The anti-apoptotic factor Bcl-2 can functionally substitute for the B cell survival but not for the marginal zone B cell differentiation activity of BAFF.

The TNF family ligand B cell-activating factor (BAFF, BLyS, TALL-1) is an essential factor for B cell development. BAFF binds to three receptors, BAFF-R, transmembrane activator and CAML interactor (TACI), and B cell maturation antigen (BCMA), but only BAFF-R is required for successful survival and maturation of splenic B cells. To test whether the effect of BAFF is due to the up-regulation of anti-apoptotic factors, TACI-Ig-transgenic mice, in which BAFF function is inhibited, were crossed with transgenic mice expressing FLICE-inhibitory protein (FLIP) or Bcl-2 in the B cell compartment. FLIP expression did not rescue B cells, while enforced Bcl-2 expression restored peripheral B cells and the ability to mount T-dependent antibody responses. However, many B cells retained immaturity markers and failed to express normal amounts of CD21. Marginal zone B cells were not restored and the T-independent IgG3, but not IgM, response was impaired in the TACI-IgxBcl-2 mice. These results suggest that BAFF is required not only to inhibit apoptosis of maturating B cells, but also to promote differentiation events, in particular those leading to the generation of marginal zone B cells.

PKCθ/β and CYLD are antagonistic partners in the NFκB and NFAT transactivation pathways in primary mouse CD3+ T lymphocytes.

In T cells PKCθ mediates the activation of critical signals downstream of TCR/CD28 stimulation. We investigated the molecular mechanisms by which PKCθ regulates NFκB transactivation by examining PKCθ/β single and double knockout mice and observed a redundant involvement of PKCθ and PKCβ in this signaling pathway. Mechanistically, we define a PKCθ-CYLD protein complex and an interaction between the positive PKCθ/β and the negative CYLD signaling pathways that both converge at the level of TAK1/IKK/I-κBα/NFκB and NFAT transactivation. In Jurkat leukemic T cells, CYLD is endoproteolytically processed in the initial minutes of stimulation by the paracaspase MALT1 in a PKC-dependent fashion, which is required for robust IL-2 transcription. However, in primary T cells, CYLD processing occurs with different kinetics and an altered dependence on PKC. The formation of a direct PKCθ/CYLD complex appears to regulate the short-term spatial distribution of CYLD, subsequently affecting NFκB and NFAT repressional activity of CYLD prior to its MALT1-dependent inactivation. Taken together, our study establishes CYLD as a new and critical PKCθ interactor in T cells and reveals that antagonistic PKCθ/β-CYLD crosstalk is crucial for the adjustment of immune thresholds in primary mouse CD3(+) T cells.

Dominant Role of CD80-CD86 Over CD40 and ICOSL in the Massive Polyclonal B Cell Activation Mediated by LAT(Y136F) CD4(+) T Cells.

Coordinated interactions between T and B cells are crucial for inducing physiological B cell responses. Mutant mice in which tyrosine 136 of linker for activation of T cell (LAT) is replaced by a phenylalanine (Lat(Y136F)) exhibit a strong CD4(+) T cell proliferation in the absence of intended immunization. The resulting effector T cells produce high amounts of T(H)2 cytokines and are extremely efficient at inducing polyclonal B cell activation. As a consequence, these Lat(Y136F) mutant mice showed massive germinal center formations and hypergammaglobulinemia. Here, we analyzed the involvement of different costimulators and their ligands in such T-B interactions both in vitro and in vivo, using blocking antibodies, knockout mice, and adoptive transfer experiments. Surprisingly, we showed in vitro that although B cell activation required contact with T cells, CD40, and inducible T cell costimulator molecule-ligand (ICOSL) signaling were not necessary for this process. These observations were further confirmed in vivo, where none of these molecules were required for the unfolding of the LAT CD4(+) T cell expansion and the subsequent polyclonal B cell activation, although, the absence of CD40 led to a reduction of the follicular B cell response. These results indicate that the crucial functions played by CD40 and ICOSL in germinal center formation and isotype switching in physiological humoral responses are partly overcome in Lat(Y136F) mice. By comparison, the absence of CD80-CD86 was found to almost completely block the in vitro B cell activation mediated by Lat(Y136F) CD4(+) T cells. The role of CD80-CD86 in T-B cooperation in vivo remained elusive due to the upstream implication of these costimulatory molecules in the expansion of Lat(Y136F) CD4(+) T cells. Together, our data suggest that CD80 and CD86 costimulators play a key role in the polyclonal B cell activation mediated by Lat(Y136F) CD4(+) T cells even though additional costimulatory molecules or cytokines are likely to be required in this process.

