A novel and selective sodium-glucose cotransporter-2 inhibitor, tofogliflozin, improves glycaemic control and lowers body weight in patients with type 2 diabetes mellitus.

AIM: To assess the efficacy, safety and tolerability of different doses of tofogliflozin, a novel, highly selective sodium-glucose cotransporter 2 (SGLT2) inhibitor, in patients with type 2 diabetes mellitus (T2DM). METHODS: In a 12-week, multicentre, multinational, randomized, double-blind, parallel-group, placebo-controlled, dose-finding study, patients with inadequate glycaemic control from diet and exercise alone, or from diet and exercise plus a stable dose of metformin, were randomized to one of five doses of tofogliflozin (2.5, 5, 10, 20, or 40 mg) or placebo. The primary efficacy endpoint was absolute change at week 12 from baseline in glycated haemoglobin (HbA1c), minus the change in the placebo group. RESULTS: Statistically significant dose-dependent reductions in HbA1c were shown in all treated groups except the 2.5-mg dose group, with a maximum reduction of 0.56% (placebo-subtracted) at the 40-mg dose, along with increased urinary glucose excretion. Metformin treatment had no substantial influence on tofogliflozin efficacy. Dose-dependent reductions in fasting plasma glucose and body weight were observed, and glucose intolerance was improved, with a trend towards blood pressure reduction. Slight increases were observed for mean ketone bodies with no abnormal change in ketone body ratio. No deaths or treatment-related serious adverse events were reported. The incidence of adverse events was similar in the placebo (37.9%) to that in the tofogliflozin group (35.9-46.3%). Withdrawal because of adverse events was rare (≤2 patients per treatment group), with similar rates of withdrawal in the placebo and tofogliflozin groups. CONCLUSIONS: A once-daily dose of tofogliflozin for 12 weeks was an effective, safe and well-tolerated treatment for T2DM.

The expression of IL-12 p40 and its homologue, Epstein-Barr virus-induced gene 3, in inflammatory bowel disease.

BACKGROUND: It has recently been suggested that Crohn's Disease (CD) is associated with an exaggerated T helper 1 cytokine response as manifest by increased production of interleukin-12 (IL-12) and interferon-gamma (IFN-gamma). Epstein-Barr virus-induced gene 3 (EBI3) encodes a 34-kDa glycoprotein that is 27% identical to the p40 unit of IL-12 and has recently been reported to be up-regulated in ulcerative colitis (UC). AIM: To determine whether mucosal expression of IL-12 p40 or EBI3 correlates with inflammatory bowel disease (IBD). PATIENTS/METHODS: mRNA expression in colonic mucosa from patients with UC, Crohn's disease (CD) and non-IBD controls was measured by reverse-transcribed real-time polymerase chain reaction (PCR). RESULTS: EBI3 was significantly increased in both involved and uninvolved colonic mucosa in patients with UC. Although IL-12 p40 was increased in some patients with CD relative to non-IBD controls, the increase was not statistically significant. However, 5-aminosalicylic acid (5-ASA) use was significantly correlated with reduced IL-12 p40 levels in the patients with CD, but not in UC cases. A similar reduction was not seen in 5-ASA-treated UC patients. CONCLUSION: IL-12 p40 expression in CD is heterogeneous. In contrast, expression of the IL-12 p40 homologue, EBI3, is up-regulated in nearly all UC cases and in a subset of CD.

Is histological investigation of polyps always necessary?

