Vascular smooth muscle cell-derived exosomes promote osteoblast-to-osteocyte transition via ß-catenin signaling.

Blood vessel growth and osteogenesis in the skeletal system are coupled; however, fundamental aspects of vascular function in osteoblast-to-osteocyte transition remain unclear. Our study demonstrates that vascular smooth muscle cells (VSMCs), but not endothelial cells, are sufficient to drive bone marrow mesenchymal stromal cell-derived osteoblast-to-osteocyte transition via ß-catenin signaling and exosome-mediated communication. We found that VSMC-derived exosomes are loaded with transcripts encoding proteins associated with the osteocyte phenotype and members of the WNT/ß-catenin signaling pathway. In contrast, endothelial cell-derived exosomes facilitated mature osteoblast differentiation by reprogramming the TGFB1 gene family and osteogenic transcription factors osterix (SP7) and RUNX2. Notably, VSMCs express significant levels of tetraspanins (CD9, CD63, and CD81) and drive the intracellular trafficking of exosomes with a lower membrane zeta potential than those from other cells. Additionally, the high ATP content within these exosomes supports mineralization mechanisms, as ATP is a substrate for alkaline phosphatase. Osteocyte function was further validated by RNA sequencing, revealing activity in genes related to intermittent mineralization and sonic hedgehog signaling, alongside a significant increase in TNFSF11 levels. Our findings unveil a novel role of VSMCs in promoting osteoblast-to-osteocyte transition, thus offering new insights into bone biology and homeostasis, as well as in bone-related diseases. Clinically, these insights could pave the way for innovative therapeutic strategies targeting VSMC-derived exosome pathways to treat bone-related disorders such as osteoporosis. By manipulating these signaling pathways, it may be possible to enhance bone regeneration and improve skeletal health in patients with compromised bone structure and function.

Toll-Like Receptor 1 Locus Re-examined in a Genome-Wide Association Study Update on Anti-Helicobacter pylori IgG Titers.

BACKGROUND & AIMS: A genome-wide significant association between anti-Helicobacter pylori (H pylori) IgG titers and Toll-like receptor (TLR1/6/10) locus on 4p14 was demonstrated for individuals of European ancestry, but not uniformly replicated. We re-investigated this association in an updated genome-wide association study (GWAS) meta-analysis for populations with low gastric cancer incidence, address potential causes of cohort heterogeneity, and explore functional implications of genetic variation at the TLR1/6/10 locus. METHODS: The dichotomous GWAS (25% individuals exhibiting highest anti-H pylori IgG titers vs remaining 75%) included discovery and replication sampls of, respectively, n = 15,685 and n = 9676, all of European ancestry. Longitudinal analysis of serologic data was performed on H pylori-eradicated subjects (n = 132) and patients under surveillance for premalignant gastric lesions (n = 107). TLR1/6/10 surface expression, TLR1 mRNA, and cytokine levels were measured in leukocyte subsets of healthy subjects (n = 26) genotyped for TLR1/6/10 variants. RESULTS: The association of the TLR1/6/10 locus with anti-H pylori IgG titers (rs12233670; ß = -0.267 ± SE 0.034; P = 4.42 × 10-15) presented with high heterogeneity and failed replication. Anti-H pylori IgG titers declined within 2-4 years after eradication treatment (P = 0.004), and decreased over time in patients with premalignant gastric lesions (P < 0.001). Variation at the TLR1/6/10 locus affected TLR1-mediated cytokine production and TLR1 surface expression on monocytes (P = 0.016) and neutrophils (P = 0.030), but not mRNA levels. CONCLUSIONS: The association between anti-H pylori IgG titers and TLR1/6/10 locus was not replicated across cohorts, possibly owing to dependency of anti-H pylori IgG titers on therapy, clearance, and antibody decay. H pylori-mediated immune cell activation is partly mediated via TLR1 signaling, which in turn is affected by genetic variation.

An 8q24 Gain in Pancreatic Juice Is a Candidate Biomarker for the Detection of Pancreatic Cancer.

