Proceedings: Characteristics of freeze-dried cells

Microorganisms have been found to be more sensitive to selective media after freeze-drying. This increased sensitivity can be measured and thus the degree of sublethal injury can be determined as a function of various processing variables. In light of this, the use of selective media for the enrichment and detection of pathogens in freeze-dried products has to be reevaluated; indeed, the literature is now becoming abundant with such evaluations. In addition, the response of freeze-dried microorganisms has been found to be dependent on the medium in which they were grown; the phenomena of “metabolic injury” and “minimal medium recovery” are observed when microorganisms are grown in a complete and minimal medium, respectively. The expression of these two phenomena also can be used to assay for injury.Observations on the effects of freeze-drying on cell viability lead to the conclusion that freeze-drying is a complex stress. Damage to the cellular membrane structure and function, RNA integrity, and, possibly, DNA have been cited. The extrapolation of these macromolecular changes to specific viability responses for the purpose of elucidating the principal site of damage is still difficult. It is our opinion that the pre- and post-freeze-drying conditions to which the microorganisms are exposed can lead to a situation in which a particular macromolecular damage can become dominant over others, depending on the physiology of the cell.This knowledge can not only be applied for the purpose of improving detection of undesirable microbes but also for the preservation of desirable cultures, such as starter cultures in the dairy industry. Finally, the finding that microorganisms leak or release nucleic acids after freeze-drying, as they do after freezing and heating, can be applied to the problem of elimination of undesirable cytoplasmic components of organisms to be used as protein sources (4, 8).     

Inhibition of the lymphocyte blastogenic response to antigen by serum-free culture supernatants of leukemic B cells

Suppressive factors were detected in culture supernatants of the guinea pig B-cell L₂C leukemia. Dialyzed culture supernatants (DCS) inhibited the blastogenic response of sensitized lymph node cells (LNC) to a wide dose range of the sensitizing antigen (ovalbumin or PPD) but failed to inhibit the proliferative response to PHA or Con A. In addition, DCS inhibited the response of blast cells to preformed T-cell growth factor (TCGF). The inhibitor(s) in DCS was noncytotoxic, heat stable (30 min at 80 °C), resistant to treatment with trypsin, and exerted its effect subsequent to activation of sensitized LNC by antigen. Washing of DCS-treated cells restored normal reactivity to a subsequent antigen challenge. The target cell for the inhibitor may be cells responding to amplification signals produced by activated T cells. KCl (3 M) extracts of L₂C cells behaved like DCS in inhibiting only antigen responses. Both undialyzed culture supernatants (UCS) and leukemic sera inhibited mitogen, allogeneic, and antigen-stimulated proliferative responses by greater than 80%. These soluble factors may participate in the depression of cell-mediated immunity associated with lymphoid malignancies.     

Inhibition of catalase activity as an early response of Arabidopsis thaliana cultured cells to the phytotoxin fusicoccin

In Arabidopsis thaliana cells, fusicoccin (FC) treatment induced an early and marked increase in the extracellular H(2)O(2) level. It also increased the huge hypo-osmotic stress-induced oxidative wave and, in addition, prevented the H(2)O(2) peak drop. These effects were apparently not linked to changes in either cytoplasmic pH or cytoplasmic free calcium concentration, since they occurred independently of the activity state of the plasma membrane (PM) H(+)-ATPase and neither influx nor efflux of (45)Ca(2+) was modified by FC. In the presence of diphenylene iodonium (DPI), inhibiting the PM NADPH oxidase presumably responsible for reactive oxygen species (ROS) production, no apoplastic H(2)O(2) development was detected either with or without FC. However, no increase in DPI-sensitive ferricyanide reduction, but rather a gradual decrease, occurred with FC. These results suggested that the H(2)O(2) increase observed with FC was not due to a overproduction of ROS but, more probably, to a reduced capability of FC-treated cells to degrade the H(2)O(2) formed. This view, at first supported by the finding that FC-treated cells failed to break down exogenously supplied H(2)O(2), was clearly confirmed by a series of measurements on exogenous catalase activity, tested in cell-free media of FC-treated samples. This assay, in fact, allowed ascertainment and partial characterization of an as yet unidentified factor increasingly accumulating in the incubation medium of FC-treated cells, behaving as a non-competitive catalase inhibitor and able to reduce markedly the cell's capability for H(2)O(2) scavenging.     

