title: On the action of methotrexate and 6-mercaptopurine on M. avium subspecies paratuberculosis.
abstract: BACKGROUND
  Clinical improvement in inflammatory bowel disease (IBD) treated with methotrexate and 6-mercaptopurine (6-MP) is associated with a decrease in pro-inflammatory cytokines. This has been presumed to indicate the mechanism of action of methotrexate and 6-MP. Although controversial, there are increasingly compelling data that Mycobacterium avium subspecies paratuberculosis (MAP) may be an etiological agent in some or all of IBD. We hypothesized that the clinical efficacy of methotrexate and 6-MP in IBD may be to simply inhibit the growth of MAP.
  
  
  METHODS
  The effect on MAP growth kinetics by methotrexate and 6-MP were evaluated in cell culture of two strains each of MAP and M. avium using a radiometric ((14)CO(2) BACTEC detection system that quantifies mycobacterial growth as arbitrary "growth index units" (GI). Efficacy data are presented as "percent decrease in cumulative GI" (% -DeltacGI).
  
  
  RESULTS
  The positive control antibiotic (clarithromycin) has >or=85% -DeltacGI at a concentration of 0.5 microg/ml. The negative control (ampicillin) has minimal inhibition at 64 microg/ml. MAP ATCC 19698 shows >or=80% -DeltacGI for both agents by 4 microg/ml. With the other three isolates, although more effective than ampicillin, 6-MP is consistently less effective than methotrexate.
  
  
  CONCLUSIONS
  We show that methotrexate and 6-MP inhibit MAP growth in vitro. Each of the four isolates manifests different % -DeltacGI. These data are compatible with the hypothesis that the clinical improvement in patients with IBD treated with methotrexate and 6-MP could be due to treating a MAP infection. The decrease in pro-inflammatory cytokines, thought to be the primary mechanism of action, may simply be a normal, secondary, physiological response. We conclude that henceforth, in clinical studies that evaluate the effect of anti-MAP agents in IBD, the use of methotrexate and 6-MP should be excluded from any control groups.
authors.affiliation: Laboratory of Molecular Surgical Research, VA Medical Center, Bronx, New York, United States of America. BGAxis@aol.com
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Greenstein
authors.forename: Liya
authors.initials: L
authors.lastname: Su
authors.forename: Vahram
authors.initials: V
authors.lastname: Haroutunian
authors.forename: Azra
authors.initials: A
authors.lastname: Shahidi
authors.forename: Sheldon T
authors.initials: ST
authors.lastname: Brown
mesh_terms: D000667:Ampicillin / Q000494:pharmacology / Q000627:therapeutic use; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology / Q000627:therapeutic use; D002417:Cattle; D016207:Cytokines / Q000276:immunology; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology* / Q000627:therapeutic use; D015212:Inflammatory Bowel Diseases / Q000188:drug therapy / Q000276:immunology / Q000382:microbiology; D015122:Mercaptopurine / Q000494:pharmacology* / Q000627:therapeutic use; D008727:Methotrexate / Q000494:pharmacology* / Q000627:therapeutic use; D008826:Microbial Sensitivity Tests; D016927:Mycobacterium avium subsp. paratuberculosis / Q000187:drug effects* / Q000276:immunology* / Q000472:pathogenicity
chemical_list: D000900:Anti-Bacterial Agents; D016207:Cytokines; D007166:Immunosuppressive Agents; D000667:Ampicillin; D015122:Mercaptopurine; D008727:Methotrexate