Recovery Of Viable Cells Research Articles

Sperm-lectin agglutination combined with swim-up leads to an efficient selection of highly motile, viable and heterogeneous ram spermatozoa

Lectins, high molecular weight glycoproteins with different sugar-binding specificity, are able to agglutinate different cell types. The recovery of high-quality spermatozoa can be facilitated by the agglutination induced by the lectin binding. The objective of this study was to combine sperm-lectin agglutination with a dextran/swim-up procedure for developing a new selection technique for ram spermatozoa. To study sperm quality, cell viability (plasma membrane integrity), the HOS-test response and progressive individual motility were assessed. Simultaneously, centrifugal countercurrent distribution (CCCD) in an aqueous two-phase system was carried out to analyze sperm surface heterogeneity. Semen from 3 mature Saltz rams was pooled, and 0.5-mL aliquots were incubated with 4 fluorescein-labelled lectins (ECL, JAC, PSA, RCA). Then, a dextran solution was gently added and overlaid with medium. The top layer of the medium containing the spermatozoa was collected and replaced by careful addition of fresh medium. The incubation sequence was repeated 3 times at 10-min intervals. The consecutive 4 top layers obtained were pooled to give the swim-up combined sample. The highest rate of improvement in sperm quality was obtained after incubation with RCA, with a 50% increase in progressive individual motility, 21.6% in HOS value and 39.5% in viability. Total cell recovery was 64% (1.56 × 10 9 cells), with a viable cell recovery rate of 86%. The obtained sample showed 82% motility, 80% HOS score and 77% viability, up from the pre-swim-up values of 51, 60 and 57 %, respectively. Comparative CCCD analysis revealed a very high heterogeneous population in the RCA/swim-up sample obtained, while a much more homogeneous population was obtained in the sample after the dextran/swim-up procedure previously developed byus. With this simple method, a large proportion of highly-motile spermatozoa with preserved plasma membrane and high heterogeneity can be obtained. These results strongly suggest that this selection procedure could result in a high fertility rate.

Development of an infusible-grade solution for non-cryopreserved hematopoietic cell storage

Various tissue culture medium formulations currently are used to transport, store and process stem and progenitor cells for transplantation, although these solutions are not manufactured for clinical use. We have developed an infusible-grade solution, STM-Sav, for short-term liquid cell storage and tested its ability to maintain viable functional hematopoietic cells, compared with commonly-used tissue culture solutions. G-CSF-stimulated normal-donor PBPC were stored in alpha-MEM, IMDM, RPMI-1640, AIM 5, X-VIVO 10, PlasmaLyte A and STM-Sav, in gas-permeable Cryocyte bags, at ambient temperature and 4 degrees C. The percentage of viable cells, percent recovery of viable mononuclear cells (MNC), percentage of CD34+ cells, CFU-GM frequency and solution pH were determined for each solution, at time points ranging from 0 to 72 h. Cells were slightly better maintained at 4 degrees C than at ambient temperature. No solution was superior to any other overall, although PlasmaLyte A was significantly inferior to all other solutions at preserving viable MNC. STM-Sav consistently maintained viable, functional cells as well as, or better than in-vitro-use-only culture media, or PlasmaLyte A. Cells could be stored in STM-Sav for at least 24 h at 4 degrees C or ambient temperature, with recovery of approximately 90% of initial viable MNC, unchanged percentage CD34+ cells and stable solution pH.

