Anatomic and functional outcome after 23-gauge vitrectomy, peeling, and intravitreal triamcinolone for idiopathic macular epiretinal membrane.

PURPOSE: To report both the functional and anatomic outcome and safety profile of 23-gauge pars plana vitrectomy combined with membrane peeling and intravitreal injection of triamcinolone acetonide in eyes with idiopathic macular epiretinal membranes. METHODS: Retrospective study of 39 consecutive patients who underwent 23-gauge transconjunctival sutureless vitrectomy, membrane peeling, and intravitreal triamcinolone acetonide injection for an idiopathic macular epiretinal membrane between February 2007 and February 2008. Minimum follow-up was 6 months. RESULTS: Thirty-nine eyes of 39 patients were included in the study. The mean follow-up was 7 +/- 2.2 months (range, 6-15 months). Twenty-two eyes (56%) were pseudophakic and 17 (44%) were phakic at the time of surgery. Five of the phakic eyes (29.4%) had worsening of cataracts during the follow-up period. Mean preoperative intraocular pressure was 14 +/- 3.5 mmHg. At the final follow-up, mean intraocular pressure was 14.5 +/- 2.7 mmHg, which did not differ significantly from the intraocular pressure at baseline (P = 0.14, two-tailed t-test). Five (13%) patients needed topical antiglaucoma treatment. Mean preoperative best-corrected visual acuity (BCVA) was 0.28 decimal equivalent (20/71 Snellen equivalent; logarithm of the minimum angle of resolution 0.54 +/- 0.2, range: 1.0-0.2) and improved significantly (P < 0.0001, two-tailed t-test) to a mean of 0.6 decimal equivalent (20/33 Snellen equivalent; logarithm of the minimum angle of resolution 0.22 +/- 0.16, range: 0.6-0) at the final follow-up. The BCVA improved by a mean of 3.2 +/- 2.1 lines (range: 0-8). Twenty-nine patients (74%) demonstrated a gain of > or =3 lines. Mean central macular thickness was 456 +/- 77 microm (mean +/- SD) at baseline, which was significantly reduced at the final follow-up to 327 +/- 79 microm (mean +/- SD; P < 0.0001, two-tailed t-test). Average central macular thickness reduction was 131 +/- 77 microm (mean +/- SD; range: 36-380 microm). A subgroup analysis of 15 selected cases, which had central macular thickness and BCVA measurements after the first postoperative week, demonstrated that 84% of the total final reduction in central macular thickness and 84% of the total final improvement in BCVA occurred already during the first postoperative week. CONCLUSION: Twenty-three-gauge sutureless transconjunctival vitrectomy is a safe and effective technique for the treatment of idiopathic macular epiretinal membranes. The concomitant administration of intravitreal triamcinolone acetonide after pars plana vitrectomy may speed up and improve the anatomic and functional outcome.

Switch-peptides: From conformational studies to Alzheimer's disease

Studies on designed peptides that exhibit high tendencies for medium-induced conformational transitions have recently attracted much attention because structural changes are considered as molecular key processes in degenerative diseases. The experimental access to these events has been limited so far mainly due to the intrinsic tendency of the involved polypeptides for self-association and aggregation, e.g. amyloid P plaque formation, thought to be at the origin of Alzheimer's disease. We have developed a new concept termed 'switch-peptides' which allows the controlled onset of polypeptide folding and misfolding in vitro and in vivo, starting from a soluble, non-toxic precursor molecule. As a major feature, the folding process is initiated by enzyme-triggered N,O-acyl migrations restoring the native peptide backbone in situ. As the folding is set off in the moment of creating the bioactive molecule ('in statu nascendi', ISN), our concept allows for the first time the investigation of the early steps of protein misfolding as relevant in degenerative diseases, opening new perspectives for the rational design of therapeutically relevant compounds.

Light-induced phosphorylation of a membrane protein plays an early role in signal transduction for phototropism in Arabidopsis thaliana.

