Role of HGF/SF in tissue regeneration

Nonalcoholic fatty liver disease (NAFLD), and the more advanced stage nonalcoholic steatohepatitis (NASH), is nowadays a major health problem with a global prevalence estimated at 25%. NAFLD patients have an increased risk to develop cirrhosis, end stage liver disease, and hepatocellular carcinoma. While there is an urgent need for effective treatment, unfortunately, there are no approved drugs for this condition. Hepatocyte apoptosis and inflammatory response are key events for NASH development. It was therefore hypothesized that combination therapy with Hepatocyte Growth Factor/Scatter Factor (HGF/SF), the most potent liver survival-promoting factor, and a macrophage-focused immunotherapeutic drug able to switch M1 macrophages to M2 phenotype can cure NASH. Unfortunately, HGF/SF is unsuitable for therapy because it has a limited tissue/organ penetration and distribution due to its high affinity for heparan sulphate proteoglycans (HSPG). Moreover, it has a short half-life and is unstable in physiological buffers. Therefore, our laboratory in collaboration with the group of Dr. Vicogne at Pasteur Institute in Lille designed and produced K1K1, a new engineered fragment of HGF/SF able to overcome the limitations of the parent molecule and to be more suitable as a drug candidate. The stability and the biological activity of K1K1 were compared to HGF/SF, highlighting K1K1 superiority. Furthermore, we investigated the activation of signaling pathways in two liver cell lines after stimulation with K1K1, HGF/SF, and other recombinant proteins, showing that K1K1 was as good as HGF/SF. In order for K1K1 treatment to work, it was important to confirm that the receptor is present, and the pathway is relevant in the diseased tissue. Assessment of MET protein expression with immunohistochemistry (IHC) in formalin-fixed paraffin-embedded (FFPE) liver samples of patients with NAFLD showed that MET receptor is expressed on the hepatocytes and therefore K1K1 could be regarded a good drug candidate for use in future therapy. Finally, we have evaluated the protective effect of K1K1 alone or in combination with macrophage-focused immunotherapeutic drugs on a NASH-on-a-chip model. The combination therapy was found to be the most effective in reducing steatosis, apoptosis, and fibrosis. These promising results highlight K1K1 potential as a new drug candidate for NASH treatment.Nonalcoholic fatty liver disease (NAFLD), and the more advanced stage nonalcoholic steatohepatitis (NASH), is nowadays a major health problem with a global prevalence estimated at 25%. NAFLD patients have an increased risk to develop cirrhosis, end stage liver disease, and hepatocellular carcinoma. While there is an urgent need for effective treatment, unfortunately, there are no approved drugs for this condition. Hepatocyte apoptosis and inflammatory response are key events for NASH development. It was therefore hypothesized that combination therapy with Hepatocyte Growth Factor/Scatter Factor (HGF/SF), the most potent liver survival-promoting factor, and a macrophage-focused immunotherapeutic drug able to switch M1 macrophages to M2 phenotype can cure NASH. Unfortunately, HGF/SF is unsuitable for therapy because it has a limited tissue/organ penetration and distribution due to its high affinity for heparan sulphate proteoglycans (HSPG). Moreover, it has a short half-life and is unstable in physiological buffers. Therefore, our laboratory in collaboration with the group of Dr. Vicogne at Pasteur Institute in Lille designed and produced K1K1, a new engineered fragment of HGF/SF able to overcome the limitations of the parent molecule and to be more suitable as a drug candidate. The stability and the biological activity of K1K1 were compared to HGF/SF, highlighting K1K1 superiority. Furthermore, we investigated the activation of signaling pathways in two liver cell lines after stimulation with K1K1, HGF/SF, and other recombinant proteins, showing that K1K1 was as good as HGF/SF. In order for K1K1 treatment to work, it was important to confirm that the receptor is present, and the pathway is relevant in the diseased tissue. Assessment of MET protein expression with immunohistochemistry (IHC) in formalin-fixed paraffin-embedded (FFPE) liver samples of patients with NAFLD showed that MET receptor is expressed on the hepatocytes and therefore K1K1 could be regarded a good drug candidate for use in future therapy. Finally, we have evaluated the protective effect of K1K1 alone or in combination with macrophage-focused immunotherapeutic drugs on a NASH-on-a-chip model. The combination therapy was found to be the most effective in reducing steatosis, apoptosis, and fibrosis. These promising results highlight K1K1 potential as a new drug candidate for NASH treatment

