Cytotoxic activity of Serratia marcescens clinical isolates

Twenty Serratia marcescens isolates from clinical specimens were examined for their cytotoxic activity on four cell lines (HEp-2, Vero, CHO, J774). Most of the isolates were found to be cytotoxic to CHO (70%), Vero (75%) and HEp-2 cells (90%). CHO cells were the most sensitive to cell-free supernatants, followed by HEp-2 and Vero cells. Two strains produced cytotonic toxins which caused elongation of CHO cells. Moreover, twelve isolates (60%) revealed cytotoxic potential to macrophage cell line J774. The results indicate that these bacteria may destroy phagocytes and epithelial cells, which may lead to spread within the host.     

A protease from Legionella pneumophila with cytotoxic and dermal ulcerative activity

Abstract Culture supernatants of Legionella pneumophila , Philadelphia 1, were found to have proteolytic activity, as well as a nondialyzable, heat-labile cytotoxin for Chinese hamster ovary (CHO) cells, and a factor which caused hemorrhagic dermal ulceration when injected intradermally into mice. A protease was purified from culture supernates by filtration on Sephacryl S-200 followed by chromatography on DEAE cellulose. Proteolytic activity had a pH optimum of 5.5, and migrated as two bands in PAGE, with molecular weights of 42 and 31 kDa. CHO cell cytotoxic, dermal ulcerative, and proteolytic activities copurified. The results are consistent with the same protein being responsible for these activities.     

On the toxinogenicity of Pseudomonas aeruginosa strains

Investigation of 367 P. aeruginosa strains primarily isolated from clinical and other biological material as well as from the environment yielded results suggesting a substantial toxinogenic potential. 92.6% of the assayed culture filtrates derived from the strains under investigation proved positive in the early skin tests on rabbits. 49.7% of the assayed material induced cytotoxic alterations on Vero cells, the rates for Y1 and CHO cells being 50.3% and 43.5% respectively. 54.3% culture filtrates caused haemolysis of rabbit RBC and 52.7% lysed horse RBC. Gelatinase activity was found in 96.3% of tested material, protease in 89.8%, lecithinase in 62.4% and elastase in 29.6%. 12.6% of tested material induced fluid accumulation in a ligated intestinal loop. None of the culture filtrates elicited a positive reaction in the suckling mice test suggesting the absence of the thermostable enterotoxin.     

Variation in nuclear DNA content in an ex-type isolate of Penicillium measured by continuous flow microfluorimetry

Abstract The effect of purified enterotoxin produced by Clostridium difficile on Chinese hamster ovary (CHO) cells was examined. In a certain concentration range (0.9–3.6 μ g/ml), purified toxin caused CHO elongation, namely a cytotonic effect, which is similar to a typical morphological change in CHO cells induced by cholera enterotoxin. At a higher concentration, purified enterotoxin had a cytotoxic effect on CHO cells which was neutralized by anti- C. difficile enterotoxin, but not by anti- C. difficile cytotoxin. Thus, enterotoxin had both cytotonic and cytotoxic effects on CHO cells. About 60 and 180 min were required for binding of enterotoxin to CHO cells, and its internalization, respectively, both times being much longer than those for C. difficile cytotoxin.     

Mechanism of action of a cytotonic enterotoxin produced by Aeromonas hydrophila

In this study, we describe the mechanism of action of a cytotonic enterotoxin produced by two isolates of Aeromonas hydrophila. Isolates SSU and Ah65 are of different origin and both are capable of producing either a cytotoxic enterotoxin or aerolysin. A cytotonic enterotoxin produced by diarrheal isolate SSU, which was purified and characterized in our laboratory, elevated intracellular cAMP and PgE2 levels in cultured Chinese hamster ovary (CHO) cells. Likewise, enterotoxic activity expressed by a cytotonic enterotoxin was detected in the culture filtrate of a fish isolate (Ah65) after cytotoxic activity was neutralized with homologous aerolysin monoclonal antibodies. This cytotonic enterotoxin also elevated intracellular cAMP and PgE2 levels in CHO cells, suggesting a cholera toxin-like mechanism of action for Aeromonas cytotonic enterotoxins.     