Secretin-stimulated pancreatic juice (PJ), collected from the duodenum, presents a valuable biomarker source for the (earlier) detection of pancreatic cancer (PC). Here, we evaluate the feasibility and performance of shallow sequencing to detect copy number variations (CNVs) in cell-free DNA (cfDNA) from PJ for PC detection. First, we confirmed the feasibility of shallow sequencing in PJ (n = 4), matched plasma (n = 3) and tissue samples (n = 4, microarray). Subsequently, shallow sequencing was performed on cfDNA from PJ of 26 cases (25 sporadic PC, 1 high-grade dysplasia) and 19 controls with a hereditary or familial increased risk of PC. 40 of the 45 PJ samples met the quality criteria for cfDNA analysis. Nine individuals had an 8q24 gain (oncogene MYC; 23%; eight cases (33%) and one control (6%), p = 0.04); six had both a 2q gain (STAT1) and 5p loss (CDH10; 15%; four cases (7%) and two controls (13%), p = 0.72). The presence of an 8q24 gain differentiated the cases and controls, with a sensitivity of 33% (95% CI 16-55%) and specificity of 94% (95% CI 70-100%). The presence of either an 8q24 or 2q gain with a 5p loss was related to a sensitivity of 50% (95% CI 29-71%) and specificity of 81% (95% CI 54-96%). Shallow sequencing of PJ is feasible. The presence of an 8q24 gain in PJ shows promise as a biomarker for the detection of PC. Further research is required with a larger sample size and consecutively collected samples in high-risk individuals prior to implementation in a surveillance cohort.

Mutation Analysis of Pancreatic Juice and Plasma for the Detection of Pancreatic Cancer.

Molecular profiling may enable earlier detection of pancreatic cancer (PC) in high-risk individuals undergoing surveillance and allow for personalization of treatment. We hypothesized that the detection rate of DNA mutations is higher in pancreatic juice (PJ) than in plasma due to its closer contact with the pancreatic ductal system, from which pancreatic cancer cells originate, and higher overall cell-free DNA (cfDNA) concentrations. In this study, we included patients with pathology-proven PC or intraductal papillary mucinous neoplasm (IPMN) with high-grade dysplasia (HGD) from two prospective clinical trials (KRASPanc and PACYFIC) for whom both PJ and plasma were available. We performed next-generation sequencing on PJ, plasma, and tissue samples and described the presence (and concordance) of mutations in these biomaterials. This study included 26 patients (25 PC and 1 IPMN with HGD), of which 7 were women (27%), with a median age of 71 years (IQR 12) and a median BMI of 23 kg/m2 (IQR 4). Ten patients with PC (40%) were (borderline) resectable at baseline. Tissue was available from six patients (resection n = 5, biopsy n = 1). A median volume of 2.9 mL plasma (IQR 1.0 mL) and 0.7 mL PJ (IQR 0.1 mL, p < 0.001) was used for DNA isolation. PJ had a higher median cfDNA concentration (2.6 ng/µL (IQR 4.2)) than plasma (0.29 ng/µL (IQR 0.40)). A total of 41 unique somatic mutations were detected: 24 mutations in plasma (2 KRAS, 15 TP53, 2 SMAD4, 3 CDKN2A 1 CTNNB1, and 1 PIK3CA), 19 in PJ (3 KRAS, 15 TP53, and 1 SMAD4), and 8 in tissue (2 KRAS, 2 CDKN2A, and 4 TP53). The mutation detection rate (and the concordance with tissue) did not differ between plasma and PJ. In conclusion, while the concentration of cfDNA was indeed higher in PJ than in plasma, the mutation detection rate was not different. A few cancer-associated genetic variants were detected in both biomaterials. Further research is needed to increase the detection rate and assess the performance and suitability of plasma and PJ for PC (early) detection.

Kinome profiling of cholangiocarcinoma organoids reveals potential druggable targets that hold promise for treatment stratification.

