Cell Survival

B-chronic lymphocytic leukaemia (B-CLL) is a heterogeneous disease characterized by an accumulation of B lymphocytes expressing CD5. To date, the biological significance of this molecule in B-CLL B cells remains to be elucidated. In this study, we have analysed the functional consequences of the binding of an anti-CD5 antibody on B-CLL B cells. To this purpose, we have measured the percentage of viability of B-CLL B cells in the presence or in the absence of anti-CD5 antibodies and also examined some of the biochemical events downstream the CD5-signalling. We demonstrate that anti-CD5 induces phosphorylation of protein tyrosine kinases and protein kinase C (PKC), while no activation of Akt/PKB and MAPKs is detected. This signalling cascade results in viability in a group of patients in which we observe an increase of Mcl-1 levels, whereas the levels of bcl-2, bcl-xL and XIAP do not change. We also report that this pathway leads to IL-10 production, an immunoregulatory cytokine that might act as an autocrine growth factor for leukaemic B cells. Inhibition of PKC prevents the induction of Mcl-1 and IL-10, suggesting that the activation of PKC plays an important role in the CD5-mediated survival signals in B cells from a subset of B-CLL patients.

Hepatocarcinogenesis is under polygenic control. We analyzed gene expression patterns of dysplastic liver nodules (DNs) and hepatocellular carcinomas (HCCs) chemically-induced in F344 and BN rats, respectively susceptible and resistant to hepatocarcinogenesis. Expression profiles were performed by microarray and validated by quantitative RT-PCR and Western blot. Cluster analysis revealed two distinctive gene expression patterns, the first of which included normal liver of both strains and BN nodules, and the second one F344 nodules and HCC of both strains. We identified a signature predicting DN and HCC progression, characterized by highest expression of oncosuppressors Csmd1, Dmbt1, Dusp1, and Gnmt, in DNs, and Bhmt, Dmbt1, Dusp1, Gadd45g, Gnmt, Napsa, Pp2ca, and Ptpn13 in HCCs of resistant rats. Integrated gene expression data revealed highest expression of proliferation-related CTGF, c-MYC, and PCNA, and lowest expression of BHMT, DMBT1, DUSP1, GADD45g, and GNMT, in more aggressi...

Introduction-Eliminating and/or inhibiting bacterial growth within the root canal system have been shown to play a key role in the regenerative outcome. The aim of this study was to synthesize and determine in vitro both the antimicrobial effectiveness and cytocompatibility of bi-mix antibiotic-containing polydioxanone (PDS)-based polymer scaffolds. Methods-Antibiotic-containing (metronidazole, MET and ciprofloxacin, CIP) polymer solutions (distinct antibiotic weight ratios) were spun into fibers as a potential mimic to the double antibiotic paste (DAP, a MET/CIP mixture). Fiber morphology, chemical characteristics, and tensile strength were evaluated by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and tensile testing, respectively. Antimicrobial efficacy was tested over time (aliquot collection) against Enterococcus faecalis (Ef), Porphyromonas gingivalis (Pg), and Fusobacterium nucleatum (Fn). Similarly, cytotoxicity was evaluated in human dental pulp stem cells (hDPSCs). Data were statistically analyzed (p<0.05). Results-SEM and FTIR confirmed that electrospinning was able to produce antibioticcontaining fibers with diameter mostly in the nanoscale. Tensile strength of 1:1MET/CIP scaffolds was significantly (p<0.05) higher than pure PDS (control). Meanwhile, all other groups presented similar strength as the control. Aliquots obtained from antibiotic-containing scaffolds inhibited growth of Ef, Pg and Fn, except pure MET that did not show an inhibitory action towards Pg or Fn. Antibiotic-containing aliquots promoted slight hDPSCs viability reduction, but none of them were considered to be cytotoxic.

Cannabinoids have been reported to provide neuroprotection in acute and chronic neurodegeneration. In this study, we examined whether they are also effective against the toxicity caused by 6-hydroxydopamine, both in vivo and in vitro, which may be relevant to Parkinson's disease (PD). First, we evaluated whether the administration of cannabinoids in vivo reduces the neurodegeneration produced by a unilateral injection of 6-hydroxydopamine into the medial forebrain bundle. As expected, 2 weeks after the application of this toxin, a significant depletion of dopamine contents and a reduction of tyrosine hydroxylase activity in the lesioned striatum were noted, and were accompanied by a reduction in tyrosine hydroxylase-mRNA levels in the substantia nigra. None of these events occurred in the contralateral structures. Daily administration of D 9 -tetrahydrocannabinol (D 9 -THC) during these 2 weeks produced a significant waning in the magnitude of these reductions, whereas it failed to affect dopaminergic parameters in the contralateral structures. This effect of D 9 -THC appeared to be irreversible since interruption of the daily administration of this cannabinoid after the 2-week period did not lead to the re-initiation of the 6-hydroxydopamine-induced neurodegeneration. In addition, the fact that the same neuroprotective effect was also produced by cannabidiol (CBD), another plant-derived cannabinoid with negligible affinity for cannabinoid CB 1 receptors, suggests that the antioxidant properties of both compounds, which are cannabinoid receptorindependent, might be involved in these in vivo effects, although an alternative might be that the neuroprotection exerted by both compounds might be due to their anti-inflammatory potential. As a second objective, we examined whether cannabinoids also provide neuroprotection against the in vitro toxicity of 6-hydroxydopamine. We found that the non-selective cannabinoid agonist HU-210 increased cell survival in cultures of mouse cerebellar granule cells exposed to this toxin. However, this effect was significantly lesser when the cannabi-noid was directly added to neuronal cultures than when these cultures were exposed to conditioned medium obtained from mixed glial cell cultures treated with HU-210, suggesting that the cannabinoid exerted its major protective effect by regulating glial influence to neurons. In summary, our results support the view of a potential neuroprotective action of cannabinoids against the in vivo and in vitro toxicity of 6hydroxydopamine, which might be relevant for PD. Our data indicated that these neuroprotective effects might be due, among others, to the antioxidant properties of certain plant-derived cannabinoids, or exerted through the capability of cannabinoid agonists to modulate glial function, or produced by a combination of both mechanisms. D

