Modified HeLa Cells Research Articles

Genetically engineered CD80-pMHC-harboring extracellular vesicles for antigen-specific CD4+ T-cell engagement.

The identification of low-frequency antigen-specific CD4+ T cells is crucial for effective immunomonitoring across various diseases. However, this task still encounters experimental challenges necessitating the implementation of enrichment procedures. While existing antigen-specific expansion technologies predominantly concentrate on the enrichment of CD8+ T cells, advancements in methods targeting CD4+ T cells have been limited. In this study, we report a technique that harnesses antigen-presenting extracellular vesicles (EVs) for stimulation and expansion of antigen-specific CD4+ T cells. EVs are derived from a genetically modified HeLa cell line designed to emulate professional antigen-presenting cells (APCs) by expressing key costimulatory molecules CD80 and specific peptide-MHC-II complexes (pMHCs). Our results demonstrate the beneficial potent stimulatory capacity of EVs in activating both immortalized and isolated human CD4+ T cells from peripheral blood mononuclear cells (PBMCs). Our technique successfully expands low-frequency influenza-specific CD4+ T cells from healthy individuals. In summary, the elaborated methodology represents a streamlined and efficient approach for the detection and expansion of antigen-specific CD4+ T cells, presenting a valuable alternative to existing antigen-specific T-cell expansion protocols.

Direct comparison of KATUSHKA far-red fluorescent protein variants with luciferase bioluminescence in xenograft mouse models of epithelial tumor studies

Background. Research in oncology assumes establishment and usage of xenograft animal models, meeting the requirements of humanity and rationality. Although praised as promising field of research, preclinical studies of low-molecular-weight inhibitors, antitumor monoclonal antibodies, vaccines, cellular therapy products, CAR-T cells entail continuous control of tumor growth dynamics. Luciferase detection of bioluminescence requires injection of a reporter substrate. However, injections not only are laborious, time-consuming and expensive, but are also stressful for animals. Thus, a vast variety of new visualization methods is employed, including proteins of the far-red spectrum.Objective. The study aimed to compare detection efficiency of tumor growth dynamics in mice models of cervical cancer, applying a commercially available line of fluorescent red protein derivatives of Katushka clade and Renilla luciferase, that is commonly used in in vivo studies. Design and methods. Xenograft mice models were derived injecting modified HeLa cell line, that expresses fluorescent reporter proteins: Katushka, Katushka2S, TurboRFP, TurboFP650 and Renilla enzyme. Results. Spectral properties and emission wavelength of far-red fluorescent protein Katushka and Katushka2S outlines these markers within RFP derivatives lineage as outstanding instrument for in vivo tumor visualization.Conclusion. Detection of fluorescent far-red reporters Katushka and Katushka2S can be considered as a credible alternative to Renilla luciferase bioluminescence in experimental models in vivo on the part of immunotherapy research.

Screening for Immunosuppressive Genes Responsible for Resistance Towards CAR-T Cell Therapy in Cancer Cells

Despite expanding areas of clinical application for CAR-T cell therapy there is still lack of complete information about genes responsible for tumor resistance to this type of therapy. Widely known anti-PD-1 antibody drug Nivolumab and anti-CTLA-4 antibody drug Ipilimumab represent immunotherapeutics that activate the immune system against advanced melanoma and have proven superior to cytotoxic chemotherapy. However, in addition to existing ligands (i.e. CD80, CD86, PD-L1, PD-L2) there are supposedly other surface-expressed ligands stipulating the ability of cancer cells to suppress CAR-T efficacy. It may turn out that tumor resistance to CAR-T therapy is caused by previously unknown immunosuppressive genes. Revealing such genes could improve clinical outcomes by engineering enhanced CAR-T cells and antibodies capable of overcoming the gene-mediated resistance.In this study we propose the use of CRISPR technique and NGS sequencing to validate this hypothesis and identify novel genes that could presumably determine resistance towards CAR-T cells. The strategy is based on genome-wide screening using human CRISPR/Cas9 Synergistic Activation Mediator (SAM) pooled library that utilize an engineered protein complex for the transcriptional activation of endogenous genes. CAR-T cells were generated from healthy donor T cells genetically modified by lentiviral vectors and expanded ex vivo.Main steps of the experiment include:Amplification of pooled SAM library and use of NGS to confirm library representation.Generation of pooled lentiviral vectors using SAM library and transduction of HeLa cells.Generation of CAR-T cells.Co-cultivation of CAR-T and modified HeLa cells, selection of resistant cells.PCR analysis of sgRNA-containing fragment, followed by cloning of amplicons and NGS of amplicon library.Validation of identified genes to confirm resistance towards CAR-T cells.We generated a polyclonal CD19-positive HeLa cell line expressing dCas9-VP64 fusion and activation helper protein. This cell line was transduced with Human CRISPR Activation Library (SAM-3 plasmid system) and incubated with anti-CD19 CAR-T cells in order to select resistant cells and identify resistance-determining genes by NGS.Work supported by RFBR grant 18-315-00049 to A.P. and performed in accordance with Program of Competitive Growth of Kazan Federal University.

