Extracellular matrix (ECM) remodeling is an inherent characteristic of invasive head and neck squamous cell carcinoma (HNSCC). We investigated the regulation of gene expression, synthesis and activation of ECM components and matrix remodeling enzymes in a co-culture model of head and neck squamous cell carcinoma (HNSCC) and in HNSCC tissue. Periodontal ligament fibroblasts (PDL) and SCC-25 HNSCC cells were co-cultured for seven days, followed by mRNA and protein expression analysis of ECM components, ECM receptors, matrix metalloproteinases (MMPs) and proteinase inhibitors. The activity of MMPs was analyzed using gelatinase zymography. MMP-9, fibronectin and “antiadhesion proteins” were investigated by immunohistochemistry in HNSCC tissue. In co-cultured fibroblasts, in addition to fibronectin (FN), the “antiadhesion proteins” tenascin C (TSC), thrombospondin 1 (TSP1) and latent TGF-beta binding protein 2 (LTBP2) were upregulated. In lower levels: FN, TSC and TSP1 were also produced by the tumor cells. In co-cultured fibroblasts, alpha 1 and 5 and beta 1 integrins, in co-cultured tumor cells alpha 3 and 5 integrins were upregulated. MMP-1 was upregulated in both co-cultured tumor cells and fibroblasts, MMP-2 in co-cultured fibroblasts, and MMP-9 in co-cultured tumor cells. MMP inhibitors were mainly produced in fibroblasts. The activation of MMP-2 required the co-culture with tumor cells. MMP-9 was exclusively produced in tumor cells. In sections of formalin-fixed paraffin embedded HNSCC tissue, FN and LTBP2 were detected in fibroblastic stroma surrounding tumor cell nests; TSP1 was detected both in tumor cell nests and in the surrounding fibroblastic stroma. Extended MMP-9 immunohistochemical staining was found in 27 of 280 HNSCC tissue samples, in a subpopulation of cells in tumor cell nests. Fibroblast – tumor cell communication is a major pathway of matrix remodeling in HNSCC tumor tissue. Carcinoma-associated fibroblasts produce new matrix elements as TSP1, TSC and LTBP2, both tumor cells and fibroblasts upregulate integrin receptors, and they also share tasks in synthesis and activation of matrix remodeling enzymes. Intensive matrix re-organization takes place at the borders of tumor cell nests.

Objectives: Paraplegia following spinal cord ischaemia represents the most severe complication of aortic surgery. Shock wave treatment (SWT) was shown to induce angiogenesis and regeneration in ischaemic tissue. In pre-clinical as well as clinical studies SWT had a favourable effect on ischaemic myocardium. We therefore hypothesised that SWT may have a beneficial effect on spinal cord ischaemia as well. Methods: Aortic cross clamp was performed between left carotid and subclavian artery in 10–12-week-old male C57/Bl6 mice. Animals were randomly divided to treatment group (SWT, 500 impulses at 0.1 mJ/mm2, 5 Hz) and untreated controls (CTR), n = 6. RNA expression of angiogenic and inflammatory cytokines was measured after 24 and 48 h. Immunofluorescence staining for degenerating neurons (Fluoro Jade B) and macrophages (Iba-1) was performed after 7 days. Results: Real-time PCR analysis revealed higher gene expression of angiogenic factors, vascular endothelial growth factor-A (VEGF-A) after 24 h (SWT 0.21 ± 0.06 vs CTR 0.07 ± 0.01, P = 0.028) and 48 h (SWT 0.11 ± 0.02 vs CTR 0.07 ± 0.01, P > 0.05) as well as hypoxia inducible factor-1α (HIF-1α) after 24 h (SWT 0.11 ± 0.04 vs CTR 0.04 ± 0.01, P > 0.05) and 48 h (SWT 0.09 ± 0.02 vs CTR 0.01 ± 0, P = 0.016). Early increase of inflammatory mRNA expression was observed after 24 h by TNF-α (SWT 0.03 ± 0.003 vs CTR 0.005 ± 0.003, P = 0.007) and TGF-β (SWT 0.57 ± 0.05 vs CTR 0.17 ± 0.08, P = 0.003). This resulted in a markedly decreased number of degenerating neurons in the treatment group 7 days after ischaemia (SWT 74.50 ± 8.14 vs CTR 250.2 ± 42.98, P = 0.0025). Conclusion: Shock wave treatment results in a marked decrease of degenerating neurons and may therefore develop as an adjunct to the treatment armamentarium for paraplegia upon aortic cross clamp.

