Effects of baseline abdominal pain and bloating on response to lubiprostone in patients with irritable bowel syndrome with constipation.

BACKGROUND: Lubiprostone (8 µg b.d.) received US Food and Drug Administration (FDA) approval in 2008 for the treatment of constipation-predominant irritable bowel syndrome (IBS-C) in women aged ≥18 years. In 2012, the FDA issued new guidance for IBS-C clinical trials, recommending a composite endpoint incorporating both abdominal pain and stool frequency. AIM: In a post hoc analysis, similar criteria were applied to data from two pivotal, phase 3, double-blind, randomised trials of lubiprostone in patients with IBS-C. METHODS: Included patients had a baseline spontaneous bowel movement (SBM) frequency <3/week and abdominal pain or bloating ratings ≥1.36 on a 5-point scale [0 (absent) to 4 (very severe)]. Responders (composite endpoint) had a mean pain reduction ≥30% compared with baseline, and an increase from baseline of ≥1 SBM/week for ≥6 of the 12 treatment weeks. Lubiprostone effects on abdominal pain alone were also evaluated, as were bloating alone and in a composite endpoint with stool frequency. RESULTS: In pooled data, 325 patients received lubiprostone and 180 received placebo. Rates of response were higher with lubiprostone vs. placebo for the composite endpoint of improved pain and stool frequency (26.3% vs. 15.3%, respectively; P = 0.008) and the composite endpoint of improved bloating and stool frequency (23.8% vs. 12.6%, respectively; P = 0.012). Response rates were also higher with lubiprostone vs. placebo for abdominal pain alone (P = 0.005) and bloating alone (P = 0.012). CONCLUSION: Lubiprostone was significantly more effective than placebo in improving abdominal pain or bloating, and also in composite endpoints that included stool frequency.

Anti-inflammatory and cartilage-protecting effects of an intra-articularly injected anti-TNF{alpha} single-chain Fv antibody (ESBA105) designed for local therapeutic use.

OBJECTIVES: (1) To show that a single-chain Fv antibody (scFv) against tumour necrosis factor alpha (TNFalpha) (ESBA105) has efficacy comparable to a full length anti-TNFalpha IgG (infliximab); (2) to evaluate whether ESBA105 has all the properties required for the local treatment of arthritis; and (3) to investigate its discriminative tissue penetration properties. METHODS: In vivo efficacy was measured in arthritis of the knee joint induced by the intra-articular injection of recombinant human TNFalpha (rhTNFalpha) in Lewis rats. Cartilage penetration of scFv (ESBA105) and full length IgG (infliximab) were studied in bovine cartilage specimens ex vivo. Tissue penetration, biodistribution and pharmacokinetics of ESBA105 were followed and compared after intra-articular and intravenous administration. RESULTS: In cell culture, ESBA105 showed similar TNFalpha inhibitory potency to infliximab. In vivo, ESBA105 inhibited rhTNFalpha-induced synovial inflammation in rats with efficacy again comparable to infliximab. An 11-fold molar excess of ESBA105 over rhTNFalpha resulted in 90% inhibition of knee joint swelling, inflammatory infiltrates and proteoglycan loss from cartilage. In ex vivo studies of bovine cartilage, ESBA105 penetrated well into the cartilage whereas infliximab remained on the surface. In vivo, rapid penetration into the synovial tissue, cartilage and surrounding tissues was observed following intra-articular injection of [(125)I]-ESBA105 into the knee joint of rabbits. CONCLUSIONS: ESBA105 potently inhibits inflammation and prevents cartilage damage triggered by TNFalpha. In contrast to a full length IgG, ESBA105 also penetrates into cartilage and can be expected to reverse the TNFalpha-induced catabolic state of articular cartilage in arthritides.

History of coronary heart disease.

Coronary heart disease, in the mid-twentieth century has become the most frequently occurring lethal disease. Coronary vessels, that is, coronary arteries tend to develop atherosclerotic plaques already at an early stage. The onset and course of this development has often been a mute one, without pain, rendering thereby the recognition of coronary artery disease difficult--far into the twentieth century. The many experiments and investigations performed during the "Renaissance" brought no results. An exact description and depiction of damaged coronary vessels as primary cause of occluded coronary arteries became possible towards the beginning of the twentieth century only.

Putting its fingers on stressful situations: the heavy metal-regulatory transcription factor MTF-1.