Measurement Properties of the Smartphone-Based B-B Score in Current Shoulder Pathologies.

NlmCategory="UNASSIGNED">This study is aimed at the determination of the measurement properties of the shoulder function B-B Score measured with a smartphone. This score measures the symmetry between sides of a power-related metric for two selected movements, with 100% representing perfect symmetry. Twenty healthy participants, 20 patients with rotator cuff conditions, 23 with fractures, 22 with capsulitis, and 23 with shoulder instabilities were measured twice across a six-month interval using the B-B Score and shoulder function questionnaires. The discriminative power, responsiveness, diagnostic power, concurrent validity, minimal detectable change (MDC), minimal clinically important improvement (MCII), and patient acceptable symptom state (PASS) were evaluated. Significant differences with the control group and significant baseline-six-month differences were found for the rotator cuff condition, fracture, and capsulitis patient groups. The B-B Score was responsive and demonstrated excellent diagnostic power, except for shoulder instability. The correlations with clinical scores were generally moderate to high, but lower for instability. The MDC was 18.1%, the MCII was 25.2%, and the PASS was 77.6. No floor effect was observed. The B-B Score demonstrated excellent measurement properties in populations with rotator cuff conditions, proximal humerus fractures, and capsulitis, and can thus be used as a routine test to evaluate those patients.

The role of the Estrogen-Responsive B box protein in skin morphogenesis, wound repair and skin disease

