Recovery Of Viable Cells Research Articles

Magnetized Carbonyl Iron and Insoluble Zirconium Hydroxide Mixture Facilitates Bacterial Concentration and Separation from Nonfat Dry Milk

A mixture of magnetized carbonyl iron and insoluble zirconium hydroxide was investigated for its ability to concentrate various foodborne pathogens from 25-ml samples of reconstituted nonfat dry milk. Each sample was artificially contaminated with 103 to 106 CFU/25 ml of representative foodborne pathogens (Salmonella enterica serovar Enteritidis, Listeria monocytogenes, and Bacillus cereus spores) and processed for bacterial concentration with high-speed centrifugation for the primary concentration followed by a secondary concentration step involving the carbonyl iron–zirconium hydroxide mixture. Bacterial recoveries, as evaluated on the basis of loss to discarded supernatants, exceeded 75% for all organisms at all inoculum levels and were usually >90%. Recovery was confirmed by direct plating of the immobilized pellet, for which the values were similar albeit more varied. Additional experiments confirmed that the magnetized carbonyl iron–insoluble zirconium hydroxide mixture was relatively nontoxic to both Salmonella Enteritidis and L. monocytogenes. Overall, the entire concentration scheme resulted in a 25-fold reduction in sample volume with the recovery of viable bacterial cells. This novel compound shows promise for facilitating inexpensive, rapid, and effective bacterial concentration in food systems.

Noncryopreserved Bone Marrow Storage in STM-Sav, an Infusible-Grade Cell Storage Solution

Liquid storage of hematopoietic stem and progenitor cells often is necessary in clinical cell engineering. The interval between cell harvest and transplantation or processing may be as long as 1 or 2 days. We previously developed an infusible-grade solution, STM-Sav, for nonfrozen storage of peripheral blood stem cells. This study addresses the validation of the storage conditions using STM-Sav for the short-term storage of bone marrow. Bone marrow from normal donors was stored in STM-Sav in gas-permeable Cryocyte and plasma transfer bags at 4°C and room temperature. Cell viability, number, and morphology, percentage of CD34+ cells, frequency of granulocyte/macrophage colony-forming units (CFU-GM), and solution pH were measured every 24 h for up to 72 h. More than 88% of viable nucleated cell recovery was observed at 72 h of liquid storage. CD34+ cell recoveries were unchanged over the 72-h storage period as well, and the frequency of CFU-GM per 105 cells was also constant. The regulation of solution pH w...

Flow cytometric CD34+ determination in stem cell transplantation: before or after cryopreservation of grafts?

Various attempts have been made to standardize and improve the reproducibility of flow cytometric determination of CD34+ hematopoietic progenitor cells. It is still not clear, however, whether the quantification of CD34+ cells in a stem cell graft should be done before or after cryopreservation. To address this issue, we investigated 78 unselected and 32 immunomagnetically selected autologous and allogeneic leukapheresis products (LA) before and after cryopreservation using pilot vials. Cell numbers were quantified within a Neubauer chamber, and CD34+ content was determined by flow cytometry; propidium iodide staining was used to exclude dead cells from analysis. Before freezing, the mean viable CD34 cell content in the unselected samples was 1.22% and increased after thawing to a mean of 2.16% of viable cells. Taking into account cell loss and cell death, the overall recovery of viable cells was 64.5%; all CD34+ cells could be recovered. Mean purity in the CD34-selected cell fraction was 85% (48-97) before and 91.3% (67-99) after thawing. The number of viable cells was 86.8% before and 86.1% after freezing with a 93.9% recovery of total cells. This leads to a mean 93.7% (SD +/- 23.1) recovery of viable cells and 100% (SD +/- 22.3) recovery of viable CD34+ cells. There was no significant difference in tolerance to freeze/thaw stress between cells from heavily pretreated autologous patients and healthy allogeneic donors. Our data show that freezing significantly increases the percentage of CD34(+) cells in unmanipulated LA, probably due to the death of granulocytes and mononuclear cells (MNCs). Nevertheless, the overall number of viable CD34+ cells in unselected as well as selected samples remains unchanged. Thus, CD34 data from different laboratories, for example, within multicenter trials, should be comparable independent of the different time points of acquisition.

