Amount Of Viable Cells Research Articles

Effects of Green Tea Compound Epigallocatechin-3-Gallate against Stenotrophomonas maltophilia Infection and Biofilm

We investigated the in vitro and in vivo activities of epigallocatechin-3-gallate (EGCg), a green tea component, against Stenotrophomonas maltophilia (Sm) isolates from cystic fibrosis (CF) patients. In vitro effects of EGCg and the antibiotic colistin (COL) on growth inhibition, survival, and also against young and mature biofilms of S. maltophilia were determined. Qualitative and quantitative changes on the biofilms were assessed by confocal laser scanning microscopy (CLSM). Further, in vivo effects of nebulized EGCg in C57BL/6 and Cftr mutant mice during acute Sm lung infection were evaluated. Subinhibitory concentrations of EGCg significantly reduced not only biofilm formation, but also the quantity of viable cells in young and mature biofilms. CLSM showed that EGCg-exposed biofilms exhibited either a change in total biofilm biovolume or an increase of the fraction of dead cells contained within the biofilm in a dose depended manner. Sm infected wild-type and Cftr mutant mice treated with 1,024 mg/L EGCg by inhalation exhibited significantly lower bacterial counts than those undergoing no treatment or treated with COL. EGCg displayed promising inhibitory and anti-biofilm properties against CF Sm isolates in vitro and significantly reduced Sm bacterial counts in an acute infection model with wild type and CF mice. This natural compound may represent a novel therapeutic agent against Sm infection in CF.

Abstract P5-09-19: Progesterone receptor isoform A as a predictive marker of antiprogestin therapy in breast cancer: A tissue culture method to evaluate drug responsiveness

Abstract Two thirds of breast cancers express steroid hormone receptors and are susceptible to an endocrine therapy usually aimed to target the estrogen receptors (ER). There is however compelling evidence suggesting that progesterone receptors (PR) participate together with ER, modulating breast cancer growth. Based on our results obtained in an experimental breast cancer model in which antiprogestins induce complete regression of carcinomas with higher levels of PR isoform A (PR-A) than isoform B (PR-B), we propose that antiprogestins such as mifepristone (MFP) might be a possible therapeutic option for patients expressing high levels of PR-A. The goal of our study is to correlate the ratio of PR isoform expression, with the in vitro responsiveness of human breast cancer samples to MFP. Preliminary studies indicated that primary cultures of breast cancer samples overexpressing PR-A were responsive to MFP (10 nM) in vitro. However, since the rate of successful primary cultures was not encouraging (7%), we decided to develop a tissue culture assay to evaluate MFP responsiveness of breast cancer samples in vitro. Forty five breast cancer samples obtained from the General Pacheco´s Hospital, Buenos Aires, were kept in culture medium at room temperature and in dry ice. The former were cut in slices of 100 μm using a tissue chopper usually used to slice mouse nervous tissue for short incubations, and were incubated on filters in the presence of growth medium (fetal calf serum 10%) with or without MFP (10 nM), for 48 hs at 37º C. The slices were then fixed, paraffin embedded, and processed for Ki-67 staining to evaluate proliferating cells. Frozen samples were processed for Western blotting and the ratio of both PR isoforms was determined in nuclear extracts. In 17/45 (38%) of the samples valuable information was obtained. This was related with the quality (size and amount of viable cells) of the sample. The number of Ki-67 positive cells over the total viable cells was evaluated by a pathologist (M. May) in at least six different control or MFP-treated slices. From nine cases in which the Ki-67 index in control slices doubled the value obtained in MFP-treated slices (p<0.05), 8 were samples in which PR-A was higher than PR-B. MFP increased (p<0.05) the Ki-67 index in one sample with higher levels of PR-B than PR-A. No significant differences were observed in the remaining cases (null, equimolar or higher levels of PR-B than PR-A). We still plan to increase the number of samples in our study. Our data suggest that the tissue culture method developed is much more efficient than the primary cultures to evaluate drug or hormone responsiveness in breast cancer tissue. The results obtained herein support the proposal of PR-A as a predictive marker of antiprogestin therapy in breast cancer patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-19.

