George Kolios

Nitric oxide in inflammatory bowel disease: a universal messenger in an unsolved puzzle

In recent years, nitric oxide (NO), a gas previously considered to be a potentially toxic chemical, has been established as a diffusible universal messenger that mediates cell–cell communication throughout the body. Constitutive and inducible NO production regulate numerous essential functions of the gastrointestinal mucosa, such as maintenance of adequate perfusion, regulation of microvascular and epithelial permeability, and regulation of the immune response. Up‐regulation of the production of NO via expression of inducible nitric oxide synthase (iNOS) represents part of a prompt intestinal antibacterial response; however, NO has also been associated with the initiation and maintenance of inflammation in human inflammatory bowel disease (IBD). Recent studies on animal models of experimental IBD have shown that constitutive and inducible NO production seems to be beneficial during acute colitis, but sustained up‐regulation of NO is detrimental. This fact is also supported by studies on mice genetically deficient in various NOS isoforms. However, the mechanism by which NO proceeds from being an indispensable homeostatic regulator to a harmful destructor remains unknown. Furthermore, extrapolation of data from animal colitis models to human IBD is questionable. The purpose of this review is to update our knowledge about the role of this universal mediator and the enzymes that generate it in the pathogenesis of IBD.

Expression of functional CXCR4 chemokine receptors on human colonic epithelial cells

In addition to their role as regulators of leukocyte migration and activation, chemokines and their receptors also function in angiogenesis, growth regulation, and HIV-1 pathogenesis--effects that involve the action of chemokines on nonhematopoietic cells. To determine whether chemokine receptors are expressed in human colonic epithelium, HT-29 cells were examined by RT-PCR for the expression of the chemokine receptors for lymphotactin, fractalkine, CCR1-10, and CXCR1-5. The only receptor consistently detected was CXCR4 (fusin/LESTR), although HT-29 cells did not express mRNA for its ligand, stromal cell-derived factor (SDF-1alpha). Flow cytometric analysis with anti-CXCR4 antibody indicated that the CXCR4 protein was expressed on the surface of roughly half of HT-29 cells. CXCR4 was also expressed in colonic epithelial cells in vivo as shown by immunohistochemistry on biopsies from normal and inflamed human colonic mucosa. The mRNA for SDF-1alpha and other CC and CXC chemokines was present in normal colonic biopsies. The CXCR4 receptor in HT-29 cells was functionally coupled, as demonstrated by the elevation in [Ca2+]i, which occurred in response to 25 nM SDF-1alpha and by the SDF-1alpha-induced upregulation of ICAM-1 mRNA. Sodium butyrate downregulated CXCR4 expression and induced differentiation of HT-29 cells, suggesting a role for CXCR4 in maintenance and renewal of the colonic epithelium. This receptor, which also serves as a coreceptor for HIV, may mediate viral infection of colonic epithelial cells.

Expression of inducible nitric oxide synthase activity in human colon epithelial cells: modulation by T lymphocyte derived cytokines

Background—Nitric oxide (NO) synthesis and inducible nitric oxide synthase (iNOS) expression are increased in colonic biopsy specimens from patients with ulcerative colitis, but the cellular source of NO production is not known. Aims—To examine the distribution of iNOS in human colonic mucosa and to explore the ability of T lymphocyte derived cytokines to regulate iNOS expression and activity in human colonic epithelial cells. Methods—iNOS expression was examined using immunohistochemistry in colonic biopsy samples from 12 patients with ulcerative colitis and three with infectious colitis and compared with 10 normal controls. In vitro iNOS expression and activity were determined in HT-29 cell cultures; nitrite levels were measured using a fluorescent substrate, iNOS mRNA expression by northern blot analysis, and iNOS protein expression by western blot analysis. Results—No iNOS expression was detected (10 of 10) in non-inflamed mucosa derived from normal controls. In 11 of 12 cases of newly diagnosed ulcerative colitis, iNOS protein was expressed in the epithelial cells, while no other positive cells were found in the lamina propria. Similar iNOS labelling was found in colonic biopsy samples from patients with infectious colitis in the acute phase, but when re-examined in samples from patients in total remission, no iNOS staining was observed. Both interleukin (IL)-13 and IL-4, but not IL-10, are potent inhibitors of iNOS expression and activity induced by an optimal combination of cytokines, namely IL-1α, tumour necrosis factor α and interferon γ. Conclusions—The data suggest that the epithelium is the major source of iNOS activity in ulcerative colitis and that IL-13 and IL-4 may act as intrinsic regulators of NO generation in intestinal inflammation.