Primitive, quiescent, Philadelphia-positive stem cells from patients with chronic myeloid leukemia are insensitive to STI571 in vitro

In clinical trials, the tyrosine kinase inhibitor STI571 has proven highly effective in reducing leukemic cell burden in chronic myeloid leukemia (CML). The overall sensitivity of CML CD34(+) progenitor cells to STI571 and the degree to which cell death was dependent on cell cycle status were determined. Stem cells (Lin(-)CD34(+)) from the peripheral blood of patients with CML in chronic phase and from granulocyte-colony-stimulating factor-mobilized healthy donors were labeled with carboxy-fluorescein diacetate succinimidyl diester dye to enable high-resolution tracking of cell division. Then they were cultured for 3 days with and without growth factors +/- STI571. After culture, the cells were separated by fluorescence-activated cell sorting into populations of viable quiescent versus cycling cells for genotyping. For healthy controls, in the presence of growth factors, STI571 affected neither cell cycle kinetics nor recovery of viable cells. In the absence of growth factors, normal cells were unable to divide. For CML samples, in the presence or absence of growth factors, the response to STI571 was variable. In the most sensitive cases, STI571 killed almost all dividing cells; however, a significant population of viable CD34(+) cells was recovered in the undivided peak and confirmed to be part of the leukemic clone. STI571 also appeared to exhibit antiproliferative activity on the quiescent population. These studies confirm that CML stem cells remain viable in a quiescent state even in the presence of growth factors and STI571. Despite dramatic short-term responses in vivo, such in vitro insensitivity to STI571, in combination with its demonstrated antiproliferative activity, could translate into disease relapse after prolonged therapy.

Transmission of Escherichia coli O157:H7 from contaminated manure and irrigation water to lettuce plant tissue and its subsequent internalization.

The transmission of Escherichia coli O157:H7 from manure-contaminated soil and irrigation water to lettuce plants was demonstrated using laser scanning confocal microscopy, epifluorescence microscopy, and recovery of viable cells from the inner tissues of plants. E. coli O157:H7 migrated to internal locations in plant tissue and was thus protected from the action of sanitizing agents by virtue of its inaccessibility. Experiments demonstrate that E. coli O157:H7 can enter the lettuce plant through the root system and migrate throughout the edible portion of the plant.

Human UC-blood banking: impact of blood volume, cell separation and cryopreservation on leukocyte and CD34+ cell recovery

UC blood represents an increasingly useful source of hematopoietic stem cells for BMT, although, currently, low cell numbers generally limit its use to pediatric patients. We have determined parameters that influence the recovery of viable cells during processing and cryopreservation, in an effort to set guidelines for determining whether a sample will yield sufficient cells to be of use in the transplant setting. UC blood was collected from donors from January 1996 to December 1999. Volume was reduced using Ficoll, followed by cryopreservation under liquid nitrogen. Total leukocyte and CD34(+)-cell counts were determined prior to processing and a subset of samples was also assessed post-processing and post-cryopreservation. Outcomes for 3816 samples were analyzed to determine the correlation between cell number, cell type, volume, and time between collection and processing. A positive relationship was observed between volume and cell number for both leukocytes and the CD34(+) cells. This correlation allowed us to determine the number of leukocytes and CD34(+) cells expected from a sample based on volume, and to set guidelines for determining the practicality of storing any given sample prior to processing and cryopreservation. Measuring blood volume gives a very useful indication of the total leukocyte and CD34(+) cell number. The majority (75%) of cord-blood samples contain sufficient leukocytes for a pediatric transplant, and the number of cells available can be determined prior to processing by measuring blood volume.

Thiram-induced cytotoxicity is accompanied by a rapid and drastic oxidation of reduced glutathione with consecutive lipid peroxidation and cell death