A MURINE TRANSGENIC MODEL OF HUMAN CLASS I MHC-MEDIATED PROGRAMMED CELL DEATH

376 Human class I MHC molecules have recently been shown to mediate programmed cell death (PCD) signals via a Fas-independent pathway, suggesting that this pathway may, like Fas/Fas ligand, be useful for conferring immune privilege. The present study was conducted to develop a murine model for PCD mediated via human class I MHC molecules. Methods: B6 mice and B6 mice expressing both HLA B27 and human β2-microglobulin transgenes (Tg++ mice) were used. PCD was evaluated using viable cell recovery, ethidium bromide/acridine orange staining/fluorescent microscopy, and propidium iodide staining/FACS. Anti-human class I MHC mAbs used included W6/32 and 5H7(specific for an α3 domain monomorphic determinant). Results: Initial studies in mice expressing a single human class I MHC transgene did not demonstrate PCD induction, however, human class I MHC transgene expression was relatively low. Mice expressing HLA B27 and human β2 microglobulin transgenes, however, demonstrated higher human class I MHC expression and underwent PCD following treatment with 5H7 mAb. Splenocytes undergoing 5H7-induced PCD demonstrated classic features of PCD: cell shrinkage, cytoplasmic vacuolization, DNA condensation, and apoptotic body formation. Similar to the human studies of 5H7-induced PCD, studies in murine Tg++ cells demonstrated a greater sensitivity of B cells than T cells to PCD induction. The murine model was also similar to the human model in that class I MHC-mediated PCD induction was profoundly influenced by MHC receptor crosslinking and also dependent on the epitope specificity of the inducing anti-class I MHC mAb. Anti-CD3 mAb enhanced PCD induction by 5H7 mAb in both murine and human cells, however, IL-2 reversed 5H7-induced PCD only in murine cells. Conclusions: These studies indicate that 1) high level expression of human class I MHC in murine cells renders them susceptible to PCD induction via anti-human class I MHC mAb, 2) similar to human lymphoid cells, crosslinking and epitope specificity of the anti-class I MHC mAb markedly influence PCD induction, 3) anti-CD3 mAb enhances human class I MHC-mediated PCD in both human and murine cells, and 4) Tg++ mice provide a useful model for study of human class I MHC-mediated PCD.

Influence of centrifugation regimes on motility, yield, and cell associations of mouse spermatozoa.

Mouse sperm are exceptionally sensitive to mechanical forces associated with pipetting and mixing. This characteristic raised the question of the sensitivity of mouse sperm to centrifugation, a step necessary in the removal of cryoprotectants and a common component in the general manipulation of sperm suspensions for experimental purpose. Epididymal spermatozoa from ICR mice were isolated and manipulated to minimize pipetting and mixing damage. The centrifugal accelerations studied were 200, 400, 600, and 800 x g (measured with a stroboscope) for 5, 10, or 15 minutes of centrifugation time. The number of cells and the number of motile cells were counted. The percent motility and longevity, total yield, and motile yield were calculated. Centrifugation at 200 and 400 x g for short times (5 minutes) caused only a small loss in either immediate or 2.5-hour motility, but centrifugation at 600 and 800 x g for 15 minutes produced up to a fivefold loss. Low speed/short time centrifugation pelleted only about half of the cells; the others were lost when the supernatant was removed. The maximum number of motile sperm (motile yield) was obtained at intermediate centrifugal forces (approximately 400 x g for 10-12 minutes), and it is the total number of motile sperm (and not the percent motility) that is important in the use of cryopreserved sperm to regenerate cryopreserved mutant lines. Relative centrifugal force and centrifugation time exhibit reciprocity (e.g., 200 x g for 10 minutes produces similar results to 400 x g for 5 minutes). The spermatozoa must be centrifuged under carefully defined conditions to minimize the damage and to maximize the recovery of viable cells.

A fundamental difference in the capacity to induce proliferation of naive T cells between CD28 and other co-stimulatory molecules.