Reduced glutamine concentration improves protein production in growth-arrested CHO-DG44 and HEK-293E cells

For most cultivated mammalian cells, glutamine is an essential medium component. However, glutamine consumption results in the production of ammonia, a cytotoxic byproduct. Here we investigated the effect of glutamine reduction on recombinant protein production and ammonia accumulation in transiently transfected CHO and HEK-293E cells maintained under conditions of growth arrest. Maximum transient recombinant protein yields were observed in HEK-293E cultures without glutamine and in CHO cultures with 2 mM glutamine. The initial concentration of glutamine correlated with the level of ammonia accumulation in each culture. For both a stable CHO-derived cell line and a polyclonal population of recombinant CHO cells grown under conditions of mild hypothermia, the highest volumetric protein productivity was observed in cultures without glutamine. Here, the level of ammonia accumulation also corresponded to the initial glutamine concentration. Our data demonstrate that reduction of glutamine in the medium is an effective approach to improve protein production in both transiently and stably transfected mammalian cells when applying conditions that reduce or arrest the growth of these cells.     

Enteropathogenic activity and invasion of HEp-2 cells by Aeromonas caviae clinical isolates

Twenty Aeromons caviae isolates from stool of children with diarrhea symptoms were examined for virulence-associated properties: production of cytotoxic and cytotonic toxins, and invasive ability. Most of A. caviae strains were cytotoxic to Vero and CHO cells and produced cytotonic toxins which caused elongation of CHO cells. Moreover, five of A. caviae strains revealed invasive ability towards HEp-2 cells.     

外源PDI基因导入CHO细胞及其对HBsAg分泌的影响

在前期的研究工作中发现,当培养基中添加1.5%DMSO(二甲基亚砜)会使CHO细胞的HBsAg(乙肝表面抗原)产量提高80%以上。与此同时,DMSO引起了HBsAg在细胞胞内的大量积累,其含量提高了8倍。同时,DMSO引起了CHO细胞中PDI(二硫键异构酶)含量降低。由此怀疑是由于PDI不足造成了HBsAg的胞内积累。根据NCBI上查阅到的中华仓鼠小肠中PDI基因序列,通过RT-PCR的方法,从CHO细胞中获得了PDI序列,并构建重组质粒pGFP-PDI。通过阳离子脂质体转染技术,导入外源PDI,使各单克隆CHO细胞的PDI酶活提高30%～90%。但分析这些克隆在1.5%DMSO下HBsAg的胞内积累情况,发现没有改善,从而排除了PDI不足而引起外源蛋白积累的可能。     

Aeromonas spp.-mediated cell-contact cytotoxicity is associated with the presence of type III secretion system

In the study we examined the production of cytotonic and cytotoxic toxins and the presence of a type III secretion system (TTSS) in 64 Aeromonas spp. strains isolated from fecal specimens of patients with gastroenteritis. We observed that contact of the bacteria with host epithelial cells is a prerequisite for their cytotoxicity at 3 h incubation. Cell-contact cytotoxic activity of the strains was strongly associated with the presence of the TTSS. Culture supernatants of the strains induced low cytotoxicity effects at the same time of incubation. Cell-free supernatants of 61 (95%) isolates expressed cytotoxic activity which caused the destruction of HEp-2 cells at 24 h. Moreover, 44% strains were cytotonic towards CHO cells and 46% of strains invaded epithelial cells.     

Intracellular vacuolation induced by culture filtrates of Plesiomonas shigelloides isolated from environmental sources

