Successful elimination of non-neural cells and unachievable elimination of glial cells by means of commonly used cell culture manipulations during differentiation of GFAP and SOX2 positive neural progenitors (NHA) to neuronal cells

<p>Abstract</p> <p>Background</p> <p>Although extensive research has been performed to control differentiation of neural stem cells – still, the response of those cells to diverse cell culture conditions often appears to be random and difficult to predict. To this end, we strived to obtain stabilized protocol of NHA cells differentiation – allowing for an increase in percentage yield of neuronal cells.</p> <p>Results</p> <p>Uncommitted GFAP and SOX2 positive neural progenitors – so-called, Normal Human Astrocytes (NHA) were differentiated in different environmental conditions to: only neural cells consisted of neuronal [MAP2+, GFAP-] and glial [GFAP+, MAP2-] population, non-neural cells [CD44+, VIMENTIN+, FIBRONECTIN+, MAP2-, GFAP-, S100β-, SOX2-], or mixture of neural and non-neural cells.</p> <p>In spite of successfully increasing the percentage yield of glial and neuronal <it>vs</it>. non-neural cells by means of environmental changes, we were not able to increase significantly the percentage of neuronal (GABA-ergic and catecholaminergic) over glial cells under several different cell culture testing conditions. Supplementing serum-free medium with several growth factors (SHH, bFGF, GDNF) did not radically change the ratio between neuronal and glial cells – i.e., 1,1:1 in medium without growth factors and 1,4:1 in medium with GDNF, respectively.</p> <p>Conclusion</p> <p>We suggest that biotechnologists attempting to enrich <it>in vitro </it>neural cell cultures in one type of cells – such as that required for transplantology purposes, should consider the strong limiting influence of intrinsic factors upon extracellular factors commonly tested in cell culture conditions.</p

Arrested neural and advanced mesenchymal differentiation of glioblastoma cells-comparative study with neural progenitors

<p>Abstract</p> <p>Background</p> <p>Although features of variable differentiation in glioblastoma cell cultures have been reported, a comparative analysis of differentiation properties of normal neural GFAP positive progenitors, and those shown by glioblastoma cells, has not been performed.</p> <p>Methods</p> <p>Following methods were used to compare glioblastoma cells and GFAP+NNP (NHA): exposure to neural differentiation medium, exposure to adipogenic and osteogenic medium, western blot analysis, immunocytochemistry, single cell assay, BrdU incorporation assay. To characterize glioblastoma cells <it>EGFR </it>amplification analysis, LOH/MSI analysis, and <it>P53 </it>nucleotide sequence analysis were performed.</p> <p>Results</p> <p><it>In vitro </it>differentiation of cancer cells derived from eight glioblastomas was compared with GFAP-positive normal neural progenitors (GFAP+NNP). Prior to exposure to differentiation medium, both types of cells showed similar multilineage phenotype (CD44+/MAP2+/GFAP+/Vimentin+/Beta III-tubulin+/Fibronectin+) and were positive for SOX-2 and Nestin. In contrast to GFAP+NNP, an efficient differentiation arrest was observed in all cell lines isolated from glioblastomas. Nevertheless, a subpopulation of cells isolated from four glioblastomas differentiated after serum-starvation with varying efficiency into derivatives indistinguishable from the neural derivatives of GFAP+NNP. Moreover, the cells derived from a majority of glioblastomas (7 out of 8), as well as GFAP+NNP, showed features of mesenchymal differentiation when exposed to medium with serum.</p> <p>Conclusion</p> <p>Our results showed that stable co-expression of multilineage markers by glioblastoma cells resulted from differentiation arrest. According to our data up to 95% of glioblastoma cells can present <it>in vitro </it>multilineage phenotype. The mesenchymal differentiation of glioblastoma cells is advanced and similar to mesenchymal differentiation of normal neural progenitors GFAP+NNP.</p

Hepatoselective Nitric Oxide (NO) Donors, V-PYRRO/NO and V-PROLI/NO, in Nonalcoholic Fatty Liver Disease: A Comparison of Antisteatotic Effects with the Biotransformation and Pharmacokinetics