T cell activation requires two signals: a signal from the TCR and a co-stimulatory signal provided by antigen-presenting cells (APC). In addition to CD28, multiple molecules on the T cell have been described to deliver co-stimulatory signals. Here, we investigated whether there exist quantitative or qualitative differences in the co-stimulatory capacity between CD28 and other molecules. Anti-CD28 monoclonal antibody (mAb) and mAb against CD5, CD9, CD2, CD44 or CD11a all induced activation of naive T cells in the absence of APC when co-immobilized with a submitogenic dose of anti-CD3 mAb. [3H]Thymidine incorporation determined 2 days after co-stimulation was all comparable. In contrast to progressive T cell proliferation induced by CD28 co-stimulation, co-stimulation by other T cell molecules led to a decrease in viable cell recovery along with the induction of apoptosis of once activated T cells. This was associated with a striking difference in IL-2 production; CD28 co-stimulation induced progressively increasing IL-2 production, whereas co-stimulation by other molecules produced limited amounts of IL-2. Addition of recombinant IL-2 to the latter cultures corrected the induction of apoptosis, resulting in levels of cellular proliferation comparable to those observed for CD28 co-stimulation. These results indicate that a fundamental difference exists in the nature of co-stimulation between CD28 and other molecules, which can be evaluated by the levels of IL-2 production, but not simply by [3H]thymidine incorporation.

Attachment and growth of anchorage-dependent cells on a novel, charged-surface microcarrier under serum-free conditions.

The present study describes a novel microcarrier substrate consisting of a swellable, copolymer of styrene and divinylbenzene, derivatized with trimethylamine. The co-polymer trimethylamine microcarriers support the growth of a number of different cell lines - Madin Darby Bovine Kidney, Madin-Darby Canine Kidney, Vero and Cos-7 - under serum-free conditions, and human diploid fibroblasts in serum-containing medium. Cells attach to the co- polymer trimethylamine microcarriers as rapidly as they attach to other charged-surface microcarriers (faster than they attach to collagen-coated polystyrene microcarriers) and spread rapidly after attachment. All of the cells examined grow to high density on the co- polymer trimethylamine microcarriers. Furthermore, cells are readily released from the surface after exposure to a solution of trypsin/EDTA. In this respect, the co-polymer trimethylamine microcarriers are different from other charged-surface microcarriers. Madin-Darby Bovine Kidney cells grown on this substrate support production of vaccine strain infectious bovine rhinotracheitis virus as readily as on other charged-surface or collagen-coated microcarriers. Thus, the co-polymer trimethylamine microcarriers combine the positive characteristics of the currently available charged-surface and adhesion-peptide coated microcarriers in a single product. The viral vaccine production industry is undergoing considerable change as manufacturers move toward complete, animal product-free culture systems. This novel substrate should find application in the industry, especially in processes which depend on viable cell recovery.

Interleukin-2 Reverses the Defect in Activation-Induced Apoptosis in T Cells from AutoimmunelprMice

Activated T cells from MRLlpr/lpr(lpr) mice have been shown to be resistant to TCR-induced apoptosis (activation-induced cell death)in vitro.We have found that this resistance is related to a defect in IL-2Rα (CD25) expression and IL-2 signaling. Following primary activation, splenic T cells from 8-week-oldlprmice failed to undergo apoptosis after the TCR was religated upon reculture with plate-bound anti-CD3 mAb. These cells had markedly reduced levels of IL-2 secretion and CD25 expression during primary activationin vitro;however, the cells still progressed through the cell cycle and were capable of cell division following TCR religation. Addition of exogenous IL-2 during the primary activation of 8-week-oldlprT cells overcame the defect in CD25 expression. Strikingly, these cells also became sensitive to apoptosis induction and died when the TCR was religated with anti-CD3 mAb. Viable cell recovery of both thelprCD4+and CD8+subsets, as well as the CD4−CD8−subsets, was dramatically reduced under these conditions. Further investigation also revealed that the defect in activation-induced apoptosis in T cells fromlprmice was age-related. Activated T cells from younglprmice (5 weeks old) underwent apoptosis in response to TCR ligation; these cells also expressed normal levels of CD25 following primary activation. However, as the mice aged from 5 to 8 weeks, susceptibility to TCR-mediated apoptosisin vitrowas progressively lost together with the ability to express CD25. Our results suggest that before the onset of severe lymphoaccumulation, activated T cells from younglprmice possess the capability to undergo TCR-induced apoptosis despite defective fas expression; IL-2 participates in sensitizing the cells to this death pathway. In older mice, this pathway breaks down and, together with the lack of fas-induced apoptosis, may account for the onset of severe lymphoaccumulation and autoimmunity.

