Significant Difference In Viability Research Articles

Modifying three-dimensional scaffolds from novel nanocomposite materials using dissolvable porogen particles for use in liver tissue engineering

Background:Although hepatocytes have a remarkable regenerative power, the rapidity of acute liver failure makes liver transplantation the only definitive treatment. Attempts to incorporate engineered three-dimensional liver tissue in bioartificial liver devices or in implantable tissue constructs, to treat or bridge patients to self-recovery, were met with many challenges, amongst which is to find suitable polymeric matrices. We studied the feasibility of utilising nanocomposite polymers in three-dimensional scaffolds for hepatocytes.Materials and methods:Hepatocytes (HepG2) were seeded on a flat sheet and in three-dimensional scaffolds made of a nanocomposite polymer (Polyhedral Oligomeric Silsesquioxane [POSS]-modified polycaprolactone urea urethane) alone as well as with porogen particles, i.e. glucose, sodium bicarbonate and sodium chloride. The scaffold architecture, cell attachment and morphology were studied with scanning electron microscopy, and we assessed cell viability and functionality.Results:Cell attachment to the scaffolds was demonstrated. The scaffold made with glucose particles as porogen showed a narrower range of pore size with higher porosity and better inter-pore communications and seemed to encourage near normal cell morphology. There was a steady increase of albumin secretion throughout the experiment while the control (monolayer cell culture) showed a steep decrease after day 7. At the end of the experiment, there was no significant difference in viability and functionality between the scaffolds and the control.Conclusion:In this initial study, porogen particles were used to modify the scaffolds produced from the novel polymer. Although there was no significance against the control in functionality and viability, the demonstrable attachment on scanning electron microscopy suggest potential roles for this polymer and in particular for scaffolds made with glucose particles in liver tissue engineering.

Effects of Sperm Conjugation and Dissociation on Sperm Viability In Vitro

Sperm conjugation is an unusual variation in sperm behavior where two or more spermatozoa physically unite for motility or transport through the female reproductive tract. Conjugation has frequently been interpreted as sperm cooperation, including reproductive altruism, with some sperm advancing their siblings toward the site of fertilization while ostensibly forfeiting their own ability to fertilize through damage incurred during conjugate break-up. Conversely, conjugation has been proposed to protect sensitive regions of spermatozoa from spermicidal conditions within the female reproductive tract. We investigated the possibility of dissociation-induced sperm mortality and tested for a protective function of conjugation using the paired sperm of the diving beetle, Graphoderus liberus. Sperm conjugates were mechanically dissociated and exposed to potentially damaging tissue extracts of the female reproductive tract and somatic tissue. We found no significant difference in viability between paired sperm and dissociated, single sperm. The results further indicate that the reproductive tract of female G. liberus might not be spermicidal and conjugation is not protective of sperm viability when damaging conditions do exist. Our results support the interpretation that, at least in some taxa, sperm conjugation is neither protective nor damaging to sperm viability.

Análise comparativa dos efeitos do ultrassom terapêutico e laser de baixa potência sobre a proliferação de células musculares durante a diferenciação celular

