TNF superfamily member TL1A elicits type 2 innate lymphoid cells at mucosal barriers.

Immune responses at mucosal barriers are regulated by innate type 2 lymphoid cells (ILC2s) that elaborate effector cytokines interleukins 5 and 13 (IL5 and IL13). IL25 and IL33 are key cytokines that support ILC2s; however, mice deficient in these pathways retain some functional ILC2s. Analysis of human and murine cells revealed that ILC2s highly express tumor necrosis factor (TNF)-receptor superfamily member DR3 (TNFRSF25). Engagement of DR3 with cognate ligand TL1A promoted ILC2 expansion, survival, and function. Exogenous protein or genetic overexpression of TL1A activated ILC2s independent of IL25 or IL33. Dr3(-/-) mice failed to control gut helminthic infections, and failed to mount ILC2 responses in the lung after nasal challenge with papain. Our data demonstrate a key role for TL1A in promoting ILC2s at mucosal barriers.

Subcutaneous abatacept versus intravenous abatacept: a phase IIIb noninferiority study in patients with an inadequate response to methotrexate.

OBJECTIVE: To compare the efficacy and safety of subcutaneous (SC) and intravenous (IV) abatacept. METHODS: In this phase IIIb double-blind, double-dummy, 6-month study, patients with rheumatoid arthritis (RA) and inadequate responses to methotrexate were randomized to receive 125 mg SC abatacept on days 1 and 8 and weekly thereafter (plus an IV loading dose [∼10 mg/kg] on day 1) or IV abatacept (∼10 mg/kg) on days 1, 15, and 29 and every 4 weeks thereafter. The primary end point for determining the noninferiority of SC abatacept to IV abatacept was the proportion of patients in each group meeting the American College of Rheumatology 20% improvement criteria (achieving an ACR20 response) at month 6. Other efficacy end points, immunogenicity, and safety were also assessed. RESULTS: Of 1,457 patients, 693 of 736 (94.2%) treated with SC abatacept and 676 of 721 (93.8%) treated with IV abatacept completed 6 months. At month 6, 76.0% (95% confidence interval 72.9, 79.2) of SC abatacept-treated patients versus 75.8% (95% confidence interval 72.6, 79.0) of IV abatacept-treated patients achieved an ACR20 response (estimated difference between groups 0.3% [95% confidence interval -4.2, 4.8]), confirming noninferiority of SC abatacept to IV abatacept. Onset and magnitude of ACR responses and disease activity and physical function improvements were comparable between the SC and IV abatacept-treated groups. The proportions of adverse events (AEs) and serious AEs over 6 months were 67.0% and 4.2%, respectively, in the SC abatacept-treated group and 65.2% and 4.9%, respectively, in the IV abatacept-treated group, with comparable frequencies of serious infections, malignancies, and autoimmune events between groups. SC injection site reactions (mostly mild) occurred in 19 SC abatacept (IV placebo)-treated patients (2.6%) and 18 IV abatacept (SC placebo)-treated patients (2.5%). Abatacept-induced antibodies occurred in 1.1% of SC abatacept-treated patients and 2.3% of IV abatacept-treated patients. CONCLUSION: SC abatacept provides efficacy and safety comparable with that of IV abatacept, with low immunogenicity and high retention rates, consistent with the established IV abatacept profile. Rates of injection site reactions were low. SC abatacept will provide additional treatment options, such as an alternative route of administration, for patients with RA.

Epstein-Barr virus latent membrane protein 2A (LMP2A) employs the SLP-65 signaling module.

In latently infected B lymphocytes, the Epstein-Barr virus (EBV) suppresses signal transduction from the antigen receptor through expression of the integral latent membrane protein 2A (LMP2A). At the same time, LMP2A triggers B cell survival by a yet uncharacterized maintenance signal that is normally provided by the antigen receptor. The molecular mechanisms are unknown as LMP2A-regulated signaling cascades have not been described so far. Using a novel mouse model we have identified the intracellular adaptor protein Src homology 2 (SH2) domain-containing leukocyte protein (SLP)-65 as a critical downstream effector of LMP2A in vivo. Biochemical analysis of the underlying signaling pathways revealed that EBV infection causes constitutive tyrosine phosphorylation of one of the two SLP-65 isoforms and complex formation between SLP-65 and the protooncoprotein CrkL (CT10 regulator of kinase like). This leads to antigen receptor-independent phosphorylation of Cbl (Casitas B lineage lymphoma) and C3G. In contrast, phospholipase C-gamma2 (PLC-gamma2) activation is completely blocked. Our data show that in order to establish a latent EBV infection, LMP2A selectively activates or represses SLP-65-regulated signaling pathways.

