The high rate of aerobic glycolysis of transformed 3T3 cells persists after cell homogenization.
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Aerobiosis↗
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Results for “persister cells”
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Dynamics of donor cell persistence and recipient cell replacement in orthotopic corneal allografts in mice.
PURPOSE: To determine the extent to which donor cells persist and recipient cells repopulate each of the three cell layers of orthotopic corneal grafts in mice. METHODS: BALB/c, C57BL/6, and enhanced green fluorescence protein (EGFP) transgenic mice (B6 background) were used as donors and recipients for orthotopic syngeneic and allogeneic corneal grafts. Graft-bearing eyes were harvested at 5, 10, 15, 28, and 56 days, stained with propidium iodide, and observed (layer by layer) by confocal microscopy. Bone marrow-derived cells in the grafts were assessed immunohistochemically. RESULTS: Donor epithelium was totally replaced by recipient epithelial cells within 15 days in both syngeneic and allogeneic grafts, whereas donor stromal keratocytes and endothelial cells were retained virtually intact in syngeneic grafts and in accepted allografts. In rejected allografts, neither donor-derived keratocytes nor endothelial cells were detected, and, instead, recipient-derived stromal fibroblasts, neovessels, and infiltrating leukocytes were heavily represented. The posterior surface of rejected grafts was devoid of corneal endothelium and was covered incompletely with bone marrow-derived cells of recipient origin. CONCLUSIONS: Whereas in mice graft-derived epithelium is largely irrelevant to corneal allograft outcome, persistence of donor-derived endothelium and keratocytes correlates perfectly with graft acceptance. Recipient endothelium is incapable of covering the posterior surface of accepted or rejected corneal grafts, whereas bone marrow-derived cells of recipient origin come to occupy this site in rejected grafts.
Animals↗
Evidence for the in vivo production and release into the serum of a T-cell lymphokine, persisting-cell stimulating factor (PSF), during graft-versus-host reactions.
The T-cell lymphokine, persisting-cell stimulating factor (PSF or interleukin-3), was detected in the serum of mice undergoing graft-versus-host reactions (GVHR). Gel filtration under non-dissociating conditions indicated that the PSF in the serum had an apparent molecular weight of 34,000, a figure identical with that of PSF generated from activated T cells in vitro, indicating that PSF was not bound by serum proteins. The GVHR was accompanied by increases in the numbers in the bone marrow and spleen of precursors of PSF-dependent mast cells, and increases in the numbers of mast cells, megakaryocytes and immature and mature neutrophils in the spleen. These effects of GVHR on haemopoietic cells paralleled those seen when haemopoietic tissues were stimulated with pure PSF in vitro and closely resembled those induced in previous studies by the presence of a tumour that secreted PSF alone. These studies are the first to show that PSF can enter the circulation during immune reactions in vivo and suggest that much of the stimulation of haemopoietic cells seen in GVHR, can be accounted for by the release of PSF from activated T cells.
Animals↗
Cells persistently infected with Newcastle disease virus. 3. Thermal stability of hemagglutinin and neuraminidase of a mutant isolated from persistently infected L cells.
Data were obtained which indicated the possible cause of the defective elution from erythrocytes of the mutant virus (NDV(pi)) isolated from L cells persistently infected with the Herts strain of Newcastle disease virus (NDV(o)). The chicken erythrocyte receptors for the mutant and wild-type viruses were equally sensitive to the action of Vibrio cholera filtrate neuraminidase; this suggests that the failure of NDV(pi) to elute from chicken erythrocytes is not due to a specific neuraminidase-resistant receptor for this virus on the erythrocyte membrane. There was no difference in the enzyme content of the intact virions of NDV(o) and NDV(pi) when tested with a soluble substrate, indicating that the inefficient elution of NDV(pi) was not due to a reduced enzyme content. The neuraminidase activity of intact NDV(pi) virions was significantly more stable at 55 C than the enzyme of NDV(o) virions, whereas the dissociated enzymes of the two viruses were inactivated at the same rate. On the basis of these findings, it seems likely there is a structural difference between the two viruses. The neuraminidase protein of the mutant NDV(pi) may be incorporated into the viral envelope in such a manner that it is prevented from reacting with the substrate in the erythrocyte membrane, although it can react with a soluble substrate. The hemagglutinin activity of both intact and disrupted NDV(pi) was significantly more resistant to thermal inactivation than that of the wild-type NDV(o). This finding suggests a genetic difference in the hemagglutinin protein of the two viruses.