The toxic effect of thiram, a widely used dithiocarbamate fungicide, was investigated in cultured human skin fibroblasts. Cell survival assays demonstrated that thiram induced a dose-dependent decrease in the viable cell recovery. Thiram exposure resulted in a rapid depletion of intracellular reduced glutathione (GSH) content with a concomitant increase in oxidized glutathione (GSSG) concentration. Alteration of glutathione levels was accompanied by a dose-dependent decrease in the activity of glutathione reductase (GR), a key enzyme for the regeneration of GSH from GSSG. Thiram-exposed cells exhibited increased lipid peroxidation reflected by enhanced thiobarbituric acid reactive substances (TBARS) production, suggesting that GSH depletion and the lower GR activity gave rise to increased oxidative processes. To investigate the role of decreased GSH content in the toxicity of thiram, GSH levels were modulated prior to exposure. Pretreatment of fibroblasts with N-acetyl- l-cysteine (NAC), a GSH biosynthesis precursor, prevented both lipid peroxidation and cell death induced by thiram exposure. In contrast, thiram cytotoxicity was exacerbated by the previous depletion of cellular GSH by l-buthionine-(S,R)-sulfoximine (BSO). Taken together, these results strongly suggest that thiram induces GSH depletion, leading to oxidative stress and finally cell death.

An evaluation of thiram toxicity on cultured human skin fibroblasts

Thiram is widely used in agriculture as a fungicide and, to a lesser extent, as a vulcanizing agent in the rubber industry. In spite of the extensive use of thiram, knowledge on its toxicity and health risk remains limited, and few investigations have been performed to assess specific damage at the cellular and subcellular level. We report here the cytotoxic effects of thiram on cultured human skin fibroblasts. Our results demonstrated that thiram exposure induced a dose- and time-dependent decrease in the viable cell recovery with 100% cell death observed with a concentration of 5.0 mg/l. As judged by morphological changes and biochemical criteria, thiram-mediated cell death was not of the apoptotic but seemed to be of the necrotic type. This cell death was not associated with a modification of gene expression of different constituents of the extracellular matrix. A late increase of lactate production was evident after thiram treatment, suggesting a mitochondrial metabolic pathway dysfunction as reported by other authors using similar compounds. However, this phenomenon appeared as a secondary response to the toxic action of thiram. The cytotoxic effect of thiram is possibly due to an oxidant effect inherent to the structure of thiram and the interaction between thiram and vital cellular molecules.

Surface expression and function of p75/AIRM-1 or CD33 in acute myeloid leukemias: engagement of CD33 induces apoptosis of leukemic cells.

p75/AIRM-1 is a recently identified inhibitory receptor expressed by natural killer and myeloid cells displaying high homology with CD33. Crosslinking of p75/AIRM-1 or CD33 has been shown to sharply inhibit the in vitro proliferation of both normal myeloid cells and chronic myeloid leukemias. In this study, we analyzed acute myeloid leukemic cells for the expression of p75/AIRM-1. p75/AIRM-1 marked the M5 (11/12) and M4 (2/2) but not the M1, M2, and M3 subtypes according to the French-American-British classification. Cell samples from 12 acute myeloid leukemias were cultured in the presence of granulocyte/macrophage colony-stimulating factor. Addition to these cultures of anti-CD33 antibody resulted in approximately 70% inhibition of cell proliferation as assessed by [(3)H]thymidine uptake or by the recovery of viable cells. Anti-p75/AIRM-1 antibody exerted a strong inhibitory effect only in two cases characterized by a high in vitro proliferation rate. After crosslinking of CD33 (but not of p75/AIRM-1), leukemic cells bound Annexin V and displayed changes in their light-scattering properties and nucleosomal DNA fragmentation, thus providing evidence for the occurrence of apoptotic cell death. Remarkably, when anti-CD33 antibody was used in combination with concentrations of etoposide insufficient to induce apoptosis when used alone, a synergistic effect could be detected in the induction of leukemic cell death. These studies provide the rationale for new therapeutic approaches in myeloid leukemias by using both chemotherapy and apoptosis-inducing mAbs.

Growth inhibitory effect of green tea extract and (−)-epigallocatechin in Ehrlich ascites tumor cells involves a cellular thiol-dependent activation of mitogenic-activated protein kinases

