Molecular pathogenesis of NASH

Nonalcoholic steatohepatitis (NASH) is the main cause of chronic liver disease in the Western world and a major health problem, owing to its close association with obesity, diabetes, and the metabolic syndrome. NASH progression results from numerous events originating within the liver, as well as from signals derived from the adipose tissue and the gastrointestinal tract. In a fraction of NASH patients, disease may progress, eventually leading to advanced fibrosis, cirrhosis and hepatocellular carcinoma. Understanding the mechanisms leading to NASH and its evolution to cirrhosis is critical to identifying effective approaches for the treatment of this condition. In this review, we focus on some of the most recent data reported on the pathogenesis of NASH and its fibrogenic progression, highlighting potential targets for treatment or identification of biomarkers of disease progression

Il cane come modello animale spontaneo di patologie neoplastiche dell'uomo: ene Ataxia-Telangiectasia Mutated (ATM) Nnella predisposizione al cancro ed importanza dei riarrangimanti genici dei geni Ig/TCR per la diagnosi e prognosi delle malattie linfoproliferative

“Naturally occurring cancers in pet dogs and humans share many features, including histological appearance, tumour genetics, molecular targets, biological behaviour and response to conventional therapies. Studying dogs with cancer is likely to provide a valuable perspective that is distinct from that generated by the study of human or rodent cancers alone. The value of this opportunity has been increasingly recognized in the field of cancer research for the identification of cancer-associated genes, the study of environmental risk factors, understanding tumour biology and progression, and, perhaps most importantly, the evaluation and development of novel cancer therapeutics”.(Paoloni and Khanna, 2008) In last years, the author has investigated some molecular features of cancer in dogs. The Thesis is articulated in two main sections. In section 1, the preliminary results of a research project aimed at investigating the role of somatic mutations of Ataxia-Telangiectasia mutated (ATM) gene in predisposing to cancer in boxer dogs, are presented. The canine boxer breed may be considered an unique opportunity to disclose the role of ATM somatic mutation since boxer dogs are known to be dramatically susceptible to cancer and since they may be considered a closed gene pool. Furthermore, dogs share with human the some environment. Overall, the abovementioned features could be considered extremely useful for our purposes. In the section 2, the results of our studies aimed at setting up accurate and sensitive molecular assays for diagnosing and assessing minimal residual disease in lymphoproliferative disorders of dogs, are presented. The results of those molecular assay may be directly translated in the field of Veterinary practice as well as the may be used to improve our objective evaluation of new investigational drugs effectiveness in canine cancer trials

Mutational Analysis of c-KIT and PDGFRA in Canine Gastrointestinal Stromal Tumors (GISTs)

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the canine gastrointestinal tract and are diagnosed by the immunohistochemical expression of the receptor tyrosine kinase (RTK) KIT. Activating mutations of the proto-oncogenes c-KIT and PDGFRA drive GIST oncogenesis and are used to predict the response to RTK-inhibitors in human oncology. Currently, the frequency and significance of these mutations in canine GIST have not been adequately explored. Therefore, we investigated the mutational status of c-KIT (exons 9, 11 and 13) and PDGFRA (exons 12 and 18) genes by PCR followed by fragment analysis for c-KIT deletions and PCR followed by screening with DHPLC and direct sequencing confirmation for single nucleotide variations in 17 formalin-fixed paraffin-embedded canine GISTs confirmed by KIT immunopositivity. c-KIT mutations were detected in 47% of cases, with a mutation detection rate significantly higher (p = 0.0004, Fisher's exact test) and always involving exon 11. A PDGFRA gene mutation (exon 18) was identified in one case. Even if follow-up data were not available for all cases, four cases with documented abdominal metastases displayed c-KIT mutations. These data confirm that c-KIT exon 11 mutations occur frequently in canine GISTs, and identify the presence of a PDGFRA mutation similar to human GISTs. This study also suggests a potential association of c-KIT mutation with more aggressive biological behavior