Animals↗
Effect of interferon on Vero cells persistently infected with Sendai virus compared to Vero cells persistently infected with SSPE virus.
Persistent infections with Sendai and SSPE virus were established in Vero cells. Sequential passages of these cells were monitored by immunofluorescence and for their sensitivity to the antiviral and antiproliferative effects of interferon (IFN). The cells rapidly developed resistance to the antiviral effect of IFN as judged by the inability of IFN to inhibit the replication of exogenous Sindbis virus. This decrease was accompanied by a reduction in the induction of the 2'-5' oligo A synthetase. Both cell lines were resistant to the antiproliferative effect of IFN. A decrease or absence of IFN receptors on the surface of the cells was not found to be the cause of their resistance to IFN.
2',5'-Oligoadenylate Synthetase↗
Cells persistently infected with newcastle disease virus: I. Properties of mutants isolated from persistently infected L cells.
The strain of Newcastle disease virus (NDV(pi)) present in persistently infected L cells differed markedly from the Herts strain (NDV(0)) used to initiate the infection. NDV(pi) produced small plaques (less than 1 mm) in chick embryo cell cultures, whereas the wild type (NDV(0)) produced large plaques (2 to 3 mm). The two viruses differed in a number of additional properties. Whereas 80% of adsorbed NDV(0) eluted from chicken red blood cells at 37 C, only about 20% of NDV(pi) was recovered under similar conditions. There was no significant difference in the neuraminidase content of the two viruses. The infectivity of NDV(0) was stable for 1 hr at 48 C, whereas 99.9% of the infectivity of NDV(pi) was destroyed. The two viruses also differed in lethality for chick embryos; NDV(pi) had significantly reduced lethality for 9-day-old chick embryos when compared to NDV(0). In contrast to NDV(0), which produced an abortive infection in L cells, NDV(pi) not only replicated effectively and destroyed these cells, but also induced significantly higher quantities of interferon than did NDV(0). These data furnished additional evidence for the lack of relationship of interferon production to abortive infection of L cells with NDV(0). In contrast, interferon was found to play a significant role in the maintenance of persistent infection.
Journal Article↗
Common cytokine receptor gamma chain (gammac)-deficient B cells persist in T cell-deficient gammac-mice and respond to a T-independent antigen.
Defects in the common cytokine receptor gamma chain (gammac) in man result in X-linked severe combined immunodeficiency disease (SCIDX1) characterized by an absence of alphabeta T cells, gammadelta T cells and NK cells, with the presence of circulating B cells. Mice made deficient for gammac lack gammadelta T cells and NK cells, but in contrast to SCIDX1 patients have appreciable numbers of alphabeta T cells, while B cells are reduced about tenfold in numbers and disappear with age. Here we show that when gammac- mice are rendered T cell deficient, B cell numbers are still reduced but the age-dependent loss of B cells does not occur. The peripheral B cells which persisted in gammac-/ nude and gammac-/TCRbeta-/- mice were able to respond to mitogen stimulation in vitro and to mount antigen-specific T-independent Ig responses in vivo. These results demonstrate that gammac- B cells are functionally competent and suggest that residual alphabeta T cells are implicated in the B cell loss in gammac mice. The gammac-/nude and gammac-/TCRbeta-/- mice provide new models to dissect the role of gammac-dependent receptors during murine B cell differentiation.
Animals↗
Persister cells and the riddle of biofilm survival.