INTRODUÇÃO: Existe um grande interesse no estabelecimento de recursos e terapias a serem utilizados na tentativa de proporcionar um processo de reparo muscular de melhor qualidade e menor duração. O ultrassom terapêutico (US) e o laser de baixa potência (LBP) são recursos muito usados na prática clínica, porém são escassas, e por vezes contraditórias, as evidências científicas que determinam com segurança os parâmetros dosimétricos e metodológicos adequados. OBJETIVOS: O objetivo do estudo foi analisar o efeito do US e do LBP sobre a proliferação celular durante a diferenciação de mioblastos C2C12. MATERIAIS E MÉTODOS: Os mioblastos foram cultivados em meio de cultura de Eagle modificado por Dulbecco, contendo 10% de soro fetal bovino (SFB), sendo induzida a diferenciação pela adição de 2% de soro de cavalo durante 96 horas. Posteriormente, as células foram irradiadas com US pulsado a 20%, 3 MHz de frequência (intensidades de 0,2 e 0,5 W/cm², durante cinco minutos) ou submetidas ao tratamento com LBP (potência de saída de 10 mW, densidade de energia de 3 e 5 J/cm², por 20 segundos). A proliferação celular foi avaliada após 24h e 72h utilizando o método de MTT. Foram realizados três experimentos independentes, em cada condição citada e células não irradiadas serviram como controle. RESULTADOS: Os resultados obtidos foram submetidos à análise estatística utilizando a Análise de Variância (ANOVA), teste Dunnet, para verificar diferenças entre o grupo controle (não tratado) e os grupos tratados com US e LBP, adotando significância de p < 0,05. Os resultados evidenciaram que não houve diferença significativa na proliferação celular entre as células musculares submetidas a tratamento com ambos os recursos terapêuticos e as células controle, nos períodos de 24h e 72h após tratamento. Além disso, foi possível verificar que não houve aumento significativo no número de células após o período de 72h quando comparado a 24h, confirmando o processo de diferenciação celular, conforme esperado. CONCLUSÕES: Conclui-se que o US e o LBP, nos parâmetros avaliados, não alteraram a proliferação de mioblastos em processo de diferenciação.

Effects of resin tapping and tree size on the purity, germination and storage behavior of Boswellia papyrifera (Del.) Hochst. seeds from Metema District, northwestern Ethiopia

Boswellia papyrifera (Del.) Hochst. is one of the tree species in dry woodlands of Ethiopia that provides several goods and services. Despite its wide economic and ecological importance, its area coverage is dwindling from time to time, and its natural regeneration is hampered. Hence, long-term prospect for a sustained supply of the goods and services from the species is becoming questionable. The objectives of this study were to investigate: (i) the effect of resin tapping and tree size (DBH) on seed susceptibility to insect attack and the production of viable seeds; and (ii) seed longevity and germination ecology of the seeds of B. papyrifera. We collected seeds from tapped and untapped B. papyrifera stands at Lemlem Terara in Metema District, northwestern Ethiopia. The result showed that both tapped and untapped stands produced comparable insect attacked seeds (tapped stands=16.6%; untapped stands=15.8%). Untapped trees yielded significantly (P<0.0001) higher viable seeds (59%) than continuously tapped trees (49.3%), and trees with medium size (20cm DBH) provided more viable seeds than bigger (30cm DBH) and younger trees (10cm DBH). Longevity of B. papyrifera seeds indicated significant difference in viability under three different temperature regimes (5, 15 and 21°C), three storage periods (6, 9 and 12months) and two tapping regimes (tapped and untapped populations). Fire that produced temperatures above 100°C was lethal to the seeds as it caused complete loss of germinability regardless of exposure time. However, heat with temperatures less than 100°C did not cause loss of germinability even after an hour of exposure. We also found that light conditions had no significant impact on the germination percentage. In general, viability of the seeds was affected by tapping and tree size but not by storage conditions and period, modest temperature and light conditions.

Efficacy of various antioxidants in the protection of the retinal pigment epithelium from oxidative stress

BackgroundOxidative stress induced retinal pigment epithelium (RPE) dysfunction is hypothesized to be fundamental in the pathogenesis of age-related macular degeneration (AMD). This study investigated whether vitamin C, vitamin C phosphate, vitamin E, propofol, betaxolol, and N-acetyl cysteine (NAC) protect human RPE cells from oxidative stress.MethodsARPE-19 cells were pretreated with the compounds under investigation. The chemical oxidant tert-butyl hydroperoxide (t-BOOH) was used to induce oxidative stress. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.ResultsExposure to t-BOOH resulted in a dose- and time-dependent reduction in ARPE-19 cell viability. Compared with cells given t-BOOH alone, vitamin E and NAC pretreated cells had significantly improved viability, propofol and betaxolol pretreated cells had no significant difference in viability, and vitamin C and vitamin C phosphate pretreated cells had significantly reduced viability.ConclusionOf the compounds studied, only vitamin E and NAC significantly mitigated the effects of oxidative stress on RPE cells. Because of their potential therapeutic value for AMD patients, these and other RPE protective compounds continue to merit further investigation.

