The N-cadherin ectodomain: fate and finction outside the cancer cell

In the present thesis N-cadherin and its cleaved form soluble N-cadherin (sN-CAD) are studied in relation to cancer. Cadherins are calcium-dependent intercellular adhesion molecules that are often deregulated in cancer. E-cadherin, which is expressed by normal epithelial cells, is downregulated when cells become cancer cells and this sometimes coincides with transactivation of another cadherin, for example N-cadherin. As a result, cells change their phenotype and become fibroblastic, motile and invasive. The role of N-cadherin during cell adhesion, differentiation, embryogenesis and cancer is reviewed, and several examples are given of N-cadherin expression in tumour cell lines and biopsies. Extracellular proteases, also known as the cancer degradome, play an important role during tumour progression; they are involved in processes as invasion and metastasis but also cell proliferation, apoptosis and angiogenesis. The proteases, such as ADAM10, MT1-MMP and plasmin are able to shed a 90 kD extracellular fragment from N-cadherin. We could establish an ELISA for the detection of this sN-CAD. We tested body fluids from persons with no evidence of disease and cancer patients for the presence of sN-CAD. Significantly elevated levels of sN-CAD were measured in the cancer patient group and a weak but significant correlation was found with prostate specific antigen, which is the most frequently used circulating tumour marker for prostate cancer. Furthermore, sN-CAD also has a biological function: it was able to stimulate the motility of N-cadherin positive cells and more specifically of the endothelial cells. By using the chorioallantoic membrane assay and the rabbit corneal micropocket assay we could identify sN-CAD as a proangiogenic molecule. Unravelling the signalling pathway, we proved that the fibroblast growth receptor is an important player in the sN-CAD mediated effects. In conclusion, sN-CAD is a potentially interesting molecule in oncology, both as a circulating tumour marker and as a target for angiostatic treatment

Phylogenetic clustering and rarity imply risk of local species extinction in prospective deep-sea mining areas of the Clarion-Clipperton Fracture Zone

An understanding of the forces controlling community structure in the deep sea is essential at a time when its pristineness is threatened by polymetallic nodule mining. Because abiotically defined communities are more sensitive to environmental change, we applied occurrence- and phylogeny-based metrics to determine the importance of biotic versus abiotic structuring processes in nematodes, the most abundant invertebrate taxon of the Clarion-Clipperton Fracture Zone (CCFZ), an area targeted for mining. We investigated the prevalence of rarity and the explanatory power of environmental parameters with respect to phylogenetic diversity (PD). We found evidence for aggregation and phylogenetic clustering in nematode amplicon sequence variants (ASVs) and the dominant genus Acantholaimus, indicating the influence of environmental filtering, sympatric speciation, affinity for overlapping habitats and facilitation for community structure. PD was associated with abiotic variables such as total organic carbon, chloroplastic pigments equivalents and/or mud content, explaining up to 57% of the observed variability and providing further support of the prominence of environmental structuring forces. Rarity was high throughout, ranging from 64 to 75% unique ASVs. Communities defined by environmental filtering with a prevalence of rarity in the CCFZ suggest taxa of these nodule-bearing abyssal plains will be especially vulnerable to the risk of extinction brought about by the efforts to extract them

Transthyretin levels in the vitreous correlate with change in visual acuity after vitrectomy

Background/aim: Little is known about biochemical markers related to change in visual acuity after vitrectomy. The potential use of transthyretin (TTR), a carrier of the retinol/retinol-binding protein, as a biochemical marker protein, was investigated. Methods: TTR was measured using immunonephelometry in a group of patients (n = 77) in longstanding (> 1 week) retinal detachment (n = 29), fresh (< 1 week) retinal detachment (n = 17), macular holes (n = 20) or diabetic retinopathy (n = 11). Vitreous samples were taken at the start of every vitrectomy procedure. For reference values, cadaver specimens (n = 73) were used. Results: Reference values for vitreous TTR (median 18 mg/l; IQR 4 to 24 mg/l) comprised 2.2% of reference values for vitreous protein levels (median 538 mg/l; IQR 269 to 987 mg/l). Vitreous TTR values of patients were comparable in all disorders. Vitreous TTR values were higher in phakic (median 22.5 mg/l; IQR 10 to 27 mg/l) than in pseudophakic patients (median 12 mg/l; IQR 8 to 19 mg/l; p = 0.06). Postoperative change in visual acuity correlated well with vitreous TTR values found peroperatively (r(s) = 0.408; p = 0.012). Both change in visual acuity and lens status were the only variables which proved to explain the variance of TTR (multiple correlation coefficient: 0.494; phakic status: t = 2.767; p = 0.0084; and change in visual acuity t = 2.924: p = 0.0056). Conclusion: Vitreous fluid concentrations of TTR can be regarded as a biochemical marker for retinal function

The Heregulin/Human Epidermal Growth Factor Receptor as a New Growth Factor System in Melanoma with Multiple Ways of Deregulation

