Camilla Recordati

Comparison of equine bone marrow-, umbilical cord matrix and amniotic fluid-derived progenitor cells

The aim of the study was to compare in vitro the stemness features of horse progenitor cells derived from bone marrow (BM-MSCs), amniotic fluid (AF-MSCs) and umbilical cord matrix (EUC-MSCs). It has been suggested that there may be a stem cell population within both umbilical cord matrix and amniotic fluid. However, little knowledge exists about the characteristics of these progenitor cells within these sources in the equine species. This study wanted to investigate an alternative and non-invasive stem cell source for the equine tissue engineering and to learn more about the properties of these cells for future cell banking. Bone marrow, umbilical cord and amniotic fluid samples were harvested from different horses. Cells were analyzed for proliferation, immunocytochemical, stem cell gene expression and multilineage plasticity. BM- and AF-MSCs took similar time to reach confluence and showed comparable plating efficiency. All cell lines expressed identical stem cell markers and capability to differentiate towards osteogenic lineage. Almost all cell lines differentiated into the adipogenic lineage as demonstrated by cytochemical staining, even if no adipose gene expression was detectable for AF-MSCs. AF- and EUC-MSCs showed a limited chondrogenic differentiation compared with BM-MSCs as demonstrated by histological and biochemical analyses. These findings suggest that AF-MSCs appeared to be a readily obtainable and highly proliferative cell line from an uninvasive source that may represent a good model system for stem cell biology. More studies are needed to investigate their multilineage potential. EUC-MSCs need to be further investigated regarding their particular behavior in vitro represented by spheroid formation.

Spatial Distribution of Helicobacter spp. in the Gastrointestinal Tract of Dogs

Background:  In dogs, the gastric Helicobacter spp. have been well studied, but there is little information regarding the other parts of the alimentary system. We sought to determine the spatial distribution of Helicobacter spp. in the gastrointestinal tract and the hepatobiliary system of dogs using culture‐independent methods.

Pharmacologic Inhibition of JAK1/JAK2 Signaling Reduces Experimental Murine Acute GVHD While Preserving GVT Effects

Purpose: Immune-mediated graft-versus-tumor (GVT) effects can occur after allogeneic hematopoietic stem cell transplantation (HSCT), but GVT is tightly linked to its main complication, graft-versus-host disease (GVHD). Strategies aimed at modulating GVHD, while maintaining the GVT effect, are needed to improve the cure rate of transplant. Given the emerging role of Janus-activated kinase (JAK) signaling in lymphoproliferative and myeloproliferative diseases and its established function at dictating T-cell differentiation, we postulated that JAKs might be potential therapeutic targets through a pharmacologic approach. Experimental Design: We examined the effect of JAK1/JAK2 modulation by ruxolitinib in a mouse model of fully MHC mismatched bone marrow transplant comprising in vivo tumor inoculation. Results: JAK1/JAK2 inhibition by ruxolitinib improved both overall survival (P = 0.03) and acute GVHD pathologic score at target organs (P ≤ 0.001) of treated mice. In addition, treatment with ruxolitinib was associated with a preserved GVT effect, as evidenced by reduction of tumor burden (P = 0.001) and increase of survival time (P = 0.01). JAK1/JAK2 inhibition did not impair the in vivo acquisition of donor T-cell alloreactivity; this observation may account, at least in part, to the preserved GVT effect. Rather, JAK1/JAK2 inhibition of GVHD was associated with the modulation of chemokine receptor expression, which may have been one factor in the reduced infiltration of donor T cells in GVHD target organs. Conclusions: These data provide further evidence that JAK inhibition represents a new and potentially clinically relevant approach to GVHD prevention. Clin Cancer Res; 21(16); 3740–9. ©2015 AACR.

An Essential Role for Liver ERα in Coupling Hepatic Metabolism to the Reproductive Cycle

Summary Lipoprotein synthesis is controlled by estrogens, but the exact mechanisms underpinning this regulation and the role of the hepatic estrogen receptor α (ERα) in cholesterol physiology are unclear. Utilizing a mouse model involving selective ablation of ERα in the liver, we demonstrate that hepatic ERα couples lipid metabolism to the reproductive cycle. We show that this receptor regulates the synthesis of cholesterol transport proteins, enzymes for lipoprotein remodeling, and receptors for cholesterol uptake. Additionally, ERα is indispensable during proestrus for the generation of high-density lipoproteins efficient in eliciting cholesterol efflux from macrophages. We propose that a specific interaction with liver X receptor α (LXRα) mediates the broad effects of ERα on the hepatic lipid metabolism.