It has been suggested that metallothioneins, discovered about 45 years ago, play a central role in heavy metal metabolism and detoxification, and in the management of various forms of stress. The metal-regulatory transcription factor-1 (MTF-1) was shown to be essential for basal and heavy metal-induced transcription of the stress-responsive metallothionein-I and metallothionein-II. Recently it has become obvious that MTF-1 has further roles in the transcriptional regulation of genes induced by various stressors and might even contribute to some aspects of malignant cell growth. Furthermore, MTF-1 is an essential gene, as mice null-mutant for MTF-1 die in utero due to liver degeneration. We describe here the state of knowledge on the complex activation of MTF-1, and propose a model with MTF-1 as an interconnected cellular stress-sensor protein involved in heavy metal metabolism, hepatocyte differentiation and detoxification of toxic agents.

Arsenite-inducible RNA-associated protein (AIRAP) protects cells from arsenite toxicity.

Exposure of cells to arsenicals activates multiple stress pathways resulting in the induction of specific genes whose identity and role in the adaptation to arsenical-induced cellular stress are poorly understood. We report here the identification of a novel gene encoding an arsenite-inducible, cysteine- and histidine-rich RNA-associated protein, AIRAP, that is conserved among mammals, Drosophila and C elegans. Immunochemistry and cell fractionation experiments indicate that, when induced, AIRAP is present in both the nucleus and the cytoplasm, and cross-linking experiments indicate that it associates with RNA in vivo. The expression of a C elegans homologue of AIRAP, aip-1, is also induced by exposure to arsenite, and expression of an aip-1::gfp transgene is most pronounced in hypodermal cells. RNA-mediated interference (RNAi) of aip-1 lowers the resistance of nematodes to arsenite yet does not appear to affect viability under standard growth conditions. These experiments suggest a role for AIRAP/AIP-1 in protecting cells from the toxic effects of arsenite.

Placenta growth factor gene expression is induced by hypoxia in fibroblasts: a central role for metal transcription factor-1.

Placenta growth factor (PlGF) is a mitogen for endothelial cells that can potentiate the growth and permeabilizing effects on endothelium of vascular endothelial growth factor. Here we report that hypoxia induces the expression of both PlGF mRNA and protein in immortalized/transformed mouse embryonic fibroblasts (mEFs) and in NIH 3T3 cells. Importantly, the magnitude of the induction of PlGF expression by hypoxia is enhanced by the presence of oncogenic Ras. To investigate the transcriptional component of hypoxia-inducible PlGF expression, we cloned and sequenced a 1350-bp fragment of the 5'-flanking region of the mouse gene. Analysis of the promoter region indicated the presence of putative consensus sequences for known hypoxia-responsive regulatory sites, including metal response elements and Sp1-like sites. In the present study, we show that the induction of PlGF expression by hypoxia is dependent on the presence of the metal response element-binding transcription factor 1 (MTF-1). Thus, in mEFs with targeted deletions of both MTF-1 alleles, hypoxia-induced increases of PIGF mRNA and protein levels were greatly attenuated compared with those in wild-type mEFs. Moreover, transient transfection of a PlGF promoter reporter gene into NIH 3T3 cells resulted in hypoxia-responsive transcriptional activation of the reporter. Finally, ectopic expression of MTF-1 resulted in increased basal transcriptional activity of a PlGF promoter reporter. Together, these findings demonstrate that the PlGF gene is responsive to hypoxia and that this response is mediated by MTF-1. It remains to be determined whether this activation is the result of direct and/or indirect transcriptional activation by MTF-1. The stimulatory effect of oncogenic Ras on the induction of PlGF expression in hypoxic cells suggests that PlGF could be an important proangiogenic factor in the tumor microenvironment.

Target gene search for the metal-responsive transcription factor MTF-1.