Zinc oxide nanoparticles (nZnO) are one of the most highly produced nanomaterials and are used in numerous applications including cosmetics and sunscreens despite reports demonstrating their cytotoxicity. Dissolution is viewed as one of the main sources of nanoparticle (NP) toxicity, however dissolution studies can be time-intensive to perform and complicated by issues such as particle separation from solution. Our work attempts to overcome some of these challenges by utilizing new methods using UV/vis and fluorescence spectroscopy to quantitatively assess nZnO dissolution in various biologically relevant solutions. All biological buffers tested induce rapid dissolution of nZnO. These buffers, including HEPES, MOPS, and PIPES, are commonly used in cell culture media, cellular imaging solutions and to maintain physiological pH. Additional studies using X-ray diffraction, FT-IR, X-ray photoelectron spectroscopy, ICP-MS and TEM were performed to understand how the inclusion of these non-essential media components impacts the behavior of nZnO in RPMI media. From these assessments, we demonstrate that HEPES causes increased dissolution kinetics, boosts the conversion of nZnO into zinc phosphate/carbonate and, interestingly, alters the structural morphology of the complex precipitates formed with nZnO in cell culture conditions. Cell viability experiments demonstrated that the inclusion of these buffers significantly decreases the viability of Jurkat leukemic cells when challenged with nZnO. This work demonstrates that biologically relevant buffering systems dramatically impact the dynamics of nZnO including dissolution kinetics, morphology, complex precipitate formation, and toxicity profiles.

Bioreactors provide a dynamic culture system for efficient exchange of nutrients and mechanical stimulus necessary for the generation of effective tissue engineered bone grafts (TEBG). We have shown that biaxial rotating (BXR) bioreactor-matured human fetal mesenchymal stem cell (hfMSC) mediated-TEBG can heal a rat critical sized femoral defect. However, it is not known whether optimal bioreactors exist for bone TE (BTE) applications. We systematically compared this BXR bioreactor with three most commonly used systems: Spinner Flask (SF), Perfusion and Rotating Wall Vessel (RWV) bioreactors, for their application in BTE. The BXR bioreactor achieved higher levels of cellularity and confluence (1.4e2.5x, p < 0.05) in large 785 mm 3 macroporous scaffolds not achieved in the other bioreactors operating in optimal settings. BXR bioreactor-treated scaffolds experienced earlier and more robust osteogenic differentiation on von Kossa staining, ALP induction (1.2e1.6Â, p < 0.01) and calcium deposition (1.3 e2.3Â, p < 0.01). We developed a Micro CT quantification method which demonstrated homogenous distribution of hfMSC in BXR bioreactor-treated grafts, but not with the other three. BXR bioreactor enabled superior cellular proliferation, spatial distribution and osteogenic induction of hfMSC over other commonly used bioreactors. In addition, we developed and validated a non-invasive quantitative micro CT-based technique for analyzing neo-tissue formation and its spatial distribution within scaffolds.

The corrosion resistance of AISI 304 stainless steel (AISI 304 SS) and manganese stainless steel (low-nickel SS) brackets in artificial saliva was investigated. The cytotoxic effects of their corrosion products on L929 cell culture were compared by two assays, crystal violet, to evaluate cell viability, and MTT (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide), for cell metabolism and proliferation. The atomic absorption spectroscopic analysis of the corrosion products demonstrated that nickel and manganese ion concentrations were higher for the AISI 304 SS-bracket immersion solution as compared with the low-nickel SS brackets. Scanning electron microscopy and energy-dispersive spectroscopy demonstrated less corrosion resistance for the AISI 304 SS brackets. Although none of the bracket extracts altered L929 cell viability or morphology, the AISI 304 SS-bracket extracts decreased cellular metabolism slightly. The results indicated that the low-nickel SS presents better i...

Preeclampsia is one of the leading causes of maternal and perinatal mortality and morbidity and its pathogenesis is not fully understood. B-cell lymphoma 6 (BCL6), a key regulator of B-lymphocyte development, is altered in preeclamptic placentas. We show here that BCL6 is present in all 3 studied trophoblast cell lines and it is predominantly expressed in trophoblastic HTR-8/SVneo cells derived from a 1 st trimester placenta, suggestive of its involvement in trophoblast expansion in the early stage of placental development. BCL6 is strongly stabilized upon stress stimulation. Inhibition of BCL6, by administrating either small interfering RNA or a specific small molecule inhibitor 79-6, reduces proliferation and induces apoptosis in trophoblastic cells. Intriguingly, depletion of BCL6 in HTR-8/SVneo cells results in a mitotic arrest associated with mitotic defects in centrosome integrity, indicative of its involvement in mitotic progression. Thus, like in haematopoietic cells and breast cancer cells, BCL6 promotes proliferation and facilitates survival of trophoblasts under stress situation. Further studies are required to decipher its molecular roles in differentiation, migration and the fusion process of trophoblasts. Whether increased BCL6 observed in preeclamptic placentas is one of the causes or the consequences of preeclampsia warrants further investigations in vivo and in vitro.