ABSTRACT Introduction Different molecular processes lead to metastatic spread and the occurrence of tumour cell resistance to therapeutic interventions. Among them, the epithelial to mesenchymal transition (EMT) process plays a key role. During EMT, epithelial tumour cells lose the expression of specific proteins and adopt the phenotype of mesenchymal cells. These structural conversions are substantially dependent on the tumour microenvironment. EMT of tumour cells can induce drug resistance and metastasis. Thus, EMT inhibition may offer a new strategy for overcoming tumour progression. Methods To evaluate EMT in a non-small cell lung cancer (NSCLC) model a 2D and a 3D- cell culture system was applied using both the human lung cancer cell line (A549) and the human lung fibroblast cell line (SV-80). For generating 3D cell spheroids, a novel system was established consisting of 96-well hanging drop microtiter plates (InSphero AG, Zurich, Switzerland). 2D co-culture assays were performed in transwell filter inserts (Costar). EMT was induced with transforming growth factor-β (TGF-β) or co-cultivation with fibroblasts in 2D/3D. The switch from epithelial to mesenchymal cells was monitored by Western Blot (WB) analyses of e-cadherin, vimentin and n-cadherin. Furthermore, immunohistochemical analyses of e-cadherin, vimentin, α-smooth muscle actin, fibronectin, KI-67 and CA-IX were done on paraffin embedded spheroids. Results EMT could be induced in the 2D not only by incubating tumour cells with TGF-β but also by co-culturing them with fibroblasts in transwell filter inserts. In A549 cells a change in morphology as well as in protein expression defined by WB analysis (down regulation: E-cadherin; up regulation; Vimentin, n-cadherin) could be detected. When cultivated in the 3D system, A549 cells showed an up regulation of the mesenchymal protein vimentin without TGF-s stimulation. Furthermore, a significant up regulation of vimentin, KI-67, CA-IX, and a slight down regulation of e-cadherin could be measured compared to monocultures. Conclusion 3D culture represents a model to study EMT in tumour cell lines without addition of growth factors and thus reflects in vivo conditions closer than 2D culture. Disclosure All authors have declared no conflicts of interest.

ABSTRACT Background Chemokines and their receptors have been shown to play a critical role in cancer growth and metastasis. In particular, recent data have suggested that the chemokine CXCL12 and its receptor CXCR7 (also known as RDC1), which has been recently identified as a chemokine receptor, have key functions in promoting tumor development and progression. However, there is little information regarding their expression and clinical relevance in gastric cancer. Here we investigated for the first time the effects of combined CXCR7 and CXCL12 expression on the prognosis of patients with gastric cancer. Methods We studied CXCL12 and CXCR7 protein expression in 221 specimens of primary gastric cancer using immunohistochemistry, and investigated the relationaship between CXCL12/CXCR7 expression and clinicopathological features and clinical outcomes. Results Patients were categorized into four groups according to CXCR7 and CXCL12 expression: low CXCR7/low CXCL12, high CXCR7/low CXCL12, low CXCR7/high CXCL12, and high CXCR7/high CXCL12. No significant differences existed in age, gender, histology, tumor location, lymphovascular invasion among the four groups. However, high CXCR7/high CXCL12 expression in tumor cells was significantly associated with invasion depth of the tumor (T status; P  5 cm (P = 0.006) compared to tumors with low CXCR7/low CXCL12 expression or high CXCR7/low CXCL12–low CXCR7/high CXCL12 expression. Furthermore, patients with high CXCR7/high CXCL12 expression had the worst prognosis (5-year survival rate 30.6%; median, 2.3 years; range, 0.1 - 5.7 years) compared to those of other patient groups (5-year survival rate, 52.4%; median, not reached; log-rank test, P = 0.008) . Conclusions CXCR7 and CXCL12 are useful prognostic factors in gastric cancer, and the combination of high CXCR7 protein expression with high CXCL12 expression suggests a dismal prognosis. Disclosure All authors have declared no conflicts of interest.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder which primarily affects motor neurons. Eight cases of ALS and seven control cases were studied with semiquantitative immunocytochemistry for chromogranin A, chromogranin B and secretogranin II that are soluble constituents of large dense core vesicles, synaptophysin as a membrane protein of small synaptic vesicles and superoxide dismutase 1. Among the chromogranin peptides, the number and staining intensity of motor neurons was highest for chromogranin A. In ALS, the staining intensity for chromogranin peptides and synaptophysin was significantly lower in the ventral horn of ALS patients due to a loss in immunoreactive motor neurons, varicose fibers and varicosities. For all chromogranins, the remaining motor neurons displayed a characteristic staining pattern consisting of an intracellular accumulation of immunoreactivity with a high staining intensity. Confocal microscopy of motor neurons revealed that superoxide dismutase 1-immunopositive intracellular aggregates also contained chromogranin A, chromogranin B and secretogranin II. These findings indicate that there is a loss of small and large dense core vesicles in presynaptic terminals. The intracellular co-occurrence of superoxide dismutase 1 and chromogranins may suggest a functional interaction between these proteins. This study should prompt further experiments to elucidate the role of chromogranins in ALS patients.