A prospective, non-randomized, no placebo-controlled, phase Ib clinical trial to study the safety of the adipose derived stromal cells-stromal vascular fraction in idiopathic pulmonary fibrosis

IntroductionRegenerative medicine and particular adult stem cells represent an alternative option with several fruitful therapeutic applications in patients suffering from chronic lung diseases including idiopathic pulmonary fibrosis (IPF). Nevertheless, lack of knowledge regarding the origin and the potential of mesenchymal stem cells (MSCs) to differentiate into fibroblasts has limited their use for the treatment of this dismal disease.Patients and methodsTo this end, we conducted a phase Ib, non-randomized, clinical trial to study the safety of three endobronchial infusions of autologous adipose derived stromal cells (ADSCs)-stromal vascular fraction (SVF) (0.5 million cells per kgr of body weight per infusion) in patients with IPF (n=14) of mild to moderate disease severity (forced vital capacity –FVC>50% predicted value and diffusion lung capacity for carbon monoxide-DLCO>35% of predicted value). Our primary end-point was incidence of treatment emergent adverse events within 12 months. Alterations of functional, exercise capacity and quality of life parameters at serial time points (baseline, 6 and 12 months after first infusion) were exploratory secondary end-points.ResultsNo cases of serious or clinically meaningful adverse events including short-term infusional toxicities as well as long-term ectopic tissue formation were recorded in all patients. Detailed safety monitoring through several time-points indicated that cell-treated patients did not deteriorate in both functional parameters and indicators of quality of life.ConclusionsThe clinical trial met its primary objective demonstrating an acceptable safety profile of endobronchially administered autologous ADSCs-SVF. Our findings accelerate the rapidly expanded scientific knowledge and indicate a way towards future efficacy trials.

Probiotics and prebiotics in inflammatory bowel disease: microflora ‘on the scope’

The intestinal microflora is a large bacterial community that colonizes the gut, with a metabolic activity equal to an organ and various functions that affect the physiology and pathology of the hosts mucosal immune system. Intestinal bacteria are useful in promotion of human health, but certain components of microflora, in genetically susceptible individuals, contribute to various pathological disorders, including inflammatory bowel disease. Clinical and experimental observations indicate an imbalance in protective and harmful microflora components in these disorders. Manipulation of gut flora to enhance its protective and beneficial role represents a promising field of new therapeutic strategies of inflammatory bowel disease. In this review, we discuss the implication of gut flora in the intestinal inflammation that justifies the role of probiotics and prebiotics in the prevention and treatment of inflammatory bowel disease and we address the evidence for therapeutic benefits from their use in experimental models of colitis and clinical trials.

Exacerbation of inflammatory bowel diseases associated with the use of nonsteroidal anti-inflammatory drugs: myth or reality?

BackgroundNonsteroidal anti-inflammatory drugs (NSAIDs), conventional and selective cyclooxygenase-2 (COX-2) inhibitors, are among the most widely used medications for the treatment of various inflammatory conditions. There is strong evidence of a possible association between the use of these drugs and the relapse of inflammatory bowel diseases (IBD).ObjectiveOur objective was to examine the literature regarding the exacerbation of IBD associated with the use of conventional NSAIDs and selective COX-2 inhibitors and the underlying pathogenetic mechanisms.Study designWe reviewed articles, including original papers, controlled trials, case reports, reviews, and editorials published in English at the PubMed, Scopus Database, and Science Direct database, searching with the following keywords: nonsteroidal anti-inflammatory drugs (NSAIDs), COX-2 inhibitors, Coxibs, inflammatory bowel diseases (IBD), ulcerative colitis (UC), Crohn’s disease (CD).ResultsThere is substantial evidence that exacerbation of IBD happens after treatment with NSAIDs, but the available data remain conflicting, and it is not clear whether selective COX-2 inhibitors are safer than traditional NSAIDs. However, there is some evidence that selective COX-2 inhibition and COX-1 inhibition (with low-dose aspirin) appear to be well-tolerated in the short term. Regarding the mechanisms of relapse, the reduction of prostaglandins appears to be the hallmark of the NSAIDs adverse effects.ConclusionsFurther randomized, double-blind, controlled trials should be performed to address this issue, and more in vitro studies to identify the pathways involved are required.

