RHB-104 triple antibiotics combination in culture is bactericidal and should be effective for treatment of Crohn's disease associated with Mycobacterium paratuberculosis.

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP) has been implicated as an etiological agent of Crohn's disease (CD), a debilitating chronic inflammatory bowel disease. Clarithromycin (CLA), clofazimine (CLO), rifabutin (RIF) and other antibiotics have been used individually or in combinations with other drugs to treat mycobacterial diseases including CD. The treatment has varied by regimen, dosage, and duration, resulting in conflicting outcomes and additional suffering to the patients. RHB-104, a drug formula with active ingredients composed of (63.3 %) CLA, (6.7 %) CLO, and (30 %) RIF, has been recently subjected to investigation in an FDA approved Phase III clinical trial to treat patients with moderate to severe CD. In this study, we determined the efficacy of RHB-104 active ingredients against MAP strains isolated from the blood, tissue, and milk of CD patients. Based on fluorescence quenching technology using the Bactec MGIT Para-TB medium, we determined the minimum inhibitory concentration (MIC) of CLA, CLO, RIF individually and in dual and triple combinations against 16 MAP clinical strains and 19 other mycobacteria. RESULTS: The MIC of all drugs against 35 different mycobacteria ranged between 0.25-20 µg/mL. However, the MIC of RHB-104 active ingredients regimen was the lowest at 0.25-10 µg/mL compared to the MIC of the other drugs at 0.5-20 µg/mL. The components of RHB-104 active ingredients at their individual concentrations or in dual combinations were not effective against all microorganisms compared to the triple combinations at MIC level. The MIC of CLA-CLO, CLA-RIF, and CLO-RIF regimens ranged between 0.5-1.25 µg/mL compared to 0.25 µg/mL of bactericidal effect of the triple combination. CONCLUSION: The data clearly demonstrated that lower concentrations of the triple combination of RHB-104 active ingredients provided synergistic anti-MAP growth activity compared to individual or dual combinations of the drugs. Consequently, this is favorable and should lead to tolerable dosage that is desirable for long-term treatment of CD and Mycobacterium avium complex disease.

Domino effect of hypomagnesemia on the innate immunity of Crohn's disease patients.

Digestive diseases play major role in development and complications of other disorders including diabetes. For example, Crohn's disease (CD) is an inflammatory bowel disease associated with Mycobacterium avium subspecies paratuberculosis. The inflammation is a complex process that involves the activity of both innate and adaptive immune responses. CD lesions are primarily due to T cell response, however; innate immune response has a significant role in initiating its pathogenesis. Toll-like receptors and NOD-like receptors promote the activity of nuclear factor (NF)-κB pathway for cytokines production. This results in the production of high levels of tumor necrosis factor-α, interleukin (IL)-1ß and IL-6. Moreover, intestinal inflammation of CD is related to increased activity of NMDA receptors and the release of substance P. Imbalanced magnesium homeostasis in CD is a frequent finding in CD, Diabetes and others. The loss of such a major mineral affects many physiological processes in the body including its role as an immunomodulator. This review aims to (1) describe the significance of hypomagnesemia in the release of pro-inflammatory mediators in CD; (2) demonstrate effects of magnesium on pathways like NF-κB; (3) address the role of hypomagnesemia in the activity of CD; and (4) examine possible future research to establish a standard magnesium supplementation strategy; helping patients with CD or other disorders to maintain a sustained remission.

Mycobacterium avium subspecies paratuberculosis causes Crohn's disease in some inflammatory bowel disease patients.

