Isolation of canine mammary cells with stem cell properties and tumour-initiating potential.

Recent data suggest that mammary carcinogenesis may be driven by cancer stem cells (CSCs) derived from mutated adult stem cells, which have acquired aberrant cell self-renewal or by progenitor cells that have acquired the capacity for cell self-renewal. Spontaneous mammary cancers in cats and dogs are important models for the understanding of human breast cancer and may represent alternative species model systems that can significantly contribute to the study of human oncogenesis. With the goal of identifying markers for isolating human breast CSCs, we have generated a canine model system to isolate and characterize normal and CSCs from dog mammary gland. Insight into the hierarchical organization of canine tumours may contribute to the development of universal concepts in oncogenesis by CSCs. Cells with stem cell properties were isolated from normal and tumoural canine breast tissue and propagated as mammospheres and tumourspheres in long-term non-adherent culture conditions. We showed that cells obtained from spheres that display self-renewing properties, have multi-lineage differentiation potential, could generate complex branched tubular structures in vitro and form tumours in NOD/SCID mice. We analysed these cells for the expression of human stem and CSC markers and are currently investigating the tumour-initiating properties of these cells and the hierarchical organization of normal and neoplastic canine mammary tissue.

The Y606C RET mutation causes a receptor gain of function.

CONTEXT: Medullary thyroid carcinoma (MTC) is the most common feature of multiple endocrine neoplasia type 2A (MEN2A) and occurs in almost all patients affected by germline RET mutations. OBJECTIVE: We identified and characterized an activating germline RET point mutation (G>A substitution leading to the heterozygous missense mutation Y606C in exon 10), in a 58-year-old female affected by MTC. DESIGN: The RET/Y606C and RET/C620Y, obtained by site-directed mutagenesis, as well as the RET/wild-type (wt) were cloned in an expression vector and transiently transfected in NIH3T3 fibroblasts. In vitro cell model was used to evaluate the effect of Y606C mutation on the RET downstream signalling pathways through Western blot analysis. RESULTS: We found that the cysteine insertion, due to the Y606C mutation, results in increased receptor dimerization, which is accompanied by an increased tyrosine phosphorylation of the Y905 residue in the RET/Y606C, demonstrating that the Y606C mutation is associated with constitutive receptor activation. As RET activation results in an intracellular signalling cascade involving extracellular signal-regulated kinases (ERKs), we investigated ERK activity in our transfected cells. Results demonstrated a significant increase in ERK2 phosphorylation in the RET/Y606C vs. the RET/wt and RET/C620Y transfected cells, suggesting an up-regulation of RET signalling. CONCLUSIONS: All these findings demonstrate that the Y606C mutation is associated with RET constitutive activation and thus has to be considered of pathogenetic relevance in the development of MTC.

Safety of celecoxib in patients with adverse skin reactions to acetaminophen (paracetamol) and other non-steroidal anti-inflammatory drugs.

BACKGROUND: Acetaminophen (paracetamol-P) is a widely used analgesic-antipyretic drug with no anti inflammatory effects and its rate of adverse hypersensitivity reactions is very low. On the contrary non-steroidal anti-inflammatory drugs (NSAIDs) are commonly involved in side effects. Celecoxib (CE) is a novel drug, with high selectivity and affinity for COX-2 enzyme. OBJECTIVE: We evaluated the tolerability of CE in a group of patients with documented history of adverse cutaneous reactions to P and to classic NSAIDs. METHODS: We studied 29 patients with hypersensitivity to P and classic NSAIDs. The diagnosis of P-induced skin reactions was based on in vivo challenge. The placebo was blindly administered at the beginning of each challenge. After three days, a cumulative dosage of 200 mg of CE in refracted doses was given. After 2-3 days, a single dose of 200 mg was administered. All patients were observed for 6 hours after each challenge, and they were controlled again after 24 hours to exclude delayed reactions. The challenge was considered positive if one or more of the following appeared: erythema, rash or urticaria-angioedema. RESULTS: No reaction was observed with placebo and twenty eight patients (96.5 %) tolerated CE. Only one patient developed a moderate angioedema of the lips. CONCLUSION: Only one hypersensitivity reaction to CE was documented among 29 P-intolerant patients. Thus, we conclude that CE is a reasonably safe alternative which can be used in subjects who do not tolerate P.

Cytocompatibility of polyurethane foams as biointegrable matrices for the preparation of scaffolds for bone reconstruction.