In Vitro Development and Characterization of a Tissue-Engineered Conduit Resembling Esophageal Wall Using Human and Pig Skeletal Myoblast, Oral Epithelial Cells, and Biologic Scaffolds

Tissue engineering represents a promising approach for esophageal replacement, considering the complexity and drawbacks of conventional techniques. To create the components necessary to reconstruct in vitro or in vivo an esophageal wall, we analyzed the feasibility and the optimal conditions of human and pig skeletal myoblast (HSM and PSM) and porcine oral epithelial cell (OEC) culture on biologic scaffolds. PSM and HSM were isolated from striated muscle and porcine OECs were extracted from oral mucosa biopsies. Myoblasts were seeded on an acellular scaffold issue from porcine small intestinal submucosa (SIS) and OEC on decellularized human amniotic membrane (HAM). Seeding conditions (cell concentrations [0.5×10(6) versus 10(6) cells/cm(2)] and culture periods [7, 14 and 21 days]), were analyzed using the methyl thiazoltetrazolium assay, quantitative PCR, flow cytometry, and immunohistochemistry. Phenotypic stability was observed after cellular expansion for PSM and HSM (85% and 97% CD56-positive cells, respectively), and OECs (90% AE1/AE3- positive cells). After PSM and HSM seeding, quantities of viable cells were similar whatever the initial cell concentration used and remained stable at all time points. During cell culture on SIS, a decrease of CD56-positive cells was observed (76% and 76% by D7, 56% and 70% by D14, 28% and 60% by D21, for PSM and HSM, respectively). Multilayered surface of α-actin smooth muscle and Desmine-positive cells organized in bundles was seen as soon as D7, with no evidence of cell within the SIS. Myoblasts fusion was observed at D21. Pax3 and Pax7 expression was downregulated and MyoD expression upregulated, at D14.OEC proliferation was observed on HAM with both cell concentrations from D7 to D21. The cell metabolism activity was more important on matrix seeded by 10(6) cells/cm(2). With 0.5×10(6) OEC/cm(2), a single layer of pancytokeratin-positive cells was seen at D7, which became pluristratified by D14, while when 10(6) OEC/cm(2) were used, a pluristratified epithelial structure was seen as soon as D7. Proliferative cells (Proliferating Cell Nuclear Antigen staining) were mainly located at the basal layer. In this model, the optimal conditions of cell seeding in terms of cell concentration and culture duration were 0.5×10(6) myoblasts/cm(2) and 10(6) OEC/cm(2), and 7 days.

Direct electrochemical determination of Candida albicans activity

Despite advances made in the field, rapid detection methods for the human pathogen Candida albicans are still missing. In this regard, bio-electrochemical systems including electrochemical sensors and biosensors satisfy the increasing demand for rapid, reliable, and direct microbial analyses. In this study, the bioelectrochemical characteristics of C. albicans were investigated for use in an analytical system that determines the viability of the organisms. The electrochemical responses of viable and non-viable cells of C. albicans and Saccharomyces cerevisiae were monitored. Cyclic voltammograms (CV) showed an irreversible oxidation peak at about 750mV that accounts for viable cells. The peak current increased at viable cell numbers ranging from 3×105 to 1.6×107cells/ml, indicating that the amount of viable cells can be accurately quantified. To elucidate the underlying electron transfer processes, the influence of electron transfer chain (ETC) – inhibitors on the electrochemical behavior of the two organisms were investigated. Inhibition of the function of classical respiratory chain (CRC) led to a decrease in the electrochemical response, whereas the oxidation current increased when the alternative oxidase (AOX) pathway was blocked by salicylhydroxamic acid (SHA). Blocking the AOX pathway improved the electrochemical performance, suggesting an involvement in the CRC, with cytochrome c oxidase (COX) as a relevant protein complex. Mutants, in which components of COX were deleted, showed a lower electro-activity than the wild-type strain. Particularly, deletion of subunit COX5a almost completely abolished the electrochemical signal. We believe that this work can be utilized for the development of early detection assays and opens the door for new technological developments in the field of C. albicans.

