Ribosome profiling of selenoproteins in vivo reveals consequences of pathogenic Secisbp2 missense mutations.

Recoding of UGA codons as selenocysteine (Sec) codons in selenoproteins depends on a selenocysteine insertion sequence (SECIS) in the 3'-UTR of mRNAs of eukaryotic selenoproteins. SECIS-binding protein 2 (SECISBP2) increases the efficiency of this process. Pathogenic mutations in SECISBP2 reduce selenoprotein expression and lead to phenotypes associated with the reduction of deiodinase activities and selenoprotein N expression in humans. Two functions have been ascribed to SECISBP2: binding of SECIS elements in selenoprotein mRNAs and facilitation of co-translational Sec insertion. To separately probe both functions, we established here two mouse models carrying two pathogenic missense mutations in Secisbp2 previously identified in patients. We found that the C696R substitution in the RNA-binding domain abrogates SECIS binding and does not support selenoprotein translation above the level of a complete Secisbp2 null mutation. The R543Q missense substitution located in the selenocysteine insertion domain resulted in residual activity and caused reduced selenoprotein translation, as demonstrated by ribosomal profiling to determine the impact on UGA recoding in individual selenoproteins. We found, however, that the R543Q variant is thermally unstable in vitro and completely degraded in the mouse liver in vivo, while being partially functional in the brain. The moderate impairment of selenoprotein expression in neurons led to astrogliosis and transcriptional induction of genes associated with immune responses. We conclude that differential SECISBP2 protein stability in individual cell types may dictate clinical phenotypes to a much greater extent than molecular interactions involving a mutated amino acid in SECISBP2.

P18-A148†Assymetrical injector for endothelium-in DMEK without the need of pull-through technique.

PURPOSE: One of latest surgical development of preloaded Descemet membrane endothelial keratoplasty (DMEK) is the delivery of the graft with the endothelium inwards, which allows for a very fast operation, but requires a pull-through surgical technique. Although the tri-folded, endo-in DMEK technique has significant advantages, the absence of proper surgical instruments that could allow their use without the 'pull-through' technique still restricts the wide use of such an operation. None of the available commercial DMEK injectors could be used for tri-folded DMEK (endothelium-inward) orientation, as it requires the graft to be intently secured within the injector. This report presents a retrospective eye bank validation study of an asymmetrical injector designed to orientally implant a tri-folded DMEK graft without needing a pull-through technique. METHODS: The injector is made from transparent plastic, allowing microscopic tissue validation directly before injection. The device is asymmetrical, so the orientation of the graft can be controlled and validated according to the best eye bank practice, which is critical for successful tri-folded DMEK graft clinical application. Four different designs of the internal compartment of the injectors were evaluated with DMEK tissues. Mates from two pairs were tested on each device type, totaling 16 grafts, all loaded with folded, endo-in grafts. The tissue was prepared, loaded into the injector, and ejected to imitate the tissue manipulation in DMEK operation. RESULTS: After graft loading the delivery of the endothelium-in grafts was performed by injection, without the need for a pull-through technique. One graft (6.25%) has double-scrolled (changed its folding) within the injector with a larger (1.5 mm) internal compartment. The loss of valuable cells was between 3-23% (13.98% average). No significant differences in cell loss were observed between injectors with different internal compartment sizes. Higher viability loss (17.3% +/- 5.7) was observed for the grafts with >20 days death to prep-days in comparison with grafts stored with less than two weeks (10.9% +/-2.1). CONCLUSION: The TissueGUARD injector is the only injector that currently allows oriented, tri-folded DMEK injection without the need for a pull-through technique. The average cell loss after loading and ejection was 13.98%, which is comparable/better than the current best practice with the precut-preloaded technique of naturally folded DMEK.

P16-A111†Factors affecting the density of corneal endothelial cell cultures obtained from donor corneas.

PURPOSE: The shortage of donor corneas represents a worldwide problem, and corneal endothelial cell (CEC) therapy might be a promising alternative approach. CEC can be implanted alone, which has shown limited efficacy, or with a scaffold that holds the cells together as a monolayer tissue, thus imitating Descemet membrane endothelial keratoplasty. We believe that endothelial cell density (ECD) >2000 cells/mm2, a cut-off value that eye banks use to provide quality tissues for transplantation to surgeons, should also be adopted as a parameter to define the quality of CECs as a new Advanced Therapy Medicinal Product for clinical applications in patients with endothelial dystrophies. METHODS: We isolated and cultured CECs from one or more corneas of elderly age donors with ECDs higher than or below 2000 cells/mm2. CEC cultures were carried out on coated plates and on hydrogels with a preformed basement membrane (from TissueGUARD, Germany). Immunofluorescence with antibodies against ZO-1 was performed to evaluate the ECDs of the CEC graft obtained. RESULTS: Our results suggest that primary cultures with ECDs>2000 cells/mm2 can be obtained on coated plated only when (1) CECs are isolated from one or more corneas of young donors; (2) CECs are isolated and pooled together from at least 2 elderly age donor corneas (if ECD>2000 cells/mm2) or 3 elderly age donor corneas (if ECD<2000 cells/mm2). Secondary cultures are all characterized by low ECDs. Hydrogels have been shown to be able to lead to increased ECDs after their release. CONCLUSION: Our protocol highlights the difficulties in obtaining cultures with ECDs>2000 cells/mm2. Despite being achievable with corneas from young donors, this becomes challenging when corneas from elderly donors are used, i.e., the overall majority of those collected by eye banks, particularly when corneas from elderly age donors with ECD<2000 cells/mm2 are considered as a source. One alternative would be to isolate CECs from more corneas, but this might raise the issue of antigenic stimulation, which could eventually lead to transplantation failure. Our strategy to overcome these challenges is the use of a preformed basement membrane as a scaffold for CECs. However, this challenging approach should be investigated more before proceeding to clinical application.

