Accuracy of a Flash Glucose Monitoring System in Diabetic Dogs.

BACKGROUND: A novel flash glucose monitoring system (FGMS) (FreeStyle Libre, Abbott, UK) was recently developed for humans. It continuously measures the interstitial glucose (IG) concentrations for 14 days. OBJECTIVES: To assess the clinical and analytical accuracy of the FGMS in diabetic dogs. ANIMALS: Ten client-owned diabetic dogs on insulin treatment. METHODS: Prospective and observational study. The FGMS was placed on the neck for up to 14 days. During the 1st-2nd, 6-7th, and 13-14th days from application, the IG measurements were compared with the plasma (EDTA) glucose (PG) concentrations analyzed by a reference hexokinase based method. RESULTS: The application and the use of the FGMS were apparently painless, easy, and well tolerated by all dogs. Mild erythema at the site of the application was found in 5/10 dogs at the end of the wearing period. A good correlation between IG and PG concentrations (rho = 0.94; P < .001) was found. The FGMS was 93, 99, and 99% accurate at low, normal, and high blood glucose concentrations. Mean ± standard deviation difference from the reference method was 2.3 ± 46.8 mg/dL. CONCLUSION AND CLINICAL IMPORTANCE: The FGMS is easy to use and is accurate for IG glucose measurement in diabetic dogs.

The role of the nuclear Akt activation and Akt inhibitors in all-trans-retinoic acid-differentiated HL-60 cells.

The pharmacological inhibitors of phosphoinositide 3-kinase (PI3K)/Akt pathway have been proposed in the treatment of leukemia based on their antiproliferative effects. However, several studies demonstrated the activation of PI3K in the nuclei of all-trans-retinoic acid (ATRA) - differentiated HL-60 cells, raising the possibility that PI3K/Akt-inhibitors may block antitumor properties of retinoids. The aim of the present study was to investigate the possible activation of nuclear Akt in ATRA-treated cells and to test the effects of Akt-inhibitors on ATRA-mediated differentiation. The Akt-activity was found to be increased in the nuclei and lysates of ATRA-differentiated HL-60 and NB4 cells. The down-modulation of the expression of Akt protein in HL-60 cells using siRNA reduces the CD11b expression in ATRA-treated cells. The treatment of both cell lines with the commercially available Akt inhibitors inhibited the growth of both control and ATRA-treated cells. Akt-inhibitors had no inhibitory effects on ATRA-mediated growth arrest and the expression of CD11b in HL-60 cells, but increased the percentage of control cells expressing CD11b. In contrast, the presence of Akt inhibitors reduced the expression of CD11b in ATRA-treated NB4 cells.

Ornithine decarboxylase, polyamines and CD11b expression in HL-60 cells during differentiation induced by retinoic acid.

Polyamines (PA) and retinoic acid affect mammalian cell growth, differentiation and apoptosis. Retinoic acid induces granulocytic differentiation of mieloid cell lines and, during this process, is responsible for the expression of CD11b, a surface antigen. In this study we investigate the effects of retinoic acid on HL-60 cells, monitoring ornithine decarboxylase (ODC) activity (enzyme rate of PA), putrescine (PUT), spermidine (SPD), spermine (SPM) levels, CD11b myeloid surface marker differentiation, cell cycle, and apoptosis. ODC activity and PUT levels are correlated with mieloid cell differentiation induced by retinoic acid treatment. Only the ODC/PUT ratio is connected with retinoic acid treated HL-60 cells. Treated cultures show a decrease of proliferation and a cell block in the G0/G1 phase, with consequent diminished S phase. The G0/G1 and S phases are significantly related to ODC activity and to PUT and SPD behavior, whereas in differentiating condition only the decrease of PUT is related to the S phase. CD11b expression, stimulated by retinoic acid treatment, is associated with the SPM trend. Total PA behavior agrees with apoptotic cell increase after 96 h of stimulation. Our data show that retinoic acid treatment modifies ODC activity and the turnover of PA. PUT, SPD and SPM, therefore, have a different role, and may be involved in the differentiative/apoptotic program of retinoic acid treated HL-60 cells.

Inositide-modifying enzymes: a cooperative role in regulating nuclear morphology during differentiation of myeloid cells.