BACKGROUND: Cholangiocarcinoma is a rare but lethal cancer of the biliary tract. Its first-line treatment is currently restricted to chemotherapy, which provides limited clinical benefit. Kinase inhibitors targeting oncogenic intracellular signaling have changed the treatment paradigm of cancer over the last decades. However, they are yet to be widely applied in cholangiocarcinoma therapy. Cholangiocarcinoma has marked molecular heterogeneity, which complicates the discovery of new treatments and requires patient stratification. Therefore, we investigated whether a commercial kinome profiling platform could predict druggable targets in cholangiocarcinoma. METHODS: Kinase activity in patient-derived cholangiocarcinoma organoids, non-tumorous adjacent tissue-derived and healthy donor-derived intrahepatic cholangiocyte organoids was determined using the PamChip® phosphotyrosine kinase microarray platform. Kinome profiles were compared and correlated with RNA sequencing and (multi-)kinase inhibitor screening of the cholangiocarcinoma organoids. RESULTS: Kinase activity profiles of individual cholangiocarcinoma organoids are different and do not cluster together. However, growth factor signaling (EGFR, PDGFRß) and downstream effectors (MAPK pathway) are more active in cholangiocarcinoma organoids and could provide potential druggable targets. Screening of 31 kinase inhibitors revealed several promising pan-effective inhibitors and compounds that show patient-specific efficacy. Kinase inhibitor sensitivity correlated to the activity of its target kinases for several inhibitors, signifying them as potential predictors of response. Moreover, we identified correlations between drug response and kinases not directly targeted by those drugs. CONCLUSIONS: In conclusion, kinome profiling is a feasible method to identify druggable targets for cholangiocarcinoma. Future studies should confirm the potential of kinase activity profiles as biomarkers for patient stratification and precision medicine.

Extracellular vesicle-derived microRNAs in pancreatic juice as biomarkers for detection of pancreatic ductal adenocarcinoma.

INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) is usually diagnosed in an advanced stage, with minimal likelihood of long-term survival. Only a small subset of patients are diagnosed with early (T1) disease. Early detection is challenging due to the late onset of symptoms and limited visibility of sub-centimeter cancers on imaging. A novel approach is to support the clinical diagnosis with molecular markers. MicroRNA derived from extracellular vehicles (EVs) in blood has shown promise as a potential biomarker for pancreatic neoplasia, but microRNA derived from pancreatic juice (PJ) may be a more sensitive biomarker, given that is in close contact with ductal cells from which PDAC arises. This study aims to evaluate and compare the performance of PJ- and serum-derived EV-miRNA for the detection of PDAC. METHODS: PJ was collected from the duodenum during EUS after secretin stimulation from 54 patients with PDAC and 118 non-malignant controls. Serum was available for a subset of these individuals. MiR-16, miR-21, miR-25, miR-155 and miR-210 derived from EVs isolated from PJ and serum were analyzed by qPCR, and serum CA19-9 levels were determined by electrochemiluminescence immunoassay. For statistical analysis, either a Mann-Whitney U test or a Wilcoxon Signed Rank test was performed. ROC curves and AUC were used to assess the sensitivity and specificity of miR expression for PDAC detection. RESULTS: Expression of EV-miR-21, EV-miR-25 and EV-miR-16 were increased in cases vs controls in PJ, while only EV-miR-210 was increased in serum. The potential to detect PC was good for a combination of PJ EV-miR-21, EV-miR-25, EV-miR-16 and serum miR-210, CA-19-9, with an area under the curve of 0.91, a specificity of 84.2% and a sensitivity of 81.5%. CONCLUSION: Detection of miRNA from EVs in PJ is feasible. A combined panel of PJ EV-miR-21, EV-miR-25, EV-miR-16, and serum EV-miR-210 and CA19-9 distinguishes cases with PDAC from controls undergoing surveillance with a specificity of 81.5% and sensitivity of 84.2%.

Protein biomarkers in pancreatic juice and serum for identification of pancreatic cancer.