Activation of genes by heavy metals, notably zinc, cadmium and copper, depends on MTF-1, a unique zinc finger transcription factor conserved from insects to human. Knockout of MTF-1 in the mouse results in embryonic lethality due to liver decay, while knockout of its best characterized target genes, the stress-inducible metallothionein genes I and II, is viable, suggesting additional target genes of MTF-1. Here we report on a multi-pronged search for potential target genes of MTF-1, including microarray screening, SABRE selective amplification, a computer search for MREs (DNA-binding sites of MTF-1) and transfection of reporter genes driven by candidate gene promoters. Some new candidate target genes emerged, including those encoding alpha-fetoprotein, the liver-enriched transcription factor C/EBPalpha and tear lipocalin/von Ebner's gland protein, all of which have a role in toxicity/the cell stress response. In contrast, expression of other cell stress-associated genes, such as those for superoxide dismutases, thioredoxin and heat shock proteins, do not appear to be affected by loss of MTF-1. Our experiments have also exposed some problems with target gene searches. First, finding the optimal time window for detecting MTF-1 target genes in a lethal phenotype of rapid liver decay proved problematical: 12.5-day-old mouse embryos (stage E12.5) yielded hardly any differentially expressed genes, whereas at stage 13.0 reduced expression of secretory liver proteins probably reflected the onset of liver decay, i.e. a secondary effect. Likewise, up-regulation of some proliferation-associated genes may also just reflect responses to the concomitant loss of hepatocytes. Another sobering finding concerns gamma-glutamylcysteine synthetase(hc) (gamma-GCS(hc)), which controls synthesis of the antioxidant glutathione and which was previously suggested to be a target gene contributing to the lethal phenotype in MTF-1 knockout mice. gamma-GCS(hc) mRNA is reduced at the onset of liver decay but MTF-1 null mutant embryos manage to maintain a very high glutathione level until shortly before that stage, perhaps in an attempt to compensate for low expression of metallothioneins, which also have a role as antioxidants.

The transcription factors MTF-1 and USF1 cooperate to regulate mouse metallothionein-I expression in response to the essential metal zinc in visceral endoderm cells during early development.

During early development of the mouse embryo, expression of the metallothionein-I (MT-I) gene is heightened specifically in the endoderm cells of the visceral yolk sac. The mechanisms of regulation of this cell-specific pattern of expression of metallothionein-I are unknown. However, it has recently been shown that MTF-1, functioning as a metalloregulatory transcription factor, activates metallothionein genes in response to the essential metal zinc. In contrast with the metallothionein genes, MTF-1 is essential for development; null mutant embryos die due to liver degeneration. We report here that MTF-1 is absolutely essential for upregulation of MT-I gene expression in visceral endoderm cells and that optimal expression also involves interactions of the basic helix-loop-helix upstream stimulatory factor-1 (USF1) with an E-box1-containing sequence at -223 bp in the MT-I promoter. Expression of MT-I in visceral endoderm cells was dependent on maternal dietary zinc. Thus, the essential metal, zinc, apparently provides the signaling ligand that activates cell-specific MT-I expression in visceral endoderm cells.

The "metal transcription factor" MTF-1: biological facts and medical implications.

Metallothioneins (MTs) are a class of small, cysteine-rich proteins that have an important function in heavy metal metabolism and detoxification and in the management of various forms of cell stress. Several lines of evidence suggest a role for metallothioneins in therapy resistance of malignant tumours, regulation of blood pressure and protection against some neurological diseases. Basal and heavy metal-induced expression of the stress-inducible metallothionein-I and -II genes and some other stress-regulated genes depends on the zinc-finger transcription factor MTF-1. MTF-1 acts as a cellular stress-sensor protein and, besides its crucial role in metallothionein expression, is essential for liver development since mice null mutant for MTF-1 die in utero due to hepatocyte degeneration. Under pathological conditions, MTF-1 seems to be involved in clinically important processes such as tumour angiogenesis and drug resistance. It thus seems generally advisable to monitor MTF-1 activity in stress-related processes including aging and carcinogenesis.

Characterization of the mouse gene for the heavy metal-responsive transcription factor MTF-1.

MTF-1 is a zinc finger transcription factor that mediates the cellular response to heavy metal stress; its targeted disruption in the mouse leads to liver decay and embryonic lethality at day E14. Recently, we have sequenced the entire MTF-1 gene in the compact genome of the pufferfish Fugu rubripes. Here we have defined the promoter sequences of human and mouse MTF-1 and the genomic structure of the mouse MTF-1 locus. The transcription unit of MTF-1 spans 42 kb (compared to 8.5 kb in Fugu) and is located downstream of the gene for a phosphatase (INPP5P) in mouse, human, and fish. In all of these species, the MTF promoter region has the features of a CpG island. In both mouse and human, the 5' untranslated region harbors conserved short reading frames of unknown function. RNA mapping experiments revealed that in these two species, MTF-1 mRNA is transcribed from a cluster of multiple initiation sites from a TATA-less promoter without metal-responsive elements. Transcription from endogenous and transfected MTF-1 promoters was not affected by heavy metal load or other stressors, in support of the notion that MTF-1 activity is regulated at the posttranscriptional level. Tissue Northern blots normalized for poly A+ RNA indicate that MTF-1 is expressed at similar levels in all tissues, except in the testes, that contain more than 10-fold higher mRNA levels.