Poly(ethylene glycol) and poly(N-vinlypyrrolidone) for improved porcine islet cryopreservation

Water-soluble polymers poly(ethylene glycol) (PEG) and poly(N-vinylpyrrolidone) (PVP) were used to study cryopreservation of porcine islets. DMSO was used as control. The effects of polymer purity, molecular weight (MW) and concentration on islet viability were investigated. The results show that both PVP and PEG are good cyroprotectant candidates for islet cryopreservation. The effects of polymer purity and concentration were significant. Increasing concentration significantly increased the islet viability. However, after the concentration reached a certain level, there was no significant difference in viability probably due to increased viscosity of the polymer solution. The effect of polymer MW was not significant. It is concluded that polymers can be a suitable cryoprotectant for porcine islet cryopreservation. The islet viability is polymer concentration-dependent. It seems that PVP is a better cryoprotectant candidate as compared to PEG because the former showed a fast dissolution rate in culture medium and lower viscosity. The polymer concentration at 30% appears to be the optimal for cryopreservation from the viewpoint of islet viability and medium viscosity.

The Achene Mucilage Hydrated in Desert Dew Assists Seed Cells in Maintaining DNA Integrity: Adaptive Strategy of Desert Plant Artemisia sphaerocephala

Despite proposed ecological importance of mucilage in seed dispersal, germination and seedling establishment, little is known about the role of mucilage in seed pre-germination processes. Here we investigated the role of mucilage in assisting achene cells to repair DNA damage during dew deposition in the desert. Artemisia sphaerocephala achenes were first treated γ-irradiation to induce DNA damage, and then they were repaired in situ in the desert dew. Dew deposition duration can be as long as 421 min in early mornings. Intact achenes absorbed more water than demucilaged achenes during dew deposition and also carried water for longer time following sunrise. After 4-d dew treatment, DNA damage of irradiated intact and demucilaged achenes was reduced to 24.38% and 46.84%, respectively. The irradiated intact achenes exhibited much higher DNA repair ratio than irradiated demucilaged achenes. Irradiated intact achenes showed an improved germination and decreased nonviable achenes after dew treatment, and significant differences in viability between the two types of achenes were detected after 1020 min of dew treatment. Achene mucilage presumably plays an ecologically important role in the life cycle of A. sphaerocephala by aiding DNA repair of achene cells in genomic-stressful habitats.

Comparative study of equine bone marrow and adipose tissue‐derived mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) represent an attractive source for regenerative medicine. However, prior to their application, fundamental questions regarding molecular characterisation, growth and differentiation of MSCs must be resolved. To compare and better understand the behaviour of equine MSCs obtained from bone marrow (BM) and adipose tissue (AT) in culture. Five horses were included in this study. Proliferation rate was measured using MTT assay and cell viability; apoptosis, necrosis and late apoptosis and necrosis were evaluated by flow cytometry. The mRNA expression levels of 7 surface marker genes were quantified using RT-qPCR and CD90 was also analysed by flow cytometry. Differentiation was evaluated using specific staining, measurement of alkaline phosphatase activity and analysis of the mRNA expression. High interindividual differences were observed in proliferation in both cell types, particularly during the final days. Statistically significant differences in viability and early apoptosis of cultured AT- and BM-MSCs were found. The highest values of early apoptosis were observed during the first days of culture, while the highest percentage of necrosis and late apoptosis and lowest viability was observed in the last days. Surface marker expression pattern observed is in accordance to other studies in horse and other species. Osteogenic differentiation was evident after 7 days, with an increasing of ALP activity and mRNA expression of osteogenic markers. Adipogenic differentiation was achieved in BM-MSCs from 2 donors with one of the 16 media tested. Chondrogenic differentiation was also observed. Proliferation ability is different in AT-MSCs and BM-MSCs. Differences in viability and early apoptosis were observed between both sources and CD34 was only found in AT-MSCs. Differences in their osteogenic and adipogenic potential were detected by staining and quantification of specific tissue markers. To provide data to better understand AT-MSCs and BM-MSCs behaviour in vitro.

