Phenotypic characterization of the epithelial and myoepithelial components in canine and feline mammary tumours

In veterinary medicine, the ability to classify mammary tumours based on the molecular profile and also determine whether the immunophenotype of the regional lymph node and/or systemic metastases is equal to that of the primary tumor may be predictive on the estimation of the effectiveness of various cancer treatments that can be scheduled. Therefore, aims, developed as projects, of the past three years have been (1) to define the molecular phenotype of feline mammary carcinomas and their lymph node metastases according to a previous modified algorithm and to demonstrate the concordance or discordance of the molecular profile between the primary tumour and lymph node metastasis, (2) to analyze, in female dogs, the relationship between the primary mammary tumor and its lymph node metastasis based on immunohistochemical molecular characterization in order to develop the most specific prognostic-predictive models and targeted therapeutic options, and (3) to evaluate the molecular trend of cancer from its primary location to systemic metastases in three cats and two dogs with mammary tumors. The studies on mammary tumours, particularly in dogs, have drawn gradually increasing attention not exclusively to the epithelial component, but also to the myoepithelial cells. The lack of complete information on a valid panel of markers for the identification of these cells in the normal and neoplastic mammary gland and lack of investigation of immunohistochemical changes from an epithelial to a mesenchymal phenotype, was the aim of a parallel research. While investigating mammary tumours, it was noticed that only few studies had focused on the expression of CD117. Therefore, it was decided to further deepen the knowledge in order to characterize the immunohistochemical staining of CD117 in normal and neoplastic mammary tissue of the dog, and to correlate CD117 immunohistochemical results with mammary histotype, histological stage (invasiveness), Ki67 index and patient survival time.

The role of interleuchin 6 (IL-6) in the promotion of an aggressive and stem cell-like phenotype in human breast cancer cells and in stem/progenitor cells expanded in vitro as mammospheres

High serum levels of Interleukin-6 (IL-6) correlate with poor outcome in breast cancer patients. However no data are available on the relationship between IL-6 and stem/progenitor cells which may fuel the genesis of breast cancer in vivo. Herein, we address this issue in mammospheres (MS), multi-cellular structures enriched in stem/progenitor cells of the mammary gland, and also in MCF-7 breast cancer cells. We show that MS from node invasive breast carcinoma tissues express IL-6 mRNA at higher levels than MS from matched non-neoplastic mammary glands. We find that IL-6 mRNA is detectable only in basal-like breast carcinoma tissues, an aggressive variant showing stem cell features. Our results reveal that IL-6 triggers a Notch-3-dependent up-regulation of the Notch ligand Jagged-1, whose interaction with Notch-3 promotes the growth of MS and MCF-7 derived spheroids. Moreover, IL-6 induces a Notch-3-dependent up-regulation of the carbonic anhydrase IX gene, which promotes a hypoxia-resistant/invasive phenotype in MCF-7 cells and MS. Finally, an autocrine IL-6 loop relies upon Notch-3 activity to sustain the aggressive features of MCF-7-derived hypoxia-selected cells. In conclusion, our data support the hypothesis that IL-6 induces malignant features in Notch-3 expressing, stem/progenitor cells from human ductal breast carcinoma and normal mammary gland.

New synthetic bile acid analogue agonists of FXR and TGR5 receptors: Analytical methodologies for the study of their physico-chemical properties, pharmacokinetic activity and metabolism.

This thesis reports an integrated analytical approach for the study of physicochemical and biological properties of new synthetic bile acid (BA) analogues agonists of FXR and TGR5 receptors. Structure-activity data were compared with those previous obtained using the same experimental protocols on synthetic and natural occurring BA. The new synthetic BA analogues are classified in different groups according also to their potency as a FXR and TGR5 agonists: unconjugated and steroid modified BA and side chain modified BA including taurine or glycine conjugates and pseudo-conjugates (sulphonate and sulphate analogues). In order to investigate the relationship between structure and activity the synthetic analogues where admitted to a physicochemical characterization and to a preliminary screening for their pharmacokinetic and metabolism using a bile fistula rat model. Sensitive and accurate analytical methods have been developed for the quali-quantitative analysis of BA in biological fluids and sample used for physicochemical studies. Combined High Performance Liquid Chromatography Electrospray tandem mass spectrometry with efficient chromatographic separation of all studied BA and their metabolites have been optimized and validated. Analytical strategies for the identification of the BA and their minor metabolites have been developed. Taurine and glycine conjugates were identified in MS/MS by monitoring the specific ion transitions in multiple reaction monitoring (MRM) mode while all other metabolites (sulphate, glucuronic acid, dehydroxylated, decarboxylated or oxo) were monitored in a selected-ion reaction (SIR) mode with a negative ESI interface by the following ions. Accurate and precise data where achieved regarding the main physicochemical properties including solubility, detergency, lipophilicity and albumin binding . These studies have shown that minor structural modification greatly affect the pharmacokinetics and metabolism of the new analogues in respect to the natural BA and on turn their site of action, particularly where their receptor are located in the enterohepatic circulation.