BACKGROUND AND AIMS: To date, surveillance of high-risk individuals for pancreatic ductal adenocarcinoma (PDAC) has not lived up to expectations, as identification of curable stages through imaging remains challenging. Biomarkers are therefore needed. Pancreatic juice (PJ) may be a promising source, because it is in direct contact with the ductal epithelial lining from which PDAC arises. We aimed to develop a panel of biomarkers from serum and PJ to detect PDAC for future surveillance purposes. METHODS: All patients who underwent PJ collection on secretin stimulation at the Erasmus MC were included. Both PJ and serum were evaluated. Protein levels were determined by the Lowry assay. Potential biomarkers (interleukin-8, interferon-γ, neutrophil gelatinase-associated lipocalin [NGAL], mucin 5, subtype AC [MUC5AC], mucin 2, phospholipase A2 group IB) were selected based on previously reported outcomes and assessed with enzyme-linked immunosorbent assay. Serum carbohydrate antigen 19-9 (CA19-9) values were determined by electrochemiluminescence immunoassay. RESULTS: This study included 59 cases and 126 surveilled control subjects (who underwent PJ collection), of whom 71 had a hereditary predisposition (35 genetic, 36 familial) and 55 had (suspected neoplastic) pancreatic cysts. CA19-9 values were available for 53 cases and 48 control subjects. Serum CA19-9, as well as PJ interleukin-8, NGAL and MUC5AC, were associated with PDAC independent of age, gender, and presence of diabetes mellitus. Serum CA19-9 had a significantly higher area under the curve (AUC; .86; 95% confidence interval [CI], .79-.94) than individual PJ markers (AUC, .62-.70). A combination of PJ markers and serum CA19-9 (panel 2: sensitivity 42% [95% CI, 29-57] and specificity 96% [95% CI, 86-100]) did not improve diagnostic performance compared with CA19-9 alone (sensitivity 70% [95% CI, 56-82] and specificity 85% [95% CI, 72-94]). CONCLUSIONS: High levels of serum CA19-9 and PJ-derived proteins are associated with PDAC. Prospective surveillance studies including individuals at risk of developing PDAC are required to validate these findings.

Constitutive programmed death ligand 1 expression protects gastric G-cells from Helicobacter pylori-induced inflammation.

INTRODUCTION: Gastric intestinal metaplasia (GIM) is a premalignant lesion, highly associated with Helicobacter pylori infection. Previous studies have shown that H. pylori is able to induce the expression of programmed death ligand 1 (PD-L1), an inhibitory immune modulator, in gastric cells. Our aim was to investigate whether tissues from GIM patients may exploit PD-L1 expression upon H. pylori infection to evade immunosurveillance. METHODS: Immunohistochemistry was performed for PD-L1 and enteroendocrine markers somatostatin and gastrin on samples derived from a cohort of patients with known GIM, both before and after H. pylori eradication. To determine the identity of any observed PD-L1-positive cells, we performed multiplex immunofluorescent staining and analysis of single-cell sequencing data. RESULTS: GIM tissue was rarely positive for PD-L1. In normal glands from GIM patients, PD-L1 was mainly expressed by gastrin-positive G-cells. While the D-cell and G-cell compartments were both diminished 2-fold (p = .015 and p = .01, respectively) during H. pylori infection in the normal antral tissue of GIM patients, they were restored 1 year after eradication. The total number of PD-L1-positive cells was not affected by H. pylori, but the percentage of PD-L1-positive G-cells was 30% higher in infected subjects (p = .011), suggesting that these cells are preferentially rescued from destruction. CONCLUSIONS: Antral G-cells frequently express PD-L1 during homeostasis. G-cells seem to be protected from H. pylori-induced immune destruction by PD-L1 expression. GIM itself does not express PD-L1 and is unlikely to escape immunosurveillance via expression of PD-L1.

Platelets in aging and cancer-"double-edged sword".

Platelets control hemostasis and play a key role in inflammation and immunity. However, platelet function may change during aging, and a role for these versatile cells in many age-related pathological processes is emerging. In addition to a well-known role in cardiovascular disease, platelet activity is now thought to contribute to cancer cell metastasis and tumor-associated venous thromboembolism (VTE) development. Worldwide, the great majority of all patients with cardiovascular disease and some with cancer receive anti-platelet therapy to reduce the risk of thrombosis. However, not only do thrombotic diseases remain a leading cause of morbidity and mortality, cancer, especially metastasis, is still the second cause of death worldwide. Understanding how platelets change during aging and how they may contribute to aging-related diseases such as cancer may contribute to steps taken along the road towards a "healthy aging" strategy. Here, we review the changes that occur in platelets during aging, and investigate how these versatile blood components contribute to cancer progression.

Genetic host factors in Helicobacter pylori-induced carcinogenesis: Emerging new paradigms.

