Preparation of reference strains for validation and comparison of mycoplasma testing methods

Aims: To optimize growth conditions for preparation of stocks of mycoplasma reference strains to obtain highly viable and disperse samples with low ratios of genomic copy (GC) number to that of colony forming units (CFU). These stocks are required for assessment of relative limits of detection (LOD) of alternative nucleic acid testing (NAT)‐based methods in comparison to the conventional microbiological methods. Methods and Results: A kinetics study was used to assess the changes in ratios between the numbers of GC and CFU at different growth phases of six different mycoplasma cultures Acholeplasma laidlawii, Mycoplasma gallisepticum, Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma orale and Mycoplasma pneumoniae. All tested mycoplasmas demonstrated low GC/CFU ratios (≤10) within the log and early stationary growth phases. A significant increase in GC/CFU ratios was observed at the very late stationary and death phases, when the titre of cultures has declined. Similar patterns of GC/CFU profiles were observed for A. laidlawii and Myc. gallisepticum co‐cultured with suspension of Chinese hamster ovary (CHO) cells. Conclusions: Tested mycoplasma strains harvested at the exponential‐early stationary phases of growth demonstrated the lowest GC/CFU ratios and low propensity to form filamentous structures or aggregates under proposed conditions and can be used for the preparation of a mycoplasma reference panel for methods comparability study. Significance and Impact of the Study: This study shows that the preparation and use of viable mycoplasma reference strains with low CG/CFU ratios is the most reliable way to adequately evaluate the LOD of alternative NAT‐based mycoplasma testing methods.     

Collaborative study for the calibration of a replacement international standard for diphtheria toxoid adsorbed

We present the results of a collaborative study for the characterization of a preparation of diphtheria toxoid adsorbed, and its calibration in terms of the 3rd International Standard (IS) for Diphtheria Toxoid Adsorbed. Calibration was performed using established World Health Organization (WHO) and European Pharmacopoeia (Ph. Eur.) protection models. Two candidate toxoid preparations were included in the study, one of which was adopted as a replacement Ph. Eur. Biological Reference Preparation (BRP, batch 4) in February 2009. The second candidate preparation was found to have a unitage of 213 IU/ampoule based on the calibration by in vivo bioassay in 19 laboratories in 16 countries, and was established as the 4th IS for Diphtheria Toxoid Adsorbed by the WHO Expert Committee on Biological Standardization (ECBS) in October 2009.The study also assessed performance of the replacement standard in mouse and guinea pig serological assays which are used as alternative procedures for diphtheria potency testing. Participants tested both candidate preparations and potency was expressed in relative terms only. Results suggest that the replacement standard is suitable for use as the reference vaccine in serological assays and that the Vero cell assay may be suitable for calibration of future replacement standards.     

Genetic and serologic relatedness between Mycoplasma fermentans strains and a mycoplasma recently identified in tissues of AIDS and non-AIDS patients

A mycoplasma previously identified in the tissues of both AIDS and non-AIDS patients dying of an acute fatal disease was earlier shown to share some biologic and genetic properties with a strain of Mycoplasma fermentans, an organism occurring infrequently in the human lower urogenital tract. More extensive genetic and serologic comparisons using DNA/DNA hybridization, DNA base composition (guanine + cytosine), restriction endonuclease DNA analysis, cellular protein patterns and metabolism inhibition serologic procedures confirm that the organism previously designated as “Mycoplasma incognitus” (Mi) is indeed very closely related to strains of M. fermentans. While the genetic and serologic features observed among the newly isolated mycoplasma and two M. fermentans strains suggest a species relationship, it now seems useful to re-examine the biological activities of other freshly isolated M. fermentans strains from man.     

Characterization of an IS Element in <Emphasis Type="Italic">Mycoplasma orale</Emphasis> That Is Highly Homologous to <Emphasis Type="Italic">M. fermentans</Emphasis> ISLE