Salicylidene acylhydrazides belong to a class of compounds shown to inhibit bacterial type III secretion (T3S) in pathogenic Gram-negative bacteria. This class of compounds also inhibits growth and replication of Chlamydiae, strict intracellular bacteria that possess a T3S system. In this study a library of 58 salicylidene acylhydrazides was screened to identify inhibitors of Chlamydia growth. Compounds inhibiting growth of both Chlamydia trachomatis and Chlamydophila pneumoniae were tested for cell toxicity and seven compounds were selected for preliminary pharmacokinetic analysis in mice using cassette dosing. Two compounds, ME0177 and ME0192, were further investigated by individual pharmacokinetic analysis. Compound ME0177 had a relatively high peak plasma concentration (C max) and area under curve and therefore may be considered for systemic treatment of Chlamydia infections. The other compound, ME0192, had poor pharmacokinetic properties but the highest anti-chlamydial activity in vitro and therefore was tested for topical treatment in a mouse vaginal infection model. ME0192 administered vaginally significantly reduced the infectious burden of C. trachomatis and the number of infected mice.

BackgroundThe environment preferred by Acanthamoeba trophozoites and the mechanism by which the amebae enters the cornea are not yet fully understood. A better understanding of the pathogenesis of this disease may help with prevention and treatment.

Percentage of viable sperms has good potential to reflect male fertilizing potential. In the present study, an attempt was made to find out the percentage of viable sperms in normal and abnormal ejaculates and influence of reactive oxygen species in affecting the viability of sperm. Malondialdehyde (MDA) in seminal plasma of normal and abnormal ejaculates was measured by Thiobarbituric Acid method and percentage of viable sperms was assessed by Eosin-Nigrosin staining. The results revealed that the abnormal semen samples have significantly lower number of viable spermatozoa (59.06 +/- 9.63% vs. 68.33 +/- 5.46%, P &lt; 0.05) and higher levels of MDA (2.53 +/- 0.66 vs. 1.66 +/- 0.37 nmol/ml, P &lt; 0.05) as compared to normal. A significant negative correlation was found between seminal MDA level and sperm viability. Based on the results obtained from the present study, we suggest that viability of sperm is an authentic parameter to assess male fertility potential and it can be affect...

We report here the potential of Pluronic tri-block copolymer micelles for the formulation of curcumin, a natural dietary compound having great therapeutic potential against many diseases including cancer. Two most commonly used Pluronic F127 and F68 were used for the formulation and analyzed for curcumin encapsulation efficiency and stability. The encapsulation of drug in micelle was highly dependent on drug-to-copolymer ratio. Pluronic F127 showed better encapsulation efficiency than Pluronic F68. In vitro release profile demonstrated slower and sustained release of curcumin from Pluronic micelles. The lyophilized form of the formulations exhibited good stability for long-term storage. The physical interaction of curcumin with Pluronic was evident by XRD analysis, UV-visible, fluorescence, and FT-IR spectroscopy. AFM study showed that the drug-encapsulated micelles were spherical in shape with diameters below 100 nm. The in vitro cytotoxicity of the drug formulations was investigated with HeLa cancer cells. Pluronic-encapsulated curcumin showed comparable anticancer activity with free curcumin.

Aloe-emodin has shown anti-neoplastic activity against some human cancer cell lines. This study aimed to explore the effects of aloe-emodin on the phagocytosis of macrophages, the activity of natural killer (NK) cells and the expression of cytokines in leukocytes from Sprague-Dawley rats. Leukocytes were collected, placed into culture plates and the functions of macrophages and NK cells and the percentage of viable cells were determined by flow cytometric analysis. Incubation of leukocytes with various concentrations of aloe-emodin caused a dose-dependent decrease of viable cells, a decrease of phagocytosis by macrophages, and a decrease of the activity of NK cells. Evaluation of cytokines in leukocytes by ELISA indicated that aloe-emodin increased the levels of interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha. The results were also confirmed by PCR assay for the mRNA expression of the examined cytokines.

Thirty-four patients with advanced Parkinson's disease participated in a prospective 24-month double-blind, placebocontrolled trial of fetal nigral transplantation. Patients were randomized to receive bilateral transplantation with one or four donors per side or a placebo procedure. The primary end point was change between baseline and final visits in motor component of the Unified Parkinson's Disease Rating Scale in the practically defined off state. There was no significant overall treatment effect ( p ‫؍‬ 0.244). Patients in the placebo and one-donor groups deteriorated by 9.4 ؎ 4.25 and 3.5 ؎ 4.23 points, respectively, whereas those in the four-donor group improved by 0.72 ؎ 4.05 points. Pairwise comparisons were not significant, although the four-donor versus placebo groups yielded a p value of 0.096. Stratification based on disease severity showed a treatment effect in milder patients ( p ‫؍‬ 0.006). Striatal fluorodopa uptake was significantly increased after transplantation in both groups and robust survival of dopamine neurons was observed at postmortem examination. Fifty-six percent of transplanted patients developed dyskinesia that persisted after overnight withdrawal of dopaminergic medication ("off"-medication dyskinesia). Fetal nigral transplantation currently cannot be recommended as a therapy for PD based on these results.

Cytocompatible, co-doped cerium oxide nanoparticles exhibited strong upconversion properties that were found to kill lung cancer cells by inducing apoptosis thereby demonstrating the potential to be used as clinical contrast agents for imaging and as therapeutic agents for treatment of cancer.