Helicobacter Pylori is a gram negative rod shaped microaerophilic bacterium that colonizes the stomach of approximately half the world's population. Infection with c may cause chronic gastritis which via a quite well described process known as Correas cascade can progress through sequential development of atrophic gastritis, intestinal metaplasia and dysplasia to gastric cancer. H. pylori is currently the only bacterium that is classified as a class 1 carcinogen by the WHO, although the exact mechanisms by which this bacterium contributes to gastric carcinogenesis are still poorly understood. Only a minority of H. pylori-infected patients will eventually develop gastric cancer, suggesting that host factors may be important in determining the outcome of H. pylori infection. This is supported by a growing body of evidence suggesting that the host genetic background contributes to risk of H. pylori infection and gastric carcinogenesis. In particular single nucleotide polymorphisms in genes that influence bacterial handling via pattern recognition receptors appear to be involved, further strengthening the link between host risk factors, H. pylori incidence and cancer. Many of these genes influence cellular pathways leading to inflammatory signaling, inflammasome formation and autophagy. In this review we summarize known carcinogenic effects of H. pylori, and discuss recent findings that implicate host genetic pattern recognition pathways in the development of gastric cancer and their relation with H. pylori.

Accuracy of upper endoscopies with random biopsies to identify patients with gastric premalignant lesions who can safely be exempt from surveillance.

INTRODUCTION: Guidelines recommend endoscopy with biopsies to stratify patients with gastric premalignant lesions (GPL) to high and low progression risk. High-risk patients are recommended to undergo surveillance. We aimed to assess the accuracy of guideline recommendations to identify low-risk patients, who can safely be discharged from surveillance. METHODS: This study includes patients with GPL. Patients underwent at least two endoscopies with an interval of 1-6 years. Patients were defined 'low risk' if they fulfilled requirements for discharge, and 'high risk' if they fulfilled requirements for surveillance, according to European guidelines (MAPS-2012, updated MAPS-2019, BSG). Patients defined 'low risk' with progression of disease during follow-up (FU) were considered 'misclassified' as low risk. RESULTS: 334 patients (median age 60 years IQR11; 48.7% male) were included and followed for a median of 48 months. At baseline, 181/334 (54%) patients were defined low risk. Of these, 32.6% were 'misclassified', showing progression of disease during FU. If MAPS-2019 were followed, 169/334 (51%) patients were defined low risk, of which 32.5% were 'misclassified'. If BSG were followed, 174/334 (51%) patients were defined low risk, of which 32.2% were 'misclassified'. Seven patients developed gastric cancer (GC) or dysplasia, four patients were 'misclassified' based on MAPS-2012 and three on MAPS-2019 and BSG. By performing one additional endoscopy 72.9% (95% CI 62.4-83.3) of high-risk patients and all patients who developed GC or dysplasia were identified. CONCLUSION: One-third of patients that would have been discharged from GC surveillance, appeared to be 'misclassified' as low risk. One additional endoscopy will reduce this risk by 70%.

Could Histamine H1 Receptor Antagonists Be Used for Treating COVID-19?

COVID-19 has rapidly become a pandemic worldwide, causing extensive and long-term health issues. There is an urgent need to identify therapies that limit SARS-CoV-2 infection and improve the outcome of COVID-19 patients. Unbalanced lung inflammation is a common feature in severe COVID-19 patients; therefore, reducing lung inflammation can undoubtedly benefit the clinical manifestations. Histamine H1 receptor (H1 receptor) antagonists are widely prescribed medications to treat allergic diseases, while recently it has emerged that they show significant promise as anti-SARS-CoV-2 agents. Here, we briefly summarize the novel use of H1 receptor antagonists in combating SARS-CoV-2 infection. We also describe the potential antiviral mechanisms of H1 receptor antagonists on SARS-CoV-2. Finally, the opportunities and challenges of the use of H1 receptor antagonists in managing COVID-19 are discussed.

Drug Discovery in Liver Disease Using Kinome Profiling.

