donor-specific antibody (DSA)). In summary, the addition of tacrolimus therapy for 2 months to a steroid-free, alemtuzumab induction and sirolimus maintenance protocol limited the previously shown acute rejection development. Nevertheless, alloantibody was present in serum and/or C4d present on 1-year biopsy in half the patients. The combination of CKA and DSA monitoring or the performance of transvivo DTH correlated with immune status of the patients.

Human allograft acceptance is associated with immune regulation.

The ultimate goal of transplantation is drug-free allograft acceptance, which is rarely encountered in transplant recipients. Using a novel human-to-mouse "trans vivo" delayed-type hypersensitivity assay, we assessed donor-reactive cell-mediated immune responses in kidney and liver transplant patients, four of whom discontinued all immunosuppression. One of these subjects (J.B.) rejected his graft after 7 years of stable function, while the others (D.S., R.D., M.L.) continue to have excellent graft function 5, 28, and 4 years after the cessation of immunosuppression. PBMCs from J.B. exhibited strong responses to both donor and recall antigens whereas PBMCs from patients D.S., R.D., and M.L. responded strongly to recall, but not donor, antigens. Furthermore, when donor and recall antigens were colocalized, the recall response in these three patients was inhibited. This donor antigen-linked nonresponsiveness was observed in four other patients who are still maintained on immunosuppression. The weakness of donor-reactive DTH responses in these patients is due to donor alloantigen-triggered regulation that relies on either TGF-beta or IL-10. In D.S., regulation is triggered by a single donor HLA Class I antigen, either in membrane-bound or soluble form. This demonstrates that allograft acceptance in humans is associated with an immune regulation pattern, which may be useful in the diagnosis and/or monitoring of transplant patients for allograft acceptance.

The incidence of deep venous thrombosis following arthroscopic knee surgery.

A prospective cohort study was undertaken at two centers to look for the incidence of deep vein thrombosis (DVT) following knee arthroscopic surgery. Eighty-four patients who had 90 arthroscopic procedures were reviewed. The relevant risk factors: past or family history of DVT, smoking, oral contraceptives, body weight, haemoglobin level, platelet count, tourniquet time and type of anaesthesia were documented. All patients were subjected to preoperative and post-operative duplex ultrasound. Only one patient (1.2 %) was noted to have DVT involving the peroneal vein. We concluded that the incidence of DVT after knee arthroscopy was very low in this study population.

Role of the X-linked gene GPR174 in autoimmune Addison's disease.

CONTEXT: Autoimmune endocrinopathies demonstrate a profound gender bias, but the reasons for this remain obscure. The 1000 genes on the X chromosome are likely to be implicated in this inherent susceptibility; various theories, including skewed X chromosome inactivation and fetal microchimerism, have been proposed. GPR174 is an Xq21 putative purinergic receptor that is widely expressed in lymphoid tissues. A single-nucleotide polymorphism, rs3827440, encoding Ser162Pro, has recently been associated with Graves' disease in Chinese and Polish populations, suggesting a role of this X chromosome gene in autoimmune disease. OBJECTIVE: We investigated the role of rs3827440 in a UK cohort of patients with autoimmune Addison's disease (AAD). Samples from 286 AAD cases and 288 healthy controls were genotyped using TaqMan single-nucleotide polymorphism genotyping assays (C_25954273_10) on the Applied Biosystems 7900HT Fast real-time PCR system. DESIGN: Using a dominant (present/absent) model, the serine-encoding T allele of rs3827440 was present in 189 of 286 AAD patients (66%) compared with 132 of 288 unaffected controls (46%) [P = .010, odds ratio 1.80 (5%-95% confidence interval 1.22-2.67)]. An allele dosage model found a significant excess of the T allele in AAD patients compared with controls [P = .03, odds ratio 1.34 (5%-95% confidence interval 1.07-1.67)]. CONCLUSION: We have demonstrated a significant association of this X chromosome-encoded immunoreceptor with AAD for the first time. This X-linked gene could have a more generalized role in autoimmunity pathogenesis: G protein-coupled receptors are promising drugable targets, and further work to elucidate the functional role of GPR174 is now warranted.