Blue light is known to cause rapid phosphorylation of a membrane protein in etiolated seedlings of several plant species, a protein that, at least in etiolated pea seedlings and maize coleoptiles, has been shown to be associated with the plasma membrane. The light-driven phosphorylation has been proposed on the basis of correlative evidence to be an early step in the signal transduction chain for phototropism. In the Arabidopsis thaliana mutant JK224, the sensitivity to blue light for induction of first positive phototropism is known to be 20- to 30-fold lower than in wild type, whereas second positive curvature appears to be normal. While light-induced phosphorylation can be demonstrated in crude membrane preparations from shoots of the mutant, the level of phosphorylation is dramatically lower than in wild type, as is the sensitivity to blue light. Another A. thaliana mutant, JK218, that completely lacks any phototropic responses to up to 2 h of irradiation, shows a normal level of light-induced phosphorylation at saturation. Since its gravitropic sensitivity is normal, it is presumably blocked in some step between photoreception and the confluence of the signal transduction pathways for phototropism and gravitropism. We conclude from mutant JK224 that light-induced phosphorylation plays an early role in the signal transduction chain for phototropism in higher plants.

No Evidence That Soluble TACI Induces Signalling via Membrane-Expressed BAFF and APRIL in Myeloid Cells.

Myeloid cells express the TNF family ligands BAFF/BLyS and APRIL, which exert their effects on B cells at different stages of differentiation via the receptors BAFFR, TACI (Transmembrane Activator and CAML-Interactor) and/or BCMA (B Cell Maturation Antigen). BAFF and APRIL are proteins expressed at the cell membrane, with both extracellular and intracellular domains. Therefore, receptor/ligand engagement may also result in signals in ligand-expressing cells via so-called "reverse signalling". In order to understand how TACI-Fc (atacicept) technically may mediate immune stimulation instead of suppression, we investigated its potential to activate reverse signalling through BAFF and APRIL. BAFFR-Fc and TACI-Fc, but not Fn14-Fc, reproducibly stimulated the ERK and other signalling pathways in bone marrow-derived mouse macrophages. However, these effects were independent of BAFF or APRIL since the same activation profile was observed with BAFF- or APRIL-deficient cells. Instead, cell activation correlated with the presence of high molecular mass forms of BAFFR-Fc and TACI-Fc and was strongly impaired in macrophages deficient for Fc receptor gamma chain. Moreover, a TACI-Fc defective for Fc receptor binding elicited no detectable signal. Although these results do not formally rule out the existence of BAFF or APRIL reverse signalling (via pathways not tested in this study), they provide no evidence in support of reverse signalling and point to the importance of using appropriate specificity controls when working with Fc receptor-expressing myeloid cells.

The gamma subunit is a specific component of the Na,K-ATPase and modulates its transport function.

The role of small, hydrophobic peptides that are associated with ion pumps or channels is still poorly understood. By using the Xenopus oocyte as an expression system, we have characterized the structural and functional properties of the gamma peptide which co-purifies with Na,K-ATPase. Immuno-radiolabeling of epitope-tagged gamma subunits in intact oocytes and protease protection assays show that the gamma peptide is a type I membrane protein lacking a signal sequence and exposing the N-terminus to the extracytoplasmic side. Co-expression of the rat or Xenopus gamma subunit with various proteins in the oocyte reveals that it specifically associates only with isozymes of Na,K-ATPase. The gamma peptide does not influence the formation and cell surface expression of functional Na,K-ATPase alpha-beta complexes. On the other hand, the gamma peptide itself needs association with Na,K-ATPase in order to be stably expressed in the oocyte and to be transported efficiently to the plasma membrane. Gamma subunits do not associate with individual alpha or beta subunits but only interact with assembled, transport-competent alpha-beta complexes. Finally, electrophysiological measurements indicate that the gamma peptide modulates the K+ activation of Na,K pumps. These data document for the first time the membrane topology, the specificity of association and a potential functional role for the gamma subunit of Na,K-ATPase.

Clinical manifestations of mucous membrane pemphigoid in a tertiary center.