The impact of temperature on the production and fitness of microsclerotia of the fungal bioherbicide Mycoleptodiscus terrestris

The impact of growth temperature was evaluated for the fungal plant pathogen Mycoleptodiscus terrestris over a range of temperatures (20–36°C). The effect of temperature on biomass accumulation, colony forming units (cfu), and microsclerotia production was determined. Culture temperatures of 24–30°C produced significantly higher biomass accumulations and 20–24°C resulted in a significantly higher cfu. The growth of M. terrestris was greatly reduced at temperatures above 30°C and was absent at 36°C. The highest microsclerotia concentrations were produced over a wide range of temperatures (20–30°C). These data suggest that a growth temperature of 24°C would optimize the parameters evaluated in this study. In addition to growth parameters, we also evaluated the desiccation tolerance and storage stability of air-dried microsclerotial preparations from these cultures during storage at 4°C. During 5 months storage, there was no significant difference in viability for air-dried microsclerotial preparations from cultures grown at 20–30°C (>72% hyphal germination) or in conidia production (sporogenic germination) for air-dried preparations from cultures grown at 20–32°C. When the effect of temperature on germination by air-dried microsclerotial preparations was evaluated, data showed that temperatures of 22–30°C were optimal for hyphal and sporogenic germination. Air-dried microsclerotial preparations did not germinate hyphally at 36°C or sporogenically at 20, 32, 34, or 36°C. These data show that temperature does impact the growth and germination of M. terrestris and suggest that water temperature may be a critical environmental consideration for the application of air-dried M. terrestris preparations for use in controlling hydrilla.

Abstract 3917: EMSY amplification and overexpression is not associated with defective homologous recombination and does not predict sensitivity to cisplatin or PARP inhibitors

Abstract EMSY is a gene amplified in 13-18% of breast and ovarian cancers, and encodes a protein reported to be a binding partner for BRCA2, that when overexpressed causes impairment of BRCA2 functions and so might constitute a mechanism for BRCA2 inactivation in non-hereditary breast and ovarian cancers. We hypothesised that if EMSY amplification abrogates BRCA2 function, cells harbouring EMSY gene amplification could have an impaired ability to elicit competent homologous recombination (HR) DNA repair in the presence of DNA double strand breaks (DSBs) and thus an increased sensitivity to DNA cross-linking agents and PARP inhibitors. EMSY amplification may therefore constitute a biomarker for response to these therapies. 59 cell lines were subjected to microarray based comparative genomic hybridisation (aCGH) with a resolution of 50Kb. Ten cell lines harbouring EMSY amplification were identified. These cell lines were matched by anatomical site and biomarker expression with cell lines lacking EMSY gene amplification. In addition, CAPAN1 (BRCA2 mutant), MDAMB436 and SUM149 (BRCA1 mutant) cells were included as controls. The associations between EMSY copy number, mRNA and protein expression were determined by aCGH and fluorescence in situ hybridisation, quantitative real-time PCR and western blotting respectively. Cell viability was assessed following transfection with validated short interfering RNAi (siRNA) against EMSY. Formation of foci of phosphorylated H2AX (γH2AX), a surrogate marker of the presence of double strand breaks, and RAD51, a surrogate marker of competent HR DNA repair, were assessed in cell lines with and without EMSY gene amplification following 10Gy of ionising radiation and treatment with cisplatin or the PARP inhibitor olaparib. Cell viability following treatment with cisplatin or olaparib was assessed to determine sensitivity of cell lines with and without EMSY amplification. EMSY is not consistently overexpressed at the mRNA and protein levels in cancer cells harbouring EMSY gene amplification. No significant difference in viability was seen in cells with or without EMSY amplification following silencing of EMSY using siRNA. Cell lines with EMSY amplification were able to elicit RAD51 foci formation in the presence of DNA double strand breaks, and did not differ from cancer cells devoid of EMSY amplification in their sensitivity to cisplatin and olaparib. These cells also showed a lower sensitivity to these drugs than CAPAN1, MDA-MB436 and SUM149 cells. EMSY amplification is not associated with an impairment of cancer cells to elicit RAD51 foci formation in the presence of DNA double strand breaks and is not associated with increased sensitivity to cisplatin or olaparib. Its potential use as a biomarker for response to cisplatin and PARP inhibitors should therefore be viewed with caution. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3917. doi:10.1158/1538-7445.AM2011-3917