Rapid and Affordable High Throughput Screening of SARS-CoV-2 Variants Using Denaturing High-Performance Liquid Chromatography Analysis

Mutations in the receptor binding domain (RBD) of SARS-CoV-2 alter the infectivity, pathogenicity, and transmissibility of new variants of concern (VOCs). In addition, those mutations cause immune escape, undermining the population immunity induced by ongoing mass vaccination programs. There is an urgent need for novel strategies and techniques aimed at the surveillance of the active emergence and spread of the VOCs. The aim of this study was to provide a quick, cheap and straightforward denaturing high-performance liquid chromatography (DHPLC) method for the prompt identification of the SARS-CoV-2 VOCs. Two PCRs were designed to target the RBD region, spanning residues N417 through N501 of the Spike protein. Furthermore, a DHPLC screening analysis was set up. The screening consisted of mixing the unknown sample with a standard sample of a known variant, denaturing at high temperature, renaturing at room temperature followed by a 2-minute run using the WAVE DHPLC system to detect the heteroduplexes which invariably form whenever the unknown sample has a nucleotide difference with respect to the standard used. The workflow was able to readily detect all the variants including B.1.1.7, P.1, B.1.585 B.1. 617.2 and lineages at a very affordable cost. The DHPLC analysis was robust being able to identify variants, even in the case of samples with very unbalanced target concentrations including those samples at the limit of detection. This approach has the potential of greatly expediting surveillance of the SARS-CoV-2 variants

A molecular study of hemotropic mycoplasmas (hemoplasmas) in cats in Iran

BackgroundThree feline hemoplasma species are recognized: Mycoplasma haemofelis, Candidatus Mycoplasma haemominutum', and Candidatus Mycoplasma turicensis'. These species can cause anemia in cats and have a worldwide distribution. ObjectivesThere was no previous information on hemotropic mycoplasma spp in cats in Iran and the Middle East. Accordingly, we investigated the molecular presence, and clinical signs and hematological profile in cats infected with these microorganisms in Iranian cats. MethodsPolymerase chain reaction (PCR) assays and cytology were performed on 100 blood samples collected from Iranian Shorthair cats. ACBC and case history were also collected for each sample. ResultsBy PCR, 22 (22%; 14-30%, 95% CI) samples were positive. The prevalence of M haemofelis, Ca M haemominutum', and Ca M turicensis' was 63.63% (14/22), 54.54% (12/22), and 18.18% (4/22), respectively. Some double and triple co-infections were also found. Using PCR as the reference method, cytology had poor sensitivity (27%) and reasonable specificity (89.74%). Male cats were at a higher risk of infection (P=.001). Cats older than 8years were more frequently infected than the younger cats (P=.0018). Lower HCT (P=.018), RBC count (P=.028) and HGB concentration (P=.003) were also associated with hemoplasma PCR-positive status. ConclusionsBased on this study, the most prevalent feline hemoplasma species in Iranian cats was M haemofelis, but double and triple co-infections are also documented. Age and sex, as well as reduced RBC parameters, were predisposing factors for hemoplasma infection

PRKG2 Splice Site Variant in Dogo Argentino Dogs with Disproportionate Dwarfism

Dwarfism phenotypes occur in many species and may be caused by genetic or environmental factors. In this study, we investigated a family of nine Dogo Argentino dogs, in which two dogs were affected by disproportionate dwarfism. Radiographs of an affected dog revealed a decreased level of endochondral ossification in its growth plates, and a premature closure of the distal ulnar physes. The pedigree of the dogs presented evidence of monogenic autosomal recessive inheritance; combined linkage and homozygosity mapping assigned the most likely position of a potential genetic defect to 34 genome segments, totaling 125 Mb. The genome of an affected dog was sequenced and compared to 795 control genomes. The prioritization of private variants revealed a clear top candidate variant for the observed dwarfism. This variant, PRKG2:XM_022413533.1:c.1634+1G>T, affects the splice donor site and is therefore predicted to disrupt the function of the PKRG2 gene encoding protein, kinase cGMP-dependent type 2, a known regulator of chondrocyte differentiation. The genotypes of the PRKG2 variant were perfectly associated with the phenotype in the studied family of dogs. PRKG2 loss-of-function variants were previously reported to cause disproportionate dwarfism in humans, cattle, mice, and rats. Together with the comparative data from other species, our data strongly suggest PRKG2:c.1634+1G>T to be a candidate causative variant for the observed dwarfism phenotype in Dogo Argentino dogs