This review addresses a long-standing puzzle in the life and death of bacterial populations--the existence of a small fraction of essentially invulnerable cells. Bacterial populations produce persisters, cells that neither grow nor die in the presence of bactericidal agents, and thus exhibit multidrug tolerance (MDT). The mechanism of MDT and the nature of persisters, which were discovered in 1944, have remained elusive. Our research has shown that persisters are largely responsible for the recalcitrance of infections caused by bacterial biofilms. The majority of infections in the developed world are caused by biofilms, which sparked a renewed interest in persisters. We developed a method to isolate persister cells, and obtained a gene expression profile of Escherichia coli persisters. The profile indicated an elevated expression of toxin-antitoxin modules and other genes that can block important cellular functions such as translation. Bactericidal antibiotics kill cells by corrupting the target function, such as translation. For example, aminoglycosides interrupt translation, producing toxic peptides. Inhibition of translation leads to a shutdown of other cellular functions as well, preventing antibiotics from corrupting their targets, which will give rise to tolerant persister cells. Overproduction of chromosomally-encoded "toxins" such as RelE, an inhibitor of translation, or HipA, causes a sharp increase in persisters. Deletion of the hipBA module produces a sharp decrease in persisters in both stationary and biofilm cells. HipA is thus the first validated persister/MDT gene. We conclude that the function of "toxins" is the exact opposite of the term, namely, to protect the cell from lethal damage. It appears that stochastic fluctuations in the levels of MDT proteins lead to formation of rare persister cells. Persisters are essentially altruistic cells that forfeit propagation in order to ensure survival of kin cells in the presence of lethal factors.
Anti-Bacterial Agents↗
Epigenetic Repression of TP53 Transcription Underlies Cancer Cell Persistence for Carboplatin Resistance in Non-Small Cell Lung Cancer.
While chemoresistance in non-small cell lung cancer (NSCLC) cells has historically been attributed to permanent genetic mutations, emerging evidence highlights the role of nongenetic transcriptional plasticity and 'drug-tolerant persister' cells. To systematically map these epigenetic vulnerabilities, we utilized a genome-wide CRISPR interference library to screen wild-type TP53 NSCLC (A549) cells under carboplatin selection. Using the DrugZ algorithm and subsequent pathway enrichment analyses, this screen revealed that transcriptional suppression of interstrand crosslink DNA repair networks, including the Fanconi anemia pathway, markedly sensitized cells to carboplatin. Unexpectedly, transcriptional silencing of TP53 and its downstream target CDKN1A emerged as the strongest drivers of resistance, enabling cells to bypass therapy-induced senescence and maintain their proliferative potential later. To validate these findings in a clinically relevant context, we established a chronic carboplatin-resistant cell model (A549CarboR cells). A549CarboR exhibited a reduction in TP53 transcripts, along with decreased H3K27 acetylation and increased DNA hypermethylation on its promoter. Epigenetic remodeling using the DNA methyltransferase inhibitor (DNMTi) was associated with unblocking TP53 transcription, restored p53 signaling, and resensitization of resistant cells to carboplatin. Conversely, histone deacetylase inhibitors induced CDKN1A transcription to bypass TP53, indicating distinct epigenetic circuits. Collectively, the results demonstrate for the first time that TP53 expression is dynamically regulated at the transcriptional level through promoter methylation related to the drug tolerance. These insights emphasize that epigenetic silencing, rather than exclusive genetic loss-of-function, contribute to platinum resistance and underscore the therapeutic potential of pairing platinum regimens with DNMTi to target the transcriptomic plasticity of persistent cancer cell populations.
CRISPR interference screening↗
Donor cell persistence and activation-induced unresponsiveness of peripheral CD8+ T cells.
We studied the impact of the duration of donor cell persistence on CD8+ T cell responsiveness after adoptive transfer of antigen-expressing lymphoid cells. Naive or immunized female mice were treated by adoptive transfer of spleen cells from mice ubiquitously expressing a lymphocytic choriomeningitis virus-derived cytotoxic T lymphocyte (CTL) epitope (gp33-41) either alone or in combination with the male H-Y antigen providing additional antigenic CTL and T helper cell determinants. Low doses of male spleen cells (or sorted B cells) primed CTL, while high doses of the same cells rendered them unresponsive. CTL unresponsiveness induced by high numbers of male spleen cells was dependent upon prolonged persistence of antigen-expressing donor cells. Unresponsive CTL reverted to a state of activation when the duration of donor cell chimerism was limited. Memory CTL could be rendered unresponsive if antigen-expressing donor cells were allowed to persist. These results suggest that, irrespective of the type of antigen-presenting cell and the functional state of the responding T cell, activation and unresponsiveness can represent two different outcomes critically determined by quantitative and kinetic differences of antigen persistence.