Manual Vs Automated Delivery of Cells for Transplantation: Accuracy, Reproducibility, and Impact on Viability

Cellular transplantation holds promise for the management of a variety of neurological disorders. However, there is great variability in cell type, preparation methods, and implantation technique, which are crucial to clinical outcomes. We compared manual injection with automated injection using a prototype device to determine the possible value of a mechanized delivery system. Neural progenitor cells and bone marrow stromal cells were injected using manual or automated methods. Consistency of injection volumes and cell number and viability were evaluated immediately or 1 day after injection. When cells were delivered as a series of 3 manual injections from the same syringe, the variation in fluid volume was greater than for single manual injections. Automated delivery of a series of 3 injections resulted in a lower variability in the amount of delivery than manual injection for both cell lines (1.2%-2.6% coefficient of variability for automated delivery vs 4.3%-24.0% for manual delivery). The amount delivered from injection 1 to injection 3 increased significantly with manual injections, whereas the amount injected did not vary over the 3 injections for the automated unit. Cell viability 1 day after injection was typically 30% to 40% of the value immediately after injection for the bone marrow stromal cells and 30% to 70% for the neural progenitor cells. There were no significant differences in viability attributed to the method of injection. The automated delivery device led to enhanced consistency of volumetric cell delivery but did not improve cell viability in the methods tested. Automated techniques could be useful in standardizing reproducible procedures for cell transplantation and improve both preclinical and clinical research.

Viability and engraftment of hematopoietic progenitor cells after long-term cryopreservation: effect of diagnosis and percentage dimethyl sulfoxide concentration

We carried out a retrospective analysis of viability by diagnosis and dimethyl sulfoxide (DMSO) concentration in patients who had undergone autologous transplants using hematopoietic progenitor cells (HPC) after long-term storage (up to 17.8 years). Viability was tested using flow cytometry for HPC that were harvested and preserved using a controlled rate freezer and 5% or 10% DMSO with human serum albumin, then stored in liquid nitrogen. Data from 262 samples were analyzed (249 myeloma patients and 13 other diagnoses): 100 consecutively thawed samples with a storage time of <1 year (all 10% DMSO), 50 consecutive samples stored for 1-4.9 years (10% DMSO), 50 samples stored for 5-9 years (5% DMSO) and all samples stored and used for transplant after >9 years (60 samples, 5% DMSO; two samples, 10% DMSO). No statistically significant difference in viability between the 5% DMSO and 10% DMSO groups was observed (P = 0.08), so the 1-4.9 years and 5-9 years were combined and the three groups (<1 year, 1-9 years and >9 years) were compared using an anova test. There was no difference in viability based on cryostorage period (P = 0.23) or between myeloma and other diagnoses (P = 0.45). No difference was seen in time to White blood cell (WBC) engraftment (P = 0.10) or to platelet engraftment between groups (P = 0.52). These data suggest that long-term storage in 5% DMSO and human serum albumin is safe.

Effects of Antidepressant Drug on Sexual Behavior and Fitness Parameters of Drosophila ananassae

Drosophila is a suitable model organism in the field of toxicological science. The objective of this study was to explore the potential of doxepin, an antidepressant drug, on sexual behavior and fitness of Drosophila ananassae. Sexual behavior such as mating latency and copulation duration was higher as concentration of antidepressant increases in both larval and adult feeding methods, decrease in the fecundity and fertility was also observed in adult feeding than larval feeding methods. According to the Chi-Square, the percentage of the hatchability is decreased in treated compared with one control and significant difference in viability in adult feeding method. Our findings show the adult one which are exposed are more sensitive comparatively with larval stage. It was also noticed that antidepressant effects some behavioral aspects of Drosophila.