Prevalence in bulk tank milk and epidemiology of Campylobacter jejuni in dairy herds in Northern Italy

ABSTRACT Thermotolerant Campylobacter spp. are frequently the cause of human gastroenteritis and have assumed more importance in Italy following the increased consumption of raw milk. Our objectives were to determine the prevalence and genotypes of Campylobacter spp. in dairy herds and to investigate the possible sources of bulk milk contamination. Bulk milk from dairy herds (n = 282) was cultured for Campylobacter spp. and Enterobacteriaceae. At three Campylobacter jejuni-positive farms, bovine feces, pigeon intestines, milk, and water points were also investigated. Isolates were identified by PCR and genotyped using multilocus sequence typing (MLST). C. jejuni was detected in 34 (12%) bulk milk samples. The strains belonged to 14 sequence types, and the most common clonal complexes were CC-21, CC-48, and CC-403. No association was demonstrated between the presence of C. jejuni and high levels of Enterobacteriaceae in bulk milk. At the three farms examined, C. jejuni was isolated from bovine feces (25/82 [30.5%]), pigeon intestines (13/60 [21.7%]), bulk milk (10/24 [41.7%]), and water points (4/16 [25%]). MLST revealed lineages that were common between milk and bovine feces but distinct between cattle and pigeons. In one herd, C. jejuni with the same genotype was isolated repeatedly from bulk milk and a cow with an udder infection. Our results showed a high prevalence of C. jejuni in bulk milk and suggested that udder excretion, in addition to fecal matter, may be a route of bulk milk contamination. MLST analysis indicated that pigeons are probably not relevant for the transmission of C. jejuni to cattle and for milk contamination.

Evaluation of White Striping prevalence and predisposing factors in broilers at slaughter

White striping ( WS: ) is an alteration of breast and thigh muscles of broiler chickens characterized by the presence of white striations parallel to the direction of muscle fibers. This study was performed to evaluate the prevalence and the predisposing factors to WS in commercial broilers of different weight reared in northern Italy. Fifty seven broiler flocks, including animals of medium- and heavy-weight, were grossly evaluated at slaughter for the presence of WS. For each flock, breeding data (mean BW at slaughter, ADG, sex, color of skin and fat, genetic line, age, antibiotic treatment, and prevalence of deep pectoral myopathy) were collected and statistically analyzed to assess their correlation with WS. Histology of breast fillets affected by different grades of WS was performed to evaluate potential differences between medium- and heavy-weight broilers. The overall prevalence of WS in medium- and heavy-weight broilers (mean BW 2.59 ± 0.13 kg and 3.64 ± 0.34 kg, respectively) was 70.2 ± 7.9% and 82.51 ± 8.5%, respectively, while the percentage of severe WS was 13.3 ± 7.1% and 25.7 ± 12.8%, respectively. A strong correlation was found between presence of WS, BW at slaughter, and ADG (Pearson correlation = 0.69, P < 0.01; Pearson correlation = 0.67, P < 0.01). WS also closely correlated with the prevalence of deep pectoral myopathy (Spearmans Rho slaughterhouse 1 = 0.74, Spearmans Rho slaughterhouse 2 = 0.51, P < 0.01). No correlation was found between genetics or sanitary status of the flock and WS. Histology confirmed that breasts with WS lesions were affected by a polyphasic degenerative and necrotizing myopathy, and that the lesions, as expected, were more severe in heavy-weight broilers. In conclusion, WS is a major concern in commercial meat poultry reared in Italy, affecting more severely heavier broilers, and it is mainly related to the BW and ADG of animals.

Metabolic effects of vitamin D active metabolites in monolayer and micromass cultures of nucleus pulposus and annulus fibrosus cells isolated from human intervertebral disc.

Intragenic polymorphisms in the vitamin D receptor gene are linked to disc degeneration features, suggesting that alterations in the vitamer-mediated signalling could be involved in the pathophysiology of the disc and that interaction of disc cells with vitamin D metabolites may be critical for disc health. The vitamer-mediated modulation of disc cells proliferation, metabolic activity, extracellular matrix (ECM) genes expression and proteins production was investigated. It was stated that disc cells express vitamin D receptor and are very sensitive to metabolic stimuli. In monolayer cultures, 1,25(OH)(2)D(3), but not 24,25(OH)(2)D(3), determined an inhibition of the proliferation and regulated also the ECM genes expression in nucleus pulposus and annulus fibrosus cells. Micromass cultures induced a more physiologic expression pattern of extracellular matrix genes. Cells Treatment with vitamin D metabolites did not result in relevant modifications of glycosaminoglycans production, except for annulus cells, whose production was reduced after 1,25(OH)(2)D(3) treatment. Moreover, a reduced glycosaminoglycans staining in both cell types and a significant reduced aggrecan gene expression in annulus cells treated with 1,25(OH)(2)D(3) were observed. A reduction of collagen I and II staining in annulus cells 1,25(OH)(2)D(3) treated, in accordance with a downregulation of collagen genes expression, was also registered. Finally, the vitamin D receptor gene expression did not show significant metabolite-mediated modification in monolayer or micromass cultures. These findings could enhance new insights on the biochemical mechanisms regulated by vitamin D in disc cartilage and possibly involved in the development of physiological/pathological modifications of the disc.