Abstract 3417: Increased anticancer effect of combining Wee1 (MK1775) and Chk1 (AZD7762) inhibitors in malignant melanoma cell lines.

Abstract Malignant melanoma is one of the most increasing forms of cancer worldwide, in addition to being notoriously resistant to therapy. Despite recent advances, there are no curative treatments once the cancer has spread (stage IV); there is thus a precarious need to find new and better treatment alternatives. Unlike normal cells, the majority of cancer cells have a defective G1/S DNA-damage checkpoint and are therefore more dependent on the G2/M checkpoint when DNA damages arise. Central in regulating the latter checkpoint is Wee1, a kinase which may stop the cell cycle by adding a negative phosphorylation on CDK1. Wee1 is regulated by Chk1 which upon DNA damage can also inhibit the complimentary counterpart of Wee1; CDC25. In recent studies, Wee1 and Chk1 have additionally been indicated as key players in regulating DNA replication in S-phase. We have previously reported that increased Wee1 protein expression was associated with malignancy in melanoma tumour samples, and that siWee1 transfection led to reduced viability in melanoma cell lines. In the present study we have combined inhibitors of Wee1 (MK1775) and Chk1 (AZD7762) in a panel consisting of both metastatic melanoma- and normal cells, including a Plexxikon resistant melanoma cell line (MelJR post3.3). We found that the combination synergistically reduced the amount of viable cells in the melanoma cell lines (∼50-80% reduction), whilst only having very moderate effect (∼10% reduction) in the normal cells. Interestingly, the reduction in number of viable cells was found in both p53WT and p53Mut cell lines, indicating that the p53 status do not predict the response to such treatment. Furthermore, combinational treatment with the inhibitors caused increased cleavage of PARP and caspases, indicative of apoptosis, as compared to MK1775 or AZD7762 treatment alone. The combination also caused a marked increase in DNA double-strand breaks, as demonstrated by augmented levels of γ-H2AX (predominantly found in S-phase cells). In line with this, preliminary results from melanoma cells grown as spheroids, revealed the same tendency, with approximately 50% reduction in spheroid volume when combining the inhibitors. In vivo experiments using melanoma cells grown in SCID mice are currently ongoing. In conclusion, our results indicate a potential new role for inhibitors of Wee1 and Chk1 in treatment of malignant melanoma. Citation Format: Gry I. Magnussen, Vivi A. Florenes, Birgit Engesæter, Elisabeth Emilsen. Increased anticancer effect of combining Wee1 (MK1775) and Chk1 (AZD7762) inhibitors in malignant melanoma cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3417. doi:10.1158/1538-7445.AM2013-3417

Evaluation of the anti-apoptotic effect of mannitol on cerebral ischemia/reperfusion injury in mice

Objective To further verify the practicality of the experimental animal model induced by mice cerebral ischemia-reperfusion injuryand to investigate the optimal time point of mannitol intervention in cerebral ischemia-reperfusion injury.Methods Thirty-six mice were randomly divided into six groups(n=6):sham operation group (group A),control group(ischemiareperfusion injury group,group B),and mannitol intervention group at 0,30 min,1 h and 1.5 h after reperfusion (group C,D,E,F).Brains were removed twenty-four hours after reperfusion.Morphological changes,cells survival and cellular degeneration in CA1 area of hippocampus were studied in each group.Results Ingroup C,most hippocampal pyramidal cells remained normal morphologically,intact structurally and no intercellular edema.The amount of viable cells was (23.01±1.23) per 100 μm in group A and no difference compared with group A (24.00±0.75).The frequency of cellular degeneration was 8.26％,significantly less than that of group B (30.59％,P＜0.05).And no difference compared with group A (7.45％).The apoptosis index (AI) was 6.50％,significantly less than that of group B (28.00％,P＜0.05).No difference of compared with that of group A (5.00％).Mannitol intervention beginning over 30 minutes after reperfusion failed to alleviate neuron injury.Conclusions Inverted osmotic pressure gradient by aggressive mannitol intervention can effectively prevent intracellular edema and avoid the subsequent irreversible cellular injury after the establishment of blood pressure gradient at the early stage of cerebral ischemia-reperfusion.The results suggest that during cardiac resuscitation,mannitol dehydration treatment should be initiated simultaneously at the recovery of autonomous heart rate. Key words: Mannitol; Cerebral ischemia/reperfusion injury; Mice; Apoptosis