Spiroplasma infection in Drosophila melanogaster: What is the advantage of killing males?

Male-killing bacteria are maternally inherited agents that cause death of sons of infected females. Their transmission rate is commonly high but imperfect and also sensitive to different environmental factors. Therefore, the proportion of infected females should be reduced in each generation. In order to explain male-killers spread and persistence in host population, a mechanism resulting in the relative increase of infected females must outweigh the losses caused by the imperfect transmission. The resource release hypothesis states that the males’ death results in increased resources available to sibling females which would otherwise be used by their male siblings. Infected females are then expected: to be larger than uninfected females in natural populations; or to have higher viability; or to have shorter development times; or any combination of these outcomes. Here, we tested the resource release hypothesis by measuring body size of infected and uninfected wild-caught Drosophila melanogaster females and carried out other fitness related measures in the laboratory. Wild-caught infected females produced more daughters than uninfected females in their first days in the laboratory. However, although no significant difference in viability was found in a controlled experiment with infected and uninfected flies from a standard laboratory strain, there was a decrease in development time probably mediated by reduced competition. Fitness effects conditioned by the host genetic background are pointed out as a possible explanation for this difference between wild and laboratory flies. Our findings are discussed in the context of the resource advantage hypothesis.

Evaluation of Porcine Pancreatic Islets Transplanted in the Kidney Capsules of Diabetic Mice Using a Clinically Approved Superparamagnetic Iron Oxide (SPIO) and a 1.5T MR Scanner

ObjectiveTo evaluate transplanted porcine pancreatic islets in the kidney capsules of diabetic mice using a clinically approved superparamagnetic iron oxide (SPIO) and a 1.5T MR scanner.Materials and MethodsVarious numbers of porcine pancreatic islets labeled with Resovist, a carboxydextran-coated SPIO, were transplanted into the kidney capsules of normal mice and imaged with a 3D FIESTA sequence using a 1.5T clinical MR scanner. Labeled (n = 3) and unlabeled (n = 2) islets were transplanted into the kidney capsules of streptozotocin-induced diabetic mice. Blood glucose levels and MR signal intensities were monitored for 30 days post-transplantation.ResultsThere were no significant differences in viability or insulin secretion between labeled and unlabeled islets. A strong correlation (r2 > 0.94) was evident between the number of transplanted islets and T2 relaxation times quantified by MRI. Transplantation with labeled or unlabeled islets helped restore normal sustained glucose levels in diabetic mice, and nephrectomies induced the recurrence of diabetes. The MR signal intensity of labeled pancreatic islets decreased by 80% over 30 days.ConclusionThe transplantation of SPIO-labeled porcine islets into the kidney capsule of diabetic mice allows to restore normal glucose levels, and these islets can be visualized and quantified using a 1.5T clinical MR scanner.

Progenitor Cell Therapy for Traumatic Brain Injury: Effect of Serum Osmolarity on Cell Viability and Cytokine Production