The liver is one of the most important organs, playing critical roles in maintaining biochemical homeostasis. Accordingly, disease of the liver is often debilitating and responsible for untold human misery. As biochemical nexus, with kinases being master regulators of cellular biochemistry, targeting kinase enzymes is an obvious avenue for treating liver disease. Development of such therapy, however, is hampered by the technical difficulty of obtaining comprehensive insight into hepatic kinase activity, a problem further compounded by the often unique aspects of hepatic kinase activities, which makes extrapolations from other systems difficult. This consideration prompted us to review the current state of the art with respect to kinome profiling approaches towards the hepatic kinome. We observe that currently four different approaches are available, all showing significant promise. Hence we postulate that insight into the hepatic kinome will quickly increase, leading to rational kinase-targeted therapy for different liver diseases.

Smoothened-dependent and -independent pathways in mammalian noncanonical Hedgehog signaling.

Hedgehog proteins are pivotal morphogens acting through a canonical pathway involving first activation of ligand binding to Patched followed by alleviation of Smoothened receptor inhibition, leading to activation of Gli transcription factors. Noncanonical Hedgehog signaling remains poorly characterized but is thought to be mainly dependent on Smoothened. However, Smoothened inhibitors have yielded only partial success in combating Hedgehog signal transduction-dependent cancer, suggesting that noncanonical Smoothened-independent pathways also are clinically relevant. Moreover, several Smoothened-dependent effects (e.g. neurite projection) do not require transcriptional activation, further suggesting biological importance of noncanonical Smoothened-dependent pathways. We comprehensively characterized the cellular kinome in Hedgehog-challenged murine WT and Smoothened-/- fibroblasts as well as Smoothened agonist-stimulated cells. A peptide assay-based kinome analysis (in which cell lysates are used to phosphorylate specific kinase substrates), along with endocytosis, Lucifer Yellow-based, and immunoblotting assays, identified an elaborate signaling network of both Smoothened-dependent and -independent pathways that mediates actin reorganization through Src-like kinases, activates various proinflammatory signaling cascades, and concomitantly stimulates Wnt and Notch signaling while suppressing bone morphogenetic protein (BMP) signaling. The contribution of noncanonical Smoothened-independent signaling to the overall effects of Hedgehog on cellular physiology appears to be much larger than previously envisioned and may explain the transcriptionally independent effects of Hedgehog signaling on cytoskeleton. The observation that Patched-dependent, Smoothened-independent, noncanonical Hedgehog signaling increases Wnt/Notch signaling provides a possible explanation for the failure of Smoothened antagonists in combating Hedgehog-dependent but Smoothened inhibitor-resistant cancer. Our findings suggest that inhibiting Hedgehog-Patched interaction could result in more effective therapies as compared with conventional Smoothened-directed therapies.

Optimization of Pancreatic Juice Collection: A First Step Toward Biomarker Discovery and Early Detection of Pancreatic Cancer.

INTRODUCTION: Imaging-based surveillance programs fail to detect pancreatic ductal adenocarcinoma at a curable stage, creating an urgent need for diagnostic biomarkers. METHODS: Secretin-stimulated pancreatic juice (PJ) was collected from the duodenal lumen during endoscopic ultrasound. The yield of biomarkers and organoids was compared for 2 collection techniques (endoscope suction channel vs catheter-based) and 3 periods (0-4 vs 4-8 vs 8-15 minutes). RESULTS: Collection through the endoscope suction channel was superior to collection with a catheter. Collection beyond 8 minutes reduced biomarker yield. PJ-derived organoid culture was feasible. DISCUSSION: The optimal protocol for secretin-stimulated PJ collection is through the endoscope suction channel for 8 minutes allowing biomarker detection and organoid culture.

Biotech-Educated Platelets: Beyond Tissue Regeneration 2.0.

The increasing discoveries regarding the biology and functions of platelets in the last decade undoubtedly show that these cells are one of the most biotechnological human cells. This review summarizes new advances in platelet biology, functions, and new concepts of biotech-educated platelets that connect advanced biomimetic science to platelet-based additive manufacturing for tissue regeneration. As highly responsive and secretory cells, platelets could be explored to develop solutions that alter injured microenvironments through platelet-based synthetic biomaterials with instructive extracellular cues for morphogenesis in tissue engineering beyond tissue regeneration 2.0.

HOXA9 mediates and marks premalignant compartment size expansion in colonic adenomas.