Brain-pancreas relative protein (BPRP) is a novel protein that mainly expresses in brain and pancreas. In our previous study, we found that various stressors significantly decreased the expression of BPRP in pancreas in vivo, accompanied by changes in insulin and glucose levels, and that expression of BPRP in pancreas also decreased significantly in diabetic rats induced by Streptozocin (STZ). All these findings suggest that BPRP may be a glucose or insulinsensitive protein. However, how the changes in insulin or glucose levels influence the expression of BPRP in hippocampus requires further study. Here, we investigated the effects of insulin or glucose on the expression of BPRP in primary cultured hippocampal neurons. We supplied hippocampal neurons with glucose, insulin, or supernatant from pancreatic b-cells, which secrete insulin into the supernatant. Our data showed that insulin had beneficial effect on the viability while no significant effect on the expression of BPRP in hippocampal neurons. On the contrary, 40 mM glucose or free glucose culture significantly decreased the expression of BPRP, while had no significant effect on the viability and apoptosis of hippocampal neurons. Further study showed that levels of insulin in the supernatant collected from pancreatic b-cells medium changed over days, and that supernatant increased the viability of hippocampal neurons, while it had no obvious effect on the expression of BPRP in hippocampal neurons. These results suggest that BPRP may be a glucose-sensitive protein.

Ischemia/reperfusion (I/R) injury is an important cause of acute renal failure because of oxidative, inflammatory, and apoptotic mechanisms. The aim of the present study was to examine any possible protective effects of levosimendan in an in vivo pig model of renal I/R injury. In 40 anesthetized pigs (eight groups of five pigs each), I/R was induced by clamping-reopening the left renal artery. During ischemia, in three groups of pigs, levosimendan and the multiorgan preservation solution Custodiol, alone or in combination with levosimendan, were infused in the renal artery. In two other groups of animals, levosimendan in combination with Custodiol was administered after the intrarenal nitric-oxide (NO) synthase blocker N -nitro-L-arginine methyl ester (L-NAME) or the mitochondrial ATP-sensitive K ϩ channel (K ATP channel) inhibitor 5-hydroxydecanoate (5-HD). In the other animals, saline, L-NAME, or 5-HD were administered alone. Throughout the experiments, urinary N-acetyl-␤-glucosaminidase (NAG) release was measured, and renal function was assessed. Moreover, renal biopsy samples were taken for the detection of apoptosis and tissue peroxidation. In pigs treated with levosimendan or the combination of levosimendan and Custodiol, NAG, peroxidation, and apoptotic markers were lower than in animals treated with Custodiol alone. In addition, renal function was better preserved, and cell survival and antioxidant systems were more activated. All beneficial effects were prevented by L-NAME and 5-HD. In conclusion, levosimendan alone or in combination with Custodiol exerted better protection against renal I/R injuries than Custodiol alone through antioxidant, antiapoptotic, and prosurvival actions depending on mitochondrial K ATP channels and NO-related mechanisms.

Oxidative stress is strictly correlated to the pathogenesis of many diseases, and a diet rich in fruits and vegetables, or adequately integrated, is currently considered to be a protective and preventive factor. This study aimed to analyze the efficacy of a 1 h preincubation with the highest nontoxic dose of a characterized Mentha longifolia extract (80 μg/mL) in protecting human keratinocytes (NCTC2544) from chemically induced oxidative stress (500 μM H 2 O 2 for 2, 16, and 24 h). As reference synthetic pure compounds rosmarinic acid (360.31 μg/mL), a major mint phenolic constituent, and resveratrol (31.95 mg/mL), a well-known antioxidant, were used. Cellular viability was significantly protected by mint, which limited protein and DNA damage, decreased lipid peroxidation, and preserved glutathione and superoxide dismutase activity in the shorter phases of oxidative stress induction, in extents comparable to or better than those of pure compounds. These data suggest that mint use as only a flavoring has to be revised, taking into consideration its enrichment in foodstuff and cosmetics.

Conventional flow cytometric methods for CD34 ؉ cell counting may be affected by the high number of nucleated red blood cells or nonviable cells in cord blood and its products. We developed a simple flow cytometric no-wash procedure that avoids these shortcomings because it provides absolute CD34 ؉ cell counts and assesses cell viability.

The DNA damage response triggered by bacterial cytolethal distending toxins (CDTs) is associated with activation of the actin-regulating protein RhoA and phosphorylation of the downstream-regulated mitogen-activated protein kinase (MAPK) p38, which promotes the survival of intoxicated (i.e. cells exposed to a bacterial toxin) cells. To identify the effectors of this CDT-induced survival response, we screened a library of 4492 Saccharomyces cerevisiae mutants that carry deletions in nonessential genes for reduced growth following inducible expression of CdtB. We identified 78 genes whose deletion confers hypersensitivity to toxin. Bioinformatics analysis revealed that DNA repair and endocytosis were the two most overrepresented signaling pathways. Among the human orthologs present in our data set, FEN1 and TSG101 regulate DNA repair and endocytosis, respectively, and also share common interacting partners with RhoA. We further demonstrate that FEN1, but not TSG101, regulates cell sur...

We examined the cytotoxic potential of nine N-[2-substituted-2-(2-thienyl) ethyl] piperazinyl quinolone derivatives on human oral epithelial mouth carcinoma (KB) and human squamous carcinoma (A431) cell lines. Phototoxic properties of these compounds were ...