The potential translation of mesenchymal stem cell (MSC) therapy into a multimodal protocol for traumatic brain injury requires evaluation of viability and cytokine production in a hyperosmolar environment. Optimization of MSC therapy requires delivery to the target area without significant loss of cellular function or viability. No model evaluating the potential efficacy of MSC therapy at varying osmolarities currently exists. Rat MSCs were characterized with flow cytometric immunophenotyping. MSCs (passage 3) were placed in culture with multipotent adult progenitor cell media at varying osmolarities (250, 270, 290, 310, 330, 350 and 370 mOsm) potentially found with hypertonic saline infusion. After culture for 24 h, cellular viability was measured using flow cytometry (n = 6). Next, brain tissue supernatant was harvested from both normal rat brains and injured brains 6 h after cortical injury. Subsequently, MSCs were placed in culture with multipotent adult progenitor cell media +/- 20% normal brain or injured brain supernatant (at the aforementioned osmolarities) and allowed to remain in culture for 24 h (n = 11). At this point, media supernatant cytokine levels were measured using a multiplex cytokine assay system. MSCs showed no clinically significant difference in viability at 24 h. MSCs cultured with 20% injured brain supernatant showed an decrease in proinflammatory cytokine production (IL-1alpha and IL-1beta) with increasing osmolarity. No difference in anti-inflammatory cytokine production (IL-4 and IL-10) was observed. Progenitor cell therapy for traumatic brain injury may require survival and activity in a hyperosmolar environment. Culture of MSCs in such conditions shows no clinically significant effect on cell viability. In addition, MSC efficacy could potentially be enhanced via a decrease in proinflammatory cytokine production. Overall, a multimodal traumatic brain injury treatment protocol based upon MSC infusion and hypertonic saline therapy would not negatively affect progenitor cell efficacy and could be considered for multicenter clinical trials.

Viability and Engraftment of Hematopoietic Progenitor Cells (HPC) After Long Term Cryopreservation: Effect of Diagnosis and % Dimethylsulfoxide (DMSO) Concentration.

Abstract 3161Poster Board III-98 IntroductionThere are few reports of HPC viability after more than 9 y of storage. It has been suggested that HPC from myeloma patients (pts) may survive storage less well than those from other diseases. The appropriate concentration of DMSO for long term storage is unclear. Our program has collected and stored HPC since November 1989, changing from 5% DMSO to 10% DMSO in August 2000. We present a retrospective analysis of viability by diagnosis and DMSO concentration in pts who underwent autologous transplant using HPC after long-term storage (up to 17.8 y). Materials and MethodsHPC were harvested and preserved using a controlled rate freezer and 5% or 10% DMSO, then stored in liquid nitrogen. Viability was tested by flow cytometry using unwashed samples and looking at propidium iodide or 7-AAD uptake within the CD34+ population. Data from 242 samples were analyzed (224 myeloma pts and 18 other diagnoses): 100 consecutively thawed samples with storage time <1 y (all 10% DMSO), 100 consecutive samples stored from 5-9 y ( 7 samples, 10% and 93 samples, 5% DMSO), and all samples stored and used for transplant after >9y (42 samples, 5% DMSO). No statistically significant difference was seen in viability between <1y and 5-9y, so these groups were combined and compared to >9y. White blood cell (WBC) and platelet engraftment were compared for the 2 groups using an unpaired t test. ResultsMean viability for the 0-9y and >9 y groups were 91.62% and 92.79% respectively, with no statistically significant difference in viability between groups (p=0.28). Using a 2-tailed t test, no difference was seen in viability based on diagnosis (p=0.19) or between 5% and 10% DMSO (p= 0.12). No difference was seen in WBC engraftment (p=0.28) or platelet engraftment between groups (p=0.57). DiscussionHPC collections remained viable after 17.8 years, the longest time reported for use after storage to date. No difference in viability was seen with 5% or 10% DMSO, or between myeloma and other diseases. Storage time did not affect engraftment. DisclosuresNo relevant conflicts of interest to declare.

High concentration of magnolol induces hepatotoxicity under serum-reduced conditions