Cytokine-induced Apoptosis in Epithelial HT-29 Cells Is Independent of Nitric Oxide Formation EVIDENCE FOR AN INTERLEUKIN-13-DRIVEN PHOSPHATIDYLINOSITOL 3-KINASE-DEPENDENT SURVIVAL MECHANISM

A combination of the pro-inflammatory cytokines interleukin (IL)-1α, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α induces nitric oxide synthase mRNA expression and nitric oxide (NO) generation in the human colon carcinoma cell line HT-29. This can be inhibited by pretreatment with IL-13 via a phosphatidylinositol (PI) 3-kinase-dependent mechanism (Wright, K., Ward, S. G., Kolios, G., and Westwick, J. (1997)J. Biol. Chem. 272, 12626–12633). Since NO has been implicated in regulating mechanisms leading to cell death, while activation of PI 3-kinase-dependent signaling cascades are thought to be involved with promoting cell survival events, we have investigated the outcome of these cytokine treatments on apoptosis and cell survival of HT-29 cells. Initiation of apoptosis can be achieved by the combinations of IFN-γ/TNF-α, IFN-γ/CD95, IL-1α/IFN-γ, and IL-1α/IFN-γ/TNF-α to varying extents. Induction of apoptotic markers by HT-29 cells in response to cytokine treatment is not dependent on NO production. Pretreatment with IL-13 protects against IL-1α/IFN-γ/TNF-α- and IFN-γ/TNF-α- as well as IFN-γ/CD95-induced (but not IL-1α/IFN-γ-induced) cell death. In addition, IFN-γ/TNF-α and IL-1α/IFN-γ/TNF-α stimulate activation of caspase-8 and caspase-3, which IL-13 pretreatment was able to partially inhibit and delay. IL-13 also stimulates activation of the major PI 3-kinase effector, protein kinase B. The PI 3-kinase inhibitors wortmannin and LY294002 inhibit IL-13 stimulation of protein kinase B as well as the cell survival effects of IL-13. These data demonstrate that cytokine-induced apoptosis of HT-29 cells is NO-independent and that the activation of a PI 3-kinase-dependent signaling cascade by IL-13 is a key signal responsible for the inhibition of apoptosis.

Inducible nitric oxide synthase activity and expression in a human colonic epithelial cell line, HT-29.

1 We have determined which cytokines regulate the expression of human inducible nitric oxide synthase (iNOS) mRNA and nitrite generation in the human colonic epithelial cell line HT‐29. 2 Growth arrested cell cultures were stimulated with the human recombinant cytokines interleukin‐lα (IL‐lα), tumour necrosisfactor‐α (TNF‐α), interferon γ (IFN‐γ) or vehicle added alone or in combination. Human iNOS mRNA was determined by Northern blot analysis and nitrite generation by the use of a fluorometric assay. 3 Unstimulated cells produced a small time‐dependent increase in nitrite generation of 50 ± 4, 75 ± 8, and 103 ±8 nM per 106 cells at 24 h, 48 h, and 72 h respectively. This nitrite generation was unaffected by cycloheximide (5 μg ml−1) pretreatment and iNOS mRNA was not detected. 4 None of cytokines alone induced either iNOS mRNA expression or an increase in nitrite generation. The combination of IL‐lα/IFN‐γ produced a highly significant (P< 0.001) 4 fold increase in nitrite production at 48 h, compared to basal values, while no other pair of cytokines was effective. 5 Time course studies with IL‐lα/IFN‐γ combination revealed significant (P< 0.001) increases in nitrite at 24 h (153 ±7), 48 h (306 ±24), and 72 h (384 ±15) compared to basal values of 50 ±4, 75 ±8, and 103 ±8 nM per 106 cells respectively. 6 Studies with IL‐lα/IFN‐γ combination demonstrated a time dependent expression of iNOS mRNA, first observed at 6 h, peaked at 24 h and was undetectable by 72 h. IL‐lα (0.3–10 ng ml−1) and IFN‐γ (10–300 u ml−1) in combination induced a concentration‐dependent expression of iNOS mRNA at 24 h. 7 Pretreatment with cycloheximide before IL‐lα/IFN‐γ stimulation reduced nitrite levels to basal values. These data suggest that the IL‐lα/IFN‐γ‐induced nitrite production by HT‐29 cells is dependent on de novo protein synthesis, probably the iNOS enzyme. 8 The addition of TNF‐α produced a significant (P< 0.001) 3 fold increase of IL‐lα/IFN‐γ‐induced nitrite generation. In marked contrast the presence of TNF‐α had no effect on IL‐lα/IFN‐γ‐induced iNOS mRNA expression by HT‐29 cells. These findings suggest that the up‐regulation by TNF‐α of IL‐lα/IFN‐γ‐induced nitrite generation is at the post‐transcriptional level. 9 These data suggest that pro‐inflammatory cytokines induce NO production in colonic epithelial cells probably due to the induction of iNOS and these cells may be a major source of NO generation in inflammatory bowel disease.