In a previous study, using a primer set designed from Mycoplasma fermentans, we amplified a PCR fragment from Mycoplasma orale similar to the 206-bp DNA fragment amplified from M. fermentans insertion-sequence-like element (ISLE). The presence of this similar ISLE fragment has the potential to cause confusion in the PCR diagnosis of M. fermentans and M. orale, which have significantly different clinical scenarios. An ISLE from three different M. orale strains was amplified by using a primer set designed from sequence within the left and right terminal stem and loop (S&L) structures flanking the ISLE of M. fermentans. Sequence analysis showed that the M. orale ISLE is 93% identical to that of M. fermentans at the nucleotide level and codes for two open reading frames also found in the M. fermentans ISLE. This is the first finding that two different mycoplasma species harbor highly homologous IS elements. This finding has great significance in clinical diagnosis and suggests a possibility of horizontal transfer of an IS element between two different mycoplasma species. Received: 17 April 2002 / Accepted: 9 July 2002     

Calibration and commutability assessment of the 1st International Standard for Diphtheria Antitoxin Human

The 1st International Standard for Diphtheria Antitoxin Human (coded 10/262) was established by the World Health Organization Expert Committee on Biological Standardization in 2012. This paper describes the production, characterization and calibration of the new standard which is intended for use in the standardization of assays used to measure diphtheria antibody responses in human serum. The new standard was calibrated in terms of the International Standard for Diphtheria Antitoxin Equine in an international collaborative study. A total of 8 participants from 8 different countries performed in vivo and/or in vitro toxin neutralization tests and returned data that was used to assign units to the proposed new standard. The new standard has a diphtheria antitoxin potency of 2 IU/ampoule and is predicted to be stable. A follow up study was performed to assess commutability of the new standard. The follow up study was an existing external quality assessment, modified to include the new standard. Results obtained suggest that the new standard is commutable, showing comparable behaviour to native human serum samples in the majority of the assays compared, and is therefore suitable for use as a reference preparation in assays used to measure the level of anti-diphtheria antibodies in human serum.     

Biological Enrichment of Mycoplasma Agents by Cocultivation with Permissive Cell Cultures

In this study, we describe our results on the evaluation of the ability of different permissive mammalian cell lines to support the biological enrichment of mycoplasma species known to be bacterial contaminants of cell substrates. The study showed that this approach is able to significantly improve the efficiency of mycoplasma detection based on nucleic acid testing or biochemical technologies (e.g., MycoAlert mycoplasma detection). Of 10 different cell lines (Vero, MDBK, HEK-293, Hep-G2, CV-1, EBTr, WI-38, R9ab, MDCK, and High Five) used in the study, only MDCK cell culture was found to support the efficient growth of all the tested mycoplasmas (Mycoplasma arginini, M. bovis, M. fermentans, M. gallinaceum, M. gallisepticum, M. synoviae, M. hominis, M. hyorhinis, M. orale, M. salivarium, and Acholeplasma laidlawii) known to be most frequently associated with contamination of cell substrates and cell lines in research laboratories or manufacturing facilities. The infection of MDCK cells with serial dilutions of each mycoplasma species demonstrated that these common cell line contaminants can be detected reliably after 7-day enrichment in MDCK cell culture at contamination levels of 0.05 to 0.25 CFU/ml. The High Five insect cell line was also found to be able to support the efficient growth of most mycoplasma species tested, except for M. hyorhinis strain DBS1050. However, mycoplasma growth in insect cell culture was demonstrated to be temperature dependent, and the most efficient growth was observed when the incubation temperature was increased from 28°C to between 35 and 37°C. We believe that this type of mycoplasma enrichment is one of the most promising approaches for improving the purity and safety testing of cell substrates and other cell-derived biologics and pharmaceuticals.     

Calibration of replacement international standard and European pharmacopoeia biological reference preparation for tetanus toxoid, adsorbed.

Here we report the characterisation of a preparation of tetanus toxoid, adsorbed, and its calibration by 27 laboratories in 19 countries in a joint international collaborative study co-sponsored by World Health Organization (WHO) Expert Committee of Biological Standardization (ECBS) and the European Biological Standardisation Programme of European Directorate for the Quality of Medicines (EDQM), Council of Europe. Calibration was in terms of the Second International Standard (I.S.) for Tetanus Toxoid, Adsorbed, by the established WHO/European Pharmacopoeia (Ph Eur) challenge methods. The replacement standard preparation was found to have a unitage of 469 IU/ampoule on the basis of its calibration in guinea-pigs and 496 IU/ampoule on the basis of its calibration in mice. Assessment, both within the collaborative study and as part of candidate characterisation, indicated satisfactory stability of the candidate preparation. This study also provided some information on the effect of mouse strain on potency testing of tetanus vaccines. A limited assessment of the impact of the replacement standard on testing of current production batches of vaccines was also carried out by four manufacturers. This study did not directly address the serological approaches to potency testing. However, one laboratory offered data from mouse serology assay, which gave comparable estimates to in vivo mouse bioassay.Based on the results of this study and with the agreement of participants, the candidate standard was established as the Third International Standard for Tetanus Toxoid, Adsorbed (coded 98/552) by the WHO Expert Committee of Biological Standardization (ECBS) in November 2000. The same preparation was also established as the second Ph Eur Biological Reference Preparation (Ph Eur BRP, batch no. 2) by the Steering Committee of the Biological Standardisation Programme of the EDQM and approved by the European Pharmacopoeia Commission.     