ABSTRACT V-PYRRO/NO [O(2)-vinyl-1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate] and V-PROLI/NO (O2-vinyl-[2-(carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate), two structurally similar diazeniumdiolate derivatives, were designed as liver-selective prodrugs that are metabolized by cytochrome P450 isoenzymes, with subsequent release of nitric oxide (NO). Yet, their efficacy in the treatment of nonalcoholic fatty liver disease (NAFLD) and their comparative pharmacokinetic and metabolic profiles have not been characterized. The aim of the present work was to compare the effects of V-PYRRO/NO and V-PROLI/NO on liver steatosis, glucose tolerance, and liver fatty acid composition in C57BL/6J mice fed a high-fat diet, as well as to comprehensively characterize the ADME (absorption, distribution, metabolism and excretion) profiles of both NO donors. Despite their similar structure, V-PYRRO/NO and V-PROLI/NO showed differences in pharmacological efficacy in the murine model of NAFLD. V-PYRRO/NO, but not V-PROLI/NO, attenuated liver steatosis, improved glucose tolerance, and favorably modified fatty acid composition in the liver. Both compounds were characterized by rapid absorption following i.p. administration, rapid elimination from the body, and incomplete bioavailability. However, V-PYRRO/NO was eliminated mainly by the liver, whereas V-PROLI/NO was excreted mostly in unchanged form by the kidney. V-PYRRO/NO was metabolized by CYP2E1, CYP2C9, CYP1A2, and CYP3A4, whereas V-PROLI/NO was metabolized mainly by CYP1A2. Importantly, V-PYRRO/NO was a better NO releaser in vivo and in the isolated, perfused liver than V-PROLI/NO, an effect compatible with the superior antisteatotic activity of V-PYRRO/NO. In conclusion, V-PYRRO/NO displayed a pronounced antisteatotic effect associated with liver-targeted NO release, whereas V-PROLI/NO showed low effectiveness, was not taken up by the liver, and was eliminated mostly in unchanged form by the kidney

PRIN 2015 - Impatto dell'infezione da virus a DNA sullo sviluppo di patologie autoimmuni: focus sulla sclerosi sistemica

La Sclerosi Sistemica (SSc) è una malattia autoimmune caratterizzata da disfunzione endoteliale e fibrosi di cute ed organi interni. Sebbene numerosi studi depongano per il coinvolgimento di alcuni virus nell’eziopatogenesi della SSc, mancano ancora dati conclusivi su una associazione tra infezione e malattia, su una possibile azione sinergica tra diversi virus e/o altri fattori in un background genetico predisponente lo sviluppo della patologia. Lo scopo di questo progetto è quello di approfondire le conoscenze sul ruolo dell’infezione da parvovirus umano B19 (B19V), citomegalovirus umano (HCMV) e herpesvirus umano 6 (HHV6) nella patogenesi della SSc, partendo dai dati disponibili in letteratura e dai risultati preliminari ottenuti dalle diverse UO partecipanti allo studio. A questo proposito si prevede di arruolare 50 pazienti con SSc seguiti presso l’UO di Reumatologia dell’Azienda Ospedaliero-Universitaria Policlinico di Modena, insieme ad un numero significativo di controlli. L’UO di Firenze ha dimostrato che il B19V persiste con elevata frequenza sia nel midollo che nella cute dei pazienti con SSc. Nella cute, il DNA ed alcuni trascritti virali sono stati individuati nei fibroblasti e nelle cellule endoteliali, che sembrano suscettibili all’infezione ma non permissivi. Utilizzando colture primarie di fibroblasti umani (FU) e di cellule endoteliali (CE) sia normali che sclerodermici verrà studiato l’effetto dell’infezione virale su queste cellule nonché la persistenza del B19V nei FU sclerodermici. Inoltre si cercherà di identificare i fattori virali eventualmente coinvolti nella patogenesi della SSc sia come induttori di risposte immuno-mediate sia come induttori di infiammazione e/o di fibrosi. Anche HCMV è stato indicato tra i possibili fattori scatenanti la SSc. Considerando l’importanza dell’apoptosi di CE e iper-proliferazione di FU nella SSc, il fatto che CE e FU siano bersagli cellulari di HCMV e la capacità del virus di alterare diversamente il ciclo cellulare di specifiche cellule (come precedentemente dimostrato dalla UO di Parma), questa UO si prefigge di studiare l’azione di HCMV sul ciclo cellulare di CE e FU in vitro ed ex vivo. Verrà inoltre verificato se specifiche associazioni genotipiche di fattori di virulenza di HCMV riscontrate negli stipiti presenti in soggetti con SSc possano rappresentarne marcatori predittivi. HHV6 è stato associato allo sviluppo di molte malattie autoimmuni, ma non è ancora stato studiato nella SSc. L’UO di Ferrara ha dimostrato l’associazione di HHV6 con la tiroidite autoimmune, spesso presente nei pazienti SSc, e il tropismo del virus per le cellule target nella SSc (FU, CE, linfociti). Si è inoltre osservato che specifici genotipi KIR aumentano il rischio di sviluppo di patologie HHV6-indotte. Saranno quindi studiati nei pazienti SSc: presenza e stato trascrizionale di HHV6; risposte immuni anti-HHV6; genotipo KIR; e alterazioni HHV6-indotte nelle cellule target SSc infettate in vitro

Simultaneous quantification of PGI_{2} and TXA_{2} metabolites in plasma and urine in NO-deficient mice by a novel UHPLC/MS/MS method