Adoptive Transfer↗
Cancer stem cells persist in many cancer cell lines.
Both stem cells and cancer cells are thought to be capable of unlimited proliferation. Paradoxically, however, some cancers seem to contain stem-like cells (cancer stem cells). To help resolve this paradox, we investigated whether established malignant cell lines, which have been maintained over years in culture, contain a subpopulation of stem cells. We have shown that four cancer cell lines contain a small side population (SP), which, in many normal tissues, is enriched for stem cells of the tissue. We have also shown that SP cells in C6 glioma cell line, but not non-SP cells, can generate both SP and non-SP cells in culture and are largely responsible for the in vivo malignancy of this cell line. We propose that many cancer cell lines contain a minor subpopulation of stem cells that is enriched in a SP, can be maintained indefinitely in culture, and is crucial for their malignancy.
Animals↗
Candida albicans biofilms produce antifungal-tolerant persister cells.
Fungal pathogens form biofilms that are highly recalcitrant to antimicrobial therapy. The expression of multidrug resistance pumps in young biofilms has been linked to increased resistance to azoles, but this mechanism does not seem to underlie the resistance of mature biofilms that is a model of in vivo infection. The mechanism of drug resistance of mature biofilms remains largely unknown. We report that biofilms formed by the major human pathogen Candida albicans exhibited a strikingly biphasic killing pattern in response to two microbicidal agents, amphotericin B, a polyene antifungal, and chlorhexidine, an antiseptic, indicating that a subpopulation of highly tolerant cells, termed persisters, existed. The extent of killing with a combination of amphotericin B and chlorhexidine was similar to that observed with individually added antimicrobials. Thus, surviving persisters form a multidrug-tolerant subpopulation. Interestingly, surviving C. albicans persisters were detected only in biofilms and not in exponentially growing or stationary-phase planktonic populations. Reinoculation of cells that survived killing of the biofilm by amphotericin B produced a new biofilm with a new subpopulation of persisters. This suggests that C. albicans persisters are not mutants but phenotypic variants of the wild type. Using a stain for dead cells, rare dark cells were visible in a biofilm after amphotericin B treatment, and a bright and a dim population were physically sorted from this biofilm. Only the dim cells produced colonies, showing that this method allows the isolation of yeast persisters. Given that persisters formed only in biofilms, mutants defective in biofilm formation were examined for tolerance of amphotericin B. All of the known mutants affected in biofilm formation were able to produce normal levels of persisters. This finding indicates that attachment rather than formation of a complex biofilm architecture initiates persister formation. Bacteria produce multidrug-tolerant persister cells in both planktonic and biofilm populations, and it appears that yeasts and bacteria have evolved analogous strategies that assign the function of survival to a small part of the population. In bacteria, persisters are dormant cells. It remains to be seen whether attachment initiates dormancy that leads to the formation of fungal persisters. This study suggests that persisters may be largely responsible for the multidrug tolerance of fungal biofilms.
Antifungal Agents↗
Phage vB_KpnM_NB cocktail synergizing with amikacin in inhibiting persister cells of Klebsiella pneumoniae.