International reference standards in coagulation

Measurement of coagulation factor activity using absolute physico-chemical techniques is not possible and estimation therefore relies on comparative bioassay relative to a reference standard with a known or assigned potency. However the inherent variability of locally prepared and calibrated reference standards can give rise to poor agreement between laboratories and methods. Harmonisation of measurement between laboratories at the international level relies on the availability of a common source of calibration for local reference standards and this is provided by the World Health Organization (WHO) International Standards which define the International Unit for the analyte. This article describes the principles, practices and problems of biological standardisation and the development and use of reference standards for assays of coagulation factors, with particular emphasis on WHO International Standards for both concentrates and plasma.     

Measurement of cytokines by bioassays: theory and application

A large number of cytokines have been characterized, of which several have proved successful in the clinic as biotherapeutic agents for malignant, infectious or autoimmune diseases. As biologically active proteins, they cannot be fully characterized by physicochemical methods alone. Thus, biological assays (bioassays) have become increasingly important for their biological characterization and potency determinations. Since cytokines exert various biological activities in vitro, cultured cell line-based bioassay methods have mainly been developed to quantify potency. Such bioassays, like all biological systems, are inherently variable. Thus, measurement of potency of a particular cytokine must be made relative to a common, stable, reference preparation of the same cytokine to permit valid inter-assay and inter-laboratory comparisons. The development and establishment of appropriate primary reference preparations as World Health Organization (WHO) International standards (IS) and reference reagents (RR) is essential for the standardization of bioassays. This review addresses the practical and statistical considerations for the development of valid bioassays, the preparation and use of WHO IS and RR and, in brief, the types of bioassay methods applicable to potency measurements of individual cytokines. More extensive details for the potency determinations of tumor necrosis factor-alpha (TNF-alpha), related cytokines, and biotherapeutic anti-TNF-alpha products are provided.     

Cultivation of Mycoplasmas on Glass

Eight Mycoplasma species of human origin were successfully cultivated on glass. Complement-fixing (CF) antigens prepared from glass-adherent mycoplasmas were potent, specific, and free from anticomplementary activity. PPLO broth medium supplemented with 1 to 5% PPLO serum fraction (bovine), 2.5% fresh yeast extract, and 1% glucose (glycolytic species) or 1% arginine (arginine-utilizing species) supported moderate to luxuriant growth of mycoplasmas on glass. The potency of CF antigens prepared from glass-adherent mycoplasmas varied with the species of Mycoplasma tested and the duration of incubation. When the potency of CF antigens prepared from glass-adherent mycoplasmas was compared with that material sedimented from the broth phase of the same culture, three patterns of growth were observed: M. hominis and M. orale type 2 grew preferentially in the broth phase; M. salivarium, M. orale types 1 and 3, M. pneumoniae, and M. lipophilum preferentially adhered to the glass; and M. fermentans was biphasic. The growth of mycoplasmas on glass provides a simple means of concentrating and purifying such organisms for immunological and biochemical studies.     

The first World Health Organization International Standard for infliximab products: A step towards maintaining harmonized biological activity

Due to the increase in the number of infliximab products, the need for global harmonization of the bioactivity of this monoclonal antibody was recognized by the World Health Organization (WHO). In response, the National Institute for Biological Standards and Control (NIBSC) developed the first international standard (IS) for infliximab, which targets tumour necrosis factor (TNF). Each ampoule is assigned values of 500 IU of TNF neutralizing activity and 500 IU of binding activity. Two preparations of infliximab were formulated and lyophilized at NIBSC prior to evaluation in a collaborative study for their suitability to serve as an IS for the in vitro biological activity of infliximab. The study involved participants using in vitro cell-based bioassays (TNF neutralization, antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity) and binding assays. The results of this study showed that the candidate preparation, coded 16/170, is suitable as an IS for infliximab bioactivity. This infliximab IS from NIBSC, is intended to support in vitro bioassay calibration and validation by defining international units of bioactivity. The proposed unitages, however, are not intended to revise product labelling or dosing requirements, as any decisions regarding this relies solely with the regulatory authorities. Furthermore, the infliximab IS is not intended for determining the specific activity of products, nor to serve any regulatory role in defining biosimilarity. We briefly discuss the future use of WHO international standards in supporting the global harmonisation of biosimilar infliximab products.     