Pet food: quality and quality improvement

Today’s pet food industry is growing rapidly, with pet owners demanding high-quality diets for their pets. The primary role of diet is to provide enough nutrients to meet metabolic requirements, while giving the consumer a feeling of well-being. Diet nutrient composition and digestibility are of crucial importance for health and well being of animals. A recent strategy to improve the quality of food is the use of “nutraceuticals” or “Functional foods”. At the moment, probiotics and prebiotics are among the most studied and frequently used functional food compounds in pet foods. The present thesis reported results from three different studies. The first study aimed to develop a simple laboratory method to predict pet foods digestibility. The developed method was based on the two-step multi-enzymatic incubation assay described by Vervaeke et al. (1989), with some modification in order to better represent the digestive physiology of dogs. A trial was then conducted to compare in vivo digestibility of pet-foods and in vitro digestibility using the newly developed method. Correlation coefficients showed a close correlation between digestibility data of total dry matter and crude protein obtained with in vivo and in vitro methods (0.9976 and 0.9957, respectively). Ether extract presented a lower correlation coefficient, although close to 1 (0.9098). Based on the present results, the new method could be considered as an alternative system of evaluation of dog foods digestibility, reducing the need for using experimental animals in digestibility trials. The second parte of the study aimed to isolate from dog faeces a Lactobacillus strain capable of exert a probiotic effect on dog intestinal microflora. A L. animalis strain was isolated from the faeces of 17 adult healthy dogs..The isolated strain was first studied in vitro when it was added to a canine faecal inoculum (at a final concentration of 6 Log CFU/mL) that was incubated in anaerobic serum bottles and syringes which simulated the large intestine of dogs. Samples of fermentation fluid were collected at 0, 4, 8, and 24 hours for analysis (ammonia, SCFA, pH, lactobacilli, enterococci, coliforms, clostridia). Consequently, the L. animalis strain was fed to nine dogs having lactobacilli counts lower than 4.5 Log CFU per g of faeces. The study indicated that the L animalis strain was able to survive gastrointestinal passage and transitorily colonize the dog intestine. Both in vitro and in vivo results showed that the L. animalis strain positively influenced composition and metabolism of the intestinal microflora of dogs. The third trail investigated in vitro the effects of several non-digestible oligosaccharides (NDO) on dog intestinal microflora composition and metabolism. Substrates were fermented using a canine faecal inoculum that was incubated in anaerobic serum bottles and syringes. Substrates were added at the final concentration of 1g/L (inulin, FOS, pectin, lactitol, gluconic acid) or 4g/L (chicory). Samples of fermentation fluid were collected at 0, 6, and 24 hours for analysis (ammonia, SCFA, pH, lactobacilli, enterococci, coliforms). Gas production was measured throughout the 24 h of the study. Among the tested NDO lactitol showed the best prebiotic properties. In fact, it reduced coliforms and increased lactobacilli counts, enhanced microbial fermentation and promoted the production of SCFA while decreasing BCFA. All the substrates that were investigated showed one or more positive effects on dog faecal microflora metabolism or composition. Further studies (in particular in vivo studies with dogs) will be needed to confirm the prebiotic properties of lactitol and evaluate its optimal level of inclusion in the diet.

Studi ed approfondimenti sulla composizione in acidi grassi del latte bovino di razza Reggiana con diverso fenotipo per k-caseina e Beta-lactoglobulina

The aim of the present study was to examine the association between milk protein polymorphism and fatty acids profiles of bovine milk. Milk samples were collected from each of 55 Reggiana cows during early, mid and late lactation, respectively, in two farms within the production area of Parmigiano Reggiano cheese. Identification and quantification of fatty acids were performed by gas chromatography. Milk fatty acid composition using cows of differing κ-casein (κ-Cn) and β-lactoglobulin (β-Lg) phenotypes was investigated. Statistically significant results regarding the associations between milk fatty acid composition and κ-Cn phenotype were found, in particular, κ-Cn BB seems to influence de novo fatty acid synthesis in the mammary gland. Also κ-Cn AB seems to have the same effect. Proportions of C10:0 (2,29a AA; 2,53b AB; 2,59b BB), C12:0 (2,77a AA; 3,17b AB; 3,20b BB) and C14:0 (9,22a AA; 10,25b AB; 10,27b BB) were higher in the milk from cows with κ-Cn phenotype AB and BB vs κ-Cn phenotype AA (p<0,05). Conversely C18:0 (7,84b AA; 7,20a,b AB; 6,94a BB) and C18:1 (19,19b AA; 16,81a AB; 16,79a BB) were lower in the milk from cows with κ-Cn phenotype AB and BB vs κ-Cn phenotype AA. The association between milk fatty acid composition and β-Lg phenotype was not statistically significant, except for some fatty acids. In particular, C12:0 (3,05a AA; 3,04a AB; 3,33b BB) was higher in the milk from cows with β-Lg phenotype BB vs β-Lg phenotype AA and AB (p<0,05). Concentrations of C18:0 (6,93a AA; 7,86b AB; 6,59a BB) and C18:1 (16,74a,b AA; 18,24b AB; 16,07a BB) were lower in the milk from cows with β-Lg phenotype AA and BB vs β-Lg phenotype AB (p<0,05). Moreover this research, carried out in farms within the Parmigiano Reggiano cheese district, analysed also the size distribution of fat globules in bulk milk of Reggiana and Frisona breed cows. In particular, the size distribution of individual milk fat globules of Reggiana cows with differing κ-Cn phenotypes was considered. From first observations, no statistically significant differences were observed.