BACKGROUND AND STUDY AIMS: To assess whether polyp histological type can be predicted by patient characteristics and endoscopic polyp findings. PATIENTS AND METHODS: 1681 polyps in 494 patients were categorized as advanced adenomas (villous component or severe dysplasia or early cancer) or insignificant polyps. Chi-squared tests were used to analyze whether polyp histological type could be predicted based on patient age (< 60 vs. > or = 60 years), gender, family history of colon polyps or cancer, presence of anemia, polyp size (< or = 5 mm vs. > 10 mm), and location (left- vs. right-sided). RESULTS: Insignificant polyp histology (n = 1337) correlated with patient age < 60 years (P = 0.0026), lack of anemia (P< 0.0001), polyp size < or = 5 mm (P < 0.0001), and right-sided location (P= 0.0058). Stepwise inclusion of these parameters demonstrated that the association of a < or = 5 mm right-sided polyp in a patient < 60 years yielded the highest combined predictive value (96.2%) for an insignificant polyp. Conversely, age > or = 60 years, presence of anemia, polyp size > 10 mm, or left-sided location, as single or combined parameters, demonstrated a maximum predictive value of only 75.4% for an advanced adenoma. CONCLUSIONS: A small right-sided polyp in a young patient is associated with a small risk (3.8 %) for advanced adenomatous tissue, indicating that histological investigation of such a polyp might not always be necessary. However, the recent recognition of flat adenomas and "mini" de novo colon carcinomas in the European population also may limit the usefulness of small polyp diameters in the exclusion of severe polyp histology.

Regulation of gastric function by endogenous gastrin releasing peptide in humans: studies with a specific gastrin releasing peptide receptor antagonist.

BACKGROUND AND AIMS: The main goal of our study was to characterise the activity of BIM26226 as a peripheral gastrin releasing peptide (GRP) receptor antagonist in healthy human subjects and to determine if endogenous GRP is a physiological regulator of gastric acid secretion and gastrin release. METHODS: Our study consisted of three parts. In part I, subjects received saline or BIM26226 followed by graded doses of intravenous human GRP in a four period crossover design. In part II, subjects received BIM26226 or saline during oral meal ingestion or modified sham feeding. In part III, subjects received an acidified meal in the presence and absence of BIM26226 in a two period crossover design. In addition, gastrin and somatostatin mRNA were measured in biopsy specimens during saline and BIM26226 infusion. RESULTS: BIM26226 dose dependently inhibited GRP induced acid output. Acid secretion after oral liquid meal intake and sham feeding was significantly inhibited by BIM26226 (p<0.01) whereas plasma gastrin release remained unchanged. Gastrin and somatostatin mRNAs were not significantly different after saline or BIM26226. CONCLUSIONS: BIM26226 is a potent GRP antagonist in humans. Endogenous GRP may be a physiological regulator of gastric acid secretion. Gastrin release does not seem to be under the control of GRP.

Staphylococcal enterotoxin B induces potent cytotoxic activity by intraepithelial lymphocytes.

In food poisoning, Staphylococcus aureus secretes staphylococcal enterotoxin B (SEB), a superantigen that causes intense T-cell proliferation and cytotoxicity. The effects of SEB on lytic activity by human intestinal intraepithelial lymphocytes (IEL) were investigated. Jejunal IEL, from morbidly obese individuals undergoing gastric bypass operations, were tested for SEB-induced cytotoxicity against C1R B-lymphoblastoid cells, HT-29 adenocarcinoma cells, or CD1d-transfected cells using the 51Cr-release assay. Fas and Fas ligand expression were detected by immunofluorescence and flow cytometry and soluble ligand by enzyme-linked immunosorbent assay (ELISA). In the presence of SEB, IEL became potently cytotoxic against C1R cells and interferon-gamma (IFN-gamma)-precultured HT-29 cells, causing 55+/-10% and 31+/-6% lysis, respectively, greater than that by phytohaemagglutinin (PHA)-, interleukin-2 (IL-2)-, or anti-T-cell receptor (TCR)-activated IEL. SEB-stimulated peripheral blood (PB) CD8+ T cells lysed similar numbers of C1R cells but fewer HT-29 cells (53+/-13% and 8+/-5%, respectively). IEL killing of C1R cells involved interaction of major histocompatibility complex (MHC) class II with TCR, CD2 with CD58, and CD11a with CD54, and was perforin mediated. SEB-induced IEL lysis of HT-29 cells, in contrast, was caused by an unknown target cell structure, not MHC class II or CD1d, and resulted from a combination of perforin and Fas-mediated events. The potent cytotoxic activities of IEL promoted by SEB utilize two different mechanisms, depending on the surface receptors expressed by the target cells.

