Whole-genome microarray analysis and functional characterization reveal distinct gene expression profiles and patterns in two mouse models of ileal inflammation.

BACKGROUND: Although a number of intestinal inflammatory conditions pertain to the ileum, whole-genome gene expression analyses in animal models of ileal inflammation are lacking to date. Therefore, we aimed to identify and characterize alterations in gene expression in the acutely inflamed ileum of two murine models of intestinal inflammation, namely intestinal schistosomiasis and TNBS-induced ileitis, compared to healthy controls. To this end, we used whole-genome microarrays, followed by bioinformatics analyses to detect over-represented Kyoto Encyclopedia of Genes and Genomes pathways and Gene Ontology categories. RESULTS: Following screening of almost all known mouse genes and transcripts represented on the array, intestinal schistosomiasis and TNBS-induced ileitis yielded 207 and 1417 differentially expressed genes, respectively, with only 30 overlapping concordantly changed genes. Functional category groups consisting of complement and coagulation cascades, extracellular matrix (ECM)-receptor interaction, Fc epsilon receptor I signaling pathways and protein activation cascade, cell adhesion categories were over-represented in the differential gene list of intestinal schistosomiasis. Antigen processing and presentation, cell adhesion molecules, ABC transporters, Toll-like receptor signaling pathways and response to chemical stimulus categories were over-represented in the differential gene list of TNBS-induced ileitis. Although cytokine-cytokine receptor interaction, intestinal immune network for IgA production, focal adhesion pathways and immune, inflammatory and defense response categories were over-represented in the differential gene lists of both inflammation models, the vast majority of the associated genes and changes were unique to each model. CONCLUSIONS: This study characterized two models of ileal inflammation at a whole-genome level and outlined distinct gene expression profiles and patterns in the two models. The results indicate that intestinal schistosomiasis involves Th2 responses, complement activation, protein activation and enhanced ECM turnover, while TNBS-induced ileitis involves Th17 responses, defective antigen processing and presentation and altered Toll-like receptor-mediated responses. Signs of an impaired epithelial barrier are apparent in both inflammation models. Furthermore, the comprehensive differential gene list and functional groups provided by this study constitute an interesting starting point to explore new targets and extended functional networks dealing with small bowel inflammation.

Intraoperative adjunctive stem cell treatment in patients with critical limb ischemia using a novel point-of-care device.

INTRODUCTION: In a prospective trial we tested whether adjunctive intraoperative stem cell treatment in patients with critical limb ischemia (CLI) can be performed safely in combination with bypass surgery and/or interventional treatment. The end point of our study was the safety and integrity of a novel point-of-care system used in patients with CLI. METHODS: We included only patients with CLI and tissue loss according to Rutherford categories 4-6. The Harvest Bone Marrow Aspirate Concentrate System consists of an automated, microprocessor-controlled dedicated centrifuge with decanting capability and the accessory BMAC Pack for processing a patient's bone marrow aspirate (BMA). The centrifuge is portable and enables BMA to be rapidly processed in the operating room to provide an autologous concentrate of nucleated cells for immediate injection. The surgeon aspirated 120 ml BMA from the iliac crest. RESULTS: Eight consecutive patients were treated according to the study protocol. The mean follow-up period was 9.2 months (range 2-18). Stem cells were always injected during the final revascularization attempt. One minor amputation and two major amputations were required. In five of eight patients there was a discrete increase in the ankle-brachial index post-stem cell treatment. The dose of stem cells after centrifugation was 17.2 (range 13.8-54.2)x10E6 CD34-positive cells and 7.8 (range 1.8-35.9)x10E6 CD133-positive cells. The injected dose of VEGFR-2-coexpressing stem cells was 0.5-5.7x10E4. CONCLUSION: We were able to show that the buffy coat preparation using a point-of-care system is a simple and fast method to enrich stem cells from BMAs. This automated system gives high recovery rates and good reproducibility.

Evaluation of bone remodeling in hemodialysis patients: serum biochemistry, circulating cytokines and bone histomorphometry.

BACKGROUND: To optimize the noninvasive evaluation of bone remodeling, we evaluated, besides routine serum markers, serum levels of several cytokines involved in bone turnover. METHODS: A transiliac bone biopsy was performed in 47 hemodialysis patients. Serum levels of intact parathyroid hormone (iPTH; 1-84), total alkaline phosphatases (tAP), calcium, phosphate and aluminum (Al) were measured. Circulating levels of interleukin-6 (IL-6), IL-1 receptor antagonist (IL-1Ra) and soluble IL-6 receptor (sIL-6r) were determined using ELISA. Circulating IL-1beta, IL-6, IL-8, IL-10, IL-12p70 and tumor necrosis factor-alpha (TNF-alpha) were simultaneously quantified by flow cytometric immunoassay. RESULTS: Patients with low/normal bone formation rate (L/N-BFR) had significantly lower serum iPTH (p<0.001) and tAP (p<0.008) and significantly higher Al (p<0.025) than patients with high BFR. Serum calcium and phosphorus, however, did not differ (p=NS). An iPTH >300 pg/mL in association with tAP >120 U/L showed low sensitivity (58.8%) and low negative predictive value (44.0%) for the diagnosis of high BFR disease. An iPTH <300 pg/mL in association with normal or low tAP, <120 U/L, was associated with low sensitivity (66.7%) but high specificity (97.1%) for the diagnosis of L/N-BFR. Serum IL-1, IL-6, IL-12p70 and TNF-alpha were positively correlated with BFR, serum IL1-Ra and IL-10 with bone area, and by multiple regression analysis, tAP and IL-6 were independently predictive of BFR. CONCLUSIONS: Significant associations were found between several circulating cytokines and bone histomorphometry in dialysis patients. The usefulness of these determinations in the noninvasive evaluation of bone remodeling needs to be confirmed in larger dialysis populations.