Inhibition of cytopathic effect of human immunodeficiency virus-1 by water-soluble extract ofGanoderma lucidum

To examine components ofGanoderma lucidum for anti-human immunodeficiency virus (HIV) activity, the aqueous extracts of its basidiocarps were separated into high-molecular-weight (HMF) and low-molecular-weight (LMF) fractions. These fractions were used in XTT [2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide] antiviral assay which can quantitatively measure cytopathic effects of HIV-1 on CEM, human T lymphoblastoid cell line. The CEM cell line added with serial diluted HMF or LMF was cultured in the absence or presence of HIV-1. The results showed that the LMF of the aqueous extract strongly inhibited cytopathic effect of the target cell induced by HIV-1. When two-fold serially diluted LMF ranging from 0.97 mug/ml to 125.00 mug/ml was added to the virus-free culture system, no toxicity on the target cells was detected in all the concentrations tested. However, when it was added to the HIV-infected culture system, the viabilities of the target cell reached a plateau recovering its viabilities to 71.7% and 82.5% in experiment-1 and-2 at 15.60 mug/ml, respectively. The cell viabilities were then gradually decreased but maintained at more than 50% above 31.20 mug/ml concentration. On the contrary, HMF did not prevent any HIV-induced cytopathic effect at any concentrations tested on this cell line. From these results, negligible toxicities were observed by both HMF and LMF ofG. lucidum, and recovery of cell viability in HIV infected target cell was induced only by LMF of the carpophores.

Class I MHC mediates programmed cell death in human lymphoid cells.

Although signaling via class I MHC molecules has been shown to suppress T-cell responses, the mechanisms by which these effects are mediated have not been delineated. Studies were conducted to examine the possibility that 5H7 (a murine anti-human mAb specific for the alpha3 domain of human class I MHC) induces programmed cell death (PCD). Normal human T cells and lymphocyte tumor lines were used. PCD was assessed by viable cell recovery (VCR) with trypan blue, ethidium bromide/acridine orange staining, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling techniques. 5H7 induced growth inhibition of lymphocyte tumor cell lines as assessed by [3H]thymidine incorporation. 5H7 also induced marked reductions in VCR of lymphocyte tumors and normal human T and B cells, with the most dramatic reductions occurring in B cells and B cell-derived tumors (JY, BeVD, and 521). Reductions in tumor growth observed with 5H7 monoclonal antibody, however, were not observed with other anti-human class I MHC monoclonal antibodies, TP2599 (alpha3 domain specific) and W6/32 (alpha2/alpha3 domain specific). Cells treated with 5H7 demonstrated typical features of PCD including cytoplasmic vacuolization, DNA condensation, and apoptotic body formation. Reduction in VCR was augmented by anti-CD3 monoclonal antibody and was not reversed by exogenous interleukin 2. Induction of PCD was not observed with soluble 5H7 F(ab)'2 fragments alone, but cross-linking of 5H7 F(ab)'2 fragments by F(ab)'2 fragments of human Ig-absorbed goat anti-mouse Ig partially restored PCD induction, indicating that anti-class I MHC monoclonal antibody can induce PCD in an FcR-independent system and that monoclonal antibody cross-linking is necessary for PCD induction. These studies provide the first evidence that class I MHC molecules mediate PCD in human T and B cells.

Expression of an inducible type of nitric oxide (NO) synthase in the thymus and involvement of NO in deletion of TCR-stimulated double-positive thymocytes.

