M cells are known as specialized epithelial cells of the follicle‐associated epithelium of the gastrointestinal tract. As M cells have a high capacity for transcytosis of a wide range of microorganisms and macromolecules, they are believed to act as an antigen sampling system. The primary physiological role of M cells seems to be the rapid uptake and presentation of particular antigens and microorganisms to the immune cells of the lymphoid follicle to induce an effective immune response. In contrast to absorptive enterocytes, M cells do not exert direct defense mechanisms to antigens and pathogens in the gut lumen. Therefore, they provide functional openings of the epithelial barrier. Although M cells represent a weak point of the epithelial barrier, even under noninflamed conditions, there seems to be a balance between antigen uptake and immunological response. The low number of M cells in the gastrointestinal tract and the direct contact to immune cells in the lamina propria usually prevent the occurrence of mucosal inflammation. During chronic intestinal inflammation we observe an increase of M cell number and apoptosis selectively in M cells. M cell damage seems to be responsible for the increase of the uptake of microorganisms that is observed during intestinal inflammation. Under inflammatory conditions in the intestine, the maintenance of the epithelial barrier is broken and M cells seem to play a major role during this process.

Background : M cells play an important role in the intestinal immune system as they have a high capacity for transcytosis of a wide range of microorganisms and macromolecules. However, little is known about the role of M cells during intestinal inflammation. Aim : We studied M cell development during indomethacin‐induced intestinal inflammation in rats. Methods : Ileitis in rats was induced by two subcutaneous injections with indomethacin (7.5 mg/kg) given 24 h apart. Rats were sacrificed after 14 days and tissue was analysed by fluorescence microscopy and electron microscopy. M cells could be visualized by using the FITC‐labelled mAb anti‐cytokeratin (CK)‐8 (clone 4.1.18), which was recently identified as specific M cell marker in rats. The number of cytokeratin‐8 positive M cells was related to the surface of the follicle associated epithelium. For morphological studies, we used both transmission electron microscopy (T.E.M.) and scanning electron microscopy (S.E.M.). Results : In non‐inflamed ileum M cells were scarce. Only 4% of the follicle associated epithelium were M cells, whereas an increase of M cells up to 11% was found in inflamed follicle associated epithelium (P < 0.001). The rate of M cell induction depended on the macroscopic degree of inflammation. T.E.M./S.E.M. studies showed that in inflamed tissue most M cells underwent apoptosis with typical morphological signs. In contrast to apoptotic M cells, the neighbouring enterocytes usually appeared intact. The number of mononuclear cells below the follicle associated epithelium was significantly increased. S.E.M. studies revealed that during induced ileitis mononuclear cells migrated from the lamina propria into the gut lumen by passing through apoptotic M cells. Conclusions : During indomethacin‐induced ileitis in rats the increase in M cell number in association with apoptosis of M cells may alter the intestinal barrier function. These observations may play a pivotal role in the pathogenesis of chronic intestinal inflammation, e.g. in inflammatory bowel disease.