SUMMARY: EBBP is a poorly characterized member of the RBCC/TRIM family (RING finger B-box coiled-coilltripartite motif). It is ubiquitously expressed, but particularly high levels are found in keratinocytes. There is evidence that EBBP is involved in inflammatory processes, since it can interact with pro-interleukin-1 ß (prolL-1 ß) in human macrophages and keratinocytes, and its downregulation results in reduced secretion of IL-1 ß. IL-1ß activation and secretion requires the proteolytic cleavage of prolL-1ß by caspase-1, which in turn is actìvated by a protein complex called the inflammasome. As it has been demonstrated that EBBP can bind two different proteins of the inflammasome (NALP-1 and caspase 1), we assumed that EBBP plays a role in the regulation of inflammation and that the inflammasome, which has as yet only been described in ínflammatory cells, may also exist in keratinocytes. Indeed, I could show in my thesis that the inflammasome components are expressed in human keratinócytes at the RNA and protein level and also in vivo in human epidermis. After irradiation with a physiological dose of UVB, keratinocytes activated prolL-1ß and secreted prolL-1 a, IL-1 ß, prolL-18 and inflammasome proteins, although all these proteins lack a classical signal peptide. The secretion was dependent on caspase-1 activity, but not on de novo protein synthesis. Knock-down of NALP1 and -3, caspase-1 and -5, EBBP and Asc strongly reduced the secretion of IL-1 ß, demonstrating that also in keratinocytes inflammasome proteins are directly involved in maturation of this cytokine. These results demonstrate for the first time the presence of an active inflammasome in non-professional immune cells. Moreover, they show that UV irradiation is a stimulus for inflammasome activation in keratinocytes. For the analysis of the ín vivo functions of EBBP, transgenic mice overexpressing EBBP in the epidermis were generated. To examine the influence of EBBP overexpression on inflammatory processes, we subjected the mice to different challenges, which induce inflammation. Wound-healing, UVB irradiation and delayed hypersensitivity were tested, but we did not observe any phenotype in the K14-EBBP mice. Besides, a conditional ebbp knockout mouse has been obtained, which will allow to determine the effects of EBBP gene deletion in different tissues and organs. RESUME: EBBP est un membre encore mal connu de la famille des RBCC/TRIM (RING finger B-box coiled-coil/tripartite motif). Il est exprimé de manière ubiquitaire, et en particulier dans les kératinocytes. EBBP étant capable d'interagir avec la prointerleukine-1 ß (prolL-1 ß) dans les macrophages et les kératinocytes humains et de réguler la sécrétion de l'IL-1 ß, il est très probable que cette protéine est impliquée dans l'inflammation. L'activation et la sécrétion de l'IL-1 ß requièrent le clivage protéolytique de son précurseur prolL-1ß par la caspase-1, qui est elle-même activée par un complexe protéique appelé l'inflammasome. Comme il a été démontré qu'EBBP peut lier deux protéines de l'inflammasome (NALP-1 et caspase-1), nous avons émis l'hypothèse qu'EBBP joue un rôle dans la régulation de l'inflammation et que l'inflammasome, jusqu'ici décrit exclusivement dans des cellules inflammatoires, existe dans les kératinocytes. En effet, j'ai pu montrer dans ma thèse que les composants de l'inflammasome sont exprimés dans les kératinocytes humains ainsi que in vivo dans l'épiderme humain. Après irradiation avec une dose, physiologique d'UVB, les kératinocytes activent la prolL-1 ß et sécrètent la prolL-1a, l'IL-1 ß, la prolL-18 et des protéines de l'inflammasome, bien que toutes ces protéines soient dépourvues de peptide signal. La sécrétion dépend de la caspase-1 mais pas de la synthèse protéique de novo. Le knock-down de NALP-1 et -3, des caspase-1 et -5, d'EBBP et d'Asc réduit de manière marquée la sécrétion d'IL-1 ß, démontrant que dans les kératinocytes également, les protéines de l'inflammasome sont impliquées directement dans la maturation de cette cytokine. Ces résultats démontrent pour la première fois la présence d'un inflammasome actif dans des cellules immunitaires non professionnelles. De plus, ils montrent que l'irradiation aux UV est un stimulus pour l'activation de l'inflammasome dans les kératinocytes. Pour l'analyse des fonctions d'EBBP in vivo, nous avons généré des souris transgéniques qui surexpriment EBBP dans l'épiderme. En vue d'examiner l'influence de la surexpression d'EBBP sur le processus inflammatoire, nous avons soumis ces souris à differents modèles d'inflammation. Nous avons testé cicatrisation, UVB et hypersensibilité retardée, mais n'avons pas observé de phénotype chez les souris transgéniques. En parallèle, nous avons également généré des souris knock-out pour ebbp qui devraient nous permettre de déterminer les effets de la suppression d'EBBP dans différents tissus et organes.

Transcriptional activation of the utrophin promoter B by a constitutively active Ets-transcription factor.

Duchenne muscular dystrophy is an X-linked genetic disease caused by the absence of functional dystrophin. Pharmacological upregulation of utrophin, the autosomal homologue of dystrophin, offers a potential therapeutic approach to treat Duchenne patients. Full-length utrophin mRNA is transcribed from two alternative promoters, called A and B. In contrast to the utrophin promoter A, little is known about the factors regulating the activity of the utrophin promoter B. Computer analysis of this second promoter revealed the presence of several conserved binding motives for Ets-transcription factors. Using electrotransfer of cDNA into mouse muscles, we demonstrate that a genetically modified beta-subunit of the Ets-transcription factor GA-binding protein potently activates a utrophin promoter B reporter construct in innervated muscle fibers in vivo. These results make the GA-binding protein and the signaling cascade regulating its activity in muscle cells, potential targets for the pharmacological modulation of utrophin expression in Duchenne patients.