Differentiation and functional response of mature myeloid cells require cytoskeleton remodelling in a dynamic system that involves subcellular organization and regional signalling. Within the myeloid lineage, neutrophils constitute a cell type in which different cell compartments, and predominantly the nucleus, undergo distinctive large changes involving actin reorganization. In the context of the progressive elucidation of the nuclear structure and composition that has been achieved in the last two decades, it is now clear that the nucleus possesses an ordered and dynamic skeletal structure which shares many properties with the cytoskeleton, and the full set of substrates and enzymes that participate in the inositol lipid metabolism. Consolidated evidence indicate that the changes in cytoskeleton assembly are regulated also by phosphoinositides in a way dependent on their local concentration and availability. Indeed, enzymes able to affect the amount and phosphorylation of inositol lipids can play fundamental roles in determining the architectural transitions of the cell. The expression pattern and the changes of activity of PLC and PI 3-K in the nucleus during differentiation of tumoral myeloid precursors suggest that these enzymes play a crucial role in modifying the intranuclear pool of phosphoinositides, which in turn induce the changes in nucleoskeleton associated to granulocytic maturation. It can be speculated that defective control of nucleoskeleton assembly is one of the causes of dysregulated cell maturation or differentiative block in the course of myeloid leukemias. Inositide modifying enzymes can thus be regarded as potential targets for molecularly designed therapeutic intervention on hematological malignancies.

Retinoic acid maintains differentiated cell morphology and functions in long-term cultured porcine hepatocytes: obtaining functional cells for prolonged treatments with bioartificial liver.

Porcine hepatocytes show several immunological characteristics and enzymatic activities of human liver, representing an ideal xenogenic source of cells as biological component of bioartificial liver (BAL). Isolated hepatocytes rapidly lose their specific metabolic activities and their typical morphology when cultured in the presence of serum. Since in BAL porcine hepatocytes are perfused by the patient's plasma, procedures able to minimize de-differentiation of cells could be useful for long-term treatment of acute liver failure (ALF). In this work we found that, in the presence of micromolar concentration of All trans-retinoic acid (ATRA), porcine parenchymal liver cells undergo to a lower extent the de-differentiating effects of long-term culture in the presence of serum. The evaluation of lidocaine metabolism showed that ATRA-treated cells retain specific hepatocyte function for a significantly longer time when compared to control hepatocytes. A tyrosine phosphorylation of PLC-gamma1 was observed in concomitance with the ATRA-induced maximal functional activity. An increased expression of PLC-beta3 and PKC-alpha and -beta2 was also evidentiated at the longer time points explored, when the effects of ATRA in preservation of the differentiated morphology were maximal. These results provide the first evidence that ATRA plays a differentiating role in adult porcine hepatocytes cultured under de-differentiating conditions. The administration of ATRA to isolated parenchymal cells from pig liver may provide functional hepatocytes for prolonged treatment with BAL.

Requirement of tyrosine-phosphorylated Vav for morphological differentiation of all-trans-retinoic acid-treated HL-60 cells.

Our previous data demonstrated that cellular and nuclear tyrosine-phosphorylated Vav associate with phosphoinositide 3-kinase during all-trans-retinoic acid-dependent granulocytic differentiation of HL-60 cells. In this study, aimed to analyze the mechanism by which Vav is recruited and activated, we report that the Src homology 2 domain of Vav interacts with tyrosine-phosphorylated proteins in a differentiation-dependent manner. Two adaptor proteins, Cbl and SLP-76, were identified, showing a discrete distribution inside the cells, with Cbl absent from the nuclei and SLP-76 particularly abundant in the nuclear compartment. Of note, Vav interacts with the tyrosine kinase Syk, which is also present in the nuclear compartment and may phosphorylate Vav in vitro when cells differentiate. Inhibition of Syk activity by piceatannol prevents both in vitro and in vivo Vav tyrosine phosphorylation, its association with the regulatory subunit of phosphoinositide 3-kinase, and the nuclear modifications typically observed during granulocytic differentiation of this cell line. These findings suggest that tyrosine-phosphorylated Vav and its association with phosphoinositide 3-kinase play a crucial role in all-trans-retinoic acid-induced reorganization of the nucleoskeleton, which is responsible for the changes in nuclear morphology observed during granulocytic differentiation of HL-60 cells.