Diamond-Blackfan syndrome: evidence against cell-mediated erythropoietic suppression

The profound anemia of Diamond-Blackfan syndrome (DBS) is due to marrow red cell failure, but the pathogenesis is not understood. Studies by others indicated cell-mediated erythropoietic suppression in this condition. To explore this mechanism further, Ficoll-Hypaque--separated peripheral blood lymphocytes (PBL) from four anemic untreated patients with DBS, or from normals were cocultured with control marrow in vitro and the growth of erythropoietin-responsive stem cell colonies (CFU-E) was dermined. CFU-E numbers obtained from cultures with added normal PBL were not significantly different from the number without PBL. Similarly, CFU-E from cultures with added DBS PBL were not significantly different from the number without PBL (215 versus 220, 229 versus 220 and 84 versus 60, 74 versus 94/10(5) cells, respectively). Mixing marrows from a control and one DBS patient in ratios of 2:1, 1:1, or 1:2 prior to culture failed to disclose a decrease of colony growth. We could not show cellular inhibition of erythropoiesis in these patients with DBS. The mechanism of anemia in this disorder remains an open question.     

Mucosal changes in ileal pouches after restorative proctocolectomy for ulcerative and crohn's colitis

PURPOSE: Inflammation and dysplasia may affect the ileal pouch after restorative proctocolectomy and ileal pouchanal anastomosis. The aim of this prospective study was to evaluate the morphologic changes and the risk of dysplasia within the pouch after ileal pouch-anal anastomosis. METHODS: Thirty-seven patients with ileal pouch-anal anastomosis underwent endoscopies and biopsies of the pouch: 21 patients were affected by ulcerative colitis and 16 by Crohn's colitis. The mucosal biopsy specimens were studied to investigate the degree of acute and chronic inflammation and the occurrence of dysplasia. A score system was calculated for each patient and correlated with the histologic diagnosis of ulcerative colitis or Crohn's colitis. RESULTS: After a median follow-up of 85 (range, 7–198) months, the inflammation histologic score evaluated was 3.8 (95 percent confidence interval, 2.4–5.1) and 3.5 (95 percent confidence interval, 2.6–4.3), respectively, in patients with Crohn's colitis and ulcerative colitis (mean and 95 percent confidence interval;P=0.74, not significant), and no patient developed mucosal dysplasia. Fifteen patients (40.5 percent) developed clinical pouchitis that occurred in Crohn's colitis (9/16 patients or 56 percent) and in ulcerative colitis (6/21 patients or 28 percent;P not significant). The score was 4.1 (95 percent confidence interval, 3.2–5) in patients with pouchitis and 3.2 (95 percent confidence interval, 2.1–4.3) in patients without clinical pouchitis (P=0.012) and was 4.1 (95 percent confidence interval, 2.6–5.5) and 4 (95 percent confidence interval, 2.9–5.3), respectively, in pouchitis patients with Crohn's colitis and ulcerative colitis. CONCLUSION: No difference in the inflammation histologic score was observed in ileal pouches after restorative proctocolectomy for ulcerative and Crohn's colitis. In our series, which includes those patients with longer follow-up (>5 years) or with chronic unremitting pouchitis, no case of dysplasia was found. The occurrence of pouchitis was higher in the case of ileal pouch-anal anastomosis for Crohn's disease than for ulcerative colitis, but no difference in the severity of the histologic score was noted.Presented at the XVIIth Biennial Congress of the International Society of University Colon and Rectal Surgeons, Malmö, Sweden, June 7 to 11, 1998.     

A competitive enzyme-linked immunosorbent assay specific for murine hepcidin-1: correlation with hepatic mRNA expression in established and novel models of dysregulated iron homeostasis

Mice have been essential for distinguishing the role of hepcidin in iron homeostasis. Currently, investigators monitor levels of murine hepatic hepcidin-1 mRNA as a surrogate marker for the bioactive hepcidin protein itself. Here, we describe and validate a competitive, enzyme-linked immunosorbent assay that quantifies hepcidin-1 in mouse serum and urine. The assay exhibits a biologically relevant lower limit of detection, high precision, and excellent linearity and recovery. We also demonstrate correlation between serum and urine hepcidin-1 values and validate the competitive enzyme-linked immunosorbent assay by analyzing plasma hepcidin response of mice to physiological challenges, including iron deficiency, iron overload, acute blood loss, and inflammation. Furthermore, we analyze multiple murine genetic models of iron dysregulation, including β-thalassemia intermedia (Hbb^th3/+), hereditary hemochromatosis (Hfe^−/−, Hjv^−/−, and Tfr2^Y245X/Y245X), hypotransferrinemia (Trf^hpx/hpx), heterozygous transferrin receptor 1 deficiency (Tfrc^+/−) and iron refractory iron deficiency anemia (Tmprss6^−/− and Tmprss6^hem8/hem8). Novel compound iron metabolism mutants were also phenotypically characterized here for the first time. We demonstrate that serum hepcidin concentrations correlate with liver hepcidin mRNA expression, transferrin saturation and non-heme liver iron. In some circumstances, serum hepcidin-1 more accurately predicts iron parameters than hepcidin mRNA, and distinguishes smaller, statistically significant differences between experimental groups.     