Evidence of T cell receptor beta-chain patterns in inflammatory and noninflammatory bowel disease states.

T cell activation, as defined by expression of relevant cell surface molecules, such as the interleukin-2 receptor (CD25), is increased in many chronic relapsing diseases, including inflammatory bowel disease (IBD). These T cells are generally activated through contact of their clonotypic T cell receptor (TCR) with a peptide antigen presented by a major histocompatibility complex molecule. One of the putative antigenic contact sites for the TCR is the third complementarity determining region (CDR3) of the TCR beta-chain variable region (TCRBV). Therefore, analysis of the TCRBV CDR3 provides insight into the diversity of antigens encountered by a given T cell population. This study evaluated the TCRBV CDR3 usage of the activated intestinal lymphocytes from human subjects with IBD, diverticulitis (inflammatory control), and a normal tissue control. Public patterns, as demonstrated by shared TCRBV CDR3 amino acid sequences of activated intestinal T cell subpopulations, were observed. In particular, a public pattern of TCRBV22, a conserved valine in the fifth position, and use of TCRBJ2S1 or TCRBJ2S5 was present in three of four Crohn's disease subjects while not present in the ulcerative colitis subjects. However, the private patterns of TCRBV CDR3 region amino acid sequences were far more striking and easily demonstrated in all individuals studied, including a normal noninflammatory control. Thus we conclude that selective antigenic pressures are prevalent among an individual's activated intestinal lymphocytes.

[Ulcer + ulcer]. / Ulcus + Ulcus.

We report on an elderly woman suffering from an acutely bleeding duodenal ulcer. Apart from old age and a history of gastrointestinal ulcer there were no risk factors detectable. However, clinical examination revealed that the patient had put a diclofenac hydroxyethylpyrrolidine plaster, which had been prescribed for lower back pain, inadvertently on a large ulcus cruris. The diclofenac serum concentration was 80 mu/l corresponding to a therapeutic serum level. This case demonstrates that false application of new pharmaceutical formulation may lead to inadvertent side-effects.

Cloning of BY55, a novel Ig superfamily member expressed on NK cells, CTL, and intestinal intraepithelial lymphocytes.

Expression of the BY55 protein has been shown to be tightly associated with NK and CD8+ T lymphocytes with cytolytic effector activity. To determine the function of this protein, we molecularly cloned BY55 cDNA. The cDNA sequence predicts a cysteine-rich, glycosylphosphatidylinositol-anchored protein of 181 amino acids with a single Ig-like domain weakly homologous to killer inhibitory receptors. Reduction and carboxyamidomethylation of immunoprecipitated BY55 gave a band of 27 kDa, whereas reduction alone led to an 80-kDa species, suggesting that BY55 is a tightly disulfide-linked multimer. RNA blot analysis revealed BY55 mRNAs of 1.5 and 1.6 kb whose expression was highly restricted to NK and T cells. BY55 was expressed on the CD56dim, CD16+ subset of NK cells, which have high cytolytic activity, but was not expressed and was not induced on the CD56bright, CD16-subset of NK cells, a subset with high proliferative, but low cytolytic, capacity. In human tissues, BY55 mRNA was expressed only in spleen, PBL, and small intestine (in gut lymphocytes). BY55 was expressed on all intestinal intraepithelial lymphocytes, which were predominantly CD3+TCRalpha/beta+CD4-CD8+CD11b+CD28-CD45RO+C D56-CD101+CD103+ (alphaEbeta7 integrin). In addition, BY55 was expressed on most CD8+CD28- peripheral blood T cells. These phenotypic relationships suggest that CD8+CD28+ precursor CTL may terminally differentiate into CD8+CD28-BY55+ effector CTL and that some of the peripheral blood CD8+CD28- subset may represent recirculation from mucosal epithelial immune sites.