Introduction to Stem Cells and Regenerative Medicine

Stem cells are a population of undifferentiated cells characterized by the ability to extensively proliferate (self-renewal), usually arise from a single cell (clonal), and differentiate into different types of cells and tissue (potent). There are several sources of stem cells with varying potencies. Pluripotent cells are embryonic stem cells derived from the inner cell mass of the embryo and induced pluripotent cells are formed following reprogramming of somatic cells. Pluripotent cells can differentiate into tissue from all 3 germ layers (endoderm, mesoderm, and ectoderm). Multipotent stem cells may differentiate into tissue derived from a single germ layer such as mesenchymal stem cells which form adipose tissue, bone, and cartilage. Tissue-resident stem cells are oligopotent since they can form terminally differentiated cells of a specific tissue. Stem cells can be used in cellular therapy to replace damaged cells or to regenerate organs. In addition, stem cells have expanded our understanding of development as well as the pathogenesis of disease. Disease-specific cell lines can also be propagated and used in drug development. Despite the significant advances in stem cell biology, issues such as ethical controversies with embryonic stem cells, tumor formation, and rejection limit their utility. However, many of these limitations are being bypassed and this could lead to major advances in the management of disease. This review is an introduction to the world of stem cells and discusses their definition, origin, and classification, as well as applications of these cells in regenerative medicine.

C-X-C and C-C chemokine expression and secretion by the human colonic epithelial cell line, HT-29: differential effect of T lymphocyte-derived cytokines

Differential chemokine production by colonic epithelial cells is thought to contribute to the characteristic increased infiltration of selected population of leukocytes cells in inflammatory bowel disease. We have previously demonstrated that IL‐13 enhances IL‐1α‐induced IL‐8 secretion by the colonic epithelial cell line HT‐29. We have now explored the C‐C chemokine expression and modulation in this system. The combination of TNF‐α and IFN‐γ was the minimal stimulation required for regulated on activation, normal T cell expressed and secreted (RANTES) and monocyte chemoattractant protein (MCP‐1) mRNA expression and secretion by HT‐29 cells. The same stimulation induced a stronger IL‐8 mRNA expression and secretion. Pretreatment with IL‐13 or IL‐4, reduced significantly the RANTES, and MCP‐1, but not IL‐8 mRNA expression and secretion. In contrast, IL‐10 had no effect on either MCP‐1, or RANTES, or IL‐8 generation. Pretreatment of HT‐29 cells with wortmannin suggested that the IL‐13‐induced inhibition of C‐C chemokine expression is via activation of a wortmannin‐sensitive phosphatidylinositol 3‐kinase. These data demonstrate that colonic epithelial cell chemokine production can be differentially regulated by T cell‐derived cytokines and suggest an interplay between epithelial cells and T lymphocytes potentially important in the intestinal inflammation.