Dendritic cells in colonic patches and iliac lymph nodes are essential in mucosal IgA induction following intrarectal administration via CCR7 interaction.

This study examined dendritic cells (DC) following intrarectal (IR) vaccination with the mucosal adjuvant cholera toxin (CT). Three rounds of IR vaccination with ovalbumin (OVA) and CT resulted in brisk levels of systemic and mucosal Ig responses. Immunohistochemical studies revealed that CD11c+ MHC class II+ cells accumulated primarily in the colonic patches (CP) and lamina propria of the large intestine (LI-LP), iliac LN (ILN) and MLN following IR vaccination with CT. Adoptively transferred CFSE-labeled OVA-specific CD4+ T cells proliferated significantly, secreting predominantly Th1-type cytokines in the CP (48 h after IR vaccination with CT) and Th2-type cytokines in the ILN (96 h after IR vaccination with CT). Following three IR vaccinations, CP-null mice that were generated by in utero treatment with anti-IL-7R Ab showed reduced levels of serum IgG and fecal IgA antibodies, suggesting a crucial role for CP in the initiation of systemic and mucosal immune responses. Of most interest, IR vaccination reduced IgA levels in fecal extracts significantly more in the CCR7-/- mice than in the wild-type mice. These results indicate that IR vaccination primarily mobilizes CD11c+ cells in the CP and ILN to induce optimal mucosal immune responses by CCR7 interaction.

A 24-kDa collagenase from Gymnorhynchus gigas elicits rat ileum hyperreactivity and is a target of humoral responses in mice previously given a single oral dose of parasite extract.

Fish declared fit to be eaten may contain plerocercoids (larvae) of the fish cestode Gymnorhynchus gigas. We showed previously that crude G. gigas larval extract given in a once-only oral dose to either mice or rats induces parasite-specific immediate-type responses and that this extract evokes increased contractile activity in normal rat ileums. We show here that a 24-kDa collagenase (24-kCol), purified from the crude extract is (1) a target of both local (intestinal) and systemic IgE responses in mice sensitized by oral G. gigas and (2) elicits considerable changes in rat ileum contractility. Exposure of rat ileum segments once to 7 microg 24-kCol significantly increased tone and amplitude, but not frequency, of contractions compared with control recordings. In all, these studies have indicated 24-kCol, an abundantly produced protein of G. gigas larvae, to be a participant in potentially serious/adverse intestinal responses in both mice and rats. Such responses are very likely to occur in "sensitized" humans also.

Impaired induction of protective immunity by highly virulent herpes simplex virus type 2 in a murine model of genital herpes.

We investigated the immune events in the vagina of mice intravaginally infected with highly virulent herpes simplex virus type 2 (HSV-2) strain 186, and compared them with those induced by HSV type 1 strain KOS, a widely known laboratory strain. Although there was no significant difference between 186 and KOS in the viral replication in the initial stage of infection, inadequate and delayed clearance of virus from the vaginal mucosa was observed in 1 86-challenged mice. The induction of antigen-presenting cells (APC) such as dendritic cells (DC) and macrophages (Mphi) in the vagina was slow in 186-challenged mice, and the number of T cells in the vagina in 186-challenged mice was much lower than that in KOS-challenged mice. Furthermore, the level of IL-12 as well as that of IFN-gamma was significantly lower in 186-challenged mice than in KOS-challenged mice, while the level of IL-4 in 186-challenged mice was higher than that in KOS-challenged mice. On the basis of these observations, we suggest that the weak activation of epithelial cells and the delayed induction of APC by 186-infection may be involved in the inadequate activation of T cells and the ineffective virus clearance from the vaginal mucosa.

Enhanced neutrophilic granulopoiesis in rheumatoid arthritis. Involvement of neutrophils in disease progression.