Use of soymilk as a storage medium for avulsed teeth

Objective. Tooth avulsion is one of the most severe forms of dental trauma. In these cases, immediate reimplantation is ideal; however, it almost never happens. The purpose of this study was to evaluate the viability of cells stored in soymilk and compare with other several storage media. Materials and methods. The media tested were: long-shelf-life coconut water, long-shelf-life whole milk, long-shelf-life soymilk, Gatorade, egg white, and Hank's Balanced Salt Solution. Cells cultured in DMEM and distilled water served as positive and negative controls, respectively. Plates containing confluent 3T3 fibroblast were soaked in the various media for 2, 12 and 24 h. After incubation at 37°C, viability of the cells was determined using the MTS assay. Data were analyzed by using one-way ANOVA and complemented by Tukey test with a significance level of 5%. Results. Statistical analysis showed that DMEM, whole milk, HBSS and soymilk were the most effective media for maintaining cell viability at all tested times (p < 0.05), followed by coconut water, egg white and Gatorade. The least amount of viable cells was observed in the distilled water group. Conclusions. The present study shows that the efficacy of soymilk in maintaining the viability of 3T3 fibroblasts is similar to that of HBSS and milk. Therefore, it can be concluded that soymilk could be a suitable alternative storage medium for avulsed teeth.

A multiparametric assay to compare the cytotoxicity of soy milk with different storage media

The aim of this study was to evaluate the cytotoxicity of soy milk compared with several other storage media [coconut water, Hank's Balanced Salt Solution (HBSS) and whole milk], assessed through a multiparametric analysis employing 3T3 cells. Plates containing confluent 3T3 fibroblasts were exposed to the various media for 24 h, at 37°C with 5% CO₂, and cell viability was evaluated by a multiparametric assay assessing sequentially, on the same cells, mitochondrial activity (XTT), membrane integrity (neutral red test) and total cell density (crystal violet dye exclusion test). Results from each test were compared by two-way analysis of variance (ANOVA). Statistical analysis showed that whole milk, HBSS and soy milk were the most effective media in maintaining cell viability at all tested times (P < 0.05). The least amount of viable cells was observed when using coconut water. This study shows that the efficacy of soy milk in maintaining the viability of 3T3 fibroblasts is similar to that of HBSS and milk, as shown by three different cell viability tests.

NAD+-dependent Sirtuin 1 and 6 Proteins Coordinate a Switch from Glucose to Fatty Acid Oxidation during the Acute Inflammatory Response

The early initiation phase of acute inflammation is anabolic and primarily requires glycolysis with reduced mitochondrial glucose oxidation for energy, whereas the later adaptation phase is catabolic and primarily requires fatty acid oxidation for energy. We reported previously that switching from the early to the late acute inflammatory response following TLR4 stimulation depends on NAD(+) activation of deacetylase sirtuin 1 (SirT1). Here, we tested whether NAD(+) sensing by sirtuins couples metabolic polarity with the acute inflammatory response. We found in TLR4-stimulated THP-1 promonocytes that SirT1 and SirT 6 support a switch from increased glycolysis to increased fatty acid oxidation as early inflammation converts to late inflammation. Glycolysis enhancement required hypoxia-inducing factor-1α to up-regulate glucose transporter Glut1, phospho-fructose kinase, and pyruvate dehydrogenase kinase 1, which interrupted pyruvate dehydrogenase and reduced mitochondrial glucose oxidation. The shift to late acute inflammation and elevated fatty acid oxidation required peroxisome proliferator-activated receptor γ coactivators PGC-1α and β to increase external membrane CD36 and fatty acid mitochondrial transporter carnitine palmitoyl transferase 1. Metabolic coupling between early and late responses also required NAD(+) production from nicotinamide phosphoryltransferase (Nampt) and activation of SirT6 to reduce glycolysis and SirT1 to increase fatty oxidation. We confirmed similar shifts in metabolic polarity during the late immunosuppressed stage of human sepsis blood leukocytes and murine sepsis splenocytes. We conclude that NAD(+)-dependent bioenergy shifts link metabolism with the early and late stages of acute inflammation.