Although magnolol is cytoprotective against warm ischemia/reperfusion injury, its effect on cold preservation has not been fully investigated. This study aimed at examining whether magnolol maintains the liver graft integrity after cold preservation and elucidating the underlying mechanisms in terms of apoptotic signaling under both normothermic and hypothermic conditions. After being preserved in Ringer's lactate (RL) at 4 degrees C for 6h ex vivo, the magnolol-treated grafts demonstrated significantly higher AST, ALT, and LDH levels in perfusates than those from negative controls. TUNEL staining showed no difference in the number of apoptotic nuclei in both groups, whereas a more intense apoptotic signal in magnolol-treated grafts was shown as compared with the controls. In vitro data showed no significant difference in viability of RL-preserved clone-9 hepatocytes between the magnolol-treated and control groups, while magnolol pretreatment at 30min before cold preservation prominently induced hepatocyte cell death. RT-PCR and Western blotting analyses revealed a suppression in Bcl-2, but an up-regulation in Bax expression in clone-9 cells after magnolol treatment. Magnolol suppressed the ratios of NF-kappaB to I-kappaBalpha protein contents and I-kappaBalpha phosphorylation induced by TNF-alpha, and potentiated mitochondrial cytochrome c release and subsequent caspase-3 cleavage. Conversely, caspase-3 inhibitor attenuated magnolol-induced hepatotoxicity. We concluded that magnolol could not protect liver grafts from cold ischemia/reperfusion injury. High concentration of magnolol under serum-reduced conditions attenuates NF-kappaB-mediated signaling and induces intrinsic apoptotic pathway, thereby inducing in vitro hepatotoxicity.

The Effects of Different Concentrations of Acetylsalicylic Acid on Proliferation and Viability of Lymphocytes in Cell Culture

Numerous studies conducted on acetylsalicylic acid (ASA, aspirin) confirmed that ASA inhibits proliferation and induces apoptosis in various types of human cells. Therefore, it was of interest to examine possible effects of different concentrations of ASA on viability and proliferation of lymphocytes in the cell culture. After separation from blood, lymphocytes were suspended in RPMI 1640 medium and cultured at 37 degrees C. Solution of ASA was added to cultures after 24 h, in final concentrations of 1, 3 and 5 mmol/l. After 48 h, proliferative response was evaluated by WST-1 assay. Significant difference in viability between controls and cell cultures treated with ASA in three different concentrations was observed (p<0.01). Percents of viable cells in cultures after application of 1, 3 and 5 mmol/l ASA were 9.9%, 2.5% and 16.9% (compared to controls), respectively. To determine whether this cytotoxic effect was result of induction of apoptosis, DNA from cell cultures was isolated and subjected to agarose gel electrophoresis. Fragmentation of DNA was not detected, excluding apoptosis as possible cause of cytotoxic effects. Addition of ASA caused change of initial extracellular pH value for each treated culture. After addition of 1 mmol/l ASA, pH of culture was 7.19, after 3 mmol/L, 6.99 and after addition of 5 mmol/l solution, pH was 6.75. Decreased lymphocyte viability could be attributed to either the effects of the added substance or possible further acidification of cell cultures during three days of incubation.

Role of Sigma B Factor in the Alkaline Tolerance Response of Listeria monocytogenes 10403S and Cross-Protection against Subsequent Ethanol and Osmotic Stress

Many of the considerable abilities of Listeria monocytogenes to persist and grow in a wide range of adverse environmental conditions are thought to be at least partly under the control of the alternative sigma factor (σB), encoded by the sigB gene. However, little is known about the role of this master regulon in the impressive ability of Listeria to persist and grow under conditions of alkaline pH. In this study, Northern blot analysis of parent Listeria mRNA revealed that alkali adaptation (pH 9.5 for 1 h) significantly increased the expression of sigB-derived mRNA. The study included a comparison of the relative survival of mid-exponential populations of adapted and nonadapted parent type (σB expressing) and mutant (not σB expressing, ΔsigB) Listeria strains during subsequent alkaline (pH 12.0), osmotic (25% NaCl, wt/vol), or ethanol (16.5%) stress. Alkali-adapted parent strains were more resistant to pH 12.0 than were adapted ΔsigB type strains, but both alkali-adapted parent and ΔsigB strains were more resistant to pH 12.0 than were nonadapted strains. Alkali-adapted parent strains were more resistant to osmotic stress than were adapted ΔsigB type strains. No significant differences in viability were observed between alkali-adapted parent and ΔsigB strains after ethanol stress, suggesting that cross-protection against osmotic stress is mediated by σB whereas cross-protection against ethanol is σB independent. Overall, alkali-induced cross-protection against osmotic and ethanol challenges may have serious implications for food safety and human health because such stress conditions are routinely used as part of food preservation and surface cleaning processes.