Validation of a Liquid Biopsy Protocol for Canine BRAFV595E Variant Detection in Dog Urine and Its Evaluation as a Diagnostic Test Complementary to Cytology

A significant proportion of canine urothelial carcinomas carry the driver valine to glutamic acid variation (V595E) in BRAF kinase. The detection of V595E may prove suitable to guide molecularly targeted therapies and support non-invasive diagnosis of the urogenital system by means of a liquid biopsy approach using urine. Three cohorts and a control group were included in this multi-step validation study which included setting up a digital PCR assay. This was followed by investigation of preanalytical factors and two alternative PCR techniques on a liquid biopsy protocol. Finally, a blind study using urine as diagnostic sample has been carried out to verify its suitability as diagnostic test to complement cytology. The digital PCR (dPCR) assay proved consistently specific, sensitive, and linear. Using the dPCR assay, the prevalence of V595E in 22 urothelial carcinomas was 90.9%. When compared with histopathology as gold standard in the blind-label cases, the diagnostic accuracy of using the canine BRAF (cBRAF) variation as a surrogate assay against the histologic diagnosis was 85.7% with 92.3% positive predictive value and 80.0% negative predictive value. In all the cases, in which both biopsy tissue and the associated urine were assayed, the findings matched completely. Finally, when combined with urine sediment cytology examination in blind-label cases with clinical suspicion of malignancy, the dPCR assay significantly improved the overall diagnostic accuracy. A liquid biopsy approach on urine using the digital PCR may be a valuable breakthrough in the diagnostic of urothelial carcinomas in dogs

Survival and Prognostic Factors in Patients with Hepatocellular Carcinoma Treated by Percutaneous Ethanol Injection: A 10-Year Experience

The treatment of early and intermediate stage hepatocellular carcinoma (HCC) is still debated. Surgical treatments are considered to be the only curative procedures available, and only for a minority of patients. Percutaneous ethanol injection (PEI) is an established technique for the ablation of HCC nodules, and shows survival rates similar to those of resection. The efficacy of PEI in patients with biopsy-proven viral cirrhosis and small to intermediate inoperable HCC was evaluated. One hundred twenty-seven patients (85 men, 42 women, mean age 63 years, range 51 to 92 years, 115 hepatitis C virus-positive, 12 hepatitis B virus-positive) were enrolled between January 1993 and December 2002. They all underwent a standard PEI procedure and were prospectively followed-up. Overall median survival rate was 28 months (range six to 112 months). The following parameters were associated with a significantly longer survival: nodule diameter smaller than 30 mm (P=0.0480), the presence of a perinodular boundary (P=0.0008), serum alpha-fetoprotein less than 20 ng/mL (P=0.0104), a Child-Pugh A class score (PÃ0.0001) or a Cancer of the Liver Italian Program score of 0 (PÃ0.0001) and the presence or absence of small esophageal varices (P=0.013). The 19 patients with all these favourable characteristics showed an overall median survival of 61 months. An alpha-fetoprotein below 20 ng/mL was associated with significantly longer disease-free survival (P=0.0009). The Child-Pugh and Cancer of the Liver Italian Program scores were effective in predicting prognosis of these patients. In conclusion, PEI still represents a safe and economically sound treatment for HCC