Simple and sensitive test for the determination of phenolic compounds in urine and its application to melanoma.

A simple procedure is outlined for the determination of phenolic compounds in urine. The method involves the diazotization of p-nitroaniline and subsequent coupling of the diazonium chloride with phenolic compounds. The diazo compound formed is determined by measuring the absorbance at 400 nm. The extinction coefficients for several compounds have been determined. Application of this method to determine the levels of phenolic compounds in urine in patients with melanoma showed that the assay may be of diagnostic value. The amounts of phenols in 24-hr urine samples from patients with melanoma, patients with various other diseases, and healthy individuals were determined. A statistical analysis of these results showed that the results for patients with melanoma were higher than those for healthy individuals and patients with other diseases at 1 percent probability level. A comparison of this test with three other methods (Thormählen test, ferric chloride test, and ferrocitrate test) for determination of melanogens showed that the diazo test is the most sensitive one for detecting melanoma.

Human liver allograft acceptance and the "tolerance assay": in vitro anti-donor T cell assays show hyporeactivity to donor cells, but unlike DTH, fail to detect linked suppression.

Human allograft acceptance is associated with immune regulation, characterized by donor-antigen-linked suppression of delayed-type hypersensitivity (DTH). We wished to determine if "classical" in vitro assays of alloreactivity could also detect linked suppression and thus be useful in the clinical diagnosis of active immune regulation. We analyzed peripheral blood mononuclear cells from a group of eight liver transplant recipients, one of whom had stopped all immunosuppression 4.5 years ago yet continues to have good graft function (graft acceptor). The regulator phenotype was defined as the ability to suppress a DTH response to a recall antigen in the presence of donor antigen. Using the trans vivo DTH test, we identified four regulators, and four nonregulators. When we tested two of the regulators for in vitro mixed lymphocyte culture (MLC) and cytotoxic T lymphocyte (CTL) responses to B-lymphoblastoid cell lines (B-LCL), we found both patients to be specifically hyporesponsive to donor compared with third-party B-LCL stimulators. However, in contrast to the linked suppression of DTH seen when a given B-LCL expressed donor-type HLA-B antigens, there was no evidence of linked suppression in vitro, either in CTL, proliferative, or interferon-gamma cytokine release assays. The primary CTL hyporesponsiveness to donor B-LCL could not be reversed by neutralizing antibodies to transforming growth factor beta or interleukin-10, which could restore a strong DTH response to donor B-LCL. We conclude that DTH analysis can readily detect donor antigen-linked suppression in liver transplant recipients. CTL and MLC tests failed to do so. These findings may be relevant to the development of a tolerance assay suitable for use in clinical trials.

Monitoring the patient off immunosuppression. Conceptual framework for a proposed tolerance assay study in liver transplant recipients.

The mission of the recently established Immune Tolerance Network includes the development of protocols for the induction of transplant tolerance in organ allograft recipients and the development of assays that correlate with and may be predictive of the tolerant state. The state of clinical organ transplant tolerance seems to already exist in a small minority of conventionally immunosuppressed liver and, more rarely, kidney transplant patients. Immunosuppressive drug therapy has been withdrawn from these patients for a variety of reasons, including protocolized weaning for a uniquely large group of liver patients at the University of Pittsburgh. In this study, we propose to evaluate the validity of a variety of in vitro immunologic and molecular biologic tests that may correlate with, and be predictive of, the state of organ transplant tolerance in stable liver patients off immunosuppression. Only peripheral blood will be available for the execution of these tests. Both adult and pediatric liver graft recipients will be studied, in comparison to appropriate controls. We shall examine circulating dendritic cell (DC) subsets [precursor (p) DC1 and p DC2] including cells of donor origin, and assess both the frequency and function of donor-reactive T cells by ELISPOT and by trans-vivo delayed-type hypersensitivity analysis in a surrogate murine model. Cytokine gene polymorphism and alloantibody titers will also be investigated. It is anticipated that the results obtained may provide physicians with a tolerance assay "profile" that may determine those patients from whom immunosuppressive therapy may be safely withdrawn.