An interleukin 12-related cytokine is up-regulated in ulcerative colitis but not in Crohn's disease.

BACKGROUND & AIMS: Interleukin 12 (IL-12) is a heterodimeric, macrophage-derived cytokine that is elevated in Crohn's disease (CD). Epstein-Barr virus-induced gene 3 (EBI3) is a recently characterized human glycoprotein that is homologous to the 40-kilodalton chain of IL-12 and forms a heterodimer with the 35-kilodalton chain of IL-12. We investigated the expression of EBI3 in colonic mucosa of normal control subjects, patients with ulcerative colitis (UC), and patients with CD. METHODS: Colonic tissue was analyzed for messenger RNA (mRNA) expression by quantitative polymerase chain reaction and for protein expression by immunohistology and Western blotting. RESULTS: EBI3 mRNA was present in intestinal biopsy specimens from healthy subjects and patients with CD but was elevated only in active UC. EBI3 levels in UC specimens correlated with histological scores of activity and T-cell infiltration. EBI3-positive cells that had a shape consistent with that of macrophages were identified in the lamina propria, and protein was detected by Western blotting. CONCLUSIONS: EBI3 is a novel IL-12-related cytokine that is expressed by macrophage-like cells in normal intestine and CD and has enhanced expression in active UC but not in active CD.

Human intestinal epithelial cell lines produce factor(s) that inhibit CD3-mediated T-lymphocyte proliferation.

Peripheral blood T lymphocytes (PBT) proliferate more to anti-CD3 stimulation than to anti-CD2 stimulation. On the other hand, fresh, but not cultivated, intestinal intraepithelial lymphocytes (iIEL) exhibit a lower response to CD3 stimulation in comparison to CD2. The goal of this study was to show that the anti-CD3 T-cell response depends on the microenvironment and is independent of the origin of the lymphocytes. Cultured T-cell lines were stimulated with either an anti-CD3 mAb or an anti-CD2 mAb. Either conditioned supernatant from intestinal epithelial cell (IEC) lines or non- conditioned medium (negative control) was added. After 2 days cytokine production and proliferation were measured. Conditioned supernatant decreased the proliferative response of small and large bowel iIEL compared to controls (P = 0.04). In the same experiments, the cytokine production was non-significantly decreased. Immortalized iIEL, that are not regularly stimulated by their CD3 pathway, showed a similar decrease in proliferation (P < 0.001) and cytokine production (P = 0.01) when incubated with conditioned supernatant. Similar results were also obtained with a non-immortalized and an immortalized PBT line (P < 0.001). In a small bowel iIEL cell line, that exhibited a significant response to anti-CD2 stimulation, the proliferative response to anti-CD2 stimulation was preserved. Active conditioned supernatant could be generated from three independent IEC lines and a liver derived epithelial cell line, but not from a non-epithelial control cell line or two extraintestinal epithelial cell lines. We conclude that supernatants of cultured IEC contain soluble factor(s) that cause cultured iIEL and extraintestinal lymphocytes to behave like fresh iIEL. These results, therefore, support and extend the studies of others which suggest that the intestinal microenvironment mucosalizes lymphocytes.

Analysis of human common bile duct-associated T cells: evidence for oligoclonality, T cell clonal persistence, and epithelial cell recognition.