The present study investigates the role of nitric oxide (NO) in the deletion of TCR-stimulated double-positive (DP) thymocytes. Fetal thymi expressed mRNA for an inducible type of NO synthase (iNOS). The levels of iNOS mRNA became maximal around gestation day 18 with a decline after birth. Administration of anti-CD3 mAb to fetal thymus organ culture (FTOC) or young mice resulted in enhanced expression of mRNAs for iNOS as well as IFN-gamma. Immunohistochemical analyses revealed that iNOS was produced in the corticomedullary junction and medulla. The effects of iNOS-induced NO on anti-CD3-unstimulated or anti-CD3-stimulated thymocytes were examined by culturing them in the presence or absence of a NO-generating compound. Stimulation of DP thymocytes with anti-CD3 alone induced the generation of CD4(low)CD8(low) thymocytes. The subsequent exposure of these anti-CD3-stimulated thymocytes to NO promoted down-regulation of CD4 and CD8 expression. The recovery of viable DP cells was considerably reduced compared with stimulation with anti-CD3 or NO alone. Even in a viable DP population, high incidences of DNA strand breaks were detected in the CD4(low)CD8(low) compartment. In contrast to DP cells, the recovery of viable single-positive cells was not decreased but rather slightly enhanced by treatment with anti-CD3 and/or NO. The recovery of anti-CD3-stimulated thymocytes were also reduced when cultured on the thymic stromal monolayer with the capacity to produce NO upon IFN-gamma stimulation. These results indicate that NO, which is generated in association with TCR stimulation in the thymus, functions to induce deletion of DP thymocytes, especially when their TCR is stimulated.

Autologous bone marrow purging by in situ IL-2 activation of endogenous killer cells.

The major cause of treatment failure in hematologic malignancies is the development of resistance to chemotherapeutic agents and the presence of clonogenic tumor cells in remission bone marrow. In the present study, we tested the optimal conditions for activation by interleukin-2 (IL-2) of endogenous bone marrow effector cells to develop cytotoxicity against solid tumor and leukemia cells and the effects of IL-2 treatment on eradication of malignant cells from the marrow product while preserving hematopoiesis. Normal donor bone marrow was contaminated with 10% A549 lung cancer cells, MCF-7 breast cancer, or EM2 leukemia cells, and then combined with peripheral blood-derived lymphokine-activated killer (LAK) cells which had been incubated in the presence of IL-2, 6000 IU/ml, for 5 days. Eradication (A549 and MCF-7) or >90% reduction in tumor cell number (EM2) was achieved upon exposure of the marrow to LAK cells but not to control, non-IL-2-exposed PBMC. The non-cross-resistance of chemotherapy and cell-mediated cytotoxicity were tested by using chemoresistant target cells for LAK cytotoxicity assays. Ara-C-resistant K562 cells were sensitive to LAK cytotoxicity, similar to sensitive controls. Similar effects were seen in daunomycin-resistant HL60 leukemia cells and VP-16-resistant K562 cells. The daunomycin-resistant K562 cells were also sensitive to LAK cell killing similar to sensitive controls. Activation of bone marrow mononuclear cells (BMMC) with IL-2, 6000 IU/ml for 24 h, significantly enhanced cytotoxicity against the NK-resistant, LAK-sensitive Daudi cell line. This procedure did not result in significant loss of total BMMC or hematopoietic colony-forming units. We then studied the possibility that the effector cell activity of the IL-2-activated BMMC which is diminished after removal of the IL-2 at 24 h could be recovered upon re-exposure of the marrow to IL-2. We found that the optimum conditions for preserving cytotoxicity were prolonged continuous exposures to IL-2 but that re-exposure of the 24-h IL-2-activated BMMC to IL-2 after a 1- or 2-week IL-2 withdrawal was associated with full recovery of cytotoxicity. These experiments also demonstrated that the overall recovery of viable cells from the BMMC was over 100%, possibly due to a proliferative effect of the IL-2 exposure on BMMC. These results demonstrate that the use of IL-2-activated marrow in patients with acute leukemia in remission undergoing autologous bone marrow transplantation (aBMT) may overcome the obstacles of clonogenic, drug-resistant minimal residual disease. The effector cell activity and hematopoietic capacity of the activated bone marrow product can also be maintained even if optimal clinical care requires an interruption in the in vivo exposure to IL-2. This therapeutic approach is currently being tested in acute leukemia patients undergoing IL-2-activated aBMT followed by prolonged IL-2 treatment before and after hematologic reconstitution.