Animal model of Mycoplasma fermentans respiratory infection

Background

Mycoplasma fermentans has been associated with respiratory, genitourinary tract infections and rheumatoid diseases but its role as pathogen is controversial. The purpose of this study was to probe that Mycoplasma fermentans is able to produce respiratory tract infection and migrate to several organs on an experimental infection model in hamsters. One hundred and twenty six hamsters were divided in six groups (A-F) of 21 hamsters each. Animals of groups A, B, C were intratracheally injected with one of the mycoplasma strains: Mycoplasma fermentans P 140 (wild strain), Mycoplasma fermentans PG 18 (type strain) or Mycoplasma pneumoniae Eaton strain. Groups D, E, F were the negative, media, and sham controls. Fragments of trachea, lungs, kidney, heart, brain and spleen were cultured and used for the histopathological study. U frequency test was used to compare recovery of mycoplasmas from organs.

Results

Mycoplasmas were detected by culture and PCR. The three mycoplasma strains induced an interstitial pneumonia; they also migrated to several organs and persisted there for at least 50 days. Mycoplasma fermentans P 140 induced a more severe damage in lungs than Mycoplasma fermentans PG 18. Mycoplasma pneumoniae produced severe damage in lungs and renal damage.

Conclusions

Mycoplasma fermentans induced a respiratory tract infection and persisted in different organs for several weeks in hamsters. This finding may help to explain the ability of Mycoplasma fermentans to induce pneumonia and chronic infectious diseases in humans.     

An international collaborative study on the First International Standard of bovine luteinizing hormone for immunoassay

An ampouled freeze-dried preparation of bovine pituitary luteinizing hormone (bLH), coded EHC-bLH-1, has been evaluated in an international collaborative study and shown to be suitable and sufficiently stable to serve as a standard for bLH. Eight laboratories provided immunoassay data, one laboratory provided receptor assay data, and bioassay data were obtained from 4 laboratories. The geometric mean potency estimate obtained by immunoassays, expressed as milliunits of the USDA bLH-B-5 preparation per ampoule, was 25.6, which is consistent with the result obtained by in-vivo bioassays. The geometric mean estimate obtained by receptor assays or by in-vitro bioassays was lower, i.e. 13.2 milliunits per ampoule. The reason for this discrepancy is currently under investigation. With the authorization of the Expert Committee on Biological Standardization of the World Health Organization this preparation was established in 1985 as the International Standard for Luteinizing Hormone, Bovine, for Immunoassay with a unitage of 25 mi.u. per ampoule.     

Simultaneous Detection and Identification of Common Cell Culture Contaminant and Pathogenic Mollicutes Strains by Reverse Line Blot Hybridization

We have developed a reverse line blot (RLB) hybridization assay to detect and identify the commonest mollicutes causing cell line contamination (Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orale, and Acholeplasma laidlawii) and human infection (Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma parvum, and Ureaplasma urealyticum). We developed a nested PCR assay with “universal” primers targeting the mollicute 16S-23S rRNA intergenic spacer region. Amplified biotin-labeled PCR products were hybridized to membrane-bound species-specific oligonucleotide probes. The assay correctly identified reference strains of 10 mollicute species. Cell cultures submitted for detection of mollicute contamination, clinical specimens, and clinical isolates were initially tested by PCR assay targeting a presumed mollicute-specific sequence of the 16S rRNA gene. Any that were positive were assessed by the RLB assay, with species-specific PCR assay as the reference method. Initially, 100 clinical and 88 of 92 cell culture specimens gave concordant results, including 18 in which two or more mollicute species were detected by both methods. PCR and sequencing of the 16S-23S rRNA intergenic spacer region and subsequent retesting by species-specific PCR assay of the four cell culture specimens for which results were initially discrepant confirmed the original RLB results. Sequencing of amplicons from 12 cell culture specimens that were positive in the 16S rRNA PCR assay but negative by both the RLB and species-specific PCR assays failed to identify any mollicute species. The RLB hybridization assay is sensitive and specific and able to rapidly detect and identify mollicute species from clinical and cell line specimens.     

