{
  "abstract": "Introduction Mycobacterium avium subspecies paratuberculosis (MAP) remains a potential contributor to the pathogenesis of Crohn’s disease (CD). Indirect demonstration of MAP in patients is well established. However, further elucidation of the role of MAP has been hampered by a lack of in-situ demonstration of this established intestinal pathogen. We have successfully optimised FISH using a IS900 specific oligonucleotide (P90CorrProbe) to demonstrate reliably the presence of MAP in gut tissue from patients with CD.Methods Formalin fixed gut biopsy samples from 21 patients with active CD were studied (12M, median age 36y, ileal = 6, ileocolonic = 11, colonic = 5). Nine patients undergoing colonoscopy for polyp surveillance were recruited as controls (5M, median age 54y)Presence of MAP was detected via FISH using the IS900-specific oligonucleotide probe P90CorrProbe (5′GTTCGGGGCCGTCGGCCTTAGG–3′) directly conjugated to fluorophore Alexa 488. Tissue from a confirmed MAP infected sheep was used as a positive staining control. Samples were viewed using a Leica Stellaris confocal microscope and MAP signal quantification determined using Fiji software. Histological inflammation was graded by Nancy scoring.Results MAP presence was clearly demonstrated in 16/21 CD patients and 2/9 controls (X 2 value p=0.005). Where positive the concentration of staining was higher in CD than controls (2.94% vs 0.96%). MAP was observed predominantly in the lamina propria of CD patients and appeared to colocalise with CD68 + macrophages although some staining within gut epithelial cells was also observed. The staining pattern was remarkably similar to that seen in MAP infected sheep. No correlation was seen between Nancy score and MAP staining in a subset of 10 CD patients.Conclusions These results in a preliminary small cohort of patients with CD demonstrate effectively for the first time the clear presence of MAP within the immune cells of the lamina propria in patients with CD. The presence does not appear to correlate with the severity of inflammation. The presence in healthy controls is infrequent and at a lower intensity but is in keeping with MAP having a ubiquitous environmental ­presence. The similarity to the staining pattern in ovine MAP infection is intriguing. A large cohort study to define MAP presence across clinical, immunological and genetic phenotypes is a key next step now in-situ detection has been demonstrated.Abstract P245 Figure 1FISH staining identifying MAP in lamina propria immune cells",
  "authors": [
    {
      "affiliations": [
        "King’s College London, London, United Kingdom"
      ],
      "name": "Marcus Barr"
    },
    {
      "affiliations": [
        "King’s College London, London, United Kingdom"
      ],
      "name": "Neil Rayment"
    },
    {
      "affiliations": [
        "King’s College London, London, United Kingdom"
      ],
      "name": "Barry Hudspith"
    },
    {
      "affiliations": [
        "Lancaster University, Lancaster, United Kingdom"
      ],
      "name": "Roger Pickup"
    },
    {
      "affiliations": [
        "UK Centre for Ecology and Hydrology, Lancaster, United Kingdom"
      ],
      "name": "Glenn Rhodes"
    },
    {
      "affiliations": [
        "Guys and St Thomas NHS Foundation Trust, London, United Kingdom"
      ],
      "name": "Gaurav Agrawal"
    },
    {
      "affiliations": [
        "Guys and St Thomas NHS Foundation Trust, London, United Kingdom"
      ],
      "name": "Baljit Gill-Barman"
    },
    {
      "affiliations": [
        "Guys and St Thomas NHS Foundation Trust, London, United Kingdom"
      ],
      "name": "Andrea Centritto"
    },
    {
      "affiliations": [
        "Guys and St Thomas NHS Foundation Trust, London, United Kingdom"
      ],
      "name": "Mark Samaan"
    },
    {
      "affiliations": [
        "Guys and St Thomas NHS Foundation Trust, London, United Kingdom"
      ],
      "name": "Jeremy Sanderson"
    }
  ],
  "title": "P245 Old story, new angle. In-situ demonstration of mycobacterium paratuberculosis in lamina propria immune cells using IS900 FISH in crohn’s disease",
  "uid": "438a00e7-82f0-529b-a07f-65f55d54687b"
}