The Flory isolated-pair hypothesis is not valid for polypeptide chains: implications for protein folding.

Using an all-atom representation, we exhaustively enumerate all sterically allowed conformations for short polyalanyl chains. Only intrachain interactions are considered, including one adjustable parameter, a favorable backbone energy (e.g., a peptide hydrogen bond). The counting is used to reevaluate Flory's isolated-pair hypothesis, the simplifying assumption that each phi,psi pair is sterically independent. This hypothesis is a conceptual linchpin in helix-coil theories and protein folding. Contrary to the hypothesis, we find that systematic local steric effects can extend beyond nearest-chain neighbors and can restrict the size of accessible conformational space significantly. As a result, the entropy price that must be paid to adopt any specific conformation is far less than previously thought.

Requirement for B cell linker protein (BLNK) in B cell development.

Linker proteins function as molecular scaffolds to localize enzymes with substrates. In B cells, B cell linker protein (BLNK) links the B cell receptor (BCR)-activated Syk kinase to the phosphoinositide and mitogen-activated kinase pathways. To examine the in vivo role of BLNK, mice deficient in BLNK were generated. B cell development in BLNK-/- mice was blocked at the transition from B220+CD43+ progenitor B to B220+CD43- precursor B cells. Only a small percentage of immunoglobulin M++ (IgM++), but not mature IgMloIgDhi, B cells were detected in the periphery. Hence, BLNK is an essential component of BCR signaling pathways and is required to promote B cell development.

An essential role for BLNK in human B cell development.

The signal transduction events that control the progenitor B cell (pro-B cell) to precursor B cell (pre-B cell) transition have not been well delineated. In evaluating patients with absent B cells, a male with a homozygous splice defect in the cytoplasmic adapter protein BLNK (B cell linker protein) was identified. Although this patient had normal numbers of pro-B cells, he had no pre-B cells or mature B cells, indicating that BLNK plays a critical role in orchestrating the pro-B cell to pre-B cell transition. The immune system and overall growth and development were otherwise normal in this patient, suggesting that BLNK function is highly specific.

Musculoskeletal manifestations of osteomalacia: report of 26 cases and literature review.

OBJECTIVE: This study was undertaken to describe the musculoskeletal manifestations in a selected population of 26 patients with biopsy-proven osteomalacia (OM) and provide a literature update. METHODS: The 26 patients with biopsy-proven OM were selected from a total number of 79 patients who underwent anterior iliac crest biopsy. The diagnosis of OM was confirmed by the presence of an osteoid volume greater than 10%, osteoid width greater than 15 microm, and delayed mineralization assessed by double-tetracycline labeling. RESULTS: OM was caused by intestinal malabsorption in 13 patients, whereas six other patients presented with hypophosphatemia of different causes. Five elderly patients presented with hypovitaminosis D, and in two patients the OM was part of renal osteodystrophy. Twenty-three patients presented with bone pain and diffuse demineralization, whereas three other patients had normal or increased bone density. Characteristic pseudofractures were seen in only seven patients. Six of the 23 patients with diffuse demineralization had an "osteoporotic-like pattern" without pseudofractures. Prominent articular manifestations were seen in seven patients, including a rheumatoid arthritis-like picture in three, osteogenic synovitis in three, and ankylosing spondylitis-like in one. Two other patients were referred to us with the diagnosis of possible metastatic bone disease attributable to polyostotic areas of increased radio nuclide uptake caused by pseudofractures. Six patients also had proximal myopathy, two elderly patients were diagnosed as having polymalgia rheumatica, and two young patients were diagnosed as having fibromyalgia. One of the patients who presented with increased bone density was misdiagnosed as possible fluorosis. CONCLUSION: OM is usually neglected when compared with other metabolic bone diseases and may present with a variety of clinical and radiographic manifestations mimicking other musculoskeletal disorders.

BLNK required for coupling Syk to PLC gamma 2 and Rac1-JNK in B cells.

