Spontaneous Extracellular Matrix Accumulation in a Human in vitro Model of Renal Fibrosis Is Mediated by αV Integrins.

BACKGROUND: Tubulointerstitial fibrosis is a key feature of chronic kidney diseases leading to renal failure. It is characterised by the infiltration of fibroblasts and aberrant accumulation of extracellular matrix (ECM) proteins, which are associated with progressive loss of renal function. Integrins play a major role in fibrosis, but the mechanisms through which they do this are not fully understood. OBJECTIVE: Using a complex cell system, we test the hypothesis that integrins are pro-fibrotic via regulation of functional interactions between tubular epithelial cells and renal fibroblasts. METHOD: Contact co-culture of human primary renal proximal tubular epithelial cells and renal fibroblasts promoted the spontaneous accumulation of a mature ECM rich in interstitial collagens, which was considerably in excess of that seen in the individual mono-cultures. Both cell types persisted throughout the culture and were capable of expressing multiple ECM components. RESULTS: While ECM accumulation was inhibited by the clinically proven anti-fibrotic, nintedanib, and was partially abrogated by transforming growth factor ß neutralisation, its levels did not return to basal, indicating additional pathways were implicated in the pro-ECM response. Application of anti-integrin blocking antibodies and small molecules demonstrated a major role of the αV integrins in the ECM accumulation during fibroblast: epithelial cell interactions. CONCLUSION: Integrin-mediated pathways can facilitate the spontaneous accumulation of ECM during fibroblast: epithelial cell interactions, and this direct renal co-culture assay system could provide a translational in vitro assay for investigating novel pathways involved in the pro-ECM response and the screening of renal anti-fibrotic agents.

Discovery of a junctional epitope antibody that stabilizes IL-6 and gp80 protein:protein interaction and modulates its downstream signaling.

Protein:protein interactions are fundamental in living organism homeostasis. Here we introduce VHH6, a junctional epitope antibody capable of specifically recognizing a neo-epitope when two proteins interact, albeit transiently, to form a complex. Orthogonal biophysical techniques have been used to prove the "junctional epitope" nature of VHH6, a camelid single domain antibody recognizing the IL-6-gp80 complex but not the individual components alone. X-ray crystallography, HDX-MS and SPR analysis confirmed that the CDR regions of VHH6 interact simultaneously with IL-6 and gp80, locking the two proteins together. At the cellular level, VHH6 was able to alter the response of endothelial cells to exogenous IL-6, promoting a sustained STAT3 phosphorylation signal, an accumulation of IL-6 in vesicles and an overall pro-inflammatory phenotype supported further by transcriptomic analysis. Junctional epitope antibodies, like VHH6, not only offer new opportunities in screening and structure-aided drug discovery, but could also be exploited as therapeutics to modulate complex protein:protein interactions.

Altered Epithelial Gene Expression in Peripheral Airways of Severe Asthma.

Management of severe asthma remains a challenge despite treatment with glucocorticosteroid therapy. The majority of studies investigating disease mechanisms in treatment-resistant severe asthma have previously focused on the large central airways, with very few utilizing transcriptomic approaches. The small peripheral airways, which comprise the majority of the airway surface area, remain an unexplored area in severe asthma and were targeted for global epithelial gene expression profiling in this study. Differences between central and peripheral airways were evaluated using transcriptomic analysis (Affymetrix HG U133 plus 2.0 GeneChips) of epithelial brushings obtained from severe asthma patients (N = 17) and healthy volunteers (N = 23). Results were validated in an independent cohort (N = 10) by real-time quantitative PCR. The IL-13 disease signature that is associated with an asthmatic phenotype was upregulated in severe asthmatics compared to healthy controls but was predominantly evident within the peripheral airways, as were genes related to mast cell presence. The gene expression response associated with glucocorticosteroid therapy (i.e. FKBP5) was also upregulated in severe asthmatics compared to healthy controls but, in contrast, was more pronounced in central airways. Moreover, an altered epithelial repair response (e.g. FGFBP1) was evident across both airway sites reflecting a significant aspect of disease in severe asthma unadressed by current therapies. A transcriptomic approach to understand epithelial activation in severe asthma has thus highlighted the need for better-targeted therapy to the peripheral airways in severe asthma, where the IL-13 disease signature persists despite treatment with currently available therapy.

Engagement of CD22 on B cells with the monoclonal antibody epratuzumab stimulates the phosphorylation of upstream inhibitory signals of the B cell receptor.

The binding of antigen to the B cell receptor (BCR) results in a cascade of signalling events that ultimately drive B cell activation. Uncontrolled B cell activation is regulated by negative feedback loops that involve inhibitory co-receptors such as CD22 and CD32B that exert their functions following phosphorylation of immunoreceptor tyrosine-based inhibition motifs (ITIMs). The CD22-targeted antibody epratuzumab has previously been shown to inhibit BCR-driven signalling events, but its effects on ITIM phosphorylation of CD22 and CD32B have not been properly evaluated. The present study therefore employed both immunoprecipitation and flow cytometry approaches to elucidate the effects of epratuzumab on direct phosphorylation of key tyrosine (Tyr) residues on both these proteins, using both transformed B cell lines and primary human B cells. Epratuzumab induced the phosphorylation of Tyr(822) on CD22 and enhanced its co-localisation with SHP-1. Additionally, in spite of high basal phosphorylation of other key ITIMs on CD22, in primary human B cells epratuzumab also enhanced phosphorylation of Tyr(807), a residue involved in the recruitment of Grb2. Such initiation events could explain the effects of epratuzumab on downstream signalling in B cells. Finally, we were able to demonstrate that epratuzumab stimulated the phosphorylation of Tyr(292) on the low affinity inhibitory Fc receptor CD32B which would further attenuate BCR-induced signalling. Together, these data demonstrate that engagement of CD22 with epratuzumab leads to the direct phosphorylation of key upstream inhibitory receptors of BCR signalling and may help to explain how this antibody modulates B cell function.

Discovery of 4-azaindoles as novel inhibitors of c-Met kinase.

A series of 4-azaindole inhibitors of c-Met kinase is described. The postulated binding mode was confirmed by an X-ray crystal structure and series optimisation was performed on the basis of this structure. Future directions for series development are discussed.

Discovery of a novel series of quinoxalines as inhibitors of c-Met kinase.

A series of quinoxaline inhibitors of c-Met kinase is described. The postulated binding mode was confirmed by an X-ray crystal structure and optimisation of the series was performed on the basis of this structure. Future directions for development of the series are discussed together with the identification of a novel quinoline scaffold.