Experimental study of mannitoi intervention on cerebral ischemia/reperfusion injury in mice

Objective To investigate the optimal timing of mannitol intervention in cerebral ischemia-reperfusion injury.Methods Thirty-six mice were randomly divided into six groups (n=6):The sham operation group (group A),the control group (ischemia-reperfusion injury group,group B),aud the mannitol intervention group at the time of 0 min,30 min,1 h and 1.5 h after reperfusion (group C,D,E,F).Taken the brain twenty-four hours after ischemia-reperfusion injury.Morphological changes,viability and degradation of cells in the area of CA1 of hippocampus were studied in each group.Results In the experimental group with mannitol intervention immediately after reperfusion,most hippocampal pyramidal cells remained normal morphology with intact structures and no intercellular edema was observed.The amount of viable cells was (22.0±1.1) per 100 μm which was not significantly different from that of the sham operation group (23.0±0.9).The incidence of cell degradation was 8.17％,which was significantly lower than that of the control group (40.17％,P＜0.05).The cell degradation difference between the experimental group and sham operation group(7.33％) was not significant(P＞0.05).Mannitol intervention beginning over 30 min after reperfusion failed to alleviate neuron injury.Conclusion After the establishment of blood pressure gradient,inverted osmotic pressure gradient by means of prompt mannitol intervention at the early stage of cerebral ischemia-reperfusion can effectively prevent the formation of intracellular edema and avoid the consequent irreversible cellular injury.Mannitol intervention beginning over 30 min after reperfusion was proved ineffective. Key words: Cerebral ischemia/reperfusion injury; Mannitol; Mice

Viability of lyophilized microorganisms after 50-year storage

The viability of lyophilized microorganisms belonging to different physiological groups was determined after 50-year storage at 2–4°C. During this period, the number of viable cells gradually decreased, in some cases, by 2–3 orders of magnitude. However, after 50-year storage, the ampoules contained considerable amounts of viable cells (in many cultures, 106–109 cells) that were quite sufficient for the culture maintenance. All the studied lyophilized microorganisms (pro- and eukaryotes) retained their viability after 50-year storage, a longer period than those known in literature.

Pathologic Response of Primary Rectal Cancer to Oxaliplatin-Based Chemotherapy

Management of stage IV rectal cancer is controversial, and different strategies may be useful. Preoperative chemotherapy for liver metastases might cause pathologic changes over the primary rectal tumor. In this study, the authors show the pathologic regression of the primary rectal tumor after neoadjuvant chemotherapy treatment. Patients suffering stage IV rectal cancer underwent surgery after oxaliplatin-based chemotherapy. Age, gender, type of surgery, carcinoembryogenic antigen (CEA) level, presence of metastatic disease in one or multiple organs, ypT, ypN, and circumferential resection margin (CRM) were evaluated. Pathologic response of the primary tumor was estimated by using three conventional grading systems and a semiquantitative system assessed by the amount of viable cells out of the total tumor area macroscopically described. Fibrosis, necrosis, and colloid response were evaluated with a semiquantitative system. A complete pathologic response (ypTO) was found in one patient. A good response was observed in the 41.6% of the cases with all grading systems. Presence of fibrosis in the primary tumor was found in six cases. No patient showed CRM involvement. One patient developed a local recurrence. Oxaliplatin-based chemotherapy for stage IV rectal cancer provides high rates of pathologic regression in the rectal tumor and may allow surgery without CRM involvement.

