Hepatic Infiltrates in Operational Tolerant Patients After Liver Transplantation Show Enrichment of Regulatory T Cells Before Proinflammatory Genes Are Downregulated.

Immunosuppression can be discontinued from selected and stable patients after liver transplantation resulting in spontaneous operational tolerance (SOT), although the underlying mechanisms remain elusive. Thus, we analyzed serial liver biopsy specimens from adult liver recipients enrolled in a prospective multicenter immunosuppression withdrawal trial that used immunophenotyping and transcriptional profiling. Liver specimens were collected before the initiation of weaning, at the time of rejection, or at 1 and 3 years after complete drug discontinuation. Unexpectedly, the tolerated grafts developed portal tract expansion with increased T cell infiltration after immunosuppression withdrawal. This was associated with transient and preferential accumulation of CD4(+) FOXP3(+) cells and a trend toward upregulation of immune activation and regulatory genes, without signs of rejection. At the same time, no markers of endothelial damage or activation were noted. Portal infiltrates persisted at 3 years but were characterized by decreased expression of genes associated with chronic immunological damage. Further, SOT was not associated with a progressive liver fibrosis up to 5 years. These data suggest that SOT involves several mechanisms: a long-lasting local immune cell persistence with a transient regulatory T cells accumulation followed by a downregulation of immune-activated genes over years. These results have important implications for designs and follow-up of weaning trials.

Clinical value and safety of liver biopsies in patients transplanted for hepatitis C virus-related end-stage liver disease.

BACKGROUND: Hepatitis C is the leading indication for liver transplantation. Differentiation between recurrent graft hepatitis C (RGH-C) and graft rejection (GR) is challenging. Liver biopsy is standard to diagnose both conditions; however, little information is available regarding this procedure in hepatitis C virus (HCV)-infected liver transplant recipients. METHODS: Liver biopsies (n = 211) from all consecutive patients (n = 138) transplanted for hepatitis C at Hannover Medical School between January 2000 and October 2011 were screened, and a final cohort of 96 patients with 196 biopsies was included. Indications, histopathological findings, and biopsy-related complications were documented. Modifications in the treatment based on the biopsy result and the biochemical outcome were analyzed. RESULTS: Most biopsies (196/211, 93%) were representative. Five patients (2.5%) developed non-fatal biopsy-related complications. Biopsy results were GR (35%), RGH-C (31%), and other diagnoses (34%). GR was independently associated with lower albumin (P = 0.025) and higher bilirubin levels (P = 0.011). Treatment was modified based on the biopsy result in 25% of cases. Alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), and bilirubin levels improved in 41%, 25%, and 31% of cases 4 weeks post biopsy respectively. ALT improvements were more significant in patients with GR than in those with RGH-C. CONCLUSION: Liver biopsy in HCV-infected liver transplant recipients is safe and representative in >90% of cases. GR is independently associated with lower albumin and higher bilirubin levels.

Naive tumour-specific CD4+ T cells were efficiently primed in acute lymphoblastic leukaemia.

The recognition and neutralization of tumour cells is one of the big challenges in immunity. The immune system has to recognize syngeneic tumour cells and has to be primed and respond in an adequate manner. Priming of a leukaemia-specific immune response is a crucial step in tumour immunology that can mislead to tumour tolerance either by T cell ignorance, deletion or Treg induction. To resemble the situation of acute lymphoblastic leukaemia (ALL) in patients, we used the murine BALB/c model with syngeneic BM185 tumour cells. We established a tumour cell line that expresses the neo-antigen ovalbumin (BM185-OVA/GFP) to allow the application of T cell receptor transgenic, antigen-specific CD4(+) T cells. Here, we demonstrate that effective anti-ALL immunity can be established by in vivo priming of CD4(+) T cells that is sufficient to differentiate into effector cells. Yet they failed to control tumour alone, but initiated a Th1 response. An efficient tumour clearance was dependent on both antigen-specific CD4(+) T cells and CD8(+) effector T cells from the endogenous repertoire. The tolerogeneic milieu was characterized by increased Tregs numbers and elevated IL-10 level. Tregs hamper effective antitumour immune response, but their depletion did not result in reduced tumour growth. In contrast, neutralization of IL-10 improved median mouse survival. Future therapies should focus on establishing a strong CD4+ T cells response, either by adjuvant or by adoptive transfer.

Treg-therapy allows mixed chimerism and transplantation tolerance without cytoreductive conditioning.

Establishment of mixed chimerism through transplantation of allogeneic donor bone marrow (BM) into sufficiently conditioned recipients is an effective experimental approach for the induction of transplantation tolerance. Clinical translation, however, is impeded by the lack of feasible protocols devoid of cytoreductive conditioning (i.e. irradiation and cytotoxic drugs/mAbs). The therapeutic application of regulatory T cells (Tregs) prolongs allograft survival in experimental models, but appears insufficient to induce robust tolerance on its own. We thus investigated whether mixed chimerism and tolerance could be realized without the need for cytoreductive treatment by combining Treg therapy with BM transplantation (BMT). Polyclonal recipient Tregs were cotransplanted with a moderate dose of fully mismatched allogeneic donor BM into recipients conditioned solely with short-course costimulation blockade and rapamycin. This combination treatment led to long-term multilineage chimerism and donor-specific skin graft tolerance. Chimeras also developed humoral and in vitro tolerance. Both deletional and nondeletional mechanisms contributed to maintenance of tolerance. All tested populations of polyclonal Tregs (FoxP3-transduced Tregs, natural Tregs and TGF-beta induced Tregs) were effective in this setting. Thus, Treg therapy achieves mixed chimerism and tolerance without cytoreductive recipient treatment, thereby eliminating a major toxic element impeding clinical translation of this approach.