Localization of Helicobacter spp. in the fundic mucosa of laboratory Beagle dogs: an ultrastructural study

In dogs Helicobacter spp. are found in all gastric regions usually localized in the surface mucus, gastric glands and parietal cells. The aim of this study was to detail the distribution of Helicobacter spp. in the fundic mucosa of asymptomatic Beagle dogs and their intracellular localization within parietal cells, in order to evaluate species-specific pathogenetic effects on gastric cells. The presence of Helicobacter spp. was investigated by immunohistochemistry, TEM, and PCR in the fundic mucosa of six Beagle dogs. Helicobacter spp. were found in all dogs examined, and H. bizzozeronii and H. felis were identified by PCR and confirmed by TEM. In the lumen of the fundic glands, co-localization was common. H. bizzozeronii was present in larger numbers than H. felis in both intraluminal and intraparietal localization. The amounts of H. bizzozeronii were similar in superficial and basal portions of the glands. H. felis was predominantly localized in the superficial portions of gastric glands but almost absent from the base. Within parietal cells, most Helicobacter organisms were intracanalicular, but intact and degenerate Helicobacter organisms were also visualized free in the cytoplasm or in secondary lysosomes. No specific degenerative lesions were found in infected parietal cells. Helicobacter organisms were also observed within macrophages in the lamina propria. In conclusion, there is a differential distribution of H. bizzozeronii and H. felis in the fundic mucosa of Beagle dogs, and their intracellular localization in parietal cells and macrophages suggests novel pathogenic scenarios for the development of immune response and maintenance of chronic gastritis in dogs.

Identification of MYC-Dependent Transcriptional Programs in Oncogene-Addicted Liver Tumors

Tumors driven by activation of the transcription factor MYC generally show oncogene addiction. However, the gene expression programs that depend upon sustained MYC activity remain unknown. In this study, we employed a mouse model of liver carcinoma driven by a reversible tet-MYC transgene, combined with chromatin immunoprecipitation and gene expression profiling to identify MYC-dependent regulatory events. As previously reported, MYC-expressing mice exhibited hepatoblastoma- and hepatocellular carcinoma-like tumors, which regressed when MYC expression was suppressed. We further show that cellular transformation, and thus initiation of liver tumorigenesis, were impaired in mice harboring a MYC mutant unable to associate with the corepressor protein MIZ1 (ZBTB17). Notably, switching off the oncogene in advanced carcinomas revealed that MYC was required for the continuous activation and repression of distinct sets of genes, constituting no more than half of all genes deregulated during tumor progression and an even smaller subset of all MYC-bound genes. Altogether, our data provide the first detailed analysis of a MYC-dependent transcriptional program in a fully developed carcinoma and offer a guide to identifying the critical effectors contributing to MYC-driven tumor maintenance. Cancer Res; 76(12); 3463-72. ©2016 AACR.

Enterohepatic Helicobacter spp. in colonic biopsies of dogs: molecular, histopathological and immunohistochemical investigations.

Enterohepatic Helicobacter spp. have been described colonizing the large intestine and liver of healthy and symptomatic subjects and are thought to have a role in the development of inflammatory bowel disease (IBD). The prevalence of enterohepatic Helicobacter spp. infection in dogs is largely unknown and to our knowledge there are no data about their potential pathogenic role. In light of these considerations, the aims of this study were (i) to assess the prevalence of enterohepatic Helicobacter spp. in colonic biopsies of symptomatic pet dogs and (ii) to evaluate a possible association between Helicobacter spp. colonization status (heavily colonized, poorly colonized and uncolonized biopsies) and histological lesions. Colonic biopsies from 27 pet dogs of different ages were evaluated by family Helicobacteraceae and enterohepatic Helicobacter spp. PCR, histology, and immunohistochemistry for the in situ detection of Helicobacter spp. organisms. 85% and 52% of colonic biopsies were positive by Helicobacteraceae and enterohepatic Helicobacter spp. PCR, respectively. Immunohistochemistry revealed Helicobacter spp. were localized both in the superficial mucus (55%) and within intestinal crypts (33%). Dogs with heavy enterohepatic Helicobacter spp. colonization were significantly younger and had a higher level of mucosal fibrosis/atrophy than dogs with uncolonized or poorly colonized biopsies (p<0.05). These findings contribute to widen current knowledge regarding canine enterohepatic Helicobacter spp., suggesting the infection is rather common in dogs and acquired at an early age. Furthermore, heavy colonization of colonic crypts is associated with chronic inflammatory lesions (fibrosis/atrophy), supporting the role of enterohepatic Helicobacter spp. in the development of canine IBD.