Anti-human class I MHC antibodies induce apoptosis by a pathway that is distinct from the Fas antigen-mediated pathway.

5H7, an anti-human class I MHC mAb that recognizes a monomorphic determinant of the alpha3 domain, profoundly inhibits T lymphocyte activation. The present study was designed to determine the role of programmed cell death in 5H7-mediated immune suppression. Incubation of PBMC with 5H7 mAb induced a marked reduction in viable cell recovery (VCR) of T cells (<10% VCR), NK cells (<1% VCR), and B cells (<1% VCR). In addition, 5H7 inhibited proliferation and VCR of JY, DBS-521, and BevD tumor lines as well as cells transfected with HLA-B27. Morphologic changes characteristic of apoptosis were induced by 5H7, including cell membrane blebbing, cytoplasmic vacuolization, condensation of nuclear chromatin, and nuclear fragmentation. Furthermore, DNA fragmentation was demonstrated in 5H7-treated PBMC using a TdT-mediated end-labeling (TUNEL) technique. 5H7, but not anti-Fas mAb, induced apoptosis of cell lines derived from patients with Niemann-Pick disease that lack acidic sphingomyelinase activity. In addition, induction of cell death by 5H7 was not inhibited by IL-1beta-converting enzyme (ICE) inhibitors under conditions that fully suppressed Fas-mediated apoptosis. These findings suggest that signal transduction through "classical" human class I MHC molecules induces apoptosis by a Fas-independent pathway that does not require acidic sphingomyelinase, is independent of ICE protease, and may represent a unique pathway of cell death in human lymphocytes.

Regulation of growth and adhesion of cultured cells by insulin conjugated with thermoresponsive polymers

We developed a new biomaterial for use in cell culture. The biomaterial enabled protein-free cell culture and the recovery of viable cells by lowering the temperature without the aid of supplements. Insulin was immobilized and a thermoresponsive polymer was grafted onto a substrate. We investigated the effect of insulin coupling on the lower critical solution temperature (LCST) of the thermoresponsive polymer, poly(N-isopropylacrylamide-co-acrylic acid), using polymers that were ungrafted, or coupled with insulin. The insulin conjugates were precipitated from an aqueous solution at high temperatures, but they were soluble at low temperatures. The LCST was not significantly affected by the insulin coupling. The thermoresponsive polymer was grafted to glow-discharged polystyrene film and covalently conjugated with insulin. The surface wettability of the conjugate film was high at low temperatures and low at high temperatures. The amounts of immobilized insulin required to stimulate cell growth were 1-10% of the amount of free insulin required to produce the same effect. The maximal mitogenic effect of immobilized insulin was greater than that of free insulin. About half of the viable cells was detached from the film only by lowering the temperature. The recovered cells proliferated normally on new culture dishes.

Regulation of growth and adhesion of cultured cells by insulin conjugated with thermoresponsive polymers

We developed a new biomaterial for use in cell culture. The biomaterial enabled protein-free cell culture and the recovery of viable cells by lowering the temperature without the aid of supplements. Insulin was immobilized and a thermoresponsive polymer was grafted onto a substrate. We investigated the effect of insulin coupling on the lower critical solution temperature (LCST) of the thermoresponsive polymer, poly(N-isopropylacrylamide-co-acrylic acid), using polymers that were ungrafted, or coupled with insulin. The insulin conjugates were precipitated from an aqueous solution at high temperatures, but they were soluble at low temperatures. The LCST was not significantly affected by the insulin coupling. The thermoresponsive polymer was grafted to glow-discharged polystyrene film and covalently conjugated with insulin. The surface wettability of the conjugate film was high at low temperatures and low at high temperatures. The amounts of immobilized insulin required to stimulate cell growth were 1–10% of the amount of free insulin required to produce the same effect. The maximal mitogenic effect of immobilized insulin was greater than that of free insulin. About half of the viable cells was detached from the film only by lowering the temperature. The recovered cells proliferated normally on new culture dishes. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 339–344, 1997.