The phenotype of T cells associated with the common bile duct (CBD) is unknown. We investigated the hypothesis that they behave like other intraepithelial lymphocytes (IEL). Thus, we sought to determine the phenotype, TCR repertoire, and epithelial recognition of T cells obtained during endoscopic retrograde cholangiopancreatography. Three subjects were studied: two with primary sclerosing cholangitis and one normal control. After establishing a short-term T cell line, cells were 1) stained with mAbs for flow cytometric analysis, 2) analyzed for TCRB chain transcript expression, and 3) used as effector cells for cytotoxicity and proliferation. Flow cytometry revealed that for all the subjects 98% of the T cells were TCR-alpha beta-positive. Immunohistology of the CBD showed that the epithelium and lamina propria contained significant numbers of CD3+ CD43+ CD45RO+ lymphocytes. Complementarity-determining region 3 length displays suggested that the CBD-derived lines were oligoclonal. This was confirmed by cloning and random sequencing of PCR amplification products using TCRBV region family-specific primers; TCRB chain sequences were reiterated in all transcripts analyzed. In one case, two expanded TCRB clones could be identified that were persistent in the bile duct over a 1-yr period. The CBD-derived lines were cytolytic in a redirected lysis assay and caused cytolysis of an intestinal epithelial cell line (Caco-2). This recognition was likely preferential for intestinal epithelial cells, since a CBD-derived line exhibited proliferation to two intestinal epithelial cell lines (HT-29 and Caco-2) but not three other lines (HepG2, human foreskin fibroblast, and KD). We conclude that the CBD contains IELs that share several characteristics with intestinal IELs.

The intestinal epithelial cell: immunological aspects.

IECs likely play an important role in immunological defense mechanism. Apart from being a passive barrier against luminal bacteria, IECs secrete protective and microbiocidal products such as ITF, complement components and cryptdins into the lumen. Moreover, IECs produce secretory component that is essential for the transport of IgA from the lamina propria into the lumen. IECs also have regulatory functions. They express adhesion molecules important in the homing of T cells and other leukocytes, and likely modulate T cell functions in a paracrine way. Furthermore, IECs secrete cytokines, either constitutively or after bacterial challenge, and they express cytokine receptors. Lastly, IECs may play an important role as non-professional antigen-presenting cells by expressing classical MHC class I and class II and nonclassical MHC class I molecules on the cell surface. This aspect is particularly intriguing in that IECs also express a FcR that may have a function in luminal antigen sampling.

Concomitant active Crohn's disease and the acquired immunodeficiency syndrome.

Symptomatic human immunodeficiency virus (HIV) infection is accompanied by depressed CD4+ T-lymphocyte counts. These cells seem to play a role in the inflammatory processes in Crohn's disease. It has even been speculated that depression of CD4+ T-lymphocytes in HIV infection may cure Crohn's disease. Here we describe a 41-year-old drug-addicted man with a 9-year history of Crohn's disease. HIV infection was diagnosed 8 years ago. At present he has stage-C3 HIV infection. He was admitted because of weight loss and chronic diarrhea with rectal blood and mucus discharge. Crohn's disease was confirmed endoscopically and histologically. Infectious diarrhea known to mimic Crohn's disease in patients with acquired immunodeficiency syndrome (AIDS) was excluded. In summary, we describe a patient with AIDS (CD4 count, 84/microliter) and active Crohn's disease, showing that both illnesses can occur simultaneously.

Assessment of gastric acid output by H2 breath test.

BACKGROUND: The standard method to measure gastric acid secretion is the aspiration of gastric juice. A noninvasive breath test after application of magnesium has been proposed. The aim of this study was to modify the method, to possibly improve the discriminatory value of the test in comparison with intubation tests. METHODS: We measured the time course of the reaction of magnesium and gastric acid in vitro and determined the gastric hydrogen kinetics in humans by insufflation of hydrogen into the stomach and measuring its reappearance in the exhaled air. Thereafter, a comparison of the breath test and the intubation test was done in 10 healthy volunteers in different secretory states. RESULTS: After hydrogen insufflation 31.4% reappeared in 90 min (16.3% exhaled, rest belched). Discriminant analysis showed that the intubation test had a good discriminatory power. On the other hand, the breath test failed to distinguish between different secretory states (stimulation, inhibition, and intermediate). CONCLUSION: Whereas the intubation test discriminated between high and low acid secretion, the breath test did not. This test therefore seems, at least as performed here, unsuitable as a diagnostic test of gastric acid secretion.