Crohn's disease (CD) is a chronic inflammatory condition that plagues millions all over the world. This debilitating bowel disease can start in early childhood and continue into late adulthood. Signs and symptoms are usually many and multiple tests are often required for the diagnosis and confirmation of this disease. However, little is still understood about the cause(s) of CD. As a result, several theories have been proposed over the years. One theory in particular is that Mycobacterium avium subspecies paratuberculosis (MAP) is intimately linked to the etiology of CD. This fastidious bacterium also known to cause Johne's disease in cattle has infected the intestines of animals for years. It is believed that due to the thick, waxy cell wall of MAP it is able to survive the process of pasteurization as well as chemical processes seen in irrigation purification systems. Subsequently meat, dairy products and water serve as key vehicles in the transmission of MAP infection to humans (from farm to fork) who have a genetic predisposition, thus leading to the development of CD. The challenges faced in culturing this bacterium from CD are many. Examples include its extreme slow growth, lack of cell wall, low abundance, and its mycobactin dependency. In this review article, data from 60 studies showing the detection and isolation of MAP by PCR and culture techniques have been reviewed. Although this review may not be 100% comprehensive of all studies, clearly the majority of the studies overwhelmingly and definitively support the role of MAP in at least 30%-50% of CD patients. It is very possible that lack of detection of MAP from some CD patients may be due to the absence of MAP role in these patients. The latter statement is conditional on utilization of methodology appropriate for detection of human MAP strains. Ultimately, stratification of CD and inflammatory bowel disease patients for the presence or absence of MAP is necessary for appropriate and effective treatment which may lead to a cure.

Exploring the role of Mycobacterium avium subspecies paratuberculosis in the pathogenesis of type 1 diabetes mellitus: a pilot study.

BACKGROUND: Although the etiology of Type 1 Diabetes mellitus (T1DM) has not been determined, genetic polymorphism in key genes, including SLC11A1, and association with Mycobacterium avium subspecies paratuberculosis (MAP) have been reported. We hypothesize that molecular mimicry between MAP Heat shock protein 65 K (Hsp65) and human Glutamic Acid Decarboxylase 65 K (GAD65) may be the trigger leading to autoimmune destruction of beta cells in patients exposed to MAP. METHOD: Peptide sequences of MAP Hsp65 and human GAD65 were investigated for amino acid sequence homology and cross reactivity. A total of 18 blood samples from T1DM and controls were evaluated for the presence of MAP. RESULTS: Peptide BLAST analysis revealed a 44% overall identity between MAP Hsp65 and GAD65 with 75% positives in a 16 amino acid region. PyMOL 3D-structural analyses identified the same 16 amino acid region as a potential epitope for antibody binding. Preliminary data suggests a cross reactivity between MAP Hsp65, and a healthy rat pancreatic tissue homogenate against plasma from T1DM patients and rabbit polyclonal anti-MAP IgG. Long-term culture of human blood resulted MAP detection in 3/10 T1DM and 4/8 controls whereas MAP IgG was detected in 5/10 T1DM samples and 3/8 non-diabetic controls. CONCLUSION: The high degree of homology between GAD65 and MAP Hsp65 in an antigenic peptide region supports a possible mycobacterial role in triggering autoimmune destruction of pancreatic cells in T1DM. Reactivity of T1DM patient sera with MAP Hsp65 supports this finding. Culture of MAP from the blood of T1DM patients is intriguing. Overall, the preliminary data are mixed and do not exclude a possible role for MAP in T1DM pathogenesis. A larger study including well-characterized controls is needed to investigate the intriguing question of whether MAP is associated with T1DM or not?

Role of ATG16L, NOD2 and IL23R in Crohn's disease pathogenesis.

Inflammatory bowel disease is a group of diseases that includes Crohn's disease (CD) and ulcerative colitis. CD is characterized as a chronic inflammatory disease of the gastrointestinal tract, ranging from the mouth to the anus. Although there are gross pathological and histological similarities between CD and Johne's disease of cattle, the cause of CD remains controversial. It is vital to understand fully the cause of this disease because it affects approximately 500,000 people in North America and Europe. It ranges from 27 to 48 cases per 100,000 people. There are many theories on the cause of CD ranging from possible association with environmental factors including microorganisms to imbalance in the intestinal normal flora of the patients. Regardless of the environmental trigger, there is strong evidence that a genetic disposition is a major key in acquiring CD. Many studies have proven the link between mutations in the ATG16L, NOD2/CARD15, IBD5, CTLA4, TNFSF15 and IL23R genes, and CD. The purpose of this review is to examine all genetic aspects and theories of CD, including up to date multiple population studies performed worldwide.