The roles of breast cancer resistance protein (BCRP/ABCG2) and multidrug resistance-associated proteins (MRPs/ABCCs) in the excretion of cycloicaritin-3-O-glucoronide in UGT1A1-overexpressing HeLa cells

Cycloicaritin is a bioactive natural phenolic compound from Epimedium species. However, the glucuronidation and excretion which would influence oral bioavailability and pharmacokinetics of cycloicaritin still remain unknown. Here we aimed to establish UGT1A1 stably transfected HeLa cells, and to determine the contributions of BCRP and MRPs transporters to excretion of cycloicaritin-3-O-glucuronide. First, β-estradiol was used to validate the expression of active UGT1A1 protein in engineered HeLa1A1 cells. Furthermore, Ko143 (5 and 20 μM) led to a significant decrease (42.4%–63.8%, p < 0.01) in CICT-3-G excretion and obvious accumulation (19.7%–54.2%, p < 0.05) of intracellular CICT-3-G, while MK571 (5 and 20 μM) caused a significant reduction (46.8%–64.8%, p < 0.05) in the excretion and obvious elevation (50.7%–85.2%, p < 0.01) of intracellular level of CICT-3-G. Furthermore, BCRP knocked-down brought marked reduction in excretion rates of CICT-3-G (26.0%–42.2%, p < 0.01), whereas MRP1 and MRP4-mediated silencing led to significant decrease in the excretion of CICT-3-G (23.8%–35.4%, p < 0.05 for MRP1 and 11.9%–16.0%, p < 0.05 for MRP4). By contrast, neither CICT-3-G excretion nor CICT-3-G accumulation altered in MRP3 knocked-down cells as compared to scramble cells. Taken together, BCRP, MRP1 and MRP4 were identified as the most important contributors for CICT-3-G excretion. Meanwhile, the UGT1A1 modified HeLa cells were a simple and practical tool to study UGT1A1-mediated glucuronidation and to characterize BCRP and MRPs-mediated glucuronide transport at a cellular level.

Modified HeLa Cells and C.elegans Multicellular Model as Unique Systems for the Study of Purinosome Formation

Purines are essential molecules for synthesis of nucleic acids, universal carriers of chemical energy and parts of signaling molecules in all living organisms. Their cellular level is maintained by the de novo purine synthesis (DNPS), effective recycling by salvage pathway and eventual degradation. DNPS includes ten reactions catalyzed by six enzymes, four of them are multifunctional in high eukaryotes. Some DNPS intermediates are unstable and/or toxic, therefore the proximity of enzymes in multienzyme complex called purinosome would be essential to ensure the vital metabolic function. The first direct evidence of its formation was demonstrated by detecting spatial overlap of signals of transiently expressed fluorescently labeled DNPS proteins in HeLa cells grown in the purine depleted medium. However, this model and its eventual utility for further research was questioned and attributed to aggregation of overexpressed proteins and stress granules resulting from the exposure of the cells to the dialyzed, and therefore nutrient reduced growth media. Another possibility for detection of purinosome complexes is immunofluorescent labeling of endogenous proteins involved in the DNPS pathway, so the artificial protein overexpression is avoided. The disadvantage of this method is the inability to study purinosome formation in vivo.In this work we detected the purinosome by combining two previously used methods in prepared purinosome non‐forming cells deficient for particular DNPS steps (CR‐DNPS cells) and in cells deficient in salvage pathway which build purinosome regardless to the purine levels (CR‐HGPRT cells). Moreover, we have prepared the model organism C. elegans with fluorescently labeled DNPS enzymes for in vivo study of the purinosome formation.We transiently transfected the CR‐DNPS and CR‐HGPRT cells with relevant vectors coding BFP labeled wild‐type proteins and observed normalization (e.g. restoration or disruption) of purinosome formation in the cells.To control if the cells undergoing transient transfection and nutrition starvation formed purinosomes or stress bodies, we transfected one type of CR‐DNPS cells with vector coding enzyme with mutation resulting in zero enzyme activity. We did not find any purinosomes regardless of the quantity of purines in the growth media.We fused DNPS proteins in C. elegans with fluorescent protein GFP or mCherry and observed the intracellular compartmentalization of purinosome by fluorescencent confocal microscopy.In conclusion, both methods, transient transfection and immunofluorescence, are useful for the detection of purinosomes in HeLa cells. Moreover, prepared cell‐based models and multicellular model organism C. elegans with translational reporters represent a unique system for purinosome assembly studies. From these models, we expect a greater understanding of the complexity of purine metabolism.Support or Funding InformationThis work was supported by grants AZV 15‐28979A (Ministry of Health, CR); PRIMUS/17/MED/6, GAUK 1102217 and PROGRES Q26/LF1 (Charles University, CR); and LQ1604 NPU II (Ministry of Education, Youth and Sports, CR).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Sensitive Cell-Based Assay to Measure the Doxycycline Concentration in Biological Samples