A collaborative study to establish a U.S. reference for tissue plasminogen activator (t-PA)

In 1987 the Second International Standard for tissue plasminogen activator (t-PA) was established by the World Health Organization following an international collaborative study. At that time, the Center for Biologics Evaluation and Research (CBER) decided to establish a national reference t-PA to be used in lot release potency testing of Alteplase, a licensed t-PA biological or of other t-PAs in development. A candidate recombinant t-PA (rt-PA) preparation was donated by Genentech, Inc. (South San Francisco, California) for this purpose and a collaborative study was launched to calibrate this material against the 2nd I.S. Four laboratories (including the Center for Biologics Evaluation and Research (CBER) and three manufacturers) participated in the study to establish the potency of the rt-PA preparation using a clot lysis assay. The results indicate that the potency of the U.S. reference for t-PA is 2900 international units (IU) per vial.     

Identification of Phosphocholine-Containing Glycoglycerolipids Purified from Mycoplasma fermentans-Infected Human Helper T-Cell Culture as Components of M. fermentans

Previously, we have reported the occurrence of novel phosphocholine-containing glycoglycerolipids (GGPLs: GGPL-I and GGPL-III) in human helper T-cell culture (MT-4 cell line) (Matsuda et al, Glycoconjugate J. 10: 340). However, the GGPLs disappeared from the MT-4 cells after treatment with an antimycoplasma agent. This disappearance suggested the involvement of microorganisms in the GGPL expression. In this paper, we show that the novel lipids are components of Mycoplasma fermentans itself. The supernatant fluid of the antimycoplasma agent-untreated MT-4 cell culture produced mycoplasma-like colonies on PPLO agar plates, and PCR and immunological methods revealed the presence of M. fermentans. GGPLs were expressed again in the treated MT-4 cells after infection with the isolated M. fermentans. The isolated M. fermentans had glycoglycerolipids corresponding to GGPL-I and GGPL-III. Thin-layer chromatography-mass spectrometry and immunological analyses showed that these glycoglycerolipids which were derived from the isolated M. fermentans were identical with GGPL-I and GGPL-III previously obtained. This is the first report that shows mycoplasma has phosphocholine-containing glycoglycerolipids.     

Neglected diseases in the news: a content analysis of recent international media coverage focussing on leishmaniasis and trypanosomiasis

Background

Although the pharmaceutical industry''s “neglect” of neglected tropical diseases (NTDs) has been investigated, no study evaluating media coverage of NTDs has been published. Poor media coverage exacerbates the neglect. This study aimed to investigate, describe, and analyse international media coverage of “neglected diseases” in general and three specific NTDs—African trypanosomiasis, leishmaniasis, and Chagas disease—from 1 January 2003 to 1 June 2007.

Methods

Archives of 11 leading international, English-language media were searched. A content analysis was done, coding for media organisation, date, author, type of report, slant, themes, and “frames”. Semi-structured interviews with journalists and key informants were conducted for further insight.

Principal Findings

Only 113 articles in a 53-month time period met the inclusion criteria, with no strong trends or increases in coverage. Overall, the BBC had the highest coverage with 20 results, followed by the Financial Times and Agence France Presse. CNN had the least coverage with one result. The term “neglected diseases” had good media currency and “sleeping sickness” was far more widely used than trypanosomiasis. The disease most covered was leishmaniasis and the least covered was Chagas. Academic researchers were most commonly quoted as a main source, while the World Health Organization (WHO) and pharmaceutical industry were the least quoted. Journalists generally agreed NTDs had not been adequately covered, but said a lack of real news development and the need to cater to domestic audiences were major obstacles for NTD reporting. All journalists said health agencies, particularly WHO, were not communicating adequately about the burden of NTDs.

Conclusions

Public health agencies need to raise priority for NTD advocacy. Innovative strategies, such as reporting grants or creating a network of voices, may be needed.     