Lactobacillus rhamnosus CNCM I-3690 and the commensal bacterium Faecalibacterium prausnitzii A2-165 exhibit similar protective effects to induced barrier hyper-permeability in mice

Impaired gut barrier function has been reported in a wide range of diseases and syndromes and in some functional gastrointestinal disorders. In addition, there is increasing evidence that suggests the gut microbiota tightly regulates gut barrier function and recent studies demonstrate that probiotic bacteria can enhance barrier integrity. Here, we aimed to investigate the effects of Lactobacillus rhamnosus CNCM I-3690 on intestinal barrier function. In vitro results using a Caco-2 monolayer cells stimulated with TNF-α confirmed the anti-inflammatory nature of the strain CNCM I-3690 and pointed out a putative role for the protection of the epithelial function. Next, we tested the protective effects of L. rhamnosus CNCM I-3690 in a mouse model of increased colonic permeability. Most importantly, we compared its performance to that of the well-known beneficial human commensal bacterium Faecalibacterium prauznitzii A2-165. Increased colonic permeability was normalized by both strains to a similar degree. Modulation of apical tight junction proteins expression was then analyzed to decipher the mechanism underlying this effect. We showed that CNCM I-3690 partially restored the function of the intestinal barrier and increased the levels of tight junction proteins Occludin and E-cadherin. The results indicate L. rhamnosus CNCM I-3690 is as effective as the commensal anti-inflammatory bacterium F. prausnitzii to treat functional barrier abnormalities.     

Increased surface expression of the membrane glycoprotein IIb/IIIa complex induced by platelet activation. Relationship to the binding of fibrinogen and platelet aggregation

Platelet activation altered the binding of three monoclonal antibodies (monovalent Fab' fragment) directed against the glycoprotein (GP) IIb/IIIa complex. An increased binding of two- to threefold occurred after stimulation with thrombin or phorbol myristate acetate (PMA), with slight but significant increase in the dissociation constants (Kd) of two antibodies (LJ-CP8 and LJ-P9). In contrast, no statistically significant changes were observed with ADP-stimulated platelets. The increased binding of LJ-CP3, but not of the other two antibodies, to activated platelets decreased by 30% to 40% in the presence of EDTA at 22 to 25 degrees C. Platelets stimulated by thrombin or PMA bound more fibrinogen than did those stimulated by ADP, and significant differences in the extent but not in the affinity of fibrinogen binding were observed with various platelet agonists. When the pool of GP IIb/IIIa molecules exposed on the surface of unstimulated platelets was reacted with the monoclonal antibody LJ-CP3 to block ADP-induced fibrinogen binding and platelet aggregation, stimulation with thrombin or PMA still induced substantial binding of antibody and fibrinogen, and aggregation ensued. Therefore, platelets exposed to "strong" agonists exhibit an increased number of surface-oriented epitopes associated with GP IIb/IIIa. The GP IIb/IIIa molecules bearing these newly exposed epitopes are functional in that they can bind fibrinogen and mediate platelet aggregation.     

Heme oxygenase-1 in macrophages controls prostate cancer progression

Innate immune cells strongly influence cancer growth and progression via multiple mechanisms including regulation of epithelial to mesenchymal transition (EMT). In this study, we investigated whether expression of the metabolic gene, heme oxygenase-1 (HO-1) in tumor microenvironment imparts significant effects on prostate cancer progression.We showed that HO-1 is expressed in MARCO-positive macrophages in prostate cancer (PCa) xenografts and human prostate cancers. We demonstrated that macrophage specific (LyzM-Cre) conditional deletion of HO-1 suppressed growth of PC3 xenografts in vivo and delayed progression of prostate intraepithelial neoplasia (PIN) in TRAMP mice. However, initiation and progression of cancer xenografts in the presence of macrophages lacking HO-1 resulted in loss of E-cadherin, a known marker of poor prognosis as well as EMT. Application of CO, a product of HO-1 catalysis, increased levels of E-cadherin in the adherens junctions between cancer cells. We further showed that HO-1-driven expression of E-cadherin in cancer cells cultured in the presence of macrophages is dependent on mitochondrial activity of cancer cells.In summary, these data suggest that HO-1-derived CO from tumor-associated macrophages influences, in part, E-cadherin expression and thus tumor initiation and progression.