Doxycycline (DOX) is widely used as a pharmacological agent and as an effector molecule in inducible gene expression systems. For most applications, it is important to determine whether the DOX concentration reaches the level required for optimal efficacy. We developed a sensitive bioassay for measuring the DOX concentration in biological samples. We used a modified HeLa cell line with the luciferase reporter gene under the control of the DOX-inducible Tet-On system for regulation of gene expression. These HeLaDOX cells constitutively express a novel variant of the rtTA transcriptional activator protein that is highly DOX-sensitive. Incubation of the cells with a DOX-containing biological sample triggers luciferase expression, which can be quantitated by standard methods. This bioassay is sensitive, with a DOX detection limit of 22 ng/ml in plasma. The assay was used to determine the DOX concentration in plasma derived from DOX-treated rhesus macaques and mice. Furthermore, we found that the DOX concentration in murine cerebrospinal fluid is 31-fold lower than the concurrent plasma DOX level. This bioassay for the quantification of DOX concentration in biological samples has several advantages over high-performance liquid chromatography-based and microbiological assays: (1) multiple samples can be assayed in a single experiment; (2) only small sample volumes are required; (3) the assay has a low detection limit; and (4) the assay can be performed in any cell culture laboratory.

Size-Uniform 200 nm Particles: Fabrication and Application to Magnetofection

We report on the fabrication of arrays of mono- and multimetallic particles via metal evaporation onto lithographically patterned posts, as well as the magnetic force calibration and successful magnetofection of iron particles grown via this method. This work represents the first instance in which metal evaporation onto post structures was used for the formation of released, shape-defined metal particles. Also, our work represents the first use of lithographically defined particles as agents of magnetofection. Using these techniques it is possible to create particles with complex shapes and lateral dimensions as small as 40 nm. Our demonstrated compositionally flexible particles are highly size-uniform due to their photolithographically defined growth substrates, with particle dimensions along two axes fixed at 200 nm; the third axis dimension can be varied from 20 nm to 300 nm during the deposition procedure. Atomic percent of metals incorporated into the particle volume is highly tunable and particles have been synthesized with as many as four different metals. We performed magnetic force calibrations on a single particle size for iron particles using an axially magnetized NeFeB permanent magnet and comparisons are made with commercially available magnetic beads. In order to evalutate their usefulness as magnetofection agents, an antisense oligonucleotide (ODN) designed to correct the aberrant splicing of enhanced green fluorescent protein mRNA, was successfully transfected into a modified HeLa cell line. Magnetically enhanced gene delivery was accomplished in vitro using antisense ODN-laden iron particles followed by application of a field gradient. Magnetically enhanced transfection resulted in a 76% and 139% increase in fluorescence intensity when compared to Lipofectamine and antisense ODN-loaded particles delivered without magnetic treatment, respectively. To our knowledge, these experiments constitute the first use of lithographically defined particles as successful agents for magnetically enhanced transfection of an antisense oligonucleotide.