The effect of green tea extract (GTE) in Ehrlich ascites tumor cells (EATC) was studied with respect to changes in the intracellular kinase system involving mitogen-activated protein kinases (MAPKs) and cellular thiol. We have previously shown a reduction in viability of EATC and tyrosine phosphorylation of 42 and 45 kDa proteins by GTE and its polyphenolic component, Epigallocatechin (EGC) (D.O. Kennedy, S. Nishimura, T. Hasuma, Y. Yoshihisa, S. Otani, I. Matsui-Yuasa, Involvement of protein tyrosine phosphorylation in the effect of green tea polyphenols on Ehrlich ascites tumor cells in vitro, Chem. Biol. Interact. 110 (1998) 159–172). Furthermore, GTE and EGC significantly decreased both cellular non-protein and protein sulfhydryl levels in EATC, but replenishing thiol stores with N-acetylcysteine (NAC) caused a recovery in cell viability, and therefore SH groups were identified as a novel target of green tea cytotoxicity (D.O. Kennedy, M. Matsumoto, A. Kojima, I. Matsui-Yuasa, Cellular thiol status and cell death in the effect of green tea polyphenols in Ehrlich ascites tumor cells, Chem. Biol. Interact. 122 (1999) 59–71). In this study, we have observed the stimulation of three forms of MAPK, namely ERK1/2, JNK/SAPK and p38, by EGC, which were dose and time-dependent. These MAPK stimulations were found to be cellular thiol status-dependent events as NAC reversed these stimulations. Furthermore, inhibition of the p38 MAPK pathway using the p38 inhibitor SB203580 caused a marked dose-dependent reduction in the decrease in cell viability caused by EGC treatment. Inhibiting the Erk1/2 MAPK pathway using the MEK inhibitor PD098059 caused a slight change in the decrease in cell viability by EGC. These may suggest that the cytotoxicity associated with EGC was more associated with the other MAPKs than with ERK1/2. This may be the first study of its kind providing a novel evidence of a role for different forms of MAPKs in the antitumoric effect of green tea polyphenols, especially EGC, in Ehrlich ascites tumor cells.

Effects of uwhangchungsimwon on cell viability, proliferation, and gene expression of human neuronal cell line IMR32.

Uwhangchungsimwon (pill, UC) is one of the traditional Korean medical prescriptions that has been most frequently used for stroke. To characterize the effects of UC on human neuronal cells, the human neuroblastoma cell line IMR32 was treated with UC, and cell viability, cell proliferation, apoptosis, and gene expression were analyzed. The effect of UC on recovery of cell viability was analyzed following stress induction by nutrient depletion or cold shock. Flow cytometric analysis of the cell cycle showed that UC inhibits cell cycle progression of IMR32 in a dose- and time-dependent manner. UC was also identified to increase cell viability and suppress apoptosis induction by a DNA-damaging agent, etoposide. Quantitative RT-PCR analysis revealed that expressions of the p53 tumor suppressor gene and its downstream effect, Waf1, are stimulated whereas expressions of positive cell cycle regulators, c-Myc, c-Fos, and Cyclin D1 were repressed by UC treatment. Moreover, while expression levels of apoptosis inhibitors, Bcl-2 and Bcl-XL were increased following UC treatment, that of an apoptosis promoter, Bax, was decreased. In addition, expression of BMP-7, which has been recently demonstrated to improve the motor neuron recovery from stroke, was induced by UC while it was not detected in untreated cells. Taken together, our data suggest that the pharmacoclinical effects of UC might be derived in part from its negative regulation of cell proliferation and apoptosis through the transcriptional control of related genes.

Apoptosis Occurs in Isolated and Banked Primary Mouse Hepatocytes.

Isolation and cryopreservation of freshly isolated hepatocytes is considered a standard procedure for the long-term storage of liver cells. However, most existing methods for banking hepatocytes do not allow sufficient recovery of viable cells to meet the needs of basic research or clinical trials of hepatocyte transplantation. The mechanisms underlying this poor rate of hepatocyte recovery are unknown. Although much of the cellular damage in freezing is caused by formation of ice crystals within the cells, this is largely prevented by the use of dimethyl sulfoxide (DMSO) and controlled rate freezing. As we demonstrated recently, necrosis does occur in primary hepatocytes following isolation and cryopreservation. In the present study, we explored the contribution of apoptosis, another form of cell death, in primary hepatocytes banked for transplantation. We evaluated apoptosis of C57BL/6J mouse primary hepatocytes using several different methods. Annexin binding and the TUNEL assay, in conjunction with flow cytometry and confocal laser scanning microscopy, revealed that the percentage of apoptotic cells was dramatically elevated in cryopreserved cells compared with that in the control group of unfrozen cells. DNA laddering detected by DNA electrophoresis in agarose gel also supported the presence of apoptosis in isolated and banked liver cells. Moreover, we found that the addition of glucose (from 10 to 20 mM) into the freezing solution (University of Wisconsin Solution) decreased the rate of apoptosis by 84% and improved the cell attachment at least fourfold in cryopreserved cells. These results suggest that apoptosis might contribute to cell death in isolated and banked primary hepatocytes.