The use of copper-based alloys for fixed dental crowns and bridges is increasingly widespread in several countries. The aim of this work is to study the dissolution of a zinc-aluminum-bronze and the cytotoxic effects of the ions released on UMR-106 osteoblastic cell line. Two sources of ions were used: (1) ions released by the metal alloy immersed in the cell culture and (2) salts of the metal ions. Conventional electrochemical techniques, atomic absorption spectroscopy [to obtain the average concentration of ions (AC) in solution], and energy dispersive X-ray (EDX) spectroscopy analysis were used to study the corrosion process. Corrosion tests revealed a strong influence of the composition of the electrolyte medium and the immersion time on the electrochemical response. The cytotoxicity was evaluated with (a) individual ions, (b) combinations of two ions, and (c) the mixture of all the ions released by a metal disc of the alloy. Importantly, synergistic cytotoxic effects were found when Al-Zn ion combinations were used at concentration levels lower than the cytotoxic threshold values of the individual ions. Cytotoxic effects in cells in the vicinity of the metal disc were also found. These results were interpreted considering synergistic effects and a diffusion controlled mechanism that yields to concentration levels, in the metal surroundings, several times higher than the measured AC value. V

Recent findings suggest that the inhibition of Aurora A (AURKA) kinase may offer a novel treatment strategy against metastatic cancers. In the current study, we determined the effects of AURKA inhibition by the small molecule inhibitor MLN8237 both as a monotherapy and in combination with the microtubule targeting drug eribulin on different stages of metastasis in triple negative breast cancer (TNBC) and defined the potential mechanism of its action. MLN8237 as a single agent and in combination with eribulin affected multiple steps in the metastatic process including migration, attachment, and proliferation in distant organs, resulting in suppression of metastatic colonization and recurrence of cancer. Eribulin application induces accumulation of active AURKA in TNBC cells providing foundation for the combination therapy. Mechanistically, AURKA inhibition induced cytotoxic autophagy via activation of the LC3B/p62 axis and inhibition of pAKT, leading to eradication of metastases, but...

The influence of epinastine hydrochloride (EP) on eosinophil survival was examined by an in vitro cell culture technique. Nasal epithelial cells (NECs) were stimulated with 25 ng/ml TNF-alpha in the presence of EP (10 to 30 ng/ml). After 24 h, the culture supernatants were obtained and used as conditioned media of NECs (CM). Eosinophils (1 x 10(3) cells/ml) prepared from healthy human peripheral blood were incubated with 25% CM and eosinophil survival was assessed by trypan blue dye exclusion test 48 h later. CM prepared from NEC cultures pre-treated with TNF-alpha and EP caused a decrease in eosinophil survival as compared with that from NEC cells pre-treated with TNF-alpha alone. The minimum concentration of EP that caused a significant decrease in eosinophil survival was 25 ng/ml. The addition of EP into eosinophil cultures did not cause inhibition of eosinophil survival, which was prolonged by stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF), even when ...

Purpose Pancreatic cancer (PC) is one of the deadliest of all tumors. Previously, we were the first to show that Thymoqui-none (TQ) derived from black seed (Nigella sativa) oil has anti-tumor activity against PC. However, the concentration of TQ required was ...

Calcium-alginate hydrogel has been widely studied as a material for cell encapsulation for tissue engineering. At present, the effect that cells have on the degradation of alginate hydrogel is largely unknown. We have shown that fibroblasts encapsulated at a density of 7.5 Â 10 5 cells ml À1 in both 2% and 5% w/v alginate remain viable for at least 60 days. Rheological analysis was used to study how the mechanical properties exhibited by alginate hydrogel changed during 28 days in vitro culture. Alginate degradation was shown to occur throughout the study but was greatest within the first 7 days of culture for all samples, which correlated with a sharp release of calcium ions from the construct. Fibroblasts were shown to increase the rate of degradation during the first 7 days when compared with acellular samples in both 2% and 5% w/v gels, but after 28 days both acellular and cell-encapsulating samples retained disc-shaped morphologies and gel-like spectra. The results demonstrate that although at an early stage cells influence the mechanical properties of encapsulating alginate, over a longer period of culture, the hydrogels retain sufficient mechanical integrity to exhibit gel-like properties. This allows sustained immobilization of the cells at the desired location in vivo where they can produce extracellular matrix and growth factors to expedite the healing process.

Previous studies have shown that high concentrations of ethanol (≥40%) cause functional damage of the gastrointestinal epithelial barrier by direct cytotoxic effect on the epithelial cells. The effects of lower noncytotoxic doses of ethanol on epithelial barrier function are unknown. A major ...

The emergence of strains of multidrug-resistant Gram-negative bacteria mandates a search for new types of antimicrobial agents. Alyteserin-2a (ILGKLLSTAAGLLSNL.NH 2) is a cationic, a-helical peptide, first isolated from skin secretions of the midwife toad, Alytes obstetricans, which displays relatively weak antimicrobial and haemolytic activities. Increasing the cationicity of alyteserin-2a while maintaining amphipathicity by the substitution Gly 11 ! Lys enhanced the potency against both Gram-negative and Gram-positive bacteria by between fourfold and 16-fold but concomitantly increased cytotoxic activity against human erythrocytes by sixfold (mean concentration of peptide producing 50% cell death; LC 50 = 24 mM). Antimicrobial potency was increased further by the additional substitution Ser 7 !Lys, but the resulting analogue remained cytotoxic to erythrocytes (LC 50 = 38 mM). However, the peptide containing D-lysine at positions 7 and 11 showed high potency against a range of Gram-negative bacteria, including multidrugresistant strains of Acinetobacter baumannii and Stenotrophomonas maltophilia (minimum inhibitory concentration = 8 mM) but appreciably lower haemolytic activity (LC 50 = 185 mM) and cytotoxicity against A549 human alveolar basal epithelial cells (LC 50 = 65 mM). The analogue shows potential for treatment of nosocomial pulmonary infections caused by bacteria that have developed resistance to commonly used antibiotics.