Prognostic impact of a 3-MicroRNA signature in cytological samples of small cell lung cancer

BACKGROUND: Small cell lung cancer (SCLC) is a highly aggressive neoplasm that accounts for approximately 10% to 15% of lung cancers. In most cases, the diagnosis relies on cytology and needs to be confirmed by immunohistochemistry. Although several genetic and molecular abnormalities have been recorded, molecular markers able to predict the prognosis are still lacking. MicroRNA (miRNA) signatures have been recently proposed as useful biomarkers in lung cancer because of their high stability during standard sample processing. METHODS: Cytological samples for 50 patients with SCLC were collected from primary tumors (n = 25) and metastases (n = 25) by means of fine-needle aspiration (FNA) or bronchial washing (BW); they were fixed in ethanol (FNA) or Duboscq-Brazil fluid (BW). The 3-miRNA panel expression (miR-192, miR-200c, and miR-205) was quantified with a TaqMan polymerase chain reaction miRNA assay and was compared with overall survival (OS) and clinicopathological data. RESULTS: All samples had sufficient RNA for the miRNA expression analysis to be performed, regardless of the sample source or the fixative medium. Patients with a low expression level of the 3-miRNA panel were associated with better OS in univariate (P = .032) and multivariate analyses (P = .022). Moreover, in the group of patients older than the mean age of our cohort (65.8 years), a significant OS advantage (P = .013) was seen for patients with a low expression level of the 3-miRNA panel. CONCLUSIONS: A specific 3-miRNA signature is potentially useful for predicting survival for patients with SCLC, and it may be feasible with cytological samples taken during standard diagnostic procedures

Human papillomavirus genotyping in high‐grade vaginal intraepithelial neoplasia: A multicentric Italian study

This study aimed to analyze the human papillomavirus (HPV) genotype distribution in a large cohort of high-grade vaginal intraepithelial neoplasia (VaIN) (vaginal HSIL, VaIN2/3) patients from two Italian referral centers. We included all patients with histologically confirmed VaIN2/3 from the Department of Surgical Sciences, Sant'Anna Hospital, University of Torino, Torino, Italy, and Ospedale Maggiore della Carità, Novara, Italy, between 2003 and 2022. After the histological evaluation of formalin-fixed paraffin-embedded samples, we performed HPV genotyping with VisionArray HPV Chip 1.0. We detected HPV DNA in 94.4% of VaIN2/3 (168/178), with HPV 16 as the most prevalent genotype, accounting for 51.8% of all infections, 41.2% of VaIN2 and 77.6% of VaIN3 cases. Other frequent genotypes were HPV 58 (8.3%, 10.9% of VaIN2 and 2.0% of VaIN3), HPV 73 (5.4%, 5.0% of VaIN2 and 6.1% of VaIN3), and HPV 31 (5.4%, 6.7% of VaIN2 and 2.0% of VaIN3). 73.2% of VaIN2/3 had a single HPV genotype infection and 26.8% a multiple infection (20.8% a double infection, 4.8% a triple infection, and 1.2% a quadruple infection). Single infection was more frequently present in VaIN3 than VaIN2 (81.6% vs. 69.8%). 69.1% of single infections and 73.3% of multiple infections had one or more genotypes covered by nine-valent HPV vaccine. HPV vaccination is expected to have a large impact on reducing the incidence of vaginal intraepithelial neoplasia