Correlation of expression of cyclooxygenase-2, vascular endothelial growth factor, and peroxisome proliferator-activated receptor delta with head and neck squamous cell carcinoma.

Cyclooxygenase (COX) is the rate-limiting enzyme in the formation of prostaglandins from arachidonic acid. COX exists in 2 isoforms, COX-1 and COX-2. These isoforms are encoded by separate genes and demonstrate cell-specific expression and regulation. Peroxisome proliferator-activated receptor delta (PPARdelta) is a nuclear transcription factor that is activated by prostacyclin. Vascular endothelial growth factor (VEGF) is a proangiogenic factor that is up-regulated in various tumors. Vascular endothelial growth factor has been shown to interact with COX-derived prostaglandins in angiogenesis. To better understand the roles of these genes in head and neck squamous cell carcinoma (HNSCCA), we examined the differential expression of the COX1, COX2, VEGF, and PPARdelta genes in these tumors. Tissue samples from patients with HNSCCA were analyzed for COX-1, COX-2, VEGF, and PPARdelta messenger RNAs (mRNAs) by in situ hybridization. COX-1 and COX-2 mRNAs were also evaluated with Northern blot hybridization. Immunohistochemistry was used to analyze for COX-2 and PPARdelta proteins. Results showed focal areas of accumulation for COX-2, VEGF, and PPARdelta but not COX-1 in human HNSCCA. Northern blot hybridization showed higher levels of COX-2 mRNA in HNSCCA than in normal tissue. This suggests a supportive role of COX-2 in development and/or progression of HNSCCA. In addition, PPARdelta may be a receptor for COX-2-produced prostaglandins in HNSCCA. There is a potential role for selective COX-2 inhibitors in the treatment of these lesions.

Liver transplantation and autoimmunity.

Autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) represent good indications for orthotopic liver transplantation (OLT). While there is effective treatment for AIH (steroids with or without azathioprine) and PBC (ursodeoxycholic acid) no such treatment is currently established for PSC. The need of transplantation can be delayed for AIH and PBC with appropriate therapies, while treatment options for PSC are still controversially discussed. Although the time point for liver transplantation can be roughly estimated for AIH by failure of immunosuppressive therapy and for PBC by prognostic models, the prediction of survival in patients with PSC is more difficult, and further complicated by the risk of developing cholangiocellular carcinoma. Long term (5-year) outcome after liver transplantation approaches 80 to 90% for autoimmune liver diseases unless cholangiocellular carcinoma complicates PSC at the time of OLT. The risk of disease recurrence has been recognised for each of these entities although its clinical relevance is controversial and not exactly determined today. As survival after liver transplantation is steadily increasing, recurrent autoimmune liver disease may become a clinical problem in the future. Recently de novo autoimmune hepatitis after liver transplantation has been reported from several transplant centres, although its importance still needs to be established.

No evidence for association between IDDMK(1,2)22, a novel isolated retrovirus, and IDDM.

In the past, endogenous retroviral sequences have been isolated from patients suffering from different kinds of autoimmune diseases. Recently, a full length retroviral genome, termed IDDMK(1,2)22, was isolated from patients with new-onset IDDM. This genome contains a major histocompatibility complex II-dependent superantigen within its envelope gene. The viral sequence was found in ten patients with new-onset IDDM, but not in age-matched control subjects (Conrad et al. [9]). We searched for the presence of this viral genome by nested reverse transcription-polymerase chain reaction (RT-PCR) in a cohort of six patients with new-onset IDDM and six control subjects of the same age. We found all samples to be positive without any differences between patients and control subjects. The same results were obtained with supernatants of activated peripheral blood mononuclear cells. We performed isopycnic ultracentrifugation in sucrose density gradients on all samples and were unable to detect particles of the new virus in any of our samples. However, positive signals were obtained from all pellet fractions. RNase, DNase treatment and nested PCRs without reverse transcription showed that the positive signals were probably derived from intracellular RNA and DNA. In summary, no correlation between a positive nested PCR signal for IDDMK(1,2)22 and diabetes was found indicating that the new sequence represents just an additional member of the human endogenous retrovirus (HERV) family with lack of an exogenous counterpart.

Muscle fiber type distribution in the normal human levator veli palatini muscle.

OBJECTIVE: This study examined the muscle fiber type distribution within the normal adult levator veli palatini muscle. METHODS: Levator veli palatini muscle tissue was harvested from the palates of 12 (seven female, five male) adult noncleft cadavers. Adjacent sections were stained for adenosine triphosphatase at pH 10.4 or 4.2. After mounting, magnifying, and photographing, Type I versus Type II fiber types were differentiated by the intensity of, or by the inhibition of, staining of matched fibers at each pH level. Type I fibers stained light at pH 10.4 and dark at pH 4.2, while Type II fibers stained light at pH 4.2 and dark at pH 10.4. MAIN OUTCOME MEASURES: The number of fibers counted for each specimen ranged from 60 to 616. The numbers of Type I and Type II stained fibers appearing in each muscle tissue sample were determined and expressed as a percentage of the total number of fibers identified. A few identified fibers could not be labelled as either Type I or Type II. RESULTS: The overall proportion of Type I fibers, averaged across all specimens, was 59.8%. Male specimens had 67.4% Type I fibers and 31.8% Type II fibers, while female specimens had 54.4% Type I fibers and 44.4% Type II fibers. CONCLUSIONS: Observed fiber type distributions were similar to those reported for other articulatory muscles, but differed slightly from previously reported distributions for normal levator veli palatini. The distributions observed in this study provide a baseline against which to relate fiber type data from the levator veli palatini of cleft palates to the functional status of the velopharyngeal mechanism.