Libraries of compounds are increasingly becoming commercially available for the use of individual academic laboratories. A high-throughput system based on a stably integrated transcriptional reporter was used to screen a library of random compounds to identify agents that conferred robust augmentation of a signal transduction pathway. A novel histone deacetylase (HDAC) inhibitor, termed scriptaid, conferred the greatest effect, a 12- to 18-fold augmentation. This facilitation of transcriptional events was generally applicable to exogenous gene constructs, including viral and cellular promoters, different cell lines and reporter genes, and stably integrated and transiently introduced sequences. Scriptaid did not interfere with a further induction provided by stimulation of the cognate signal transduction pathway (transforming growth factor beta/Smad4), which implied the functional independence of ligand-stimulated transcriptional activation and histone acetylation states in this syst...

Tuberculosis (TB) is one of the leading infectious causes of death due to single infectious agent after HIV/AIDS. Rifampicin (RIF), Isoniazid (INH), Ethambutol (EMB), Pyrazinamide (PZA) and/or their combinations are extensively prescribed to treat TB. Despite several therapeutic implications, these drugs also produce several toxic effects at cellular level. MTT assay and Ames test were adopted in this study for the determination of cytotoxic and mutagenic potential of these anti-TB drugs. Among all tested drugs, cytotoxic potential of RIF was strongest with highly significant decline (p < 0.001) in cell numbers at the concentration of 250 g/ml with LC 50 at 325 g/ml, while significant decline (p < 0.01) in cell count was observed in INH treated group at the concentration 500 g/ml with LC 50 at 1000 g/ml. Moreover, combination RIPE demonstrated significant reduction (p < 0.01) in cell number at the concentration of 25-500-500-500 g/ml with LC 50 at 60-1200-1200-1200 g/ml. It is apparent from the data that almost all drugs represented identical mutagenic pattern i.e., more significant results were achieved in TA100 with metabolic activation (+S9). RIF proved to be highly mutagenic of all tested drugs with significant mutagenicity (p < 0.01) at 0.0525 g/plate against TA98 strain with S9. The combination RIPE exhibited highly significant mutagenic activity (p < 0.01) at concentration 0.125-3-3-3 g/plate without S9, while addition of S9 resulted in similar activity at lower doses, i.e., 0.0525-1-1-1 g/plate. It was concluded from the data that all anti-TB drugs possess significant cytotoxic and mutagenic potential, especially in combination, making TB patient more vulnerable to cytotoxic and mutagenic effects of anti-TB drugs, which could produce further health complications in TB patients.

Limited recovery of islets post-cryopreservation influences graft survival and transplantation efficiency during diabetes treatment. As curcumin, a potent antioxidant/radical scavenging compound, protects islets against beta cell toxins, we hypothesized that inclusion of curcumin during cryopreservation or during post-thaw culture or both may rescue islets from cryoinjury. To test the effect of curcumin inclusion on islet recovery murine islets were isolated by the collagenase digestion, cultured for 48 h, cryopreserved using dimethylsulphoxide as cryoprotectantwith or without curcumin (10 μM)and then slow cooled to −40°C before immersing them in liquid nitrogen for 7 days. Following rapid thawing with sucrose gradient and 24 h post-thaw culturein presence or absence of curcumin (10 μM)islet viability and functionality were determined. Islet recovery in curcumin treated groups was significantly higher than in groups where islets were cryopreserved without curcumin. Islets cryopreserved with curcumin also showed more intact islets as well as better morphology as compared to islets cryopreserved without curcumin. Curcumin treated islets also showed significant inhibition of ROS generation as compared to islets cryopreserved without curcumin. Glucose responsiveness and insulin secretion in islets cryopreserved with curcumin was equal to that of the freshly isolated islets as against islets cryopreserved without curcumin. Elevated level of Hsp 70 and HO-1 were observed in islets cryopreserved with curcumin and may contribute to curcumin-induced islet rescue. Hence, we conclude that inclusion of curcumin into cryopreservation medium inhibits ROS generation and corresponding islet damage and dysfunction.

Phosphorodiamidate morpholino oligomers (PMO) are synthetic antisense molecules that interfere with translation, pre-mRNA splicing and RNA synthesis. Like other gene-silencing technologies, PMO are poorly taken up by primary leukocytes without the use of physical or chemical delivery techniques. We sought an alternative delivery mechanism of PMO into immune cells that eliminates the need for such manipulations. Here we demonstrate the first use of arginine-rich cell-penetrating peptides (CPPs) to deliver PMO (P-PMO) directly into primary murine leukocytes for inhibition of gene expression and promotion of altered pre-mRNA splicing. We compared the P-PMO delivery efficacy of four arginine-rich CPPs including HIV Tat and penetratin, and one histidine rich CPP, and found that the (RXR) 4 peptide was the most efficacious for PMO delivery and targeted antisense effect. The delivery and antisense effects of P-PMO are time-and dose-dependent and influenced by the activation and maturation states of T cells and dendritic cells, respectively. Targeted expression of several genes using P-PMO is shown including surface signaling proteins (CD45 and OX-40), a cytokine (interleukin-2), and a nuclear transcription factor (Foxp3). Considering the abundance of naturally occurring alternatively spliced gene products involved in immune regulation, P-PMO offer an effective method for modulating gene activity for immunological research and applications beyond traditional antisense approaches.