UNLABELLED: The emergence of multidrug-resistant Klebsiella pneumoniae (KPN) and antibiotic-tolerant persister cells poses a significant challenge to existing anti-infection therapies. Given the urgent need for sustainable alternatives to antibiotics, phage cocktails are emerging as a promising alternative to control K. pneumoniae infections. We isolated three lytic phages vB_KpnM_NB (1-3) from Ningbo environmental samples, classified them into the Drexlerviridae family, and determined the biological characteristics of two representative phages. Genomic analysis confirmed that these phages are closely related and lack resistance and virulence genes, ensuring biosafety. Subsequently, a stable KPN persister model was established using amikacin, with a biphasic killing pattern observed during treatment. At a multiplicity of infection of 10, the phage cocktail eliminated 99.00% of persister cells, while individual phages were less effective. The phage cocktail also inhibited persister-derived biofilm formation, showing improved results when combined with amikacin. This combination significantly reduced capsule polysaccharide production in persisters, weakening the outer membrane barrier. These findings demonstrate that the phage cocktail-amikacin combination effectively targets planktonic cells, persister cells, and biofilms, providing a promising strategy against persisters and recurrent K. pneumoniae infections. IMPORTANCE: This study fills the critical gap in understanding how phage cocktails synergize with amikacin against K. pneumoniae persister cells. By constructing a highly specific phage vB_KpnM_NB cocktail, establishing a stable persister model, and performing in vitro bactericidal and biofilm assays, we demonstrate that the cocktail effectively eliminates planktonic cells, persisters, and biofilms. We clarify the core synergistic mechanism: inhibiting capsular polysaccharide synthesis, improving phage adsorption, and disrupting the bacterial outer membrane barrier. These findings provide experimental evidence for the prevention and control of multidrug-resistant and carbapenem-resistant K. pneumoniae persister infections, establishing a safe and effective phage-antibiotic combination therapy. The results are crucial for addressing antibiotic tolerance and controlling chronic, recurrent infections. They hold significant theoretical and translational value for the treatment of refractory infections in clinical settings and offer new insights into the development of novel antimicrobial strategies.
Klebsiella pneumoniae↗
Persister cells mediate tolerance to metal oxyanions in Escherichia coli.
Bacterial cultures produce subpopulations of cells termed 'persisters', reputedly known for high tolerance to killing by antibiotics. Ecologically, antibiotics produced by competing microflora are only one potential stress encountered by bacteria. Another pressure in the environment is toxic metals that are distributed ubiquitously by human pollution, volcanic activity and the weathering of minerals. This study evaluated the time- and concentration-dependent killing of Escherichia coli planktonic and biofilm cultures by the water-soluble metal(loid) oxyanions chromate (CrO4(2-)), arsenate (AsO4(2-)), arsenite (AsO2-), selenite (SeO3(2-)), tellurate (TeO4(2-)) and tellurite (TeO3(2-)). Correlative to previous reports in the literature, control antibiotic assays indicated that a small proportion of E. coli biofilm populations remained recalcitrant to killing by antibiotics (even with 24 h exposure). In contrast, metal oxyanions presented a slow, bactericidal action that eradicated biofilms. When exposed for 2 h, biofilms were up to 310 times more tolerant to killing by metal oxyanions than corresponding planktonic cultures. However, by 24 h, planktonic cells and biofilms were eradicated at approximately the same concentration in all instances. Coloured complexes of metals and chelators could not be generated in biofilms exposed to CrO4(2-) or TeO3(2-), suggesting that the extracellular polymeric matrix of E. coli may have a low binding affinity for metal oxyanions. Viable cell counts at 2 and 24 h exposure revealed that, at high concentrations, all of the metal oxyanions had killed 99 % (or a greater proportion) of the bacterial cells in biofilm populations. It is suggested here that the short-term survival of <1 % of the bacterial population corresponds well with the hypothesis that a small population of persister cells may be responsible for the time-dependent tolerance of E. coli biofilms to high concentrations of metal oxyanions.
Anions↗
Detection of anti-rabies virus cytotoxic T lymphocytes in mice of four distinct H-2 haplotypes using target cells persistently infected with ERA rabies virus.
Cells persistently infected with Evelyn-Rokitnicki-Abelseth (ERA) rabies virus were established. The cells were used as stimulator and target cells to compare H-2 restricted cytotoxic T lymphocyte (CTL) responses specific for rabies virus in A/WySnJ (H-2a), C57BL/6J (H-2b), BALB/cByJ (H-2d), A.SW/SnJ (H-2s) and SJL/J (H-2s) mice. Using a 51chromium release assay, it was determined that an effector/target (E/T) ratio of 5:1 was necessary to demonstrate specific lysis of ERA virus persistently infected mouse neuroblastoma (MNB) (H-2a), EL-4 (H-2b) and P815 (H-2d) cells. Effectors at an E:T ratio of only 0.05:1 specifically lysed an SV-40 transformed SJL/J mouse fibroblast (SSSV) (H-2s) target monolayer. The CTL destruction of the SSSV monolayer was observed visually following Giemsa staining. This is the first instance in which a detailed method for detection of murine anti-rabies virus CTLs has been reported. Furthermore, it is the first time target cells persistently infected with rabies virus were used as stimulator cells to amplify CTLs in vitro and as target cells in the CTL assay. It also is the initial report in which rabies specific CTLs were characterized in H-2d and H-2s rabies virus infected mice.