Effects of antidepressants on GluR2 Q/R site-RNA editing in modified HeLa cell line

Marked reduction of RNA editing at the glutamine (Q)/arginine (R) site of the glutamate receptor subunit type 2 (GluR2) in motor neurons may be a contributory cause of neuronal death specifically in sporadic ALS. It has been shown that deregulation of RNA editing of several mRNAs plays a causative role in diseases of the central nervous system such as depression. We analyzed the effects of eight antidepressants on GluR2 Q/R site-RNA editing in a modified HeLa cell line that stably expresses half-edited GluR2 pre-mRNA. We also measured changes in RNA expression levels of adenosine deaminase acting on RNA type 2 (ADAR2), the specific RNA editing enzyme of the GluR2 Q/R site, and GluR2, in order to assess the molecular mechanism causing alteration of this site-editing. The editing efficiency at the GluR2 Q/R site was significantly increased after treatment with seven out of eight antidepressants at a concentration of no more than 10 μM for 24 h. The relative abundance of ADAR2 mRNA to GluR2 pre-mRNA or to β-actin mRNA was increased after treatment with six of the effective antidepressants, whereas it was unchanged after treatment with milnacipran. Our results suggest that antidepressants have the potency to enhance GluR2 Q/R site-editing by either upregulating the ADAR2 mRNA expression level or other unidentified mechanisms. It may be worth investigating the in vivo efficacy of antidepressants with a specific therapeutic strategy for sporadic ALS in view.

A theta-defensin composed exclusively of D-amino acids is active against HIV-1.

The ability of certain theta-defensins, including retrocyclin-1, to protect human cells from infection by HIV-1 marks them as potentially useful molecules. Theta-defensins composed of L-amino acids are likely to be unstable in environments that contain host and microbial proteases. This study compared the properties of two enantiomeric theta-defensins, retrocyclin-1, and RC-112. Although these peptides have identical sequences, RC-112 is composed exclusively of D-amino acids, whereas retrocyclin-1 contains only L-amino acids. We compared the ability of these peptides to protect JC53-BL human cells from infection by 30 primary HIV-1 isolates. JC53-BL cells are modified HeLa cells that express surface CD4, CXCR4, and CCR5. They also contain reporter cassettes that are driven by the HIV-1 LTR, and express beta-galactosidase and luciferase. The HIV-1 isolates varied in co-receptor specificity and included subtypes A, B, C, D, CRF01-AE, and G. RC-112 was several fold more potent than retrocyclin-1 across the entire HIV-1 panel. Although RC-112 bound immobilized gp120 and CD4 with lower affinity than did retrocyclin-1, surface plasmon resonance experiments performed with 1 microg/mL of RC-112 and retrocyclin-1 revealed that both glycoproteins were bound to a similar extent. The superior antiviral performance of RC-112 most likely reflected its resistance to degradation by surface-associated or secreted proteases of the JC53-BL target cells. Theta-defensins composed exclusively of D-amino acids merit consideration as starting points for designing microbicides for topical application to the vagina or rectum.

Establishment of an HIV cell–cell fusion assay by using two genetically modified HeLa cell lines and reporter gene

Infection of human cells with the human immunodeficiency virus type I (HIV-1) can be mimicked by a fusion process between cells expressing the HIV envelope protein (Env) and cells expressing both human CD4 together with the appropriate human chemokine receptors. In this study, a T-tropic HIV cell–cell fusion assay was established that utilized CD4, human CXCR4 and HIV NL4-3 gp160 as fusion components and a T7 polymerase-activated luciferase as a reporter system. The HeLa T4 cells used, expressed CD4 and CXCR4, and the applied HeLa KS386 cells expressed HIV NL4-3 gp160. By combining HeLa T4 cells with HeLa KS386 cells, an approximately about 100- to 300-fold increase in luciferase activity could be elicited relative to the control. The addition of anti-CD4 monoclonal antibody (Mab) (RPA-T4) or anti-CXCR4 Mab (12G5) in the assay significantly inhibited the fusion event; in contrast, an anti-CCR5 Mab (2D7) had no effect, indicating that the fusion assay was CD4 and CXCR4 dependent. In this report, fusion events could be monitored by both the luciferase reporter system and syncytia formation. Fusion events were monitored and compared using these two approaches. The luciferase reporter system was found to be more sensitive than syncytia formation. Moreover, compared with previous HIV fusion models, such as using recombinant vaccinia viruses, this system has several advantages, including simplicity and sensitivity. Finally, the system provides a powerful tool to study fusion mechanisms mediated by T-tropic HIV gp160, as well as to screen for fusion-blocking antibodies and antiviral agents.