A Novel HGF/SF Receptor (MET) Agonist Transiently Delays the Disease Progression in an Amyotrophic Lateral Sclerosis Mouse Model by Promoting Neuronal Survival and Dampening the Immune Dysregulation

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease with no effective treatment. The Hepatocyte Growth Factor/Scatter Factor (HGF/SF), through its receptor MET, is one of the most potent survival-promoting factors for motor neurons (MN) and is known as a modulator of immune cell function. We recently developed a novel recombinant MET agonist optimized for therapy, designated K1K1. K1K1 was ten times more potent than HGF/SF in preventing MN loss in an in vitro model of ALS. Treatments with K1K1 delayed the onset of muscular impairment and reduced MN loss and skeletal muscle denervation of superoxide dismutase 1 G93A (SOD1G93A) mice. This effect was associated with increased levels of phospho-extracellular signal-related kinase (pERK) in the spinal cord and sciatic nerves and the activation of non-myelinating Schwann cells. Moreover, reduced activated microglia and astroglia, lower T cells infiltration and increased interleukin 4 (IL4) levels were found in the lumbar spinal cord of K1K1 treated mice. K1K1 treatment also prevented the infiltration of T cells in skeletal muscle of SOD1G93A mice. All these protective effects were lost on long-term treatment suggesting a mechanism of drug tolerance. These data provide a rational justification for further exploring the long-term loss of K1K1 efficacy in the perspective of providing a potential treatment for ALS

Bitter taste receptor (TAS2R) 46 in human skeletal muscle: expression and activity

: Bitter taste receptors are involved not only in taste perception but in various physiological functions as their anatomical location is not restricted to the gustatory system. We previously demonstrated expression and activity of the subtype hTAS2R46 in human airway smooth muscle and broncho-epithelial cells, and here we show its expression and functionality in human skeletal muscle cells. Three different cellular models were used: micro-dissected human skeletal tissues, human myoblasts/myotubes and human skeletal muscle cells differentiated from urine stem cells of healthy donors. We used qPCR, immunohistochemistry and immunofluorescence analysis to evaluate gene and protein hTAS2R46 expression. In order to explore receptor activity, cells were incubated with the specific bitter ligands absinthin and 3ß-hydroxydihydrocostunolide, and calcium oscillation and relaxation were evaluated by calcium imaging and collagen assay, respectively, after a cholinergic stimulus. We show, for the first time, experimentally the presence and functionality of a type 2 bitter receptor in human skeletal muscle cells. Given the tendentially protective role of the bitter receptors starting from the oral cavity and following also in the other ectopic sites, and given its expression already at the myoblast level, we hypothesize that the bitter receptor can play an important role in the development, maintenance and in the protection of muscle tissue functions

Table1_Bitter taste receptor (TAS2R) 46 in human skeletal muscle: expression and activity.DOCX

Bitter taste receptors are involved not only in taste perception but in various physiological functions as their anatomical location is not restricted to the gustatory system. We previously demonstrated expression and activity of the subtype hTAS2R46 in human airway smooth muscle and broncho-epithelial cells, and here we show its expression and functionality in human skeletal muscle cells. Three different cellular models were used: micro-dissected human skeletal tissues, human myoblasts/myotubes and human skeletal muscle cells differentiated from urine stem cells of healthy donors. We used qPCR, immunohistochemistry and immunofluorescence analysis to evaluate gene and protein hTAS2R46 expression. In order to explore receptor activity, cells were incubated with the specific bitter ligands absinthin and 3ß-hydroxydihydrocostunolide, and calcium oscillation and relaxation were evaluated by calcium imaging and collagen assay, respectively, after a cholinergic stimulus. We show, for the first time, experimentally the presence and functionality of a type 2 bitter receptor in human skeletal muscle cells. Given the tendentially protective role of the bitter receptors starting from the oral cavity and following also in the other ectopic sites, and given its expression already at the myoblast level, we hypothesize that the bitter receptor can play an important role in the development, maintenance and in the protection of muscle tissue functions.</p