Mucous membrane pemphigoid (MMP) is a progressive inflammatory disease of autoimmune etiology. We performed a retrospective analysis of clinical signs and treatment on 16 patients. Conjunctival biopsies were performed in all patients and showed typical immuno-deposits at the basement membrane zone. The mean age at presentation was 69 years, 60 % were female.12 patients demonstrated ocular involvement (11 bilaterally). At the time of referral to our hospital, 92 % had reached an advanced stage III or IV. All patients presented conjunctival fibrosis with resultant fornix foreshortening. Trichiasis and symblepharon were found in 11 patients. Keratitis was found in 11 patients resulting in ulceration in 5 cases. Complications required surgical interventions included: entropion surgery (n = 2), tarsorrhaphy (n = 1), amniotic membrane transplantation (n = 2), keratoplasty (n = 1). Systemic immunomodulatory therapy is the treatment of choice. Dapsone (n = 8), steroids (n = 8), azathioprine (n = 5), cyclophosphamide (n = 2), mycophenolate mofetil (n = 4) and methotrexate (n = 1) were used concomitantly or consecutively. Early diagnosis can prevent ocular complications. Immunomodulatory therapy has provided an avenue for preserving vision. The management of MMP requires a multidisciplinary approach.

Productive HIV-1 infection of primary CD4+ T cells induces mitochondrial membrane permeabilization leading to a caspase-independent cell death.

We have explored in vitro the mechanism by which human immunodeficiency virus, type 1 (HIV-1) induces cell death of primary CD4+ T cells in conditions of productive infection. Although HIV-1 infection primed phytohemagglutinin-activated CD4+ T cells for death induced by anti-CD95 antibody, T cell death was not prevented by a CD95-Fc decoy receptor, nor by decoy receptors of other members of the TNFR family (TNFR1/R2, TRAILR1/R2/OPG, TRAMP) or by various blocking antibodies, suggesting that triggering of death receptors by their cognate ligands is not involved in HIV-induced CD4 T cell death. HIV-1 induced CD4 T cell shrinkage, cell surface exposure of phosphatidylserine, loss of mitochondrial membrane potential (Deltapsim), and mitochondrial release of cytochrome c and apoptosis-inducing factor. A typical apoptotic phenotype (nuclear chromatin condensation and fragmentation) only occurred in around half of the dying cells. Treatment with benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, a broad spectrum caspase inhibitor, prevented nuclear chromatin condensation and fragmentation in HIV-infected CD4+ T cells and in a cell-free system (in which nuclei were incubated with cytoplasmic extracts from the HIV-infected CD4+ T cells). Nevertheless, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone did not prevent mitochondrial membrane potential loss and cell death, suggesting that caspases are dispensable for HIV-mediated cell death. Our findings suggest a major role of the mitochondria in the process of CD4 T cell death induced by HIV, in which targeting of Bax to the mitochondria may be involved.

Antitumour effects of single or combined monoclonal antibodies directed against membrane antigens expressed by human B cells leukaemia.

Background: The increasing availability of different monoclonal antibodies (mAbs) opens the way to more specific biologic therapy of cancer patients. However, despite the significant success of therapy in breast and ovarian carcinomas with anti-HER2 mAbs as well as in non-Hodkin B cell lymphomas with anti-CD20 mAbs, certain B cell malignancies such as B chronic lymphocytic leukaemia (B-CLL) respond poorly to anti-CD20 mAb, due to the low surface expression of this molecule. Thus, new mAbs adapted to each types of tumour will help to develop personalised mAb treatment. To this aim, we analyse the biological and therapeutic properties of three mAbs directed against the CD5, CD71 or HLA-DR molecules highly expressed on B-CLL cells. Results: The three mAbs, after purification and radiolabelling demonstrated high and specific binding capacity to various human leukaemia target cells. Further in vitro analysis showed that mAb anti-CD5 induced neither growth inhibition nor apoptosis, mAb anti-CD71 induced proliferation inhibition with no early sign of cell death and mAb anti-HLA-DR induced specific cell aggregation, but without evidence of apoptosis. All three mAbs induced various degrees of ADCC by NK cells, as well as phagocytosis by macrophages. Only the anti-HLA-DR mAb induced complement mediated lysis. Coincubation of different pairs of mAbs did not significantly modify the in vitro results. In contrast with these discrete and heterogeneous in vitro effects, in vivo the three mAbs demonstrated marked anti-tumour efficacy and prolongation of mice survival in two models of SCID mice, grafted either intraperitoneally or intravenously with the CD5 transfected JOK1-5.3 cells. This cell line was derived from a human hairy cell leukaemia, a type of malignancy known to have very similar biological properties as the B-CLL, whose cells constitutively express CD5. Interestingly, the combined injection of anti-CD5 with anti-HLA-DR or with anti-CD71 led to longer mouse survival, as compared to single mAb injection, up to complete inhibition of tumour growth in 100% mice treated with both anti-HLA-DR and anti-CD5. Conclusions: Altogether these data suggest that the combined use of two mAbs, such as anti-HLA-DR and anti-CD5, may significantly enhance their therapeutic potential.