Constitutive expression of human coagulating factor IX in HeLa cells by homologous recombination of the promoter

Constitutive expression of hFIX protein in nonhepatocytes was studied. The gene targeting vector was constructed and transferred into HeLa cells. With the detection system of PCR, we demonstrated that the endogenoushFIX promoter was replaced with anhCMV promoter when targeted insertion of the constructor was directed by the sequence homology. The expression ofhFIX in the modified HeLa cells, 11.2 ng/10(6) cell/24 h, strongly suggested thathFIX gene could be activated by a powerful promoter in nonhepatocytes. The results would make it possible to examine the feasibility of re-regulate gene expression by promoter replacement.

Inhibition of angiopoietin-1 expression in tumor cells by an antisense RNA approach inhibited xenograft tumor growth in immunodeficient mice.

Angiopoietin-1 (Ang1) is an angiogenic growth factor that functions through activation of its endothelium-specific tyrosine kinase receptor Tie2; it mediates the interaction between endothelial and surrounding cells to promote the remodeling, maturation and stabilization of blood vessels. Although Ang1 is expressed constitutively in many adult tissues, its role in tumor growth and metastasis is not clear. Here we describe experiments in which Ang1 expression was inhibited in HeLa cells by an antisense RNA approach. The modified HeLa cells produced significantly less Ang1 protein both in cultured cells and in tumors formed when these cells were injected into immunodeficient mice. The Ang1 antisense tumors grew much more slowly, with significantly reduced tumor angiogenesis compared with control tumors. Furthermore, they also had substantially increased tumor cell apoptosis and decreased tumor necrosis. Our results indicate that the perturbation of Ang1 expression in tumors could be an effective method to control tumor growth by inhibiting tumor angiogenesis and that antisense RNA is an efficient way to inhibit Ang1 protein production in tumor cells.

Autoantibody recognition of distinctly modified forms of the U1-70-kd antigen is associated with different clinical disease manifestations.

To examine whether autoantibody recognition of modified forms of the U1-70-kd RNP antigen correlates with manifestations of rheumatic disease. Blinded to clinical disease manifestations, sera from 27 rheumatic disease patients with U1-70-kd antibodies were used to immunoblot control, apoptotic, and oxidatively modified HeLa cell lysates. Using densitometry, recognition of antigen fragments was quantitated. The presence or absence of 1) lupus skin disease and 2) Raynaud's phenomenon (RP) was determined for each patient by chart review. The ability of patient sera to recognize the different fragments was compared for patients with and without skin disease and with and without RP. Patients with lupus skin disease had higher recognition of apoptotic U1-70 kd than did patients without skin disease (mean +/- SD fragment recognition index [FRI] 1.35 +/- 0.57 versus 0.95 +/- 0.25; P < 0.024, by Student's t-test). Patients with RP had higher recognition of oxidatively modified U1-70 kd than did patients without RP (mean +/- SD FRI 0.95 +/- 0.80 versus 0.24 +/- 0.22; P < 0.048). Recognition of apoptotically and oxidatively modified forms of the U1-70-kd autoantigen are associated with distinct clinical rheumatic disease manifestations. This finding provides in vivo evidence for the hypothesis that immune recognition of modified forms of self antigens may be relevant to the pathogenesis of systemic rheumatic diseases. Understanding the antigenic modifications to which patients react may help predict the expression of rheumatic syndromes.

Studies on the Enzymology of Aldehyde Dehydrogenase-2 in Genetically Modified HeLa Cells

The Asian flush reaction is known to result from a lysine for glutamine substitution in the aldehyde dehydrogenase 2 (ALDH2) gene. This mutation was studied in HeLa cells transduced with retroviruses carrying the ALDH2E (wild-type) and ALDH2K (variant) cDNAs. The cells expressed high levels of the enzyme protein that had the same pl and enzymatic characteristics as the corresponding liver enzyme. When cells expressing ALDH2E were transduced with ALDH2K expressing retrovirus, the ALDH2 activity was reduced roughly in proportion to the amount of ALDH2K mRNA expressed, indicating a dominant suppression of activity. The half-life of the wild-type enzyme was 22 hr, while that of variant enzyme was 14 hr. The half-lie of the enzyme in cells expressing both cDNAs was 13 hr, indicating that the shorter half-life of the variant was also dominant. These data are consistent with a model in which the wild-type homodimers have normal activity and turnover, while the heterodimers have reduced to virtually no activity and increased turnover. The model predicts residual ALDH2 activity in heterozygotes of about 13% of that in individuals homozygous for ALDH2*1, in good agreement with experimentally determined values.