Stabilization and Preservation of Lactobacillus acidophilus in Saccharide Matrices

Lyophilization and vacuum- or spray-drying are some of the most useful techniques for preserving foods, agricultural products, and pharmaceuticals. Biological materials, however, can be irreversibly damaged during these treatments. Therefore, it is essential to design protective agents to preserve protein activity and cell viability. In this paper we examine the use of α,α-trehalose–borate systems as protectants for Lactobacillus acidophilus during freeze- and vacuum-drying. Trehalose was found to be an effective protectant for freeze-dried and vacuum-dried samples, and it is equivalent to a protective formulation which is in current industrial use. It is known from our previous work on enzymes that the presence of borate can dramatically enhance the protective ability of trehalose. In this work, the addition of trehalose–borate to bacterial concentrate greatly improves the recovery of viable cells after storage. This improvement was seen in freeze-dried samples stored at 37°C as well as for vacuum-dried samples held at room temperature. A tailored buffering strategy was tested to counteract the high pH resulting from the addition of borate to the mixture. Use of citric or lactic acids in combination with ammonium hydroxide gave a protectant solution with high pH (resulting in effective crosslinking between trehalose and borate) but a dry product with reduced pH upon rehydration (conducive to cell survival). These results raise exciting possibilities for protection of more labile prokaryotic species as well as simple eukaryotes.

The generation of dendritic-like cells with increased allostimulatory function from acute myeloid leukemia cells of various FAB subclasses

To increase the immunogenicity of leukemic cells, attempts were made to generate dendritic-like antigen presenting cells (DC) from AML blasts from 14 patients with AML FAB classifications M0-M5. Leukemic cells were cultured in the presence or absence of various cytokines including GM-CSF, SCF, TNF-α, IL-4, and γ-interferon. After various intervals recovery of viable cells was measured and expression of CD80, CD86, CD40, CD54, CD58, and CD11a was analyzed by flow cytometry. Functionally, DC derived from six AML samples were tested in a mixed lymphocyte response (MLR) using HLA-DR mismatched donor T cells as responder cells. Proliferation (5/14) or increased survival (7/14) of AML cells was observed in the presence of GM-CSF, SCF, and TNF-α. Only in the AML M2, M3, and M4 FAB subtypes proliferation was found. GM-CSF, SCF, and TNF-α induced morphologic changes typical for DC and increased the expression of costimulatory and adhesion molecules. No significant effect of IL-4 or γ-interferon was observed. The day of maximal expression of these molecules varied. In cases with minor upregulation of CD80 or CD86, no further stimulation using CD40-L activation was observed. In the three cases tested, the DC-like cells retained the chromosomal abnormalities present in the original AML cells. In five out of six cases tested an increase in allostimulatory capacity was found at the day of maximal expression of costimulatory and adhesion molecules. In two patients a decrease in stimulatory capacity was found at day 7 compared with day 2 correlating with a decreased expression of these molecules. In conclusion, AML cells can be induced to increase their stimulatory capacity by upregulating costimulatory and adhesion molecules.

Interaction of merosin (laminin 2) with very late activation antigen-6 is necessary for the survival of CD4+ CD8+ immature thymocytes.

The laminin alpha2-chain is a component of merosin, a member of the laminin family molecules, which is mainly expressed in the basement membranes of striated muscle. It is known that laminin alpha2 gene (lama2) null mutant mice (dy3k/dy3k) exhibit congenital muscular dystrophy (CMD). Because the laminin alpha2-chain is also expressed in the thymus, the role of merosin in the thymus was examined. In association with the onset of muscular dystrophy, CD4+ CD8+ double-positive (DP) thymocytes disappear by apoptotic cell death, while CD4+ CD8- or CD4- CD8+ thymocytes remain. In order to study the mechanisms leading to the selective death of DP cells in the absence of merosin, the role of the interaction between very late activation antigen-6 (VLA-6), a candidate merosin ligand in the thymus, and merosin was examined. The in vitro survival of thymocytes from normal mice was maintained by the addition of either anti-VLA-6 monoclonal antibodies (mAbs) or merosin. Furthermore, when the normal thymocytes were cultured on thymic epithelial cell lines, viable DP cell recoveries on wild-type epithelial cells were better than on cells from null mutant mice. The results suggest that DP cells are more sensitive to an uncharacterized apoptotic death signal, and that survival is supported by the interaction between VLA-6 and merosin.