Determinants for membrane association of the hepatitis C virus NS2 protease domain.

Hepatitis C virus (HCV) nonstructural protein 2 (NS2) is required for HCV polyprotein processing and particle assembly. It comprises an N-terminal membrane domain and a C-terminal, cytosolically oriented protease domain. Here, we demonstrate that the NS2 protease domain itself associates with cellular membranes. A single charged residue in the second α-helix of the NS2 protease domain is required for proper membrane association, NS2 protein stability, and efficient HCV polyprotein processing.

Descemet membrane detachment after nonpenetrating filtering surgery.

PURPOSE: To make surgeons performing nonpenetrating filtering surgery aware of an unusual complication namely Descemet membrane detachment. METHODS: We retrospectively reviewed nine eyes of nine patients seen in our hospital with Descemet membrane detachment occurring after nonpenetrating filtering surgery from January 1994 to December 2000. RESULTS: Both planar and nonplanar detachments were reported. Neither scrolls nor tears in the Descemet membrane were observed in any patient. After viscocanalostomy (four patients), the detachment was generally noticed shortly after the procedure and the cornea maintained its clarity. After deep sclerectomy with a collagen implant (five patients), it developed weeks to months postoperatively with adjacent corneal edema. Four patients had descemetopexy. None required more than one procedure. However, at the last visit, two detachments persisted although they had diminished in size: one after viscocanalostomy and conservative treatment and one after descemetopexy after deep sclerectomy with a collagen implant. To date otherwise, no signs of significant corneal damage could be observed clinically nor by specular microscopy and pachymetry. CONCLUSIONS: The diagnosis of Descemet membrane detachment can be easily overlooked or misdiagnosed. The clinical presentation, clinical course, and pathogenesis depend on the type of nonpenetrating filtering surgery performed. Ophthalmologists should be aware of this unusual complication, which is likely to be more common after nonpenetrating filtering surgery than after trabeculectomy. A period of observation before attempting descemetopexy is recommended.

Structural determinants for membrane association and dynamic organization of the hepatitis C virus NS3-4A complex.

Hepatitis C virus (HCV) NS3-4A is a membrane-associated multifunctional protein harboring serine protease and RNA helicase activities. It is an essential component of the HCV replication complex and a prime target for antiviral intervention. Here, we show that membrane association and structural organization of HCV NS3-4A are ensured in a cooperative manner by two membrane-binding determinants. We demonstrate that the N-terminal 21 amino acids of NS4A form a transmembrane alpha-helix that may be involved in intramembrane protein-protein interactions important for the assembly of a functional replication complex. In addition, we demonstrate that amphipathic helix alpha(0), formed by NS3 residues 12-23, serves as a second essential determinant for membrane association of NS3-4A, allowing proper positioning of the serine protease active site on the membrane. These results allowed us to propose a dynamic model for the membrane association, processing, and structural organization of NS3-4A on the membrane. This model has implications for the functional architecture of the HCV replication complex, proteolytic targeting of host factors, and drug design.

H-2Kd-restricted antigenic peptides share a simple binding motif.