The promise of advanced imaging techniques for the detection of hepatitis C virus antigens in the infected liver.

Injection drug use before and after liver transplantation: a retrospective multicenter analysis on incidence and outcome. Clin Transplant 2009 DOI: 10.1111/j.1399-0012.2009.01121.x. Background and aims: Injecting drug use (IDU) before and after liver transplantation (LT) is poorly described. The aim of this study was to quantify relapse and survival in this population and to describe the causes of mortality after LT. Methods: Past injection drug users were identified from the LT listing protocols from four centers in Switzerland and France. Data on survival and relapse were collected and used for uni- and multivariate analysis. Results: Between 1988 and 2006, we identified 59 patients with a past history of IDU. The mean age at transplantation was 42.4 yr and the majority of patients were men (84.7%). The indication for LT was for the vast majority viral cirrhosis accounting for 91.5% of cases, while alcoholic cirrhosis was 5.1%. There were 16.9% of patients who had a substitution therapy before and 6.8% who continued after LT. Two patients (3.4%) relapsed into IDU after LT and died at 18 and 41 months. The mean follow-up was 51 months. Overall survival was 84%, 66%, and 61% at 1, 5, and 10 yr after transplantation. Conclusions: Documented IDU was rare in liver transplanted patients. Past IDU was not associated with poorer survival after LT, and relapse after LT occurred in 3.4%.

BAFF and the regulation of B cell survival.

The TNF family member BAFF is a fundamental survival factor for B cells. BAFF binds to three receptors, only one of which, BAFF-R, does not cross-react with the BAFF-related ligand APRIL. The survival function of BAFF on B cells is mediated mainly by BAFF-R and is particularly effective in transitional B cells. BAFF depletion leads to a considerable decrease in mature B cells, without apparent effect on B cell genesis. Consistently, BAFF overexpression results in an expanded B cell compartment and autoimmunity in mice. Elevated amounts of BAFF can be found in the serum of patients suffering from autoimmune diseases. The BAFF system is a promising target for the treatment of autoimmune diseases.

The NF-κB signaling protein Bcl10 regulates actin dynamics by controlling AP1 and OCRL-bearing vesicles.

The protein Bcl10 contributes to adaptive and innate immunity through the assembly of a signaling complex that plays a key role in antigen receptor and FcR-induced NF-κB activation. Here we demonstrate that Bcl10 has an NF-κB-independent role in actin and membrane remodeling downstream of FcR in human macrophages. Depletion of Bcl10 impaired Rac1 and PI3K activation and led to an abortive phagocytic cup rich in PI(4,5)P(2), Cdc42, and F-actin, which could be rescued with low doses of F-actin depolymerizing drugs. Unexpectedly, we found Bcl10 in a complex with the clathrin adaptors AP1 and EpsinR. In particular, Bcl10 was required to locally deliver the vesicular OCRL phosphatase that regulates PI(4,5)P(2) and F-actin turnover, both crucial for the completion of phagosome closure. Thus, we identify Bcl10 as an early coordinator of NF-κB-mediated immune response with endosomal trafficking and signaling to F-actin remodeling.

Safety, pharmacokinetics, and preliminary clinical activity of inotuzumab ozogamicin, a novel immunoconjugate for the treatment of B-cell non-Hodgkin's lymphoma: results of a phase I study.