Interferon-lambda serum levels in hepatitis C.

BACKGROUND & AIMS: Dendritic cells (DCs) trigger adaptive immune responses and are an important source of antiviral cytokines. In hepatitis C virus (HCV) infection DC function is markedly impaired. Thus far, studies have focused on types I and II interferon (IFN). We studied IFN-lambda1 (IL-29) and IFN-lambda2/3 (IL-28A/B) serum levels in patients with different outcomes of HCV infection. METHODS: IFN-lambdas were measured by ELISAs detecting IL-29 or IL-28A and IL-28B, respectively. Results were stratified with respect to the recently discovered rs12979860 T/C polymorphism upstream of the IL-28B gene. RESULTS: In general IL-29 serum levels exceeded IL-28A/B at least twofold, with IL-29 and IL-28A/B levels being significantly higher in carriers of the rs12979860 C allele than in TT homozygous individuals (p<0.02). IL-29 levels were substantially lower in patients with chronic hepatitis C than in healthy controls (p=0.005) and patients with spontaneously resolved hepatitis (p=0.001). Patients with acute hepatitis C showed IL-29 levels intermediate between chronic hepatitis C and normal controls; and IL-29 serum levels were higher in patients who spontaneously resolved hepatitis C than in those who became chronic. In vitro HCV proteins NS3 and E2 directly inhibited IL-29 production in poly I:C-stimulated purified DCs. CONCLUSIONS: Our data suggest that HCV proteins modify IFN-lambda production in DCs. Carriers of the rs12979860 C allele associated with resolution of HCV infection exhibited increased IFN-lambda levels. Moreover, high IFN-lambda levels predisposed to spontaneous resolution of HCV infection. Thus, IFN-lambdas seem to play an important role in the control of hepatitis C.

Core-specific adaptive regulatory T-cells in different outcomes of hepatitis C.

CD4+ Treg-cells (regulatory T-cells) probably contribute to the impaired virus-specific T-cell responses in chronic HCV (hepatitis C virus) infection; however, their antigen-specificity has remained elusive. In the present study, we analysed peripheral blood CD4+ Treg-cells in patients with chronic hepatitis C and subjects with self-limited HCV infection and characterized individual Treg-cell clones obtained from both groups at the phenotypic and functional level. Foxp3 (forkhead box p3)+CD25+CD4+ Treg-cells were detected more frequently in patients with chronic hepatitis C than self-limited HCV infection, which responded to HCV core stimulation and inhibited proliferation of reporter cells. Cloning under limiting dilution conditions resulted in 14 and six hypoproliferative Foxp3+CD25+CD127-CD4+ T-cell clones from patients with chronic hepatitis C and subjects with self-limited HCV infection respectively. All clones expressed Treg-cell markers and produced IL (interleukin)-10 upon mitogen stimulation. However, exclusively Treg-cell clones from chronic hepatitis C produced IL-10 in response to HCV core and inhibited proliferation of reporter T-cells. These core-specific Treg-cell clones recognized epitopes in two regions of HCV core (amino acids 1-44 and 79-113). Co-culture inhibition assays demonstrated Treg-cells to inhibit reporter T-cells via secretion of IL-10 and IL-35 rather than cell-contact-dependent mechanisms. Finally, the HCV-specific Treg-cell clones lost their functional capacity, along with Foxp3 expression, if kept in culture without HCV core exposure. In conclusion, we identified functionally active HCV core-specific Treg-cells in patients with chronic hepatitis C, which share their epitopes with conventional T-cells and require the continued presence of antigen to maintain their functional differentiation. Thus HCV core-specific Treg-cells may contribute to the immunoregulatory balance in chronic hepatitis C.

Ribavirin exerts differential effects on functions of Cd4+ Th1, Th2, and regulatory T cell clones in hepatitis C.

Ribavirin improves outcomes of therapy in chronic hepatitis C but its mode of action has still remained unclear. Since ribavirin has been proposed to modulate the host's T cell responses, we studied its direct effects on CD4(+) T cell clones with diverse functional polarization which had been generated from patients with chronic hepatitis C. We analysed in vitro proliferation ([(3)H] thymidine uptake) and cytokine responses (IL-10, IFN-gamma) at varying concentrations of ribavirin (0-10 µg/ml) in 8, 9 and 7 CD4(+) TH1, TH2 and regulatory T cell (Treg) clones, respectively. In co-culture experiments, we further determined effects of ribarivin on inhibition of TH1 and TH2 effector cells by Treg clones. All clones had been generated from peripheral blood of patients with chronic hepatitis C in the presence of HCV core protein. Ribavirin enhanced proliferation of T effector cells and increased production of IFN-gamma in TH1 clones, but had only little effect on IL-10 secretion in TH2 clones. However, ribavirin markedly inhibited IL-10 release in Treg clones in a dose dependent fashion. These Treg clones suppressed proliferation of T effector clones by their IL-10 secretion, and in co-culture assays ribavirin reversed Treg-mediated suppression of T effector cells. Our in vitro data suggest that--in addition to its immunostimulatory effects on TH1 cells--ribavirin can inhibit functions of HCV-specific Tregs and thus reverses Treg-mediated suppression of T effector cells in chronic hepatitis C.