Interleukin-2 and interleukin-7 augment the cytolytic activity and expand the antitumor killing spectrum of alpha CD3-induced activated killer cells: potential use in the immunotherapy of non-immunogenic tumors.

This study investigates the effect of different cytokines on the growth and antitumor activity of the alpha CD3-induced killer cells CD3-AK, and the potential of the use of CD3-AK cells in cancer immunotherapy. Eight cytokines were tested. Only three (interleukin-2, -4 and -7) were able to support the growth of CD3-AK cells, which selectively killed different tumor targets of diversified origin. Culturing in interleukin-4 (IL-4) or IL-7 alone could maintain the growth of CD3-AK cells for 6-8 days. Only IL-2 could maintain long-term growth, but further addition of IL-4 exerted an inhibitory effect, which terminated the cell growth in 2 weeks. In contrast, despite the fact that IL-7 inhibited the proliferation of CD3-AK cells cultured in IL-2, as determined by [3H]thymidine uptake, the recovery of viable cells was not reduced. In 10 days, CD3-AK cells cultured in IL-2 alone or IL-2 plus IL-7 increased 160- or 176-fold respectively. There is an inverse relationship between the in vitro growth ability and Fas expression on the CD3-AK cells. Further, IL-7 increased the cytolytic activity of the CD3-AK cells two- to threefold. CD3-AK cells could be maintained in IL-2 or IL-2 plus IL-7 for 60-240 days or more. The long-term-cultured CD3-AK cells not only possessed a high level of cytolytic activity, but also showed a wide spectrum of killing with different tumor targets; the normally "resistant" targets, such as EL-4 lymphoma, fibrosarcoma, or melanoma, became susceptible. When the in vivo antitumor activity of the CD3-AK cells against a non-immunogenic tumor. EL-4, was tested by tumor-neutralization experiments, we found that only the long-term-cultured cells gave significant protection, with those maintained in both IL-2 and IL-7 giving the highest degree of protection. Thus, these long-term-cultured CD3-AK cells may have the potential to be used for immunotherapy of a variety of tumors whatever their immunogenicity.

INFLAMMATION AND CELL DAMAGE INDUCED BY REPEATED EXPOSURE OF HUMANS TO OZONE

Humans exposed to ozone a single time develop decrements in lung function and an inflammatory reaction in the lung characterized by increased numbers of neutrophils and increased amounts of soluble mediators of inflammation such as interleukin (IL)-8, IL-6, prostaglandin (PG) E, lactate dehydrogenase, elastase, and lung protein in bron2 choalveolar lavage fluid (BALF). However, humans who undergo repeated daily expo- sures to ozone have decrements in lung function on days 1 and 2 of exposure, followed by progressive diminution or even complete disappearance on following days, a process termed "attenuation" or "tolerance." It is not known whether repeated exposure of humans to ozone also results in attenuation of the inflammatory response. In this study, 16 healthy young adult males were exposed to air and 0.4 ppm ozone for 2 h/day on 5 consecutive days, and again a single time 10 or 20 days after the initial 5-day ozone exposure. Bronchoalveolar lavage (BAL) was performed after 5 days of ozone exposure, after 5 days of air exposure, and a third time after the 10- or 20-day follow-up ozone exposure. Ozone-induced increases in percent polymorphonuclear leukocytes (PMNs) and BALF levels of IL-6, PGE, elastase, and fibronectin were diminished after 5 days of expo2 sure. In addition, ozone-induced decreases in recovery of viable BALF cells and alveolar macrophage phagocytosis of yeast were also blunted. However, lactate dehydrogenase (LDH), elastase, IL-8, BALF total protein, 1-antitrypsin, and percent epithelial cells were not diminished. Reversal of ozone-induced attenuation was complex. Some mediators (PMNs, IL-6, PGE, fibronectin, recovery of viable cells) showed at least partial, if not complete, reversal within 10-20 days, while others (total protein, 1-antitrypsin) did not return to the normal response to ozone even after 20 days. We conclude from this study that increases in many cellular and biochemical mediators indicative of inflammation seen after a single exposure of humans to ozone are attenuated after 5 consecutive days of exposure. This attenuation is similar to attenuation of symptoms and lung function reported previously. However, markers of cell injury (increased epithelial cells, LDH, total protein) are not attenuated, and may indicate a persistent but not perceived effect of ozone.