Determine the Potency of BCG Vaccines by Flow Cytometer

ABSTRACTThe Bacille Calmette-Guerin (BCG) vaccine is a live attenuated vaccine prepared from a strain of Mycobacterium bovis. The potency of this vaccine is dependent on the amount of viable cells. The conventional method to determine the potency of the BCG vaccine is to measure the number of colony forming units (CFU) produced after the vaccine has been reconstituted in normal saline and the cells cultured on Löwenstein-Jensen (LJ) medium. However, as M. bovis is a slow growing organism, it needs to grow at least four weeks to be detected in the potency assay. The results of the potency assay could also vary due to manual counting. Therefore, in order to increase the assay efficiency and decrease the variation of the results, we developed an alternative method by using flow cytometer combined with cell counter (viable counts calculated from flow cytometer and counter, VFC) to determine the potency of BCG vaccines. To further test the accuracy of the assay we developed, the viable counts of ten batches of BCG vaccine determined by the VFC assay and conventional CFU method were compared. Wilcoxon statistical analysis showed that there were no statistically significant differences between the VFC assay and the conventional CFU method. Moreover, the results of the VFC assay are all read out from instruments, which could reduce the variation from different operators in order to overcome the limitation of the conventional CFU method. In summary, the results demonstrated that the potency of BCG vaccines could be determined by the VFC assay within four hours instead of four weeks cultivation by the conventional CFU method. On the whole, compared to the conventional CFU method, the VFC assay is a rapid, efficient and reliable method to determine the potency of BCG vaccines.

Impact of MAGE-A antigens on taxane response in oral squamous cell carcinoma

MAGE-A antigens are a subgroup of cancer/testis antigens that are exclusively expressed in malignant cells. Only scarce information on the function of MAGE-A antigens is available. There is some evidence that they may influence the response to chemotherapeutic drugs. This study aimed to evaluate the impact of the MAGE-A antigen subgroups MAGE-A2, -A3, -A4 and -A6 on oral squamous cell carcinoma cell lines treated with docetaxel and paclitaxel. Five oral squamous cell carcinoma cell lines were characterized for their quantitative expression of MAGE-A2, -A3, -A4 and -A6. The cell lines were treated with concentrations ranging from 0.025 to 0.8 μM of docetaxel and paclitaxel. The amount of viable cells after 24 and 48 h was measured. The measurements were statistically correlated with MAGE-A expression. All cell lines responded to docetaxel and paclitaxel. One cell line showed a statistically significant weaker response to the taxane treatment. This cell line was the only one that expressed MAGE-A4. MAGE-A4 has a statistically significant impact on the tumour response to docetaxel and paclitaxel in oral squamous cell carcinoma. This may influence treatment options and the course of the disease. Therefore, patients should be evaluated for MAGE-A4 expression before treatment with taxanes.

Fas activation increases neural progenitor cell survival

Although there is a sizable amount of research focusing on adult neural progenitor cells (NPCs) as a therapeutic approach for many neurodegenerative diseases, including multiple sclerosis, little is known about the pathways that govern NPC survival and apoptosis. Fas, a member of the death receptor superfamily, plays a well-characterized role in the immune system, but its function in neural stem cells remains uncertain. Our study focuses on the effects of Fas on NPC survival in vitro. Activation of Fas by recombinant Fas ligand (FasL) did not induce apoptosis in murine NPCs in culture. In fact, both an increase in the amount of viable cells and a decrease in apoptotic and dying cells were observed with FasL treatment. Our data indicate that FasL-mediated adult NPC neuroprotection is characterized by a reduction in apoptosis, but not increased proliferation. Further investigation of this effect revealed that the antiapoptotic effects of FasL are mediated by the up-regulation of Birc3, an inhibitor of apoptosis protein (IAP). Conversely, the observed effect is not the result of altered caspase activation or FLIP (Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitory protein) up-regulation, which is known to inhibit caspase-8-mediated cell death in T cells. Our data indicate that murine adult NPCs are resistant to FasL-induced cell death. Activation of Fas increased cell survival by decreasing apoptosis through Birc3 up-regulation. These results describe a novel pathway involved in NPC survival.