Primary ciliary dyskinesia (PCD) is an autosomal recessive inherited disease characterized by abnormal ciliary motion and impaired mucociliary clearance. The prevalence of PCD is approximately 1 : 15 000 - 1 : 20 000 in live births. Cilia dysfunction is also implicated in a wider spectrum of diseases due to impaired organ genesis and body symmetry. Cilia are highly conserved in animals and show complex structures containing more than 250 proteins for their formation. Recent studies have begun to locate the PCD genes in the genome and characterize functional mutations. Specific diagnosis of the ciliary dysfunction requires physiological measurements as well as light- and electron microscopy. Abnormalities in ciliary motion and ultrastructural studies can be performed with nasal mucosal epithelium.

Primary ciliary dyskinesia (PCD) is an autosomal recessive inherited disease characterized by abnormal ciliary motion and impaired mucociliary clearance. The prevalence of PCD is approximately 1 : 15 000 - 1 : 20 000 in live births. Cilia dysfunction is also implicated in a wider spectrum of diseases due to impaired organ genesis and body symmetry. Cilia are highly conserved in animals and show complex structures containing more than 250 proteins for their formation. Recent studies have begun to locate the PCD genes in the genome and characterize functional mutations. Specific diagnosis of the ciliary dysfunction requires physiological measurements as well as light- and electron microscopy. Abnormalities in ciliary motion and ultrastructural studies can be performed with nasal mucosal epithelium.

The human vagal/nucleus solitary complex is a primary visceral relay station and an integrative brain stem area which displays a high density of chromogranin B- and secretoneurin-like immunoreactivity. In this study, we localized and biochemically identified these proteins during prenatal development. At prenatal week 11, 15, 20 and 37, we performed a chromatographic analysis to identify the molecular forms of PE-11, a peptide within the chromogranin B sequence, and secretoneurin, a peptide within secretogranin II. Their localization was studied with immunocytochemistry, and was compared to that of substance P which is well established as a functional neuropeptide in the vagal/nucleus solitary complex. At prenatal week 11, chromogranin B-, secretoneurin- and substance P-like immunoreactivities were detected consisting of varicosities, varicose fibers and single cells. At the same time, PE-11 and secretoneurin appeared as a single peak in chromatographic analysis. Prohormone convertases PC1- and PC2-like immunoreactivities were also present at week 11. In general, the density for each peptide increased during later fetal stages with the highest density at week 37. These results demonstrate that each chromogranin peptide is expressed during human fetal life in neurons of the vagal/nucleus solitary complex indicating that these peptides could be important during prenatal development.

Chromogranin A (CgA) belongs to the family of chromogranin peptides which are contained in large dense-core vesicles. The novel CgA fragment catestatin (bovine CgA(344-364); RSMRLSFRARGYGFRGPGLQL) is a potent inhibitor of catecholamine release by acting as a nicotinic cholinergic antagonist. Catestatin is a recently characterized neuropeptide, consisting of 21 amino acids, which might play an autocrine regulatory role in neuroendocrine secretion through its interaction with different nicotinic acetylcholine receptor subtypes. This study investigates for the first time the distribution of this peptide in the human auditory system using immunohistochemistry. A high density of catestatin-like immunoreactivity (catestatin-LI) is located in the spiral ganglion cells. In the dorsal cochlear nucleus, a high density of catestatin-LI consists of varicose fibers, immunoreactive varicosities and immunoreactive neurons. A moderate density is detected in the ventral cochlear and the medial vestibular nucleus. A low density is found in the inferior colliculus and superior olivary complex. The study indicates that catestatin is distinctly distributed in the auditory system, suggesting a role as a neuromodulatory peptide. Further studies should elucidate a possible interaction with other neurotransmitters in the auditory system.