PURPOSE Inotuzumab ozogamicin (CMC-544) is an antibody-targeted chemotherapy agent composed of a humanized anti-CD22 antibody conjugated to calicheamicin, a potent cytotoxic agent. This was a phase I study to determine the maximum-tolerated dose (MTD), safety, and preliminary efficacy of inotuzumab ozogamicin in an expanded MTD cohort of patients with relapsed or refractory CD22(+) B-cell non-Hodgkin's lymphoma (NHL). PATIENTS AND METHODS Inotuzumab ozogamicin was administered intravenously as a single agent once every 3 or 4 weeks at doses ranging from 0.4 to 2.4 mg/m(2). Outcomes included MTD, safety, pharmacokinetics, response, progression-free survival (PFS), and overall survival. Results Seventy-nine patients were enrolled. The MTD was determined to be 1.8 mg/m(2). Common adverse events at the MTD were thrombocytopenia (90%), asthenia (67%), and nausea and neutropenia (51% each). The objective response rate at the end of treatment was 39% for the 79 enrolled patients, 68% for all patients with follicular NHL treated at the MTD, and 15% for all patients with diffuse large B-cell lymphoma treated at the MTD. Median PFS was 317 days (approximately 10.4 months) and 49 days for patients with follicular NHL and diffuse large B-cell lymphoma, respectively. CONCLUSION Inotuzumab ozogamicin has demonstrated efficacy against CD22(+) B-cell NHL, with reversible thrombocytopenia as the main toxicity.

Analysis of the Cytoprotective Role of α-Crystallins in Cell Survival and Implication of the αA-Crystallin C-Terminal Extension Domain in Preventing Bax-Induced Apoptosis.

α-Crystallins, initially described as the major structural proteins of the lens, belong to the small heat shock protein family. Apart from their function as chaperones, α-crystallins are involved in the regulation of intracellular apoptotic signals. αA- and αB-crystallins have been shown to interfere with the mitochondrial apoptotic pathway triggering Bax pro-apoptotic activity and downstream activation of effector caspases. Differential regulation of α-crystallins has been observed in several eye diseases such as age-related macular degeneration and stress-induced and inherited retinal degenerations. Although the function of α-crystallins in healthy and diseased retina remains poorly understood, their altered expression in pathological conditions argue in favor of a role in cellular defensive response. In the Rpe65(-/-) mouse model of Leber's congenital amaurosis, we previously observed decreased expression of αA- and αB-crystallins during disease progression, which was correlated with Bax pro-death activity and photoreceptor apoptosis. In the present study, we demonstrated that α-crystallins interacted with pro-apoptotic Bax and displayed cytoprotective action against Bax-triggered apoptosis, as assessed by TUNEL and caspase assays. We further observed in staurosporine-treated photoreceptor-like 661W cells stably overexpressing αA- or αB-crystallin that Bax-dependent apoptosis and caspase activation were inhibited. Finally, we reported that the C-terminal extension domain of αA-crystallin was sufficient to provide protection against Bax-triggered apoptosis. Altogether, these data suggest that α-crystallins interfere with Bax-induced apoptosis in several cell types, including the cone-derived 661W cells. They further suggest that αA-crystallin-derived peptides might be sufficient to promote cytoprotective action in response to apoptotic cell death.

SOST expression is restricted to the great arteries during embryonic and neonatal cardiovascular development.

Spatial-temporal regulation of bone morphogenetic protein (BMP) and Wnt activity is essential for normal cardiovascular development, and altered activity of these growth factors causes maldevelopment of the cardiac outflow tract and great arteries. In the present study, we show that SOST, a Dan family member reported to antagonize BMP and Wnt activity, is expressed within the medial vessel wall of the great arteries containing smooth muscle cells. The ascending aorta, aortic arch, brachiocephalic artery, common carotids, and pulmonary trunk were all associated with SOST expressing smooth muscle cells, while the heart itself, including the valves, and more distal arteries, that is, pulmonary arteries, subclavian arteries, and descending aorta, were negative. SOST was expressed from embryonic day 15.5 up to the neonatal period. SOST expression, however, did not correspond with inhibition of Smad-dependent BMP activity or beta-catenin-dependent Wnt activity in the great arteries. Activity of both signaling pathways was already down-regulated before induction of SOST expression.