RNA Interference-Mediated Inhibition of Erythropoietin Receptor Expression Suppresses Tumor Growth and Invasiveness in A2780 Human Ovarian Carcinoma Cells

Although recombinant human erythropoietin (rHuEpo) has revolutionized the treatment of anemia, recent clinical trials suggested that rHuEpo use may be associated with decreased survival in cancer patients. Although the expression of erythropoietin (Epo) receptor (EpoR) has been demonstrated in various human cancers, the effect of exogenous Epo on the growth and therapy resistance of EpoR-bearing tumor cells is unclear at present. In the current study, we examined the hypothesis that EpoR may contribute to tumor growth independent of Epo in A2780 human ovarian carcinoma cells. A2780 human ovarian carcinoma cells showed high levels of EpoR expression, but lacked expression of Epo mRNA and biologically active Epo protein under both normoxic and hypoxic conditions. Exogenous Epo did not stimulate EpoR-mediated signaling, proliferation, invasiveness, or resistance to cytotoxic drugs in A2780 cells. In contrast, specific inhibition of EpoR expression using a short hairpin RNA (shRNA) expression plasmid resulted in markedly reduced proliferation and invasiveness in vitro. In addition, inhibition of EpoR expression led to abrogated in vivo ovarian cancer cell growth in a tumor xenograft system and resulted in decreased EpoR signaling. Our findings suggest that EpoR may be constitutively active in some cancer cells in the absence of Epo and provide the first evidence for a potential role of an Epo-independent, EpoR-mediated pathway in the growth of some human cancers.

Cytotoxic Effect of Sodium Hypochlorite 0.5% (NaOCl) on Ocular Melanoma Cells In Vitro

Purpose: Excision with or without adjuvant cryotherapy or brachytherapy is the treatment of choice in conjunctival melanoma. Adjuvant rinsing with alcohol or sodium hypochlorite peroperatively (Dakin's solution) is used in some centers to prevent seeding of melanoma cells. The purpose of this research is to compare the cytotoxicity of sodium hypochlorite (NaOCl) with other potential cytotoxic solutions in the treatment of conjunctival melanoma. Methods: Three uveal melanoma cell lines (OCM8, Mel285, and Mel270) and one conjunctival melanoma cell line (CM2005.1) were tested in a proliferation test (CellTiter 96 AQueous One Solution Cell Proliferation Assay, Promega, Madison, WI). The 96-well plates were coated with melanoma cells and treated with sodium hypochlorite 0.5%, sodium bicarbonate (1.4% and 8.4%), ethanol 99%, or sodium chlorite during 3, 5, or 15 minutes. Each solution was tested in several dilutions. Results: In all cell lines, no surviving cells were observed after treatment of 3 minutes with sodium hypochlorite. Ethanol 99% had a similar effect. A reduction of 70% of viable cells could be reached using sodium bicarbonate 1.4% or 8.4%. Water reduced the amount of viable cells by 40%. Conclusions: Sodium hypochlorite is cytotoxic for melanocytic cells in vitro. Its use may reduce local seeding of tumor cells and may decrease metastasis after extirpation of an extended ocular tumour. Further in vivo evaluation of sodium hypochlorite is required.

Effects of selective somatostatin analogs and cortistatin on cell viability in cultured human non-functioning pituitary adenomas