The heavy metal-responsive transcription factor-1 (MTF-1) is not required for neural differentiation.

The zinc finger transcription factor MTF-1 is essential for proper response to heavy metal load and other stress conditions in vertebrates, and also contributes to the maintenance of the cellular redox state. Target genes include metallothioneins (MT-I and MT-II) and gamma-glutamylcysteine synthetase (gamma-GCS), an enzyme involved in glutathione biosynthesis. Although MTF-1 is expressed ubiquitously, the primary defect in null mutant mice is hepatocyte necrosis, which results in embryonic lethality around day E14 and prevents the analysis of delayed effects on other organs. To assess the impact of MTF-1 deficiency on the function of the mature central nervous system, we employed the neural grafting strategy. Neuroectodermal brain tissue obtained from transgenic mouse embryos at gestational day 12.5 was transplanted into the caudoputamen of adult wild-type mice. 33 days later, grafts derived from MTF-1 deficient mice consisted of fully differentiated neuroectodermal tissue and showed no differences to heterozygous control grafts. This indicates that MTF-1 is dispensable for the development and differentiation of the nervous system. Such transplants devoid of MTF-1 may provide a useful tool for the further investigation of the effect of cell stress, including oxidative stress.

Analysis of arrhythmias in the Circadian Antiischemia Program in Europe (CAPE) study.

The aim of this study was to analyze whether, in patients with long-standing (>4 years) coronary artery disease (CAD), the addition of the long-acting calcium channel blocker (CCB) amlodipine to conventional treatment [beta-blockers (BBLs) and nitrates] during anginal attacks would have a proarrhythmic effect. This was tested by analyzing data from patients who had taken part in the Circadian Anti-ischemia Program in Europe (CAPE) trial. After a 2-week, single-blind, run-in period (Phase 1), patients were randomized to amlodipine, 5 mg/day (first 4 weeks) and 10 mg/day (second 4 weeks), or placebo for 8 weeks (Phase 2). The 48-h Holter data were analyzed for 167 amlodipine-treated patients and 83 placebo patients based on a 2:1 randomization scheme. Sixty-three per cent of amlodipine patients and 67% of placebo patients were receiving concomitant BBLs, and >90% had taken sublingual nitrates during anginal attacks, as basic antiischemic therapy. After 7 weeks of therapy, when 48-h Holter monitoring was repeated, there were no significant changes in the frequency of ventricular arrhythmias in the placebo or amlodipine groups for all patients or subgroups of patients with or without BBLs. Also, between-group comparisons showed no significant differences in arrhythmias between amlodipine and placebo patients. In summary, amlodipine (5-10 mg/day) given to patients with severe, chronic CAD receiving conventional antiischemic therapy, did not produce any proarrhythmic effects.

Unstable angina in 1998.

Unstable angina (UA) and non-Q-wave myocardial infarction (NQWMI) are acute coronary syndromes with repeated, severe ischemic events of short duration. These events are mainly due to a rapid decrease in coronary blood flow, and to a rapid, reversible reduction of the arterial lumen in localized areas. Episodes often are a mixture of thrombus formation due to platelet aggregation and localized spasm, leading to vasoconstriction. Due to the short interval (minutes) of ischemic events, usually no or minimal irreversible myocardial damage takes place. The main goal of treatment is to prevent progression of this unstable situation into a myocardial infarction. In the majority of cases, this is possible with adequate treatment of vasodilatory substances like nitrates, long-acting dihydropyridines like amlodipine and betablockers. In addition heparin and particular antiaggregatory drugs inhibiting platelet activation by blocking the GPIIb/IIIa receptor, the common pathway for platelet aggregation, are applied to prevent thrombus formation. This, in the majority of cases allows a passivation of the acute situation, leaving time to undertake possible further steps as coronary angiography, eventually followed by PTCA of the culprit lesion or, in advanced cases of CAD, by CABG with complete revascularization.

Comparison of ECG variables of dispersion of ventricular repolarization with direct myocardial repolarization measurements in the human heart.