AIMS: Potential virulence factors produced by culture filtrates of Plesiomonas shigelloides isolated from water were investigated. METHODS AND RESULTS: Culture filtrates of P. shigelloides strains were assayed for cytotoxic activity in CHO (Chinese hamster ovary), Vero (African green monkey kidney), HeLa (human cervix), HT29 (human epithelial intestinal) and SK6 (swine epithelial kidney) cells. Microscopic analyses revealed intensive cytoplasmic vacuolation including cell rounding and swelling, with gradual destruction of the monolayer in filtrate-treated cells. Neutral red assays showed that CHO, HeLa and Vero cells were the most sensitive to the vacuolating activity, which was evident within 30 min of culture filtrate exposure. This activity was inactived by heating at 56 degrees C for 15 min and partially neutralized by antiserum to the cytotoxin of Aeromonas hydrophila. All P. shigelloides strains had a cell-associated haemolysin in the agar plate assay. Three isolates were found to produce a cell-free haemolytic activity at 37 degrees C. In the suckling mouse test, two P. shigelloides culture supernatants were positive for enterotoxic activity. CONCLUSIONS: P. shigelloides culture filtrates isolated from aquatic environment cause intracellular vacuolation on mammalian cells, and produce haemolytic and enterotoxic activities. SIGNIFICANCE AND IMPACT OF THE STUDY: This work revealed the presence of putative virulence factors that could be associated with human infections involving Plesiomonas strains.     

Clonal analysis of human tumor infiltrating lymphocytes reactive with autologous tumor cells: different target cell specificities of NK-like and cytotoxic T-cell clones

Lymphocytes, derived from surgically resected lung carcinoid tissue, were stimulated in mixed culture with irradiated autologous tumor cells (MLTC). The autologous MLTC-stimulated lymphocytes were found to have killing activity against both autologous tumor cells and NK-sensitive target cells. The lymphoblasts generated during MLTC were isolated and cloned under limiting dilution conditions in the presence of interleukin 2. The cloned cell lines were analyzed for cell phenotype and tested for cytotoxic activity. Three cloned cell lines, out of 19 tested, were found to be cytotoxic either against NK-sensitive target cells (natural killers) or the autologous tumor cells. Two clones, having OKT8 phenotype, caused no lysis of the autologous tumor cells, though both exerted NK-like activity against K562 cells. Only one clone with OKT4 phenotype showed specific cytotoxic activity against the autologous tumor, but no NK-like activity against a panel of tumor target cells. These results suggest the coexistence of two types of antitumor cytotoxic lymphocytes at the tumor site: precursors of NK-like cells and specific cytotoxic T cells. Target cell specificity provided a means of distinguishing between the two types.     

Effect of environmental factors on expression and activity of chitinase genes of vibrios with special reference to Vibrio cholerae

AIMS: The aim of this study was to investigate the distribution and inducibility of chitinase genes in vibrios and the effect of environmental factors on the expression level and activity of chitinase genes in Vibrio cholerae strains. METHODS AND RESULTS: Chitin agar plate assays showed that V. cholerae strains were more chitinolytic than non-cholerae vibrios. All of the identified or putative chitinase genes were expressed in V. cholerae (four strains) but not in non-cholerae vibrios (seven species/strains) under standard laboratory growth conditions. In non-cholerae vibrios, these genes were induced by chitin, its monomer N-acetyl-d-glucosamine and on exposure to rabbit intestine, while in V. cholerae strains, these genes showed significant variation in expression levels. To study the effects of environmental factors on the expression and activity of chitinase genes in V. cholerae, bacteria were cultured in different pH, temperature, sodium chloride and nutrients. RT-PCR analysis showed that lower temperatures and higher pH, salinity and nutrition favoured expression of these genes, while their activity increased under higher nutrition content and salinity. CONCLUSIONS: Chitinase genes are distributed in all the relatively small number of strains studied here, and biotic and abiotic factors have significant role in the induction, expression level and activity of this gene family in vibrios. SIGNIFICANCE AND IMPACT OF THE STUDY: Chitinases have important applications especially in recycling of chitin. Vibrios can be used as chitinolytic agents, using suitable culture conditions that maximize the expression and activity of these genes.     

Conditions for Legionella pneumophila cultivation that affect cytotoxin production

Experiments in CHO cell cultures have demonstrated the cytotoxic action of culture filtrates obtained after growing L. pneumophilia strain Philadelphia I, serogroup 1, in a liquid culture medium. This cytotoxic activity has been found to differ in its character from that described in earlier works. The dynamics of the accumulation of cytotoxin in the culture medium has been studied, and the work aimed at the determination of the limits of some cultivation parameters, within which the maximum toxin formation is observed, has been carried out.     

