Mycobacterium avium subspecies paratuberculosis (MAP) is suspected to be a causative agent in Crohn's disease. Recent evidence suggests that MAP can induce the expression of Matrix Metalloproteinases (MMPs), which are the main proteases in the pathogenesis of mucosal ulcerations in inflammatory bowel disease (IBD). Within the present study, we analysed whether oral MAP exposure can induce colonic MMP expression in vivo. In MAP exposed mice MAP and spheroplasts were visualized in intramucosal leukocyte aggregates. MAP exposed mice exhibited a higher colonic expression of Mmp-2, -9, -13, -14, Timp-1, Tlr2, Tlr6, Il-1β, and Tnf-α. Cell clusters of MMP-9 positive cells adjacent to intramucosal leukocyte aggregates and CD45(+) leukocytes were identified as the major cellular sources of MMP-9. Enhanced TLR2 expression was visualized on the luminal side of colonic enterocytes. Although MAP exposure did not lead to macroscopic intestinal inflammation, the observed MAP spheroplasts in intramucosal leukocyte aggregates together with increased colonic expression of toll-like receptors, pro-inflammatory cytokines, and MMPs upon MAP exposure represents a part of the host immune response towards MAP.

During self-limiting acute rejection preceding chronic vasculopathy, large amounts of leukocytes, predominantly monocytes, interact with the endothelium of renal allografts. We aim to characterize them and to identify targets for functional and interventional studies. Leukocytes were harvested by vascular perfusion from Fischer 344 to Lewis renal allografts or Lewis isografts, followed by flow cytometry, quantitative RT-PCR and genome-wide transcriptional profiling. Leukocyte accumulation peaked in allografts on day 9. The percentage of monocytes expressing MHC class II and CD161 was increased whereas CD4, CD11a, CD43, and CD71 expression remained unchanged. IFN-γ, IL-1β, IL-2, IL-10, TNF-α, and iNOS mRNA increased in allograft leukocytes but IL-4, IL-6, IL-12, TGF-β, and tissue factor did not. During acute rejection, 1783 genes were differentially expressed. In conclusion, graft blood leukocytes display a unique state of partial activation during self-limiting rejection. Numerous differentially expressed genes deserve further investigation as potential factors in deciding the fate of the allograft.

Einleitung: Es gibt starke Hinweise dafür, dass Mycobacterium avium Subspezies paratuberculosis (MAP) an der Ätiologie des Morbus Crohn (MC) beteiligt ist 1,2. Jüngsten Untersuchungen zufolge können pathogene Mykobakterien und MAP die Expression von Matrix Metalloproteinasen (MMP)3, die die wichtigsten Proteasen in der Pathogenese der mucosalen Ulzerationen bei chronisch entzündlichen Darmerkrankungen (CED) bilden4,5, induzieren. Im der vorliegenden Studie haben wir untersucht, ob eine kontrollierte MAP-Infektion Darmpathologien und Entzündungen hervorrufen und ob MAP die MMP-Expression in vivo im Colon induzieren.

BACKGROUND:Mycobacterium avium subspecies paratuberculosis (MAP) is suspected to be a causative agent in human Crohn's disease (CD). Recent evidence suggests that pathogenic mycobacteria and MAP can induce the expression of Matrix Metalloproteinases (MMP), which are the main proteases in the pathogenesis of mucosal ulcerations in inflammatory bowel disease (IBD). Within this study we assessed the prevalence of intestinal MAP specific DNA in patients with Crohn's disease, ulcerative colitis (UC), and healthy controls. We further analysed regulation patterns of MMPs in mucosal tissues of UC patients with and without intestinal MAP DNA detection.METHODS:Colonic biopsy samples were obtained from 63 Norwegian and German IBD patients and 21 healthy controls. RNA was quantified by quantitative real-time polymerase chain reaction (PCR) to study MMP gene expression in both pathological and healthy mucosal specimens. The presence of MAP DNA in colonic mucosa was examined using MAP specific PCR.RESULTS:MAP DNA was detected in 20% of UC patients and 33% of healthy controls but only in 7% of patients with CD. UC patients treated with corticosteroids exhibited a significantly increased frequency of intestinal MAP DNA compared to those not receiving corticosteroids. Expression of MMP-1, -2, -7, -9, -13, -19, -28 and TNF-α did not differ between UC patients with presence of intestinal MAP DNA compared to those without. MMP-2, MMP-9 and MMP-13 were significantly decreased in UC patients receiving corticosteroids.CONCLUSIONS:The presence of intestinal MAP specific DNA is not associated with altered MMP expression in UC in vivo. Corticosteroids are associated with increased detection of intestinal MAP DNA and decreased expression of certain MMPs. Frequent detection of MAP DNA in healthy controls might be attributable to the wide environmental distribution of MAP and its presence in the food-chain.

Leukocytes interacting with endothelia of lung allografts probably play a seminal role in acute rejection, but have not been characterized before. Transplantation was performed in the Lewis to Lewis and in the Dark Agouti to Lewis rat strain combinations. DNA replication was detected in T-cells on day 2 after pulse-labelling in vivo with 5-bromo-2'-deoxyuridine (BrdU). On day 5, leukocytes were isolated by intensive perfusion the graft, subject to flow cytometry and to quantitative RT-PCR. About 34 million leukocytes accumulated in allograft vessels, but only 10 and 6 million cells in isografts and control lungs, respectively. During rejection, IFN-gamma, IL-1beta and IL-10 mRNA expression increased, IL-12 mRNA decreased, whereas IL-2, IL-6, TNF-alpha, and TGF-beta mRNA did not change. The phenotype of graft monocytes was partially activated and intravascular T-cells proliferated. In conclusion, during rejection, monocytes with unusual properties accumulate and T-lymphocytes are activated in lung allograft blood vessels.

Exogenous application of keratinocyte growth factor protects the lung against a variety of injurious stimuli. KGF-treatment leads to pronounced hyperplasia of alveolar epithelial type II cells and to stabilization of surfactant homeostasis after lung injury. Epidermal fatty acid-binding protein is involved in the synthesis of surfactant phospholipids and acts as an antioxidant scavenging reactive lipids. We treated adult rats with recombinant human keratinocyte growth factor (Palifermin) via intratracheal instillation and analyzed the expression of epidermal fatty acid-binding protein mRNA and protein by quantitative RT-PCR, immunoblotting as well as immunohistochemistry. Keratinocyte growth factor-treatment in vivo leads to an increased expression of epidermal fatty acid-binding protein mRNA and protein in the total lung. Epidermal fatty acid-binding protein mRNA expression per alveolar epithelial type II cell remains constant as shown in isolated type II cells. Epidermal fatty acid-binding protein immunoreactivity is seen in most if not all hyperplastic alveolar epithelial type II cells, and is mainly localized to the cytoplasm. The increase in epidermal fatty acid-binding protein gene expression associated with type II cell hyperplasia might contribute to the molecular mechanisms mediating lung protection by keratinocyte growth factor.