Semi-synthetic bile acids as novel drug candidate in liver diseases: physico-chemical characterization and HPLC-ES-MS/MS methods for their quali-quantitative analysis in different experimental animal models

The physico-chemical characterization, structure-pharmacokinetic and metabolism studies of new semi synthetic analogues of natural bile acids (BAs) drug candidates have been performed. Recent studies discovered a role of BAs as agonists of FXR and TGR5 receptor, thus opening new therapeutic target for the treatment of liver diseases or metabolic disorders. Up to twenty new semisynthetic analogues have been synthesized and studied in order to find promising novel drugs candidates. In order to define the BAs structure-activity relationship, their main physico-chemical properties (solubility, detergency, lipophilicity and affinity with serum albumin) have been measured with validated analytical methodologies. Their metabolism and biodistribution has been studied in “bile fistula rat”, model where each BA is acutely administered through duodenal and femoral infusion and bile collected at different time interval allowing to define the relationship between structure and intestinal absorption and hepatic uptake ,metabolism and systemic spill-over. One of the studied analogues, 6α-ethyl-3α7α-dihydroxy-5β-cholanic acid, analogue of CDCA (INT 747, Obeticholic Acid (OCA)), recently under approval for the treatment of cholestatic liver diseases, requires additional studies to ensure its safety and lack of toxicity when administered to patients with a strong liver impairment. For this purpose, CCl4 inhalation to rat causing hepatic decompensation (cirrhosis) animal model has been developed and used to define the difference of OCA biodistribution in respect to control animals trying to define whether peripheral tissues might be also exposed as a result of toxic plasma levels of OCA, evaluating also the endogenous BAs biodistribution. An accurate and sensitive HPLC-ES-MS/MS method is developed to identify and quantify all BAs in biological matrices (bile, plasma, urine, liver, kidney, intestinal content and tissue) for which a sample pretreatment have been optimized.

New analytical LC-mass spectrometry methodologies for the quali-quantitative determination of natural substances and drugs in complex matrices

This thesis reports an integrated analytical and physicochemical approach for the study of natural substances and new drugs based on mass spectrometry techniques combined with liquid chromatography. In particular, Chapter 1 concerns the study of Berberine a natural substance with pharmacological activity for the treatment of hepatobiliary and intestinal diseases. The first part focused on the relationships between physicochemical properties, pharmacokinetics and metabolism of Berberine and its metabolites. For this purpose a sensitive HPLC-ES-MS/MS method have been developed, validated and used to determine these compounds during their physicochemical properties studies and plasma levels of berberine and its metabolites including berberrubine(M1), demethylenberberine(M3), and jatrorrhizine(M4) in humans. Data show that M1, could have an efficient intestinal absorption by passive diffusion due to a keto-enol tautomerism confirmed by NMR studies and its higher plasma concentration. In the second part of Chapter 1, a comparison between M1 and BBR in vivo biodistribution in rat has been studied. In Chapter 2 a new HPLC-ES-MS/MS method for the simultaneous determination and quantification of glucosinolates, as glucoraphanin, glucoerucin and sinigrin, and isothiocyanates, as sulforaphane and erucin, has developed and validated. This method has been used for the analysis of functional foods enriched with vegetable extracts. Chapter 3 focused on a physicochemical study of the interaction between the bile acid sequestrants used in the treatment of hypercholesterolemia including colesevelam and cholestyramine with obeticolic acid (OCA), potent agonist of nuclear receptor farnesoid X (FXR). In particular, a new experimental model for the determination of equilibrium binding isotherm was developed. Chapter 4 focused on methodological aspects of new hard ionization coupled with liquid chromatography (Direct-EI-UHPLC-MS) not yet commercially available and potentially useful for qualitative analysis and for “transparent” molecules to soft ionization techniques. This method was applied to the analysis of several steroid derivatives.