Adjuvant effect of poly(A:U) upon T cell-mediated in vitro cytotoxic allograft responses

The effect of complexes of polyadenylic acid and polyuridylic acid [poly(A:U)] on thymus-processed lymphocytes was studied using a tissue culture system in which T cells responded to cell bound alloantigens. The in vitro activation of T cells into cytotoxic lymphocytes was assessed with the aid of the ⁵¹Cr cytotoxic assay. Introduction of poly(A:U) into cultures or pretreatment of thymus cells prior to culture resulted in a reduction in the time required for the development of maximal cytotoxic activity as well as a reduction in the dose of allogeneic cells required for maximum stimulus. Poly(A:U) had no influence on the ability of differentiated cytotoxic T cells to lyse ⁵¹Cr-labeled target cells. The amplification of cytotoxic activity caused by poly(A:U) was specific to the antigens used to activate the thymus lymphocytes.     

Evaluation of cytotoxic activity in fecal filtrates from patients with Campylobacter jejuni or Campylobacter coli enteritis

We sought to determine the prevalence of cytotoxic activity in fecal filtrates from persons with C. jejuni or C. coli enteritis. Stool specimens were collected from 20 persons with C. jejuni or C. coli enteritis, 20 persons with acute diarrheal illnesses of other causes, and 9 healthy, asymptomatic persons. Fecal filtrates were then incubated with Chinese hamster ovary (CHO) or HeLa cells. The fecal filtrate from 1 of the 20 (5%) persons with Campylobacter enteritis was cytotoxic for HeLa cells at a titer of 1:40, and 10 (50%) were cytotoxic for CHO cells at maximum titers of 1:20. Cytotoxic activity for CHO cells at a median titer of 1:20 was also present in 40% of the fecal filtrates from persons with diarrhea due to causes other than Campylobacter enteritis, and in 33% of filtrates from healthy, asymptomatic persons. The observed low level of cytotoxicity in fecal filtrates from all patient groups studied likely resulted from non-specific factors, unrelated to the pathogenesis of Campylobacter enteritis.     

Induction of aldose reductase expression in rat kidney mesangial cells and Chinese hamster ovary cells under hypertonic conditions

Rat kidney cortex mesangial cells (MES) and Chinese hamster ovary cells (CHO) responded to hypertonicity (600 mosmol/kg) in culture by accumulating sorbitol. The accumulation of sorbitol was due to increased aldose reductase (AR) activity, apparently brought about by increased levels of AR mRNA and protein. The levels of AR mRNA increased approximately 60-fold in MES cells and 30-fold in CHO cells by 24 h in culture media (300 mosmol/kg supplemented with 150 mM NaCl, 600 mosmol/kg total). AR activity also markedly increased (14- to 16-fold above control), but MES took 4 days and CHO 6 days to reach this maximum. Other osmolytes, raffinose and sorbitol (at concentrations of 250 to 300 mM) elicited the same response as that of 150 mM NaCl. These data show that AR expression is induced in MES and CHO cells under hypertonic conditions. Of special interest is the induction of large amounts of AR in rat kidney cortex mesangial cells, a target tissue of diabetes and a site where excessive accumulation of sorbitol is suspected to be a critical factor in diabetic nephropathy.     

Culture media optimization for Chinese hamster ovary cell growth and expression of recombinant varicella-zoster virus glycoprotein E