The balance between vascular prostacyclin (PGI2) generated mainly via cyclooxygenase-2 (COX-2) and its physiological antagonist platelet-derived thromboxane A2 (TXA2) formed by cyclooxygenase-1 (COX-1) determines cardiovascular homeostasis. In the present work, a novel bioanalytical method for simultaneous quantification of stable plasma and urinary metabolites of PGI2 (6-keto-PGF1α, 2,3-dinor-6-keto-PGF1α) and TXA2 (TXB2, 2,3-dinor-TXB2) using ultra high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC/MS/MS) was developed. The method was validated using artificial plasma and urine and linearity range, intra- and inter-day precision and accuracy, recovery of analytes, relative and absolute matrix effect and stability of analytes were determined. The use of artificial biofluids improved the method sensitivity as it eliminated the contribution of endogenous metabolites present in mice plasma and urine to validation procedure. The newly developed and validated method allowed to quantify 6-keto-PGF1α and TXB2 in mice plasma as well as 2,3-dinor-6-keto-PGF1α and 2,3-dinor-TXB2 in urine samples with high sensitivity and accuracy. The calibration range was established from 0.1 to 100 ng/mL for all analytes using artificial biofluids and the recoveries were greater than 89.9%. All validated parameters met the criteria of acceptance specified in FDA and EMA guidance. This method was successfully employed for profiling of the changes in PGI2 and TXA2 generation in NO-deficient mice. This work demonstrated that NO-deficiency induced by L-NAME, evidenced by a fall in nitrite in plasma and urine, was associated with platelet activation, robust increase in TXB2 and mild increase in 6-keto-PGF1α concentration in plasma. Changes in 2,3-dinor-6-keto-PGF1α and 2,3-dinor-TXB2 concentration in urine were less evident suggesting that the measurements in plasma better reflect modest changes in PGI2/TXA2 homeostasis than measurements in urine

Effects of 1-Methylnicotinamide (MNA) on Exercise Capacity and Endothelial Response in Diabetic Mice.

1-Methylnicotinamide (MNA), which was initially considered to be a biologically inactive endogenous metabolite of nicotinamide, has emerged as an anti-thrombotic and anti-inflammatory agent with the capacity to release prostacyclin (PGI2). In the present study, we characterized the effects of MNA on exercise capacity and the endothelial response to exercise in diabetic mice. Eight-week-old db/db mice were untreated or treated with MNA for 4 weeks (100 mg·kg-1), and their exercise capacity as well as NO- and PGI2-dependent response to endurance running were subsequently assessed. MNA treatment of db/db mice resulted in four-fold and three-fold elevation of urine concentrations of MNA and its metabolites (Met-2PY + Met-4PY), respectively (P<0.01), but did not affect HbA1c concentration, fasting glucose concentration or lipid profile. However, insulin sensitivity was improved (P<0.01). In MNA-treated db/db mice, the time to fatigue for endurance exercise was significantly prolonged (P<0.05). Post-exercise Δ6-keto-PGF1α (difference between mean concentration in the sedentary and exercised groups) tended to increase, and post-exercise leukocytosis was substantially reduced in MNA-treated animals. In turn, the post-exercise fall in plasma concentration of nitrate was not affected by MNA. In conclusion, we demonstrated for the first time that MNA improves endurance exercise capacity in mice with diabetes, and may also decrease the cardiovascular risk of exercise

Post-exercise plasma concentration of 6-keto-PGF_1α (A) nitrite (B) and nitrate (C) in untreated and MNA-treated db/db mice.

<p>Delta (Δ) denotes the difference between the mean concentration of a given metabolite determined at rest in the sedentary group and in the exercised group of mice after completing the fatiguing run. Data are presented as the mean ±SEM. Statistical analysis was performed using the Mann-Whitney test or unpaired t-test depending on the results of the normality test. *P<0.05, **P<0.01, ***P<0.001 vs. corresponding sedentary group (n = 6–12).</p

Effect of 4 weeks treatment with MNA on diabetic profile.

<p>Intraperitoneal glucose tolerance test (IPGTT) (A) (n = 18–20), blood glucose area under the curve (AUC) for IPGTT (B) (n = 18–20), blood HbA_1c concentration (C) (n = 7), lipid profile (D) (n = 7). TC (total cholesterol), LDL (low-density lipoprotein), HDL (high-density lipoprotein), and TG (triglycerides). MNA-treated db/db mice were supplemented with MNA in drinking water for 4 weeks at a dose of 100 mg·kg^-1. The effects of MNA on blood HbA_1c concentration and lipid profile were evaluated in sedentary db/db mice. Data are presented as the mean ±SEM. Statistical analysis was performed using the Mann-Whitney test or unpaired t-test depending on the results of the normality test. ***P<0.001 vs. control db/db mice at 8 weeks of age, ##P<0.01 vs. control db/db mice at 12 weeks of age.</p