Simultaneous cognate epitope recognition by bovine CD4 and CD8 T cells is essential for primary expansion of antigen-specific cytotoxic T-cells following ex vivo stimulation with a candidate Mycobacterium avium subsp. paratuberculosis peptide vaccine.

Studies in cattle show CD8 cytotoxic T cells (CTL), with the ability to kill intracellular bacteria, develop following stimulation of monocyte-depleted peripheral blood mononuclear cells (mdPBMC) with antigen presenting cells (APC, i.e. conventional dendritic cells [cDC] and monocyte-derived DC [MoDC]) pulsed with MMP, a membrane protein from Mycobacterium avium subsp. paratuberculosis (Map) encoded by MAP2121c. CTL activity was diminished if CD4 T cells were depleted from mdPBMC before antigen (Ag) presentation by APC, suggesting simultaneous cognate recognition of MMP epitopes presented by MHC I and MHC II molecules to CD4 and CD8 T cells is essential for development of CTL activity. To explore this possibility, studies were conducted with mdPBMC cultures in the presence of monoclonal antibodies (mAbs) specific for MHC class I and MHC class II molecules. The CTL response of mdPBMC to MMP-pulsed APC was completely blocked in the presence of mAbs to both MHC I and II molecules and also blocked in the presence of mAbs to either MHC I or MHC II alone. The results demonstrate simultaneous cognate recognition of Ag by CD4 and CD8 T cells is essential for delivery of CD4 T cell help to CD8 T cells to elicit development of CTL.

Microbial Population Differentials between Mucosal and Submucosal Intestinal Tissues in Advanced Crohn's Disease of the Ileum.

Since Crohn's disease is a transmural disease, we hypothesized that examination of deep submucosal tissues directly involved in the inflammatory disease process may provide unique insights into bacterial populations transgressing intestinal barriers and bacterial populations more representative of the causes and agents of the disease. We performed deep 16s microbiota sequencing on isolated ilea mucosal and submucosal tissues on 20 patients with Crohn's disease and 15 non-inflammatory bowel disease controls with a depth of coverage averaging 81,500 sequences in each of the 70 DNA samples yielding an overall resolution down to 0.0001% of the bacterial population. Of the 4,802,328 total sequences generated, 98.9% or 4,749,183 sequences aligned with the Kingdom Bacteria that clustered into 8545 unique sequences with <3% divergence or operational taxonomic units enabling the identification of 401 genera and 698 tentative bacterial species. There were significant differences in all taxonomic levels between the submucosal microbiota in Crohn's disease compared to controls, including organisms of the Order Desulfovibrionales that were present within the submucosal tissues of most Crohn's disease patients but absent in the control group. A variety of organisms of the Phylum Firmicutes were increased in the subjacent submucosa as compared to the parallel mucosal tissue including Ruminococcus spp., Oscillospira spp., Pseudobutyrivibrio spp., and Tumebacillus spp. In addition, Propionibacterium spp. and Cloacibacterium spp. were increased as well as large increases in Proteobacteria including Parasutterella spp. and Methylobacterium spp. This is the first study to examine the microbial populations within submucosal tissues of patients with Crohn's disease and to compare microbial communities found deep within the submucosal tissues with those present on mucosal surfaces. Our data demonstrate the existence of a distinct submucosal microbiome and ecosystem that is not well reflected in the mucosa and/or downstream fecal material.

Crohn's disease may be differentiated into 2 distinct biotypes based on the detection of bacterial genomic sequences and virulence genes within submucosal tissues.

OBJECTIVE: To determine whether bacterial pathogens can be detected within the diseased submucosal tissues of patients with Crohn's disease by molecular techniques independent of cultural methods. DESIGN: We designed a quantitative polymerase chain reaction to detect 32 virulence genes and transposons within submucosal tissues of patients with Crohn's disease and controls and compared the microbiome of the submucosa with mucosal bacterial populations. RESULTS: Within submucosal tissues, the bacterial invasion/adherence genes eaeA and invA were detected in 43% of patients (P=0.01 and 0.008 vs. mucosa and controls, respectively) and the Mycobacterium-specific IS900 and 251F genes detected in 50% of patients (P=0.03 vs. mucosa and controls). These findings were mutually exclusive: invasion/adhesion genes and Mycobacterium-associated transposons were not detected in the same patient. Metagenomic sequencing and quantitative polymerase chain reaction results confirmed effective separation of the submucosal and mucosal microbiome and the existence of a submucosal bacterial population within diseased tissues. CONCLUSIONS: This study is the first to examine the microbial populations of submucosal tissues during intestinal disease and provide evidence of a distinct submucosal microbiome and biotypes within Crohn's disease. These data suggests that Crohn's disease may not be a single disease, but a spectrum that can be divided into distinct biotypes based on the presence of invasion/adherence genes or Mycobacterium-associated transposons. If corroborated by larger population studies, these findings could revolutionize the diagnosis, management, and treatment of Crohn's disease by the identification of patient biotypes and the application of targeted chemotherapeutic treatments that go beyond supportive in nature.

Crohn's disease and the mycobacterioses: a quarter century later. Causation or simple association?

It has been more than 25 years since Mycobacterium paratuberculosis was first proposed as an etiologic agent in Crohn's disease based on the isolation of this organism from several patients. Since that time, a great deal of information has been accumulated that clearly establishes an association between M. paratuberculosis and Crohn's disease. However, data are conflicting and difficult to interpret and the field has become divided into committed advocates and confirmed skeptics. This review is an attempt to provide a thorough and objective summary of current knowledge from both basic and clinical research from the views and interpretations of both the antagonists and proponents. The reader is left to draw his or her own conclusions related to the validity of the issues and claims made by the opposing views and data interpretations. Whether M. paratuberculosis is a causative agent in some cases or simply represents an incidental association remains a controversial topic, but current evidence suggests that the notion should not be so readily dismissed. Remaining questions that need to be addressed in defining the role of M. paratuberculosis in Crohn's disease and future implications are discussed.

Integrating theories of the etiology of Crohn's disease. On the etiology of Crohn's disease: questioning the hypotheses.

The most prominent theory describes the Crohn's Syndrome as a dysregulated, inappropriate immune response to otherwise innocuous bowel flora in a genetically susceptible host. The autoimmune theory assumes that a specific infectious agent does not exist. Data from mouse models, impairment of the mucosal epithelial barrier and the influence of gut flora are used to support the autoimmune theory. Critics claim that the dysregulated immune responses are not the primary disorder but secondary to an underlying infection. Two other theories are also consistent with the same data. The immunodeficiency theory hypothesizes that defects in innate immunity leading to compensatory immune processes underlie the Crohn's phenotype and suggests that therapy should stimulate immunity rather than suppress it. The mycobacterial theory proposes that Mycobacterium avium subspecies paratuberculosis is one of the causes of the Crohn's Disease syndrome. Mycobacterial molecules dysregulate immune signaling pathways as part of the organisms'evolved survival strategy. If MAP were to initiate the dysregulated immune response then the necessity to hypothesize that commensal gut flora provide the antigenic stimulus would be moot. Commensal bacteria would be relegated to a secondary role of modifying the immune response rather than occupying the central role of providing the initiating antigens. Critics claim that MAP is merely a secondary invader. The three theories differ primarily by emphasizing different aspects of the same overall process.