Herpes zoster (HZ) is caused by reactivation of varicella-zoster virus (VZV) latent in the sensory ganglia and causes severe pain, often leading to postherpetic neuralgia (PHN). Two prophylactic vaccines against HZ are currently licensed for human use, a live attenuated vaccine and a subunit vaccine containing recombinant VZV glycoprotein E (gE) as antigen. The latter has superior protective efficacy against HZ and PHN. During HZ subunit vaccine development, we obtained Chinese hamster ovary (CHO) cell clones expressing VZV gE. This study was performed to optimize culture media conditions for CHO cell growth and gE production. Using a high-throughput culture system, three CHO cell clones were cultured in microtiter plates containing 24 different basal media, and three basal media were selected. The clone with the highest gE expression was fed-batch cultured in each of the three basal media in combination with 13 different feed media. A pair of media, BalanCD CHO Growth A and EX-CELL Advanced CHO Feed 1, with the highest productivity was selected for gE production. Scale-up fed-batch cultures of the selected clone cultured in a wave bag bioreactor containing the optimized media yielded 2440 mg gE protein/L culture, a 11.5-fold increase compared to original culture conditions (batch culture in CD OptiCHO medium). The optimized media condition is used to produce VZV gE antigen for an HZ subunit vaccine, which is under phase I clinical trial. This study would provide valuable insights on culture media optimization for CHO cells expressing a recombinant vaccine antigen.

     

Production of a membrane-bound proteins,the human gamma-glutamyl transferase,by CHO cells cultivated on microcarriers,in aggregates and in suspension

Recombinant Chinese Hamster Ovary (CHO) cells, engineered for the production of human gamma-glutamyl transferase (GGT), have been grown on Cytodex 1 microcarriers, as aggregates, or as single cells in suspension after adaptation. GGT is a membrane bound enzyme which was not secreted during the culture period. The maximal enzyme activity was found to be directly related to the achieved maximal cell density. Culture of CHO on microcarriers yielded the fastest growth, with a specific growth rate of 0.04 h^–1, the highest cell density (near 1.3×10⁶ cells ml^–1), and the highest enzyme activity around 300 mU ml^–1, which corresponded to a specific cellular level of 20 mU 10^–5 cells. GGT could also be produced by growing CHO cells in suspension as single cells or as aggregates. Under these conditions, however, the specific CHO growth rate was significantly slower and the GGT level per cell was divided by a factor 6. Growing CHO cells without microcarriers also resulted in differences in cell metabolism, with a higher conversion yield of glutamine into ammonia, and a higher cell lysis. The catalytic kinetic constants of the enzyme were found identical for the three culture systems.     

Enterotoxin and cytotoxin production by Salmonella enteritidis strains isolated from gastroenteritis outbreaks

Seventy-six Salmonella enteritidis , three Salmonella virchow and one Salmonella braedenrup strains were screened for enterotoxigenicity by using the Chinese hamster ovary (CHO), Y1 adrenal, Vero and HeLa cell tests. All the strains gave positive reactions for enterotoxin production, except one, and the relative sensitivity to the toxin exhibited by the different cell lines was evaluated. An enterotoxic activity has been identified in sonicated extracts of Salm. enteritidis: This enterotoxin was purified on Agarose A-5m (Bio-Rad) and Superose 12 HR 10/30 column. The enterotoxic activity was eluted from the Superose column in the first peak. Like Vibrio cholerae toxin CT and Escherichia coli enterotoxin LT, it was blocked by GM₁ ganglioside, but at a higher concentration. In addition, a cytotoxic factor has been partially identified. The procedure for isolating the cytotoxin included ammonium sulphate precipitation, size-exclusion chromatography and anion exchange chromatography. This cytotoxin factor caused inhibition of protein synthesis in cultured cells, as determined by flow cytometry and [³H]-leucine incorporation. Flow cytometry analysis also showed an activation of CHO cells when exposed to this cytotoxic factor resulting in a state of active growth. Cytotoxic activity was not blocked by gangliosides.     

In vitro sensitization of human lymphoid cells to antigens on cultured melanoma cells. I. Culture conditions resulting in augmented cell-mediated cytotoxicity

Culture conditions have been established that result in the sensitization of normal human peripheral blood lymphoid cells on allogeneic melanoma monolayers. Optimal culture conditions require 2 to 8 × 10⁶ mitomycin C treated stimulator melanoma cells to sensitize 5 to 10 × 10⁶ responder lymphoid cells. Neither rocking nor refeeding of the culture is necessary for the sensitization procedure. Stimulator cells grown in either fetal calf serum or human serum will serve as effective stimulator or target cells. Peak cytotoxic activity was detected at 44 hr in a microcytotoxicity assay, although some cytotoxic activity was detectable at 24 hr.