Vitamins A & D Inhibit the Growth of Mycobacteria in Radiometric Culture

The role of vitamins in the combat of disease is usually conceptualized as acting by modulating the immune response of an infected, eukaryotic host. We hypothesized that some vitamins may directly influence the growth of prokaryotes, particularly mycobacteria. complex).Vitamins A and D cause dose-dependent inhibition of all three mycobacterial species studied. Vitamin A is consistently more inhibitory than vitamin D. The vitamin A precursor, β-carotene, is not inhibitory, whereas three vitamin A metabolites cause inhibition. Vitamin K has no effect. Vitamin E causes negligible inhibition in a single strain.We show that vitamin A, its metabolites Retinyl acetate, Retinoic acid and 13-cis Retinoic acid and vitamin D directly inhibit mycobacterial growth in culture. These data are compatible with the hypothesis that complementing the immune response of multicellular organisms, vitamins A and D may have heretofore unproven, unrecognized, independent and probable synergistic, direct antimycobacterial inhibitory activity

On the Action of Methotrexate and 6-Mercaptopurine on M. avium Subspecies paratuberculosis

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. METHODOLOGY: 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” (% −ΔcGI). PRINCIPAL FINDINGS: The 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. CONCLUSIONS: We 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

On the Action of Cyclosporine A, Rapamycin and Tacrolimus on M. avium Including Subspecies paratuberculosis

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP) may be zoonotic. Recently the "immuno-modulators" methotrexate, azathioprine and 6-MP and the "anti-inflammatory" 5-ASA have been shown to inhibit MAP growth in vitro. We concluded that their most plausible mechanism of action is as antiMAP antibiotics. The "immunosuppressants" Cyclosporine A, Rapamycin and Tacrolimus (FK 506) treat a variety of "autoimmune" and "inflammatory" diseases. Rapamycin and Tacrolimus are macrolides. We hypothesized that their mode of action may simply be to inhibit MAP growth. METHODOLOGY: The effect on radiometric MAP (14)CO(2) growth kinetics of Cyclosporine A, Rapamycin and Tacrolimus on MAP cultured from humans (Dominic & UCF 4) or ruminants (ATCC 19698 & 303) and M. avium subspecies avium (ATCC 25291 & 101) are presented as "percent decrease in cumulative GI" (%-DeltacGI.) PRINCIPAL FINDINGS: The positive control clofazimine has 99%-DeltacGI at 0.5 microg/ml (Dominic). Phthalimide, a negative control has no dose dependent inhibition on any strain. Against MAP there is dose dependent inhibition by the immunosuppressants. Cyclosporine has 97%-DeltacGI by 32 microg/ml (Dominic), Rapamycin has 74%-DeltacGI by 64 microg/ml (UCF 4) and Tacrolimus 43%-DeltacGI by 64 microg/ml (UCF 4) CONCLUSIONS: We show heretofore-undescribed inhibition of MAP growth in vitro by "immunosuppressants;" the cyclic undecapeptide Cyclosporine A, and the macrolides Rapamycin and Tacrolimus. These data are compatible with our thesis that, unknowingly, the medical profession has been treating MAP infections since 1942 when 5-ASA and subsequently azathioprine, 6-MP and methotrexate were introduced in the therapy of some "autoimmune" and "inflammatory" diseases

Bowel associated dermatosis – arthritis syndrome: a case report

We report a rare case of Bowel Associated Dermatosis – Arthritis Syndrome in a young patient with complex Crohn's disease who presented with fever, arthritis, rash and worsening of diarrhea with abdominal pain, who promptly responded to a short course of steroids

Where Are All the Mycobacterium avium Subspecies paratuberculosis in Patients with Crohn's Disease?

Mycobacterium avium subspecies paratuberculosis (MAP) causes a chronic granulomatous inflammation of the intestines, Johne's disease, in dairy cows and every other species of mammal in which it has been identified. MAP has been identified in the mucosal layer and deeper bowel wall in patients with Crohn's disease by methods other than light microscopy, and by direct visualization in small numbers by light microscopy. MAP has not been accepted as the cause of Crohn's disease in part because it has not been seen under the microscope in large numbers in the intestines of patients with Crohn's disease. An analysis of the literature on the pathology of Crohn's disease and on possible MAP infection in Crohn's patients suggests that MAP might directly infect endothelial cells and adipocytes and cause them to proliferate, causing focal obstruction within already existing vessels (including granuloma formation), the development of new vessels (neoangiogenesis and lymphangiogenesis), and the “creeping fat” of the mesentery that is unique in human pathology to Crohn's disease but also occurs in bovine Johne's disease. Large numbers of MAP might therefore be found in the mesentery attached to segments of intestine affected by Crohn's disease rather than in the bowel wall, the blood and lymphatic vessels running through the mesentery, or the mesenteric fat itself. The walls of fistulas might result from the neoangiogenesis or lymphangiogenesis that occurs in the bowel wall in Crohn's disease and therefore are also possible sites of large numbers of MAP. The direct visualization of large numbers of MAP organisms in the tissues of patients with Crohn's disease will help establish that MAP causes Crohn's disease

Rapid and Sensitive Detection of an Intracellular Pathogen in Human Peripheral Leukocytes with Hybridizing Magnetic Relaxation Nanosensors

Bacterial infections are still a major global healthcare problem. The quick and sensitive detection of pathogens responsible for these infections would facilitate correct diagnosis of the disease and expedite treatment. Of major importance are intracellular slow-growing pathogens that reside within peripheral leukocytes, evading recognition by the immune system and detection by traditional culture methods. Herein, we report the use of hybridizing magnetic nanosensors (hMRS) for the detection of an intracellular pathogen, Mycobacterium avium spp. paratuberculosis (MAP). The hMRS are designed to bind to a unique genomic sequence found in the MAP genome, causing significant changes in the sample’s magnetic resonance signal. Clinically relevant samples, including tissue and blood, were screened with hMRS and results were compared with traditional PCR analysis. Within less than an hour, the hMRS identified MAP-positive samples in a library of laboratory cultures, clinical isolates, blood and homogenized tissues. Comparison of the hMRS with culture methods in terms of prediction of disease state revealed that the hMRS outperformed established culture methods, while being significantly faster (1 hour vs 12 weeks). Additionally, using a single instrument and one nanoparticle preparation we were able to detect the intracellular bacterial target in clinical samples at the genomic and epitope levels. Overall, since the nanoparticles are robust in diverse environmental settings and substantially more affordable than PCR enzymes, the potential clinical and field-based use of hMRS in the multiplexed identification of microbial pathogens and other disease-related biomarkers via a single, deployable instrument in clinical and complex environmental samples is foreseen

Large clones of pre-existing T cells drive early immunity against SARS-COV-2 and LCMV infection

T cell responses precede antibody and may provide early control of infection. We analyzed the clonal basis of this rapid response following SARS-COV-2 infection. We applied T cell receptor (TCR) sequencing to define the trajectories of individual T cell clones immediately. In SARS-COV-2 PCR+ individuals, a wave of TCRs strongly but transiently expand, frequently peaking the same week as the first positive PCR test. These expanding TCR CDR3s were enriched for sequences functionally annotated as SARS-COV-2 specific. Epitopes recognized by the expanding TCRs were highly conserved between SARS-COV-2 strains but not with circulating human coronaviruses. Many expanding CDR3s were present at high frequency in pre-pandemic repertoires. Early response TCRs specific for lymphocytic choriomeningitis virus epitopes were also found at high frequency in the preinfection naive repertoire. High-frequency naive precursors may allow the T cell response to respond rapidly during the crucial early phases of acute viral infection