Clinically "non-functioning" human pituitary adenomas (NFPA) constitute about 35% of pituitary adenomas. Somatostatin receptors (SSTR) expression in these adenomas has previously been described both in vitro and in vivo, without evidence for a correlation with tumor volume or the therapeutic efficacy of somatostatin analogs. This study was performed on 13 surgically removed pituitary macroadenomas, diagnosed before surgery as "non-functioning". In addition, 3 growth hormone (GH)-secreting adenomas served as controls. A specimen from each tumor was dispersed and digested to isolate and culture the tumor cells, and the in vitro effects of SSTR2 and SSTR5 selective analogs and Cortistatin (CST) (100nM) on cell viability were studied. The quantity of viable cells was estimated using the XTT method. RNA purification of tumor samples and subsequent RT-PCR studies for SSTR2 and SSTR5 expression were performed. Somatostatin analog with high affinity for SSTR2 reduced cell viability by 20-80% in 8 of 13 NFPAs studied, all expressing the SSTR2. The inhibitory effect on cell viability of SSTR5-selective analog was 15-80% in 10 of 13 NFPAs studied, all but three expressing the SSTR5. CST, however, effectively reduced cell viability in only 6 NFPAs. Cell viability was inhibited by all peptides studied in 2 out of 3 GH-secreting adenomas, expressing both receptors. The third adenoma responded to SSTR2 analog and expressed only SSTR2. These results suggest the involvement of SSTR2 and SSTR5 in the anti-proliferative effects of somatostatin; however, CST is less potent in reducing cell viability in these tumors.

Effects of different preservation temperatures and periods menisci cellularity in rabbits

PURPOSE: Quantify the progressive decrease of the cellular viability of rabbit meniscus preserved for transplants over a 30 day period at different freezing temperatures. METHODS: 180 menisci were removed from 45 rabbits. Menisci were frozen from 2 to 30 days at -7.2°Celsius -21.4°Celsius and -73°Celsius. Four menisci from each temperature were thawed every two days and the number of present cells was quantified. RESULTS: On the 14th freezing day at -7.2°Celsius, there were 92.38% mean viable cells. However, as from the 16th day, there has been a significant 12% decrease (p = 0.001), as compared to 14th day mean. Mean cell viability at -21.4°Celsius and -73ºCelsius, until the 16th day was statistically similar. As from the 18th day at -21.4°Celsius, there has been significant cell count decrease (p &lt; 0.001), especially from the 28th (54.5%) to the 30th (30%) freezing day. The number of viable cells at -73 0Celsius has shown insignificant 2.3% decrease (p = 1.000) from the 28th (40.2%) to the 30th (37.9%) day. CONCLUSION: The amount of viable meniscus cells is similar up to the 14th day of preservation, under any of the used temperatures. However, from the 14th day to the 30th day, the amount of viable cells is smaller in lower temperatures.

The effect of selective sst1, sst2, sst5 somatostatin receptors agonists, a somatostatin/dopamine (SST/DA) chimera and bromocriptine on the “clinically non-functioning” pituitary adenomas in vitro

The aim of the work was to investigate the effects of somatostatin analogs acting selectively on sst1 (BIM-23926), sst2 (BIM-23120) and sst5 (BIM-23206) receptor subtypes on the viability of “clinically non-functioning” pituitary adenomas in vitro. The effects of native SST (SST-14), a SST/DA chimera (BIM-23A387) and a D 2-dopamine receptor agonist bromocriptine (BC) were also examined. The study was performed on 10 surgically removed pituitary macroadenomas, diagnosed before surgery as “non-functioning”. A part of each tumor was mechanically dispersed and digested with collagenase to isolate the tumoral cells. Another part of each tumor was fixed, embedded in paraffin and immunostained to reveal the pituitary hormones and SST receptor subtypes (sst1, sst2A, sst2B, sst3, sst4, sst5). The tumoral cell suspensions were incubated for 24 h with the substances mentioned above. The quantity of viable cells was estimated using the EZ4U system. The results were compared with the immunohistochemical evaluation of the hormonal profile of adenoma and the sst receptor subtype immunoreactivities present. The findings indicate that selective sst1, sst2 and sst5 receptors agonists, SST/DA chimera and D 2-dopamine receptor agonist bromocriptine affect the viability of some, but not all, “clinically non-functioning” pituitary adenomas in vitro. The most effective was bromocriptine. The investigated somatostatin analogs including SST/DA chimera exerted roughly similar inhibitory effects. Further studies are needed to fully evaluate the potential usefulness of these compounds in the pharmacological treatment of “non-functioning” pituitary tumors.