Ultrasonic enhancement of gene transfection in murine melanoma tumors

The enhancement of gene transfection by ultrasound (US) was evaluated in vitro and in vivo using the B16 mouse melanoma model. Cultured cells were either exposed in suspensions in vitro or implanted subcutaneously in female C57BL/6 mice for 10–14 days and, subsequently exposed, in vivo. For comparison to results with a luciferase plasmid, a reporter plasmid for green fluorescent protein (GFP) was used to evaluate transfection efficiency. US was supplied by a system, similar to a Dornier HM-3 lithotripter, that produced shock waves (SW) of 24.4 MPa peak positive and 5.2 MPa peak negative pressure amplitudes at the focus. The plasmids were mixed with the suspensions to achieve 20 μL mL −1, or were injected intratumorally to provide 0.2 mg DNA per mL of tumor. Acoustic cavitation was promoted by retaining 0.2 mL of air in the 1.2-mL exposure chambers in vitro and by injecting air at 10% of tumor volume in vivo. In vitro, cell counts declined to 5.3% of shams after 800 SW exposure, with 1.4% of the cells expressing GFP after 2 days of culture. In vivo, 2 days after 400 SW exposure, viable-cell recovery from excised tumors was reduced to 4.2% of shams and cell transfection was enhanced by a factor of about 8, reaching 2.5% of cell counts ( p < 0.005 in t-test). These results show that strong tumor ablation induced by US shock wave treatment can be coupled with simultaneous enhancement of gene transfection.

Recovery of Escherichia coli Biotype I and Enterococcus spp. during Refrigerated Storage of Beef Carcasses Inoculated with a Fecal Slurry

Three beef front quarters/carcasses were inoculated with a slurry of cattle manure. During storage at 4°C, two sponge samples from each of three sites (i.e., 100 cm2 from each of two fat surfaces and 100 cm2 from a lean surface) were taken from each of the three carcasses on days 0, 1, 3, 7, and 10 after inoculation. The initial numbers of Escherichia coli averaged 2.0 log10 CFU/cm2 (1.21 to 2.47 log10 CFU/cm2) using the Petrifilm method and 2.09 log10 most probable number (MPN)/cm2 (0.88 to 2.96 log10 MPN/cm2) using the MPN method. The initial numbers of enterococci averaged 3.34 log10 CFU/cm2 (3.07 to 3.79 log10 CFU/cm2) using kanamycin esculin azide agar. In general, an appreciable reduction in the numbers of E. coli occurred during the first 24 h of storage; for the Petrifilm method an average reduction of 1.37 log10 CFU/cm2 (0.69 to 1.71 log10 CFU/cm2) was observed, and for the MPN method an average reduction of 1.52 log10 MPN/cm2 (0.47 to 2.08 log10 MPN/cm2) was observed. E. coli were not detected (<−0.12 log10 CFU/cm2) using Petrifilm on day 7 of the storage period on two (initial counts of 1.21 and 2.29 log10 CFU/cm2) of the three carcasses. However, viable E. coli cells were recovered from these two carcasses after a 24-h enrichment at 37°C in EC broth. Viable E. coli cells were detected at levels of −0.10 log10 CFU/cm2 on the third carcass (initial count of 2.47 log10 CFU/cm2) after 7 days at 4°C. No significant difference in recovery of viable cells was observed between the MPN and Petrifilm methods on days 0, 1, and 3 (P > 0.05). However, viable E. coli cells were recovered from all three carcasses by the MPN method on day 7 at an average of −0.29 log10 MPN/cm2 (−0.6 to −0.1 log10 MPN/cm2). On day 10, viable cells were recovered by the MPN method from two of the three carcasses at −0.63 and −0.48 log10 MPN/cm2 but were not recovered from the remaining carcass (<−0.8 log10 MPN/cm2). Similar to E. coli, the greatest reduction (average of 1.26 log10 CFU/cm2, range = 1.06 to 1.45 log10 CFU/cm2) in the numbers of enterococci occurred during the first 24 h of storage. Because of higher initial numbers and a slightly slower rate of decrease, the numbers of Enterococcus spp. were significantly higher (P < 0.017) than the numbers of E. coli Biotype I after 3, 7, and 10 days of storage. These results suggest that enterococci may be useful as an indicator of fecal contamination of beef carcasses.