Antimicrobial Effect of Spices on the Growth ofYersinia enterocolitica

ABSTRACT Ground spices were examined in vitro for antibacterial effects on the growth of Yersinia enterocolitica. All spices showed bacteriostatic effects after 12 h when added at concentrations 4.1 to 4.7 percent (w/v) to Trypticase Soya Broth. The most effective spices were cloves, allspice, sage, cinnamon, rosemary, and oregano of which ethanol extracts reduced bacterial cell counts in a concentration dependent manner. Cloves extract at a concentrations of 2000 ppm revealed bactericidal activities and prevented recovery of viable cells of Yersinia enterocolitica within 1 min. An agar diffusion technique indicated that among the species evaluated, the eugenol-containing cloves and allspice had the strongest inhibitory activity.

NMR Water Relaxation, Water Activity and Bacterial Survival in Porous Media

Working from the simple assumption that bulk, surface and bound water in a heterogeneous system are characterised by different intrinsic nuclear magnetic resonance (NMR) relaxation rates and water activities several non-trivial relationships are derived between NMR water relaxation times and water activity. These relationships are tested for randomly packed beds of silica and Sephadex particles both in the fast and slow diffusive exchange regimes. It is shown that Archies law for electrical conductivity is obeyed by these porous beds and this observation is used to establish further relationships between NMR relaxation rates and electrical conductivity. By comparing the rates of recovery of viable cells of Salmonella typhimurium with the NMR, sorption isotherm and conductivity data it is shown that the bacterial cells are monitoring the local water activity in the fluid immediately surrounding them and not the experimental, global water activity coefficient. The implications of this observation for food preservation are considered.

Resuscitation of Vibrio cholerae O1 strain TSI-4 from a viable but nonculturable state by heat shock

Vibrio cholerae strain TSI-4 was incubated in an M9 salt solution at 15 °C for more than 100 days. The plate counts showed no viable cells on day 30, but a broth culture from that day showed the growth of bacteria. However, after 35 days the bacteria entered the nonculturable state, based on the assessment of both the plate counts and broth culture. A portion of the culture was heated at 45 °C for 1 min in a water bath and subsequently plated onto a nutrient agar plate. More than 1000 colonies were recovered after this heat-shock treatment. The recovered cells showed the same chromosomal DNA pattern in the restriction map and the same outer membrane protein pattern in SDS-PAGE. Recovery of viable cells by heat-shock was achieved in cultures grown on M9 salt but not from cultures grown in phosphate-buffered saline. This suggests that the presence of NH 4Cl in the M9 salt solution may support the growth of the bacteria in a low nutrient medium, while also playing an important role in resuscitation.

Resuscitation of Vibrio cholerae O1 strain TSI-4 from a viable but nonculturable state by heat shock.

Vibrio cholerae strain TSI-4 was incubated in an M9 salt solution at 15 degrees C for more than 100 days. The plate counts showed no viable cells on day 30, but a broth culture from that day showed the growth of bacteria. However, after 35 days the bacteria entered the nonculturable state, based on the assessment of both the plate counts and broth culture. A portion of the culture was heated at 45 degrees C for 1 min in a water bath and subsequently plated onto a nutrient agar plate. More than 1000 colonies were recovered after this heat-shock treatment. The recovered cells showed the same chromosomal DNA pattern in the restriction map and the same outer membrane protein pattern in SDS-PAGE. Recovery of viable cells by heat-shock was achieved in cultures grown on M9 salt but not from cultures grown in phosphate-buffered saline. This suggests that the presence of NH4Cl in the M9 salt solution may support the growth of the bacteria in a low nutrient medium, while also playing an important role in resuscitation.