Animals↗
Expression of constitutive and inducible HSP70 and HSP47 is enhanced in cells persistently spread on OPN1 or collagen.
Cells persistently spread on OPN or collagen survive heat shock better than cells transiently spread on fibronectin or tissue culture plates. Thus, a central question is whether constitutively or inducible stress proteins are enhanced in cells grown on adhesive proteins that maintain a persistent spread cell shape. Levels of Hsp 72,73, and colligin/Hsp47 were determined by Western blot analyses. The inducible Hsp 72 was prominently expressed following heat shock in cells grown on OPN or collagen, but not in cells plated on fibronectin coated substratum or on tissue culture plates. Colligin/Hsp 47 and Hsp 73 manifested a similar pattern of expression indicating that these adhesive attachment proteins accommodate cell function through organization of cell architecture.
Cell Adhesion↗
Cells persistently infected with Newcastle disease virus. II. Ribonucleic acid and protein synthesis in cells infected with mutants isolated from persistently infected L cells.
A comparison of the replication patterns in L cells and in chick embryo (CE) cell cultures was carried out with the Herts strain of Newcastle disease virus (NDV(o)) and with a mutant (NDV(pi)) isolated from persistently infected L cells. A significant amount of virus progeny, 11 plaque-forming units (PFU)/cell, was synthesized in L cells infected with NDV(o), but the infectivity remained cell-associated and disappeared without being detectable in the medium. In contrast, in L cells infected with NDV(pi), progeny virus (30 PFU/cell) was released efficiently upon maturation. It is suggested that the term "covert" rather than "abortive" be used to describe the infection of L cells with NDV(o). In both L and CE cells, the latent period of NDV(pi) was 2 to 4 hr longer than for NDV(o). The delay in synthesis of viral ribonucleic acid (RNA) in the case of NDV(pi) coincided with the delay in the inhibition of host RNA and protein synthesis. Although both NDV(o) and NDV(pi) produced more progeny and more severe cell damage in CE cells than in L cells, the shut-off of host functions was significantly less efficient in CE cells than in L cells. Paradoxically, no detectable interferon was produced in CE cells by either of the viruses, whereas in L cells most of the interferon appeared in the medium after more than 90% of host protein synthesis was inhibited. These results suggest that the absence of induction of interferon synthesis in CE cells infected with NDV is not related to the general shut-off of host cell synthetic mechanisms but rather to the failure of some more specific event to occur. In spite of the fact that NDV(pi) RNA synthesis commenced 2 to 4 hr later than that of NDV(o), interferon was first detected in the medium 8 hr after infection with both viruses. This finding suggests that there is no relation between viral RNA synthesis and the induction of interferon synthesis.
Animals↗
Attachment defect in mouse fibroblasts (L cells) persistently infected with Chlamydia psittaci.
Almost all the cells in populations of mouse fibroblasts (L cells) persistently infected with the 6BC strain of Chlamydia psittaci were immune to superinfection with high multiplicities of C. psittaci, whether or not the L cells contained visible chlamydial inclusions. As ascertained by experiments with 14C-labeled C. psittaci, immunity to superinfection resulted from the failure of added chlamydiae to attach to persistently infected host cells. However, when exogenous C. psittaci was introduced into persistently infected L cells by centrifuging the inoculum onto host cell monolayers or by pretreating the monolayers with diethylaminoethyl-dextran, these chlamydiae produced expected numbers of infectious progeny. Persistently infected L cells were associated in an unknown way with a C. psittaci population that entered the host cells only with the aid of centrifugation or pretreatment with diethylaminoethyl-dextran. Inclusion-free, persistently infected L cells appeared to present at least two separate hindrances to chlamydial activity: blockage of the attachment of exogenous elementary bodies to persistently infected host cells and prevention of the initiation of chlamydial multiplication by means of a normal developmental cycle in the absence of added C. psittaci.
Animals↗