We have defined structural features that are apparently important for the binding of four different, unrelated antigenic epitopes to the same major histocompatibility complex (MHC) class I molecule, H-2Kd. The four epitopes are recognized in the form of synthetic peptides by cytotoxic T lymphocytes of the appropriate specificity. By analysis of the relative potency of truncated peptides, we demonstrated that for each of the four epitopes, optimal antigenic activity was present in a peptide of 9 or 10 amino acid residues. A comparison of the relative competitor activity of the different-length peptides in a functional competition assay, as well as in a direct binding assay based on photoaffinity labeling of the Kd molecule, indicated that the enhanced potency of the peptides upon reduction in length was most likely due to a higher affinity of the shorter peptides for the Kd molecule. A remarkably simple motif that appears to be important for the specific binding of Kd-restricted peptides was identified by the analysis of peptides containing amino acid substitutions or deletions. The motif consists of two elements, a Tyr in the second position relative to the NH2 terminus and a hydrophobic residue with a large aliphatic side chain (Leu, Ile, or Val) at the COOH-terminal end of the optimal 9- or 10-mer peptides. We demonstrated that a simple peptide analogue (AYP6L) that incorporates the motif can effectively and specifically interact with the Kd molecule. Moreover, all of the additional Kd-restricted epitopes defined thus far in the literature contain the motif, and it may thus be useful for the prediction of new epitopes recognized by T cells in the context of this MHC class I molecule.

The efficiency of antigen recognition by CD8+ CTL clones is determined by the frequency of serial TCR engagement.

Using H-2Kd-restricted CTL clones, which are specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS(252-260) (SYIPSAEKI) and permit assessment of TCR-ligand interactions by TCR photoaffinity labeling, we have previously identified several peptide derivative variants for which TCR-ligand binding and the efficiency of Ag recognition deviated by fivefold or more. Here we report that the functional CTL response (cytotoxicity and IFN-gamma production) correlated with the rate of TCR-ligand complex dissociation, but not the avidity of TCR-ligand binding. While peptide antagonists exhibited very rapid TCR-ligand complex dissociation, slightly slower dissociation was observed for strong agonists. Conversely and surprisingly, weak agonists typically displayed slower dissociation than the wild-type agonists. Acceleration of TCR-ligand complex dissociation by blocking CD8 participation in TCR-ligand binding increased the efficiency of Ag recognition in cases where dissociation was slow. In addition, permanent TCR engagement by TCR-ligand photocross-linking completely abolished sustained intracellular calcium mobilization, which is required for T cell activation. These results indicate that the functional CTL response depends on the frequency of serial TCR engagement, which, in turn, is determined by the rate of TCR-ligand complex dissociation.

Sequential analysis of trans-SNARE formation in intracellular membrane fusion.

SNARE complexes are required for membrane fusion in the endomembrane system. They contain coiled-coil bundles of four helices, three (Q(a), Q(b), and Q(c)) from target (t)-SNAREs and one (R) from the vesicular (v)-SNARE. NSF/Sec18 disrupts these cis-SNARE complexes, allowing reassembly of their subunits into trans-SNARE complexes and subsequent fusion. Studying these reactions in native yeast vacuoles, we found that NSF/Sec18 activates the vacuolar cis-SNARE complex by selectively displacing the vacuolar Q(a) SNARE, leaving behind a Q(bc)R subcomplex. This subcomplex serves as an acceptor for a Q(a) SNARE from the opposite membrane, leading to Q(a)-Q(bc)R trans-complexes. Activity tests of vacuoles with diagnostic distributions of inactivating mutations over the two fusion partners confirm that this distribution accounts for a major share of the fusion activity. The persistence of the Q(bc)R cis-complex and the formation of the Q(a)-Q(bc)R trans-complex are both sensitive to the Rab-GTPase inhibitor, GDI, and to mutations in the vacuolar tether complex, HOPS (HOmotypic fusion and vacuolar Protein Sorting complex). This suggests that the vacuolar Rab-GTPase, Ypt7, and HOPS restrict cis-SNARE disassembly and thereby bias trans-SNARE assembly into a preferred topology.