The transformation of normal colonic epithelium to colorectal cancer (CRC) involves a relatively ordered progression, and understanding the molecular alterations involved may aid rational design of strategies aimed at preventing or counteracting disease. Homeobox A9 (HOXA9) is an oncogene in leukemia and has been implicated in CRC pathology, although its role in disease etiology remains obscure at best. We observe that HOXA9 expression is increased in colonic adenomas compared with location-matched healthy colon epithelium. Its forced expression results in dramatic genetic and signaling changes, with increased expression of growth factors IGF1 and FLT3, super-activity of the AKT survival pathway and a concomitant increase in compartment size. Furthermore, a reduced mRNA expression of the epithelial to mesenchymal transition marker N-cadherin as well as reduced activity of the actin cytoskeletal mediator PAK was seen, which is in apparent agreement with an observed reduced migratory response in HOXA9-overexpressing cells. Thus, HOXA9 appears closely linked with adenoma growth while impairing migration and metastasis and hence is both a marker and driver of premalignant polyp growth. Colonic polyps grow but remain premalignant for up to decades. Here, we show that HOXA9 drives growth in premalignant polyps, but simultaneously prevents further transformation.

Human mesenchymal stromal cells in adhesion to cell-derived extracellular matrix and titanium: Comparative kinome profile analysis.

The extracellular matrix (ECM) physically supports cells and influences stem cell behaviour, modulating kinase-mediated signalling cascades. Cell-derived ECMs have emerged in bone regeneration as they reproduce physiological tissue-architecture and ameliorate mesenchymal stromal cell (MSC) properties. Titanium scaffolds show good mechanical properties, facilitate cell adhesion, and have been routinely used for bone tissue engineering (BTE). We analyzed the kinomic signature of human MSCs in adhesion to an osteopromotive osteoblast-derived ECM, and compared it to MSCs on titanium. PamChip kinase-array analysis revealed 63 phosphorylated peptides on ECM and 59 on titanium, with MSCs on ECM exhibiting significantly higher kinase activity than on titanium. MSCs on the two substrates showed overlapping kinome profiles, with activation of similar signalling pathways (FAK, ERK, and PI3K signalling). Inhibition of PI3K signalling in cells significantly reduced adhesion to ECM and increased the number of nonadherent cells on both substrates. In summary, this study comprehensively characterized the kinase activity in MSCs on cell-derived ECM and titanium, highlighting the role of PI3K signalling in kinomic changes regulating osteoblast viability and adhesion. Kinome profile analysis represents a powerful tool to select pathways to better understand cell behaviour. Osteoblast-derived ECM could be further investigated as titanium scaffold-coating to improve BTE.

Low dose Naltrexone for induction of remission in inflammatory bowel disease patients.

BACKGROUND: Around 30% of patients with inflammatory bowel disease (IBD) are refractory to current IBD drugs or relapse over time. Novel treatments are called for, and low dose Naltrexone (LDN) may provide a safe, easily accessible alternative treatment option for these patients. We investigated the potential of LDN to induce clinical response in therapy refractory IBD patients, and investigated its direct effects on epithelial barrier function. METHODS: Patients not in remission and not responding to conventional therapy were offered to initiate LDN as a concomitant treatment. In total 47 IBD patients prescribed LDN were followed prospectively for 12 weeks. Where available, endoscopic remission data, serum and biopsies were collected. Further the effect of Naltrexone on wound healing (scratch assay), cytokine production and endoplasmic reticulum (ER) stress (GRP78 and CHOP western blot analysis, immunohistochemistry) were investigated in HCT116 and CACO2 intestinal epithelial cells, human IBD intestinal organoids and patient samples. RESULTS: Low dose Naltrexone induced clinical improvement in 74.5%, and remission in 25.5% of patients. Naltrexone improved wound healing and reduced ER stress induced by Tunicamycin, lipopolysaccharide or bacteria in epithelial barriers. Inflamed mucosa from IBD patients showed high ER stress levels, which was reduced in patients treated with LDN. Cytokine levels in neither epithelial cells nor serum from IBD patients were affected. CONCLUSIONS: Naltrexone directly improves epithelial barrier function by improving wound healing and reducing mucosal ER stress levels. Low dose Naltrexone treatment is effective and safe, and could be considered for the treatment of therapy refractory IBD patients.