Inhibition of H-thymidine incorporation by hydroxyurea: Atypical response of mycoplasma-infected cells in culture

Contamination with Mycoplasma hyorhinis was demonstrated in long-term cultures of HeLa, BICR/M1R_K rat mammary tumor, and NV1C rat neurinoma cells, by microbiological, equilibrium sedimentation, and autoradiographic techniques. In non-infected DNA-synthesizing cells, hydroxyurea (HU) in concentrations 10⁻⁴ M typically inhibits ³H-thymidine (³H-TdR) incorporation into acid-insoluble material. This effect was lacking in the contaminated cell lines, although HU did block nuclear DNA replication, as shown by pulse-cytophotometric analyses. The response to HU could be restored to normal by supplementing the culture medium either with the anti-mycoplasma agent Tylosin or with fresh rat serum. The total ³H-activity in non-infected (or anti-mycoplasma treated) versus infected cells, in the absence of HU, was up to four times higher in the former. The data indicate that (i) incorporation of ³H-TdR into the nuclear DNA of contaminated cells was strongly reduced, probably due to a ‘scavenger effect’ (i.e. utilisation and rapid cleavage) by the mycoplasma; (ii) mycoplasmal ³H-TdR incorporation, contrary to nuclear DNA replication, was insensitive to HU in concentrations 10⁻² M. If equally valid for other species of mycoplasma, the observed phenomenon provides a criterion (together with the possibility of a rapid test) for the presence of mycoplasmal contamination in cell cultures.     

Inhibition of the delayed-type hypersensitivity response by staphylococcal enterotoxin B-induced suppressor T cells

Staphylococcal enterotoxin B (SEB) is a member of a family of gram-positive bacterial exotoxins which act as superantigens in both mouse and man. The administration of this toxin has been shown to inhibit antibody responses in vivo. We have previously shown that SEB is a potent inducer in vitro of multiple T suppressor cell populations. The present studies show that administration of microgram quantities of this toxin result in a reduced capacity to manifest a delayed-type hypersensitivity (DTH) response. In addition, we find that the failure to generate a normal DTH response appears to be due to the generation of a T suppressor cell population following SEB administration. Adoptive transfer studies show that the suppressor cells bear the CD5+ I-J+ CD4- CD8- Thy 1+ surface phenotype. The relationship of these cells to suppressor T cell populations generated following in vitro activation by SEB is discussed.     

Inhibition of the graft-versus-host response by BCGcw-induced suppressor cells or prostaglandin E1

Immunization of C57BL/6 mice with BCGcw stimulated a population of "suppressor cells" which had a decreased capacity to induce the graft-versus-host response. The graft-versus-host response was quantitated using the Simonsen splenomegaly assay. F1 mice (C57BL/6 X CBA) were inoculated intraperitoneally with 1 X 10(8) parental (C57BL/6) or (CBA) spleen cells. The F1 mice were sacrificed 13 days later and the resulting splenomegaly was 3-4 times the normal amount. F1 mice which were injected with parental BCGcw-primed C57BL/6 spleen cells had a 50% inhibition of splenomegaly, whereas BCGcw-primed CBA spleen cells (a strain which does not develop suppressor cells) did not show this inhibition. In vitro results also confirmed that only C57BL/6 mice and not CBA mice developed suppressor cells after BCGcw immunization. A second study showed that X-irradiated (1000 R) BCGcw-primed "suppressor cells" could inhibit splenomegaly caused by the inoculation of normal parental C57BL/6 cells into F1 mice. The mechanism by which BCGcw-primed "suppressor cells" caused this inhibition of splenomegaly was delineated and found to be dependent upon the secretion of prostaglandin (PGE-1). Indomethacin and aspirin, potent inhibitors of prostaglandin synthesis, blocked the activity of C57BL/6 BCGcw "suppressor cells" and splenomegaly resulted. Systemic administration of the prostaglandin (15S)-15-methyl PGE-1 reduced splenomegaly approximately 50% in F1 mice which were injected with C57BL/6 or CBA cells. These results indicated that immunization with BCGcw stimulated a population of "suppressor cells" which could cause a decrease in graft-versus-host response and that the secretion of prostaglandin was responsible for this inhibition.     

Inhibition of erythroid cell growth in irradiated mice by allogeneic lymphoid cells: Specificity of the response

Allogeneic lymphocytes can inhibit proliferation of erythroid cells in the spleens of irradiated mice grafted with syngeneic bone marrow cells. Since there is a linear relationship between the number of injected lymphocytes and erythroid activity, the method is frequently used as a graft-vs-host assay. In this investigation we show that the reduction of erythroid activity is due to both a reaction of the lymphocytes against the tissues of the host and against the grafted bone marrow cells. Thus, reduced erythropoietic activity does not necessarily indicate that the bone marrow targets possess antigens against which the lymphocytes are reactive.     

Firing properties of accessory olfactory bulb mitral/tufted cells in response to urine delivered to the vomeronasal organ of gray short-tailed opossums

In comparison with many mammals, there is limited knowledge of the role of pheromones in conspecific communication in the gray short-tailed opossum. Here we report that mitral/tufted (M/T) cells of the accessory olfactory bulb (AOB) of male opossums responded to female urine but not to male urine with two distinct patterns: excitation followed by inhibition or inhibition. Either pattern could be mimicked by application of guanosine 5'-O-3-thiotriphosphate and blocked by guanosine 5'-O-2-thiodiphosphate, indicating that the response of neurons in this pathway is through a G-protein-coupled receptor mechanism. In addition, the inhibitor of phospholipase C (PLC), U73122, significantly blocked urine-induced responses. Male and female urine were ineffective as stimuli for M/T cells in the AOB of female opossums. These results indicate that urine of diestrous females contains a pheromone that directly stimulates vomeronasal neurons through activation of PLC by G-protein-coupled receptor mechanisms and that the response to urine is sexually dimorphic.     