Cellular thiols status and cell death in the effect of green tea polyphenols in Ehrlich ascites tumor cells

Epidemiological studies suggest that the consumption of green tea may help prevent cancers in humans, and also breast and prostate cancers in animal models are reduced by green tea, and several mechanisms have been proposed for these effects. In this study the relationship between cellular sulfhydryl (SH) groups and the cytotoxicity of green tea polyphenols in Ehrlich ascites tumor cells was examined. It was found that in the presence of green tea extract (GTE) (100 μg/ml) and one of its polyphenolic components, epigallocatechin (EGC; 100 μM), both cellular non-protein (GSH) and protein-sulfhydryl (PSH) levels were significantly decreased and this was associated with a decrease in cell viability. Replenishing the thiol levels by using N-acetylcysteine (NAC) caused a recovery in cell viability, but this recovery was dependent on the time of thiol replenishment in the presence of EGC (initial 15 min). These results identify SH groups as a novel target of green tea polyphenols cytotoxicity in tumor cells, and a regulatory role for green tea in terms of reducing sulfhydryls in tumor inhibition.

Recovery of viable but non-culturable Campylobacter jejuni cells in two animal models

As in many bacteria, including such pathogens as Salmonella enteritidis, enterotoxicEscherichia coli, Vibrio vulnificus, Vibrio cholerae , the occurrence of a viable but non-culturable (VBNC) state has been described for Campylobacter jejuni. This bacteria has been recognized as a leading foodborne pathogen. In this study, VBNC cells from three C. jejuni human isolates were suspended in microcosm water at 4°C and entered the VBNC state. The metabolic activity of these VBNC cells was monitored by CTC reduction. Once in VBNC state, the strains were inoculated per os into newborn mice and 1-day-old chicks so as to evaluate and compare these animal models of VBNC cell recovery. The three strains used in this experiment were revived using the murine model, whereas only two strains were revived with the chick model. The results show that the murine model permits better recovery than the 1-day chick model. All three strains revived exhibited an associative index very similar to that measured in the culturable state. This study indicates that the VBNC state should be considered as playing a role in the epidemiology of Campylobacter infection.

Umbilical cord blood banking for unrelated transplantation: Evaluation of cell separation and storage methods

Cost-efficient umbilical cord blood (UCB) banking requires well-standardized methods of volume reduction and storage. To compare UCB fractionation using a technique of hydroxyethyl starch (HES) sedimentation with the Ficoll (double) and Percoll methods, 50 whole units was allocated randomly to each procedure. HES resulted in a significantly better recovery of mononuclear cells (87.5%), granulocyte/macrophage colony-forming units (CFU-GM) (88.4%), and CD34 + cells (87.4%) and lesser volume reduction (85.5%). HES was the least laborious, time consuming, and expensive of the three procedures, costing 3.4- and 4.4-fold less than the Ficoll and Percoll methods, respectively. Five units processed by each method was frozen in 4.5-mL cryotubes under optimal conditions. After thawing, the greatest degree of recovery of viable nucleated cells and number of CFU-GM per unit were obtained using the HES procedure. Using 4.5-mL cryotubes, the calculated number of units that could be stored in 600-L containers was 3.8- and 2.2-fold higher for Ficoll- and Percoll-separated than for HES-separated units, respectively. Nevertheless, the higher direct costs of the density gradient separation procedures outweighed their lower storage cost. For long-term cryopreservation, we assessed the freezing of HES-processed units in 50-mL cryobags and their specifically designed canisters. We found cell recoveries similar to those obtained with cryotubes, but storage capacity was decreased. Special racks designed for these canisters resulted in a 5-fold increase over the number of units stored in standard cryobags. This system also is feasible for Percoll- and Ficoll-separated units, resulting in comparable storage costs for the three separation methods. We conclude that this HES procedure and the 50-mL cryobags constitute a cost-efficient system for large-scale UCB banking.