Studies on the Enzymology of Aldehyde Dehydrogenase‐2 in Genetically Modified HeLa Cells

The Asian flush reaction is known to result from a lysine for glutamine substitution in the aldehyde dehydrogenase 2 (ALDH2) gene. This mutation was studied in HeLa cells transduced with retroviruses carrying the ALDH2E (wild-type) and ALDH2K (variant) cDNAs. The cells expressed high levels of the enzyme protein that had the same pI and enzymatic characteristics as the corresponding liver enzyme. When cells expressing ALDH2E were transduced with ALDH2K expressing retrovirus, the ALDH2 activity was reduced roughly in proportion to the amount of ALDH2K mRNA expressed, indicating a dominant suppression of activity. The half-life of the wild-type enzyme was 22 hr, while that of variant enzyme was 14 hr. The half-life of the enzyme in cells expressing both cDNAs was 13 hr, indicating that the shorter half-life of the variant was also dominant These data are consistent with a model in which the wild-type homodimers have normal activity and turnover, while the heterodimers have reduced to virtually no activity and increased turnover. The model predicts residual ALDH2 activity in heterozygotes of about 13% of that in individuals homozygous for ALDH2*1, in good agreement with experimentally determined values.

Study of enhanced immunogenecity of B7-1 gene transfected human hela cell line

Although cervical carcinoma cells may express the human papillomavirus protein E6 and E7, they fail to induce an effective specific cytotoxic T lymphocyte response. Recent studies suggest that expression of CD-80 (B7-1) on tumor cells is effective to induce antitumor immune responses.1,2 In our study, CD-80 gene was transfected into human Hela cell line with a CD-80 expression plasmid (B7-l*pcDNA3) by electroporation, then the immunogenecity of the modified Hela cell was tested in TLMC (tumor lymphocyte mixed culture) system. [3H]thymidine lymphocyte proliferation assays showed that the response of human peripheral blood lymphocytes (PBLS) to CD-80 positive Hela cells demonstrated a substantial increase in cell proliferation compared to the response to control cells. Cocultivation of allogeneic PBLs with CD-80 positive tumor cells for three days can induce an increased secretion of IL-2. Our results demonstrate an immunostimulatory effect of CD-80 expression on cervical cancer cells, which provides a basis for the development of a therapeutic tumor vaccine.

Effect of RU 38486 on TNF production and toxicity

Gluracarticoid steroids provide considerable protection against the systemic toxicity or tumor necrosis factor-α (TNF-α, cachexin). In animal experiments RU 38486 (mircpristone), a steroid antagonist, increased the synthesis of TNF and sensitized the animals to the cytotoxic action of TNF. As compared to the control and methylprednisolone-treated groups, mifepristone significantly increased the level of TNF in the serum, liver and spleen or lipopolysaccharide (LPS)-treated animals. In tissue cultures F.U. 38486 induced the TNF synthesis of mycloid cells and increase the TNF production or genetically modified HeLa cells, which synthesize TNF constitutively. Normal and tumor cell cultures exhibited increased sensitivity toward TNF in the presence of mifepristone.

Comparative evaluation of three tests for the detection of Escherichia coli cytotoxic necrotizing factors (CNF1 and CNF2) using filtrates of cultures treated with mitomycin C

Necrotizing Escherichia coli (NTEC) strains grown in the presence of mitomycin C released cell associated necrotizing factors CNF1 and CNF2 to culture medium. Using culture filtrates from 96 mitomycin C treated E. coli strains, we have found that a modified HeLa cell assay was a more sensitive and specific method for the detection of CNF1 and CNF2 than the Vero cell assay and the rabbit skin test.

Comparative evaluation of three tests for the detection ofEscherichia colicytotoxic necrotizing factors (CNF1 and CNF2) using filtrates of cultures treated with mitomycin C

Necrotizing Escherichia coli (NTEC) strains grown in the presence of mitomycin C released cell associated necrotizing factors CNF1 and CNF2 to culture medium. Using culture filtrates from 96 mitomycin C treated E. coli strains, we have found that a modified HeLa cell assay was a more sensitive and specific method for the detection of CNF1 and CNF2 than the Vero cell assay and the rabbit skin test.