a b s t r a c t Gastroretentive drug delivery system is a promising option for the treatment of Helicobacter pylori infection, which can prolong gastric residence time and supply high drug concentration in the stomach. In the present study, a low density system of metronidazole-loaded porous Eudragit ® RS microparticle with high drug loading capacity (>25%) was fabricated via electrospray method. The porous structure and size distribution of microparticles were affected by polymer concentration and flow rate of solution. FTIR and XRD analyses indicated that drug has been entrapped into the porous microparticles. In addition, sustained release profiles and slight cytotoxicity in vitro were detected. Gamma scintigraphy study in vivo demonstrated that 131 I-labeled microparticles retained in stomach for over 8 h, and about 65.50% radioactive counts were finally detected in the region of interest. The biodistribution study confirmed that hotspot of radioactivity was remaining in the stomach. Furthermore, metronidazole-loaded porous microparticles can eradicate H. pylori completely with lower dose and administration frequency of antibiotic compared with pure drug, which were also more helpful for the healing of mucosal damages. These results suggest that prepared porous microparticle has the potential to provide better treatment for H. pylori infection.

We analyzed the survival responses and downstream signaling elicited by GDNF on sympathetic neurons from different Ret knockin mice. Lack of tyrosine 1062, a multidocking site in Ret, completely prevented GDNF-mediated survival. Importantly, lack of tyrosine 981, although abrogating Akt phosphorylation, had no effect on neuronal survival, indicating that the PI 3-K/Akt pathway is not necessary for survival of sympathetic neurons. In contrast, silencing of B-Raf completely prevented not only GDNF-mediated but also NGF-mediated cell survival, independently of MEK-1/2. We identified IKKs as the main effectors of the protective effects of B-Raf. First, B-Raf interacted with and activated IKKs. Second, knockdown of IKKs reversed the protection afforded by a constitutively active form of B-Raf. Third, knockdown of IKKs prevented both NGF-and GDNF-mediated survival. In conclusion, our data delineate a novel survival pathway for sympathetic neurons linking B-Raf to IKKs, independently of both PI 3-K and MEK-1/2 pathways.

Background: 1E10 monoclonal antibody is a murine anti-idiotypic antibody that mimics N-glycolyl-GM3 gangliosides. This antibody has been tested as an anti-idiotypic cancer vaccine, adjuvated in Al(OH) 3 , in several clinical trials for melanoma, breast, and lung cancer. During early clinical development this mAb was obtained in vivo from mice ascites fluid. Currently, the production process of 1E10 is being transferred from the in vivo to a bioreactor-based method.

Plant-derived phenolic compounds, including polyphenols (e.g., tannins), flavonoids, and phenolic acids, have been under investigation for their anticancer therapeutic and chemoprevention properties. Recently, certain mechanisms underlying the differential effects of GTPPs on tumor versus normal cells have been determined. These suggest that GTPPs may simultaneously activate multiple pathways. However, existing screening methods are insufficient for the identification of agents that possess both a cytotoxic effect on tumor cells and a protective effect on normal cells. The current study describes the establishment of an in vitro survival/apoptosis testing system based on detecting these mechanisms by a double-fluorescence method. This system is able to screen potential chemopreventive or therapeutic agents from (but not limited to) plant-derived compounds based on the pathways differentially activated by the agents. Tumor cell death and normal cell survival are detected simultaneously, in a device that co-cultures normal human cells adjacent to human tumor cells.

We report a procedure to prepare highly 14 monodisperse copper telluride nanocubes, nanoplates, and 15 nanorods. The procedure is based on the reaction of a 16 copper salt with trioctylphosphine telluride in the presence 17 of lithium bis(trimethylsilyl)amide and oleylamine. CuTe 18 nanocrystals display a strong near-infrared optical 19 absorption associated with localized surface plasmon 20 resonances. We exploit this plasmon resonance for the 21 design of surface-enhanced Raman scattering sensors for 22 unconventional optical probes. Furthermore, we also 23 report here our preliminary analysis of the use of CuTe 24 nanocrystals as cytotoxic and photothermal agents. 25 C opper-based chalcogenides are used in a wide range of 26

The antiviral drug acyclovir [9-(2-hydroxyethoxymethyl)guanine (ACV)], its 7-isomer (7-ACV) and its two derivatives: N2-acetyl ACV (ac-ACV) and N2,O-diacetyl ACV (diac-ACV) were examined for their potential in vitro lymphotoxicity and in vivo immunotoxicity in mice. In vitro lymphotoxicity of ACV and its acetylated derivatives was low, whereas the 7-ACV isomer enhanced the in vitro cell proliferation in PHA-stimulated cultures. Addition of 2'-deoxyguanosine (dGuo) did not exhibit any inhibitory potential of ACV. However, reduction in the absolute number of CD3+, CD8+, and CD25+ cells, but not Ig+ cells, was noted at high concentrations of ACV and its derivatives, suggesting a selective T cell cytotoxicity. Similarly, the in vivo exposure revealed selective T cell immunotoxicity of ACV and its derivatives since the reduced number of Thy 1.2+ and CD8+ cells was not accompanied with any marked changes in the Ig+ population. The CD4+/CD8+ ratio was affected both in vitro and in vivo by high concentrations of ACV.

