BACKGROUND: Despite research, there are still controversial areas in the management of Crohn\'s disease (CD).
OBJECTIVE: To establish practical recommendations on using anti-tumour necrosis factor (TNF) drugs in patients with moderate-to-severe CD.
METHODS: Clinical controversies in the management of CD using anti-TNF therapies were identified. A comprehensive literature review was performed, and a national survey was launched to examine current clinical practices when using anti-TNF therapies. Their results were discussed by expert gastroenterologists within a nominal group meeting, and a set of statements was proposed and tested in a Delphi process.
RESULTS: Qualitative study. The survey and Delphi process were sent to 244 CD-treating physicians (response rate: 58%). A total of 14 statements were generated. All but two achieved agreement. These statements cover: (1) use of first-line non-anti-TNF biological therapy; (2) role of HLA-DQA1*05 in daily practice; (3) attitudes in primary non-response and loss of response to anti-TNF therapy due to immunogenicity; (4) use of ustekinumab or vedolizumab if a change in action mechanism is warranted; (5) anti-TNF drug level monitoring; (6) combined therapy with an immunomodulator.
CONCLUSIONS: This document sought to pull together the best evidence, experts\' opinions, and treating physicians\' attitudes when using anti-TNF therapies in patients with CD.

Cell and tissue stiffness have been known to contribute to both developmental and pathological signalling for some time, but the underlying mechanisms remain elusive. Integrins and their associated adhesion signalling complexes (IACs), which form a nexus between the cell cytoskeleton and the extracellular matrix, act as a key force sensing and transducing unit in cells. Accordingly, there has been much interest in obtaining a systems-level understanding of IAC composition. Proteomic approaches have revealed the complexity of IACs and identified a large number of components that are regulated by cytoskeletal force. Here we review the function of the consensus adhesome, an assembly of core IAC proteins that emerged from a meta-analysis of multiple proteomic datasets, in the context of mechanosensing. As IAC components have been linked to a variety of diseases involved with rigidity sensing, the field is now in a position to define the mechanosensing function of individual IAC proteins and elucidate their mechanisms of action.

Integrin receptor activation initiates the formation of integrin adhesion complexes (IACs) at the cell membrane that transduce adhesion-dependent signals to control a multitude of cellular functions. Proteomic analyses of isolated IACs have revealed an unanticipated molecular complexity; however, a global view of the consensus composition and dynamics of IACs is lacking. Here, we have integrated several IAC proteomes and generated a 2,412-protein integrin adhesome. Analysis of this data set reveals the functional diversity of proteins in IACs and establishes a consensus adhesome of 60 proteins. The consensus adhesome is likely to represent a core cell adhesion machinery, centred around four axes comprising ILK-PINCH-kindlin, FAK-paxillin, talin-vinculin and α-actinin-zyxin-VASP, and includes underappreciated IAC components such as Rsu-1 and caldesmon. Proteomic quantification of IAC assembly and disassembly detailed the compositional dynamics of the core cell adhesion machinery. The definition of this consensus view of integrin adhesome components provides a resource for the research community.

Aggregatibacter actinomycetemcomitans produces a repeats-in-toxin (RTX) leukotoxin (LtxA) that selectively kills human immune cells. Binding of LtxA to its β2 integrin receptor (lymphocyte function-associated antigen-1 (LFA-1)) results in the clustering of the toxin·receptor complex in lipid rafts. Clustering occurs only in the presence of LFA-1 and cholesterol, and LtxA is unable to kill cells lacking either LFA-1 or cholesterol. Here, the interaction of LtxA with cholesterol was measured using surface plasmon resonance and differential scanning calorimetry. The binding of LtxA to phospholipid bilayers increased by 4 orders of magnitude in the presence of 40% cholesterol relative to the absence of cholesterol. The affinity was specific to cholesterol and required an intact secondary structure. LtxA contains two cholesterol recognition/amino acid consensus (CRAC) sites; CRAC(336) ((333)LEEYSKR(339)) is highly conserved among RTX toxins, whereas CRAC(503) ((501)VDYLK(505)) is unique to LtxA. A peptide corresponding to CRAC(336) inhibited the ability of LtxA to kill Jurkat (Jn.9) cells. Although peptides corresponding to both CRAC(336) and CRAC(503) bind cholesterol, only CRAC(336) competitively inhibited LtxA binding to this sterol. A panel of full-length LtxA CRAC mutants demonstrated that an intact CRAC(336) site was essential for LtxA cytotoxicity. The conservation of CRAC(336) among RTX toxins suggests that this mechanism may be conserved among RTX toxins.

The interaction between the cytoskeletal proteins talin and vinculin plays a key role in integrin-mediated cell adhesion and migration. Three vinculin binding sites (VBS1-3) have previously been identified in the talin rod using a yeast two-hybrid assay. To extend these studies, we spot-synthesized a series of peptides spanning all the alpha-helical regions predicted for the talin rod and identified eight additional VBSs, two of which overlap key functional regions of the rod, including the integrin binding site and C-terminal actin binding site. The talin VBS alpha-helices bind to a hydrophobic cleft in the N-terminal vinculin Vd1 domain. We have defined the specificity of this interaction by spot-synthesizing a series of 25-mer talin VBS1 peptides containing substitutions with all the commonly occurring amino acids. The consensus for recognition is LXXAAXXVAXX- VXXLIXXA with distinct classes of hydrophobic side chains at positions 1, 4, 5, 8, 9, 12, 15, and 16 required for vinculin binding. Positions 1, 8, 12, 15, and 16 require an aliphatic residue and will not tolerate alanine, whereas positions 4, 5, and 9 are less restrictive. These preferences are common to all 11 VBS sequences with a minor variation occurring in one case. A crystal structure of this variant VBS peptide in complex with the vinculin Vd1 domain reveals a subtly different mode of vinculin binding.

Metastatic diseases of prostate cancer reveal high expression of alpha6 integrin and the activation of mitogen-activated protein kinases (MAP kinase). Therefore, the present study was conducted to examine whether MAP kinase pathway is involved in the alpha6 integrin gene expression in androgen-independent prostate cancer cell lines. alpha6 integrin mRNA expression, the alpha6 integrin promoter-induced luciferase activities and MAP kinase enzyme activities in androgen-independent LNCaP and PC-3 cell lines were higher than those in androgen-dependent LNCaP. Deletion and mutation analysis showed that Sp1 consensus sequence at -48 to -43 bp from the transcription start site was necessary for basal promoter activity. Binding of Sp1 to its consensus sequence in three cell lines was confirmed by electrophoretic mobility shift assays. Sp1 binding to its consensus sequence, as well as promoter activity and mRNA expression, were found to be inhibited by an inhibitor of MAP kinase kinase 1 and 2, U0126, in the androgen-independent cell lines. Our results indicate that the proximal Sp1 is necessary for basal promoter activity of the alpha6 integrin, suggesting that signal transduction from MAP kinases to activation of Sp1 might be involved in alpha6 integrin gene expression in androgen-independent prostate cancer cell lines.