BackgroundIntroduced in 1942, sulfasalazine (a conjugate of 5-aminosalicylic acid (5-ASA) and sulfapyridine) is the most prescribed medication used to treat “inflammatory” bowel disease (IBD.) 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 have shown that two other agents used in the therapy of IBD (methotrexate and 6-MP) profoundly inhibit MAP growth. We concluded that their most plausible mechanism of action is as antiMAP antibiotics. We herein hypothesize that the mechanism of action of 5-ASA and/or sulfapyridine may also simply be to inhibit MAP growth.MethodologyThe effect on MAP growth kinetics by sulfasalazine and its components were evaluated in bacterial culture of two strains each of MAP and M. avium, using a radiometric (14CO2 BACTEC®) detection system that quantifies mycobacterial growth as arbitrary “growth index units” (GI). Efficacy data are presented as “percent decrease in cumulative GI” (%−ΔcGI).Principal FindingsThere are disparate responses to 5-ASA and sulfapyridine in the two subspecies. Against MAP, 5-ASA is inhibitory in a dose-dependent manner (MAP ATCC 19698 46%−ΔcGI at 64 µg/ml), whereas sulfapyridine has virtually no effect. In contrast, against M. avium ATCC 25291, 5-ASA has no effect, whereas sulfapyridine (88%−ΔcGI at 4 µg/ml) is as effective as methotrexate, our positive control (88%−ΔcGI at 4 µg/ml). Conclusions5-ASA inhibits MAP growth in culture. We posit that, unknowingly, the medical profession has been treating MAP infections since sulfasalazine's introduction in 1942. These observations may explain, in part, why MAP has not previously been identified as a human pathogen. We conclude that henceforth in clinical trials evaluating antiMAP agents in IBD, if considered ethical, the use of 5-ASA (as well as methotrexate and 6-MP) should be excluded from control groups.

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BackgroundClinical 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.MethodologyThe 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 (14CO2 BACTEC®) detection system that quantifies mycobacterial growth as arbitrary “growth index units” (GI). Efficacy data are presented as “percent decrease in cumulative GI” (% −ΔcGI).Principal FindingsThe positive control antibiotic (clarithromycin) has ≥85% −ΔcGI at a concentration of 0.5 µg/ml. The negative control (ampicillin) has minimal inhibition at 64 µg/ml. MAP ATCC 19698 shows ≥80% −ΔcGI for both agents by 4 µg/ml. With the other three isolates, although more effective than ampicillin, 6-MP is consistently less effective than methotrexate.ConclusionsWe show that methotrexate and 6-MP inhibit MAP growth in vitro. Each of the four isolates manifests different % −ΔcGI. 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.

Careful studies of the antibiotic susceptibilities of mixtures of bacteria likely to be encountered in clinical cultures have shown that the results obtained are completely unreliable. Mixtures of resistant and sensitive species appeared either as "resistant" or "sensitive" depending upon the organisms and the drug. A number of sensitive species gave reactions interpreted as resistant when tested in combination. Since reactions of bacterial mixtures are completely unpredictable, the authors emphasize that antibiotic susceptibility testing be limited to pure cultures.

We established a cell culture system for the replication of hepatitis C virus (HCV) by using human T and B leukemia cell lines. These 2 cell lines were infected in vitro by using HCV-positive pooled patient serum samples. HCV RNA was extracted from infected cell lines at different times after infection, and a sequence of the virus 5' untranslated region was analyzed. Hepatitis C minus-strand RNA was detected in the infected cell lines by highly strand-specific rTth (recombinant Thermus thermophilus DNA polymerase)-based reverse transcription followed by a novel, highly sensitive, single-tube nested polymerase chain reaction (PCR) method. PCR products were analyzed by direct DNA sequencing. These results indicate that the HCV can replicate in T and B lymphocytes. This model should represent a valuable tool for the detailed study of the initial steps of the HCV replication cycle and for the evaluation of antiviral molecules.