Lateral growth limitation of corneal fibrils and their lamellar stacking depend on covalent collagen cross-linking by transglutaminase-2 and lysyl oxidases, respectively.

Corneal stroma contains an extracellular matrix of orthogonal lamellae formed by parallel and equidistant fibrils with a homogeneous diameter of ~35 nm. This is indispensable for corneal transparency and mechanical functions. However, the mechanisms controlling corneal fibrillogenesis are incompletely understood and the conditions required for lamellar stacking are essentially unknown. Under appropriate conditions, chick embryo corneal fibroblasts can produce an extracellular matrix in vitro resembling primary corneal stroma during embryonic development. Among other requirements, cross-links between fibrillar collagens, introduced by tissue transglutaminase-2, are necessary for the self-assembly of uniform, small diameter fibrils but not their lamellar stacking. By contrast, the subsequent lamellar organization into plywood-like stacks depends on lysyl aldehyde-derived cross-links introduced by lysyl oxidase activity, which, in turn, only weakly influences fibril diameters. These cross-links are introduced at early stages of fibrillogenesis. The enzymes are likely to be important for a correct matrix deposition also during repair of the cornea.

Characterization and Validation of Antibodies for Immunohistochemical Staining of the Chemokine CXCL12.

Chemokines and their receptors have been implicated in cancer biology. The CXCL12/CXCR4 axis is essential for the homing and retention of hematopoietic stem cells in bone marrow niches, and has a significant role in neonatal development. It is also implicated in multiple facets of cancer biology including metastasis, angiogenesis/neo-vasculogenesis, and immune cell trafficking at the tumor microenvironment (TME). Immunohistochemistry (IHC) is an ideal method for investigating involvement of CXCL12 in the TME. Three antibodies were evaluated here for their suitability to stain CXCL12. Both D8G6H and K15C gave apparent specific staining in both lymphoid and tumor tissue, but with converse staining patterns. D8G6H stained cells in the parafollicular zone whereas K15C showed staining of lymphoid cells in the interfollicular zone of tonsil tissue. Using a cell line with high CXCL12 expression, TOV21G, as a positive control, it was found that D8G6H gave strong staining of TOV21G cells whereas no staining was observed with K15C indicating that D8G6H specifically stains CXCL12. Significant staining of CXCL12 in the ovarian TME using tissue microarray was observed using D8G6H. These data demonstrate the importance of antibody characterization for IHC applications, and provide further evidence for the involvement of CXCL12 in ovarian cancer biology.

Unilateral nephrectomy leads to up-regulation of syndecan-2- and TGF-beta-mediated glomerulosclerosis in syndecan-4 deficient male mice.

Syndecan-4 is an ubiquitous, plasma membrane-spanning heparan sulfate proteoglycan involved in proliferation, differentiation, adhesion and migration of cells in vitro. Syndecan-4 knockout (KO) mice show no obvious defects but respond abnormally to experimental stress conditions. In the adult, syndecan-4 is the most abundant syndecan of renal tissue. We therefore investigated the consequences of syndecan-4 deficiency during progression of kidney disease using unilaterally nephrectomized mice, a model of glomerular hyperfiltration and renal hypertrophy. 60 days after unilateral nephrectomy (UNX), mesangial expansion, enhanced matrix production (collagens I and IV, fibronectin) and focal segmental glomerulosclerosis, resembling early stages of diabetic nephropathy, was apparent in male but not female syndecan-4 KO mice. No defect was detected in wild type UNX males. Syndecan-2 mRNA and protein were not detectable in renal glomeruli of wild type mice, but were induced specifically in the glomeruli of the syndecan-4 deficient kidneys after unilateral nephrectomy. Due to the structural similarities of syndecans-2 and -4 we hypothesize that de novo-production of syndecan-2 in kidneys after unilateral nephrectomy reflects a compensatory response. However, this response is counterproductive since syndecan-2 supports the pro-sclerotic activity of TGF-beta1 which is increased in parallel with syndecan-2 synthesis. By contrast, signaling through syndecan-4 negatively controls the production of pro-sclerotic TGF-beta1.

Surgical procedures and postsurgical tissue processing significantly affect expression of genes and EGFR-pathway proteins in colorectal cancer tissue.

An understanding of tissue data variability in relation to processing techniques during and postsurgery would be desirable when testing surgical specimens for clinical diagnostics, drug development, or identification of predictive biomarkers. Specimens of normal and colorectal cancer (CRC) tissues removed during colon and liver resection surgery were obtained at the beginning of surgery and postsurgically, tissue was fixed at 10, 20, and 45 minutes. Specimens were analyzed from 50 patients with primary CRC and 43 with intrahepatic metastasis of CRC using a whole genome gene expression array. Additionally, we focused on the epidermal growth factor receptor pathway and quantified proteins and their phosphorylation status in relation to tissue processing timepoints. Gene and protein expression data obtained from colorectal and liver specimens were influenced by tissue handling during surgery and by postsurgical processing time. To obtain reliable expression data, tissue processing for research and diagnostic purposes needs to be highly standardized.