Inhibition of the mitogenic response to lipopolysaccharide (LPS) in mouse spleen cells by polymyxin B.

The addition of low doses of the cationic polypeptide antibiotic, polymyxin B (PB), to cultures of mouse spleen cells inhibits lipopolysaccharide-(LPS) induced DNA synthesis but not that stimulated by PPD, PHA, or Con A. Inhibition is stoichiometric; the mitogenic response is suppressed by 50% at a weight ratio of PB:LPS of 0.055 to 1. Furthermore, PB-LPS complexes have a much reduced mitogenic capacity. These complexes inhibit the mitogenic response of spleen cells to unmodified LPS but not to PPD, Con A, or PHA. The inhibitory activity of PB is less effective when added after LPS is mixed with responding cells, achieving 50% inhibition when addition is made at 4 to 6 hr. Time course experiments indicate that partial inhibition is a reflection of a lower rate of DNA synthesis. Thus, PB inhibition of LPS mitogenesis apparently occurs as a result of formation of PB-LPS complexes with reduced mitogenic capacity. Specific inhibition by the complexes of mitogenesis induced by native LPS suggests that the inactive complex may bind to B cells but is unable to trigger them.     

Magnitude-matching: the measurement of taste and smell

In the method of magnitude-matching, subjects try to judge intensitiesof sensations from two or more modalities on a single, commonscale. Using responses to one modality as a standard makes itpossible to compare subjects' suprathreshold perceptions onthe other, test modality. A series of ten experiments revealedthe following: (i) magnitude-matching ‘works’: withboth loudness of tones and lightness of grays as standards,tasters versus nontasters of 6-n-prophylthiouracil (PROP) (asdefined by a threshold criterion) show much greater responceto suprathreshold PROP and slightly greater response to surcose;(ii) though superior to rating-scale judgements of sensory intensitymade without reference to a second modality, magnitude-matchingis not, however, flawless: the cross-modality matching relationproduced by a set of magnitude-matches depends systematicallyon the contextual sets of stimulus levels presented for judgement;(iii) with taste as the standard, old versus young subjectsshowed only a 25% decrement in responce to the odor intensityof butanol when both groups recieved the same physical (concentration)levels, but a >50% decrement in responce when both groupsrecieved about the same perceptual levels; (iv) magnitude-matchesare much the same whether subjets make their judgements on abounded rating-scale or an open-ended magnitude-estimation scale:and (v) loudness, lightness and odor intensity serve about equallyin magnitude-matching with taste intensity.     

Circadian rhythms: timing the sense of smell

It is well established that Drosophila olfaction is under circadian control, yet the mechanisms underlying this temporal regulation have remained elusive. The kinase GPRK2 has now been identified as a critical link between the circadian clock and olfactory responses.     

Percutaneous mitral valve repair: the necessity to redefine secondary mitral regurgitation

Netherlands Heart Journal - Interest in percutaneous mitral valve repair has increased during recent years. This is mainly driven by the significant number of patients being declined for mitral...     

Inhibition of an vitro cell-mediated response: further experiments on the cell lineage and target of suppressor cells

The contribution of lymphotoxin to guinea pig leukocyte natural cytotoxicity was evaluated with [³H]TdR release and colony-inhibition assays of 104C1 benzo(a)pyrene in vitro-transformed and tumorigenic, tumor-specific transplantation antigen-negative, syngeneic strain 2/ N fibroblasts. Cytolethal ³H-release activities of mitogen (PHA)¹-stimulated nonimmune and ovalbumin (OA) immune as well as OA-stimulated OA immune unfractionated, adherent (macrophage-enriched) and nonadherent peritoneal leukocytes are qualitatively similar. ³H release is maximal by 48 hr, increases with antigen or mitogen concentration, is greatest with unfractionated leukocytes, and is least with adherent macrophages. Lymphotoxin produced by peritoneal leukocytes, alone or in combination with the leukocytes does not or only minimally induces ³H release even after 6 days of incubation with guinea pig target cells although guinea pig lymphotoxin possesses cytolytic activity as indicated by ³H release from αL929 mouse tumor cells. In contrast to the absent or very weak cytolytic activity of guinea pig lymphotoxin for the guinea pig target cells nonimmune macrophages, nonadherent leukocytes, and lymphotoxin all exhibit readily detectable colony-inhibitory (CI) activity for the syngeneic tumor cells. Macrophage and lymphotoxin CI, moreover, are additive, whereas nonadherent leukocyte and lymphotoxin CI are synergistic. The latter may be due to additional lymphotoxin induced by target cell antigens or other mechanisms of target cell stimulation of effector lymphoid cells and result from very high local levels of lymphotoxin released by the effector cells. Lymphotoxin CI, furthermore, can be cytostatic or cytolethal as indicated by resumption of 104C1 but not αL929 colony growth following removal of lymphotoxin, indicating that natural cell-mediated cytotoxicity consists of lymphotoxin-dependent and -independent cytostatic and cytolethal effector mechanisms.