Evaluation of cell substrates for the production of biologicals: Revision of WHO recommendations: Report of the WHO Study Group on Cell Substrates for the Production of Biologicals, 22–23 April 2009, Bethesda,USA

Evaluating cell substrates for producing vaccines and other biologicals is one of the critical aspects in assuring quality and safety of these products. As part of its mission in setting standards for biological products, WHO provides recommendations for manufacturing and evaluating biologicals. Regular updates of the guidance documents are important to manufacturers and regulators worldwide. WHO Expert Committee on Biological Standardization (ECBS) identified a need for revising the requirements for cell substrates (WHO TRS 878, annex 1). In response, WHO established a Study Group (SG) in 2006 that prepared an updated set of recommendations for using cell substrates for the production of biologicals. A summary of the proposed changes that the SG made in 2007 is available at WHO web site (http://www.who.int/biologicals/publications/meetings/areas/vaccines/cells/en/index.html). Draft revised recommendations were circulated to regulators, manufacturers and other experts for comments in April 2009.The SG held its third meeting on 22–23 April 2009 to review progress in the revision and to propose further improvements. In addition, the experts discussed the need for reference preparations, reference cell banks, and standardization of testing methodologies. The SG proposed clarifications of the rationale for in vivo testing as well as the potential for applying new methods for in vitro testing for detecting microbial agents. In line with this, WHO should conduct review of the current manufacturers' practice in using tests for microbial agents and interpreting these results. Additionally, WHO should take a lead in developing an International Standard for nucleic acid amplification test (NAT) for detecting mycoplasma contamination in cell substrates. WHO Collaborating Centers will lead this initiative, involving other relevant institutions in this area. Finally, advice on the replacement of the WHO Vero reference cell bank 10–87 with respect to the source of cells and re-characterization of the bank was provided. The intended use of the replacement cell bank would be the same as for the current cell bank, which is to serve as a source of well-characterized cells for establishing master cell banks for the production of biologicals. The SG will report outcomes of its discussion to the ECBS at its next meeting in October 2009 for further considerations and advice regarding the proposed course of action.     

The DprE1 enzyme, one of the most vulnerable targets of Mycobacterium tuberculosis

The re-emergence of tuberculosis in recent years led the World Health Organization (WHO) to launch the Stop TB Strategy program. Beside repurposing the existing drugs and exploring novel molecular combinations, an essential step to face the burden of tuberculosis will be to develop new drugs by identifying vulnerable bacterial targets. Recent studies have focused on decaprenylphosphoryl-d-ribose oxidase (DprE1) of Mycobacterium tuberculosis, an essential enzyme involved in cell wall metabolism, for which new promising molecules have proved efficacy as antitubercular agents. This review summarizes the state of the art concerning DprE1 in terms of structure, enzymatic activity and inhibitors. This enzyme is emerging as one of the most vulnerable target in M. tuberculosis.     

Mycoplasma fermentans Inhibits the Activity of Cellular DNA Topoisomerase I by Activation of PARP1 and Alters the Efficacy of Its Anti-Cancer Inhibitor

To understand the effects of the interaction between Mycoplasma and cells on the host cellular function, it is important to elucidate the influences of infection of cells with Mycoplasma on nuclear enzymes such as DNA Topoisomerase type I (Topo I). Human Topo I participates in DNA transaction processes and is the target of anti-cancer drugs, the camptothecins (CPTs). Here we investigated the mechanism by which infection of human tumor cells with Mycoplasma fermentans affects the activity and expression of cellular Topo I, and the anti-cancer efficacy of CPT. Human cancer cells were infected or treated with live or sonicated M. fermentans and the activity and expression of Topo I was determined. M. fermentans significantly reduced (by 80%) Topo I activity in the infected/treated tumor cells without affecting the level of Topo I protein. We demonstrate that this reduction in enzyme activity resulted from ADP-ribosylation of the Topo I protein by Poly-ADP-ribose polymerase (PARP-1). In addition, pERK was activated as a result of the induction of the MAPK signal transduction pathway by M. fermentans. Since PARP-1 was shown to be activated by pERK, we concluded that M. fermentans modified the cellular Topo I activity by activation of PARP-I via the induction of the MAPK signal transduction pathway. Moreover, the infection of tumor cells with M. fermentans diminished the inhibitory effect of CPT. The results of this study suggest that modification of Topo I activity by M. fermentans may alter cellular gene expression and the response of tumor cells to Topo I inhibitors, influencing the anti-cancer capacity of Topo I antagonists.