OBJECTIVE: To investigate enhanced granulopoiesis in bone marrow of patients with rheumatoid arthritis (RA), and the role of neutrophils in RA pathogenesis. METHODS: Aspirated bone marrow cells and peripheral blood leukocytes from patients with RA and non-RA patient controls were analyzed morphologically and by 2 color flow cytometry. Thirteen iliac bones (8 RA, 5 non-RA) were examined by light and transmission electron microscope (TEM). RESULTS: The percentage of CD15+CD16- cells (immature neutrophils) in RA bone marrow (64.3+/-13.4%, mean +/- SD) increased significantly compared to that of non-RA controls (43.2+/-14.3%), whereas the fraction of CD15+CD]6+ cells (mature neutrophils)was greatly decreased (RA 21.8+/-10.1%; non-RA 38.1+/-8.9%). The absolute number of CD15+CD16- cells also increased markedly in RA bone marrow. The ratio of immature cells to the total granulocytes (% CD15+CD16- to % CD15+) correlated with the Lansbury Index score (R = 0.76, p<0.0001). TEM observations revealed that abundant immature neutrophils adhered closely to the trabeculae of the iliac bone. Margins of trabeculae were mostly irregular, especially in severe RA, and collagenous fibers frequently disappeared in those trabeculae with ragged margins. CONCLUSION: In RA bone marrow, immature neutrophils (CD15+CD16-) were markedly increased in number; by contrast, no changes were found for mature cells. Augmented production of immature neutrophils (at the promyelocyte-to-myelocyte stage) might lead to the destruction of collagenous fibers in RA bone trabeculae, as revealed by TEM. Generalized bone destruction in RA might, at least in part, be caused by enhanced production of immature neutrophils.

Cytokine RNA levels in transiliac bone biopsies from healthy early postmenopausal women.

The cytokines interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and IL-6 induce osteoclast formation and may contribute to the development of postmenopausal osteoporosis. Cross-sectional studies have suggested that both IL-1 and IL-1ra secretion increase on estrogen withdrawal, and that postmenopausal osteoporosis is associated with an inadequate increase in monocyte IL-1ra secretion with age. We measured cytokine mRNA (IL-1beta, IL-1ra, IL-6, and TNF-alpha) directly in bone biopsies from early postmenopausal women to determine if a lower compensatory increase in IL-1ra mRNA could be demonstrated in women with rapid bone loss after the menopause. Biopsies were obtained from 23 early postmenopausal women (mean age 53.9 years) who participated in a randomized study of hormone replacement therapy (HRT) and risk factors for osteoporosis. Bone mineral density was assessed by duel energy X-ray absorptiometry at 0, 1, 2, and 5 years. Women in the control group were recruited to the biopsy study based on their observed rate of bone loss (upper or lower tertile). Consent was also obtained from 11 participants receiving HRT. Biopsies were taken at 2 years, frozen in nitrogen, and homogenized. Cytokine mRNA was measured by competitive reverse transcriptase polymerase chain reaction. The IL-1ra/IL-1beta mRNA slope for the slow-loss group was steeper (deltaF = 23.3, p < 0.01) than that observed in the fast-loss group, indicating that slower bone loss was associated with higher IL-1ra mRNA levels relative to IL-1beta. During HRT, the IL-1beta mRNA level was inversely correlated with serum estradiol (log r2 = 0.77, p < 0.01), and women with a serum estradiol below 200 pmol/L during HRT had IL-1beta, mRNA levels identical to the control group. In contrast, IL-1ra mRNA was independent of serum estradiol. Histomorphometric analysis revealed weak correlations between IL-1beta mRNA and activation frequency (r2 = 0.26, p = 0.06) and between IL-1ra and volume referent bone resorption rate (r2 = 0.19, p = 0.11). TNF-alpha was not associated with the bone loss rates or with serum estradiol, and only three samples were positive for IL-6 mRNA. The findings support the hypothesis that IL-1beta production within bone increases with declining estrogen levels, and that an increase in II-1ra protects against accelerated bone loss.

Homing of mononuclear cells from iliac lymph nodes to the genital and rectal mucosa in non-human primates.

The route of immunization may affect the type of immunity that is induced. The objectives of this investigation were to establish in the non-human primate if the internal iliac lymph nodes (LN) function as an inductive site of immunity from which mononuclear cells home to the rectal and cervico-vaginal mucosa. Rhesus macaques were immunized with simian immunodeficiency virus (SIV) core antigen p27 in the proximity of the iliac lymph nodes, and compared with the intramuscular (i.m.) (deltoid or gluteal), and axillary LN routes of immunization. The macaques were then challenged rectally or vaginally by a particulate SIVp27 antigen which was applied to the mucosal surface. The tracking dye PKH26 was injected near the immunizing LN or i.m. site and a week later the mucosal and lymphoid tissues were examined at autopsy. Preferential homing of PKH26-labeled cells from the internal iliac LN to the rectal and vaginal mucosa was demonstrated by flow cytometry after targeted iliac LN (TILN) but not after intramuscular (deltoid) or axillary LN immunization. Homing of the subsets of cells revealed that labeled CD4, CD8 and B cells, as well as monocytes were found in the rectum, colon, vagina or cervix. The results of this investigation shows that the route of immunization may affect regional mucosal immunity. Furthermore, the internal iliac LN may function as an inductive immunological site from which CD4, CD8 and B cells may home preferentially to the rectal, cervical and vaginal mucosa, as well as to the related regional but not the unrelated distal LN.