Signaling through the B cell receptor (BCR) is essential for B cell function and development. Despite the key role of Syk in BCR signaling, little is known about the mechanism by which Syk transmits downstream effectors. BLNK (B cell LiNKer protein), a substrate for Syk, is now shown to be essential in activating phospholipase C (PLC)gamma 2 and JNK. The BCR-induced PLC gamma 2 activation, but not the JNK activation, was restored by introduction of PLC gamma 2 membrane-associated form into BLNK-deficient B cells. As JNK activation requires both Rac1 and PLC gamma 2, our results suggest that BLNK regulates the Rac1-JNK pathway, in addition to modulating PLC gamma 2 localization.

A potential smoothing algorithm accurately predicts transmembrane helix packing.

Potential smoothing, a deterministic analog of stochastic simulated annealing, is a powerful paradigm for the solution of conformational search problems that require extensive sampling, and should be a useful tool in computational approaches to structure prediction and refinement. A novel potential smoothing and search (PSS) algorithm has been developed and applied to predict the packing of transmembrane helices. The highlight of this method is the efficient manner in which it circumvents the combinatorial explosion associated with the large number of minima on multidimensional potential energy surfaces in order to converge to the global energy minimum. Here we show how our potential smoothing and search method succeeds in finding the global minimum energy structure for the glycophorin A (GpA) transmembrane helix dimer by optimizing interhelical van der Waals interactions over rigid and semi-rigid helices. Structures obtained from our ab initio predictions are in close agreement with recent experimental data.

Regulation of PAK activation and the T cell cytoskeleton by the linker protein SLP-76.

Tyrosine phosphorylation of linker proteins enables the T cell antigen receptor (TCR)-associated protein tyrosine kinases to phosphorylate and regulate effector molecules that generate second messengers. We demonstrate here that the SLP-76 linker protein interacts with both nck, an adaptor protein, and Vav, a guanine nucleotide exchange factor for Rho-family GTPases. The assembly of this tri-molecular complex permits the activated Rho-family GTPases to regulate target effectors that interact through nck. In turn, assembly of this complex mediates the enzymatic activation of the p21-activated protein kinase 1 and facilitates actin polymerization. Hence, phosphorylation of linker proteins not only bridges the TCR-associated PTK, ZAP-70, with downstream effector proteins, but also provides a scaffold to integrate distinct signaling complexes to regulate T cell function.

Accuracy of side-chain prediction upon near-native protein backbones generated by Ab initio folding methods.

The ab initio folding problem can be divided into two sequential tasks of approximately equal computational complexity: the generation of native-like backbone folds and the positioning of side chains upon these backbones. The prediction of side-chain conformation in this context is challenging, because at best only the near-native global fold of the protein is known. To test the effect of displacements in the protein backbones on side-chain prediction for folds generated ab initio, sets of near-native backbones (< or = 4 A C alpha RMS error) for four small proteins were generated by two methods. The steric environment surrounding each residue was probed by placing the side chains in the native conformation on each of these decoys, followed by torsion-space optimization to remove steric clashes on a rigid backbone. We observe that on average 40% of the chi1 angles were displaced by 40 degrees or more, effectively setting the limits in accuracy for side-chain modeling under these conditions. Three different algorithms were subsequently used for prediction of side-chain conformation. The average prediction accuracy for the three methods was remarkably similar: 49% to 51% of the chi1 angles were predicted correctly overall (33% to 36% of the chi1+2 angles). Interestingly, when the inter-side-chain interactions were disregarded, the mean accuracy increased. A consensus approach is described, in which side-chain conformations are defined based on the most frequently predicted chi angles for a given method upon each set of near-native backbones. We find that consensus modeling, which de facto includes backbone flexibility, improves side-chain prediction: chi1 accuracy improved to 51-54% (36-42% of chi1+2). Implications of a consensus method for ab initio protein structure prediction are discussed.

The early folding kinetics of apomyoglobin.

The folding pathway of apomyoglobin has been experimentally shown to have early kinetic intermediates involving the A, B, G, and H helices. The earliest detected kinetic events occur on a ns to micros time scale. We show that the early folding kinetics of apomyoglobin may be understood as the association of nascent helices through a network of diffusion-collision-coalescence steps G + H <--> GH + A <--> AGH + B <--> ABGH obtained by solving the diffusion-collision model in a chemical kinetics approximation. Our reproduction of the experimental results indicates that the model is a useful way to analyze folding data. One prediction from our fit is that the nascent A and H helices should be relatively more helix-like before coalescence than the other apomyoglobin helices.