This work reports preliminary results on the development of biointegrable scaffolds, composed of biostable 3D polymer matrices and bioabsorbable inorganic salts, to be used for cell anchorage in bone regeneration. Three crosslinked polyurethane foams (PUFs), prepared by one-step bulk polymerisation from a polyether-polyol mixture, polymeric MDI and water as expanding agent, were tested for their ability to promote adhesion and growth of bone-derived cells. The open porosity of these foams ranged from 16 to 31% with an average pore size of 470 /600 microm, compressive strength (at 10% ε ) of 0.28/0.38 MPa and elastic moduli of 4.88/6.61 MPa. The human osteosarcoma line Saos-2, and primary cultures of normal human articular chondrocytes and bone marrow-derived (HBM) stromal cells were used for in vitro cytocompatibility tests. For cell adhesion and proliferation analysis, DNA synthesis was evaluated by 3 H-thymidine uptake. Osteoblastic differentiation of Saos-2 adherent cells was determined by measuring the enzymatic activity of alkaline phosphatase (ALP). All cell types were able to adhere to all tested PUFs and to synthesize DNA. At 48 hr culture, HBM stromal cells showed the maximal rate of adhesion with the highest rate of proliferation onto PUFs with the largest pore size, whereas both chondrocytes and Saos-2 appeared to adhere preferentially onto foams exhibiting the highest percentage of open porosity. Up to 8 days in culture Saos-2 cells were able to proliferate into all PUFs, with a time-dependent increase of DNA synthesis and ALP activity. At SEM, the morphology of cells adherent to PUF pores was spread with cytoplasmatic extroflessions, indicating a good metabolic activation. These results demonstrate a good cytocompatibility of the proposed 3D matrices, suggesting that their use in the preparation of composite scaffolds is worth further investigation. (Journal of Applied Biomaterials & Biomechanics 2003; 1: 58-66)ABSTRACT: This work reports preliminary results on the development of biointegrable scaffolds, composed of biostable 3D polymer matrices and bioabsorbable inorganic salts, to be used for cell anchorage in bone regeneration. Three crosslinked polyurethane foams (PUFs), prepared by one-step bulk polymerisation from a polyether-polyol mixture, polymeric MDI and water as expanding agent, were tested for their ability to promote adhesion and growth of bone-derived cells. The open porosity of these foams ranged from 16 to 31% with an average pore size of 470 /600 microm, compressive strength (at 10% ε ) of 0.28/0.38 MPa and elastic moduli of 4.88/6.61 MPa. The human osteosarcoma line Saos-2, and primary cultures of normal human articular chondrocytes and bone marrow-derived (HBM) stromal cells were used for in vitro cytocompatibility tests. For cell adhesion and proliferation analysis, DNA synthesis was evaluated by 3 H-thymidine uptake. Osteoblastic differentiation of Saos-2 adherent cells was determined by measuring the enzymatic activity of alkaline phosphatase (ALP). All cell types were able to adhere to all tested PUFs and to synthesize DNA. At 48 hr culture, HBM stromal cells showed the maximal rate of adhesion with the highest rate of proliferation onto PUFs with the largest pore size, whereas both chondrocytes and Saos-2 appeared to adhere preferentially onto foams exhibiting the highest percentage of open porosity. Up to 8 days in culture Saos-2 cells were able to proliferate into all PUFs, with a time-dependent increase of DNA synthesis and ALP activity. At SEM, the morphology of cells adherent to PUF pores was spread with cytoplasmatic extroflessions, indicating a good metabolic activation. These results demonstrate a good cytocompatibility of the proposed 3D matrices, suggesting that their use in the preparation of composite scaffolds is worth further investigation. (Journal of Applied Biomaterials & Biomechanics 2003; 1: 58-66).

Dose-dependent inhibition of mitochondrial ATP synthase by 17 beta-estradiol.

Mitochondria produce energy through oxidative phosphorylation. A key enzyme in this pathway is F0F1-ATP synthase, catalyzing ATP production from ADP and inorganic phosphate. Recently a subunit of F0F1-ATP synthase, oligomycin sensitivity-conferring protein, was identified as a new estradiol-binding protein. Estradiol could directly modulate mitochondrial ATP synthase activity through this subunit. In addition, intracellular ATP levels play a role in apoptotic death, which is an energy-dependent process requiring functioning mitochondria. Here we examined the effect of 17 beta-estradiol on F0F1-ATP synthase directly (in permeabilized cells) and in intact osteoclastic FLG 29.1 cells, a model of inducible apoptosis. The baseline F0F1-ATP synthase activity of FLG 29.1 cells was 4.485 nmol/min per mg. Estradiol rapidly inhibited F0F1-ATP synthase activity in the physiological range (half-inhibition concentration, IC50, of 30 nmol/l). With 1 nmol/l of estradiol, the inhibition was already significant (8-10% inhibition, p < 0.01) and with 100 nmol/l residual enzyme activity was only 15% (85% inhibition, p < 0.01). In addition, the effect of estradiol appeared to be directed towards F0F1-ATP synthase, since succinate-sustained respiration, uncoupled from the electron transport chain, was unaffected by estradiol. We assayed F0F1-ATP synthase activity in FLG 29.1 cells during inducible apoptosis. No significant difference of ATP synthesis was detected in apoptotic cells versus controls. In conclusion, we showed a new non-genomic effect of estradiol on a key mitochondrial enzyme, which thereby directly modulates cellular energy metabolism.

Lack of evidence for the Th2 predominance in patients with chronic hepatitis C.

A T helper (Th)1 to Th2 shift has been proposed to be a critical pathogenic determinant in chronic hepatitis C. Here, we evaluated mitogen-induced and hepatitis C virus (HCV) core antigen-induced cytokine production in 28 patients with biopsy-proven chronic hepatitis C. Flow cytometry demonstrated that after mitogenic stimulation the percentage of Th2 cells (IL-4 + or IL-13 +) and Th0 cells (IFN-gamma/IL-4 + or IL-2/IL-13 +) did not differ between patients and controls. In contrast, the percentage of Th1 cells (IFN-gamma + or IL-2 +) was significantly increased in CD4 +, CD8 +, 'naive'-CD45RA + and 'memory'-CD45RO + T-cell subsets from patients versus controls. Similar results were obtained by ELISA testing supernatants from mitogen-stimulated, unfractionated peripheral blood mononuclear cell (PBMC) cultures. Interferon-alpha treatment was associated with a reduction in the mitogen-induced Th1 cytokine response in those patients who cleared their plasma HCV-RNA. Analysis of cytokine expression by CD4 + T cells after HCV core antigen stimulation in a subgroup of 13 chronic hepatitis C patients demonstrated no cytokine response in 74% of these patients and an IFN-gamma-restricted response in 26%. Finally, no Th2 shift was found in lipopolysaccharide-stimulated monocytes. These data indicate that a Th1 to Th2 shift does not occur in chronic hepatitis C.