Keywords: hAChE hBuChE Dual AChE inhibitors 6-Chloro-pyridonepezils In vitro blood brain barrier Molecular modeling ADME Alzheimer's disease a b s t r a c t 6-Chloro-pyridonepezils are chloropyridineedonepezil hybrids designed by combining the N-benzylpiperidine moiety present in donepezil with the 2-chloropyridine-3,5-dicarbonitrile heterocyclic ring system, both connected by an appropriate polymethylene linker. 6-Chloro-pyridonepezils 1e8 were prepared by reaction of 2,6-dichloro-4-phenylpyridine-3,5-dicarbonitrile (13) [or 2,6-dichloropyridine-3,5-dicarbonitrile ] with suitable 2-(1-benzylpiperidin-4-yl)alkylamines (9e12). The biological evaluation showed that these new compounds are cholinesterase inhibitors, in the submicromolar range, one of them (6) being a potent hBuChE inhibitor (IC 50 ¼ 0.47 AE 0.08 mM). 6-Chloro-pyridonepezils 4, 7 and 8 are potent hAChE inhibitors showing IC 50 in the 0.013e0.054 mM range. Particularly, 6-chloro-pyridonepezil 8 is 625-fold more selective for hAChE than for hBuChE and compared to donepezil is equipotent for the inhibition of hAChE. Molecular modeling investigation on 6-chloro-pyridonepezils 4, 6e8 supports its dual AChE inhibitory profile, by binding simultaneously at the catalytic active and at peripheral anionic sites of the enzyme. The in vitro Blood Brain Barrier (BBB) and theoretical ADME analysis of 6-chloro-pyridonepezils 1e8 have been carried out. Overall, compound 8, is a permeable potent and selective dual AChEI that can be considered as a good candidate with potential impact for further pharmacological development in Alzheimer's therapy.

Transformation with ras oncogenes results in increased radiation survival in many but not all cells. In addition, prenyltransferase inhibitors, which inhibit ras proteins by blocking posttranslational modification, radiosensitize cells with oncogenic ras. These findings suggest that oncogenic ras contributes to intrinsic radiation resistance.

AbstractöThere is growing evidence to suggest that growth hormone plays a role in the growth and development of the CNS. Speci¢cally, growth hormone has been implicated in promoting brain growth, myelination, neuronal arborisation, glial di¡erentiation and cognitive function. Here we investigate if growth hormone has a role in the recovery from an unilateral hypoxic^ischaemic brain injury. Using moderate (15 min hypoxia) and severe (60 min hypoxia) models of hypoxic^ischaemia in juvenile rats and standard immunohistochemical techniques, we found intense growth hormone-like immunoreactivity present within regions of cell loss by 3 days (P 6 0.05). Growth hormone-like immunoreactivity was observed on injured neurones, myelinated axons, glial cells within and surrounding infarcted tissue and on the choroid plexus plus ependymal cells within the injured hemisphere. The pattern of immunoreactivity suggests that (a) growth hormone (or a growth hormone-like substance) is transported via the cerebrospinal £uid and (b) that growth hormone (or a growth hormone-like substance) is acting in a neurotrophic manner speci¢cally targeted to injured neurones and glia.

in minimal GVHD with retention of antitumor activity In vivo trafficking and survival of cytokine-induced killer cells resulting http://bloodjournal.hematologylibrary.org/content/112/6/2563.full.html Updated information and services can be found at: (1882 articles) Transplantation Articles on similar topics can be found in the following Blood collections http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests

The dynamics of sperm DNA fragmentation (sDF) and sperm viability were analyzed in frozen-thawed sperm samples of Equus asinus (Zamorano-Leonés), a breed at risk of extinction. Sperm DNA fragmentation was assessed using an adaptation of the sperm chromatin dispersion test developed for stallions in five different frozen samples. Sperm were thawed and incubated at different temperatures (37 8C, 25 8C, and 4 8C) and sDF was assessed at different times and compared. The mean sDF after thawing at the beginning of the experiment was 18.20 AE 14.77% and did not differ significantly from the results of a neutral comet assay (22.0 AE 19.34%). The tendency in the sDF of all donkeys indicated that sperm DNA is more sensitive to breakage when incubated at 37 8C than when incubated at 25 8C or 4 8C. Interestingly, the tendency was not the same when different animals were compared, and differences in sDF dynamics were established among individuals. sDF correlated negatively with sperm viability in some individuals but not in others. From a conservation perspective, sDF analysis may offer a new way to assess sperm quality in endangered breeds in order to identify and select the best semen samples for artificial reproduction purposes. In particular, we recommend for artificial insemination the use of semen samples with a slow increase in sDF with time after thawing. #

Recent evidence suggests that ageing-related diseases could result in an accelerated loss of selfrenewal capability of adult stem cells, normally involved in replacing damaged cellular elements. In previous works, we highlighted that a specific treatment, named tissue optimization-regenerative (TO-RGN), of radioelectric asymmetric conveyer (REAC) technology, influenced gene expression profiles controlling stem cell differentiation and pluripotency of human skin-derived fibroblasts in vitro. The purpose of the present work was to verify whether TO-RGN may also be effective in counteracting the expression of the senescence marker beta-galactosidase and of senescence-associated gene expression patterning, engaged during prolonged culture of human adipose-derived stem cells (hADSCs). Following TO-RGN exposure, we observed a significant downregulation in beta-galactosidase staining and in the expression of the senescence mediator genes p16INK4, ARF, p53, and p21 CIP1 . Moreover, differently formed untreated cells, TO-RGN-exposed hADSCs maintained their typical fibroblast-like morphology and exhibited a multilineage potential even at late passages, as shown by the remarkable preservation of commitment to osteogenic, adipogenic, chondrogenic, and vasculogenic fates, both at morphologic and gene expression levels.