Hematopoietic progenitor cells are rich in aldehyde dehydrogenase (ALDH) activity, allowing their identification using fluorogenic substrates (Aldefluor®, StemCo Biomedical, Durham, North Carolina) and Fluorescence-activated cell sorting (FACS). We compared the numbers of ALDH+ cells in peripheral blood and progenitor cell harvests with the numbers of CD34-positive cells. Furthermore, we compared the numbers of ALDH+ cells with the kinetics of hematopoietic engraftment following high-dose chemotherapy (HDCT) and transplantation of autologous stem cell harvests (SCT). 25 Patients (Multiple Myeloma, n=10, Hodgkin’s disease, n=3, mantle cell lymphoma, n=3, follicular lymphoma, n=2, T-cell lymphoma, n=3, Burkitt-like lymphoma, n=3) were included in treatment protocols involving high-dose chemotherapy, and received mobilization chemotherapy and G-CSF (10 μg/kg/d s.c.). The numbers of CD34-positive cells were determined daily, and peripheral blood progenitor cell apheresis was initiated when adequate. PBPC collections were performed on an AS 104 cell separator (Fresenius AG, St. Wendel, Germany). Samples of peripheral blood and of progenitor cell harvests were routinely tested for the numbers of CD34-positive cells and ALDH+ cells. The enrichment of CD34-positive cells was calculated and compared to the numbers of ALDH+ cells. 20 patients (Multiple Myeloma, n=10, Hodgkin’s disease, n=3, mantle cell lymphoma, n=3, follicular lymphoma, n=2, T-cell lymphoma, n=2) proceeded to HDCT followed by reinfusion of progenitor cell harvests. The enrichment of ALDH+ cells in the course of apheresis exceeded the enrichment of CD34-positive cells slightly (18,3fold +/−12,8 vs. 15,7fold +/−10,2). The percentage of CD34-negative cells among ALDH+ cells was comparable in peripheral blood and in the harvest, whereas the population of CD34-positive, ALDH−negative cells varied substantially in the peripheral blood (CD34−/ ALDH+: 7,53% +/−5,2% (pB) vs. 6,52% +/−3,9 (harvest); CD34+/ALDH−: 24,6% +/−12,3% (pB) vs. 11,9% +/−9,3% (harvest). Following HDCT and SCT, the numbers of ALDH+ cells and of CD34+ cells in the peripheral blood on the day of apheresis and in the harvests were compared with the reconstitution of the peripheral blood count. In a regression analysis, the number of ALDH+ cells in the peripheral blood on the day of apheresis (p=0,005), the number of ALDH+ cells transfused (p=0,01) and the number of CD34-positive cells transfused (p=0,012) were independent predictors of early recovery of the leukocyte counts. CD34-positive and ALDH+ cells appear to comprise partially different subsets of hematopoietic progenitor cells. The quantitation of ALDH+ cells may allow a more reliable prediction of the numbers of early hematopoietic progenitor cells than the assessment of CD34-positive cells and thus may be of predictive value for the recovery of leukocytes following SCT.

The rare association of pulmonary hypertension and myeloproliferative syndromes (MPS) has been described previously (Garcia-Manero G et al., Am J Hematol1999; 60(2): 130-5, Dingli D et al., Chest2001, 120(3): 801-8), but the mechanisms contributing to this potentially hazardous condition await further elucidation. Bone morphogenetic protein receptors (BMPR) modulate the size of the hematopoetic niche (Zhang J et al., Nature2003, 425(6960): 836-41), with an inhibitory effect on myeloproliferation. In the pulmonary vascular bed, a decreased or deficient expression of BMPR has been shown to result in the proliferation of vascular smooth-muscle cells, asymmetric neointimal hyperplasia in small pulmonary arteries and subsequent pulmonary hypertension (Lane KB et al., Nat Genet2000, 26(1):81-4; Du L et al, N Engl J Med2003, 348(6): 500-9). We hypothesized that, in patients suffering from MPS and pulmonary hypertension, changes in the expression of BMPR and subsequent signalling molecules Angiopoietin-1 (Ang-1) and its receptor, TIE-2, may occur not only in the lung, but also in the bone marrow and correspond with enhanced myeloproliferation in vitro. Bone marrow stromal cells were cultured frompatients with pulmonary hypertension and MPS (n=2),patients with MPS and no evidence of pulmonary hypertension (n=3) andhealthy controls (n=3).The cultured cells were subjected to Western Blot analysis for the expression of BMP receptors BMPR-1A and BMPR-2, Angiopoietin-1 and TIE-2. Furthermore, a modified long-term culture initiating cell (LTC-IC) assay was established using the cultured bone marrow stromal cells as layers for autologous and allogeneic progenitor cell assays. The two patients suffering from both, MPS and pulmonary hypertension, showed a diminished expression of BMPR-1A and an enhanced expression of Ang-1 and TIE-2 in cultured stromal cells when compared to patients with MPS alone and to healthy controls. In one of the two patients, a three- to fourfold increase in the number of long-term culture-initiating cells after seeding of CD34-positive cells and of bone marrow mononuclear cells from healthy donors and from the other patients included was observed in modified LTC-IC assays. Our observations argue in favour of changes in BMP receptor expression and signalling with impact not only on pulmonary hypertension as described before, but also on the emergence of a myeloproliferative state.