In a screening for new growth factors released by melanoma cells, we found that the p185-phosphorylating capacity of a medium conditioned by a melanoma cell line was due to the secretion of heregulin, a ligand for the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases. Expression of heregulin, including a new isoform, and secretion of functionally active protein was found in several cell lines. Receptor activation by heregulin, either autocrine or paracrine, resulted in a potent growth stimulation of both melanocytes and melanoma cells. Heregulin receptor HER3 and coreceptor HER2 were the main receptors expressed by these cells. Nevertheless, none of the cell lines in our panel overexpressed HER2 or HER3. In contrast, loss of HER3 was found in two cell lines, whereas one cell line showed loss of functional HER2, both types of deregulations resulting in unresponsiveness to heregulin. This implies the heregulin/HER system as a possible important physiologic growth regulatory system in melanocytes in which multiple deregulations may occur during progression toward melanoma, all resulting in, or indicating, growth factor independence

Local immunoglobulin E in the nasal mucosa : clinical implications

Immunoglobulin E (IgE) can be highly elevated in the airway mucosa independently of IgE serum levels and atopic status. Mostly, systemic markers are assessed to investigate inflammation in airway disease for research or clinical practice. A more accurate but more cumbersome approach to determine inflammation at the target organ would be to evaluate markers locally. We review evidence for local production of IgE in allergic rhinitis (AR) and chronic rhinosinusitis with nasal polyps (CRSwNP). Diagnostic and therapeutic consequences in clinical practice are discussed. We describe that the airway mucosa has the intrinsic capability to produce IgE. Moreover, not only do IgE-positive B cells reside within the mucosa, but all tools are present locally for affinity maturation by somatic hypermutation (SHM), clonal expansion, and class switch recombination to IgE. Recognizing local IgE in the absence of systemic IgE has diagnostic and therapeutic consequences. Therefore, we emphasize the importance of local IgE in patients with a history of AR or CRSwNP

The HMI™ module: a new tool to study the host-microbiota interaction in the human gastrointestinal tract in vitro

Background: Recent scientific developments have shed more light on the importance of the host-microbe interaction, particularly in the gut. However, the mechanistic study of the host-microbe interplay is complicated by the intrinsic limitations in reaching the different areas of the gastrointestinal tract (GIT) in vivo. In this paper, we present the technical validation of a new device - the Host-Microbiota Interaction (HMI) module - and the evidence that it can be used in combination with a gut dynamic simulator to evaluate the effect of a specific treatment at the level of the luminal microbial community and of the host surface colonization and signaling. Results: The HMI module recreates conditions that are physiologically relevant for the GIT: i) a mucosal area to which bacteria can adhere under relevant shear stress (3 dynes cm-2); ii) the bilateral transport of low molecular weight metabolites (4 to 150 kDa) with permeation coefficients ranging from 2.4 x 10(-6) to 7.1 x 10(-9) cm sec(-1); and iii) microaerophilic conditions at the bottom of the growing biofilm (PmO2 = 2.5 x 10(-4) cm sec(-1)). In a long-term study, the host's cells in the HMI module were still viable after a 48-hour exposure to a complex microbial community. The dominant mucus-associated microbiota differed from the luminal one and its composition was influenced by the treatment with a dried product derived from yeast fermentation. The latter - with known anti-inflammatory properties induced a decrease of pro-inflammatory IL-8 production between 24 and 48 h. Conclusions: The study of the in vivo functionality of adhering bacterial communities in the human GIT and of the localized effect on the host is frequently hindered by the complexity of reaching particular areas of the GIT. The HMI module offers the possibility of co-culturing a gut representative microbial community with enterocyte-like cells up to 48 h and may therefore contribute to the mechanistic understanding of host-microbiome interactions

Metabarcoding free-living marine nematodes using curated 18S and CO1 reference sequence databases for species-level taxonomic assignments

High‐throughput sequencing has the potential to describe biological communities with high efficiency yet comprehensive assessment of diversity with species‐level resolution remains one of the most challenging aspects of metabarcoding studies. We investigated the utility of curated ribosomal and mitochondrial nematode reference sequence databases for determining phylum‐specific species‐level clustering thresholds. We compiled 438 ribosomal and 290 mitochondrial sequences which identified 99% and 94% as the species delineation clustering threshold, respectively. These thresholds were evaluated in HTS data from mock communities containing 39 nematode species as well as environmental samples from Vietnam. We compared the taxonomic description of the mocks generated by two read‐merging and two clustering algorithms and the cluster‐free Dada2 pipeline. Taxonomic assignment with the RDP classifier was assessed under different training sets. Our results showed that 36/39 mock nematode species were identified across the molecular markers (18S: 32, JB2: 19, JB3: 21) in UClust_ref OTUs at their respective clustering thresholds, outperforming UParse_denovo and the commonly used 97% similarity. Dada2 generated the most realistic number of ASVs (18S: 83, JB2: 75, JB3: 82), collectively identifying 30/39 mock species. The ribosomal marker outperformed the mitochondrial markers in terms of species and genus‐level detections for both OTUs and ASVs. The number of taxonomic assignments of OTUs/ASVs was highest when the smallest reference database containing only nematode sequences was used and when sequences were truncated to the respective amplicon length. Overall, OTUs generated more species‐level detections, which were, however, associated with higher error rates compared to ASVs. Genus‐level assignments using ASVs exhibited higher accuracy and lower error rates compared to species‐level assignments, suggesting that this is the most reliable pipeline for rapid assessment of alpha diversity from environmental samples