INTRODUCTION: QT dispersion (QTD) from the 12-lead ECG has been widely adopted as a noninvasive index of dispersion of ventricular repolarization (DVR). QTD, however, has never been validated by direct comparison with myocardial DVR in the human heart. METHODS AND RESULTS: Monophasic action potential (MAP) recordings obtained in an earlier study were retrospectively matched with 12-lead ECGs available from within 24 hours of the invasive procedure. MAPs were available from an average of 8+/-3 left endocardial sites in 4 patients with left ventricular hypertrophy (LVH) and 7 patients with normal ECGs, and 6+/-2 epicardial sites in 3 patients of each group during normal ventricular activation. Local repolarization time (RT) was determined as MAP duration at 90% repolarization plus the local activation time. Dispersion of RT was calculated as the difference between the earliest and latest RT. ECGs were digitized and analyzed with recently described interactive QTD analysis software. In addition to standard QTD (defined as QTmax-QTmin), all currently proposed ECG dispersion variables were compared and correlated with the invasive measurements of DVR. QTD exhibited a reasonable correlation with dispersion of RT (R = 0.67; P < 0.01). Several other variables designed to measure DVR exhibited a similar, but not better, correlation. Among them, the QT peak/QT end ratio in V3 (R = -0.72; P < 0.01) and averaged over all analyzable leads (R = -0.59; P < 0.01) exhibited a good correlation with dispersion of RT, which was further improved when endocardial measurements were considered alone. T area measures did not correlate with dispersion of RT, but discriminated LVH. CONCLUSION: DVR can be assessed by means of a 12-lead surface ECG. Several of the variables under study exhibit a similar accuracy in determination of true myocardial dispersion of repolarization. Variables involving the terminal part of repolarization, such as the QT peak/QT ratio, even from a single lead, may add to the determination of DVR from the human heart.

Embryonic lethality and liver degeneration in mice lacking the metal-responsive transcriptional activator MTF-1.

We have shown previously that the heavy metal-responsive transcriptional activator MTF-1 regulates the basal and heavy metal-induced expression of metallothioneins. To investigate the physiological function of MTF-1, we generated null mutant mice by targeted gene disruption. Embryos lacking MTF-1 die in utero at approximately day 14 of gestation. They show impaired development of hepatocytes and, at later stages, liver decay and generalized edema. MTF-1(-/-) embryos fail to transcribe metallothionein I and II genes, and also show diminished transcripts of the gene which encodes the heavy-chain subunit of the gamma-glutamylcysteine synthetase, a key enzyme for glutathione biosynthesis. Metallothionein and glutathione are involved in heavy metal homeostasis and detoxification processes, such as scavenging reactive oxygen intermediates. Accordingly, primary mouse embryo fibroblasts lacking MTF-1 show increased susceptibility to the cytotoxic effects of cadmium or hydrogen peroxide. Thus, MTF-1 may help to control metal homeostasis and probably cellular redox state, especially during liver development. We also note that the MTF-1 null mutant phenotype bears some similarity to those of two other regulators of cellular stress response, namely c-Jun and NF-kappaB (p65/RelA).

Fetuses from preeclamptic mothers show reduced hepatic erythropoiesis.

The fetal liver is the main hematopoietic organ during intrauterine life. Morphometrical studies were performed on liver sections to detect changes occurring with intrauterine growth retardation and preeclampsia. Compared with the controls (n = 10), fetuses from preeclamptic mothers showed a severe reduction of erythroid cells by 60% on average (n = 18). Closer examination revealed that the erythroid cells at early stages of differentiation were more affected (80% reduction) than at later stages (55%). Seven out of 18 fetuses from preeclamptic mothers did not show growth retardation but exhibited severely reduced hepatic erythropoiesis. We suggest that the prime factor for impaired red blood cell production is preeclampsia itself rather than intrauterine growth retardation. Regulation of erythropoiesis in utero might depend on the interaction of many hematopoietic growth factors, and preeclampsia might alter the balance. To test this notion, we quantitated erythropoietin in fetal blood and various cytokines in the amniotic fluid. An elevation of erythropoietin and interleukin (IL)-3 levels was seen in babies born under the conditions of preeclampsia, whereas the concentrations of granulocyte/macrophage-colony-stimulating factor (CSF), granulocyte-CSF, and IL-1 beta were reduced, and the levels of IL-6 and IL-8 remained constant. With preeclampsia, a discrepancy between elevation of erythrocyte numbers in peripheral blood and depression of hematopoiesis at the main production site, the fetal liver, is seen. Concomitantly, there is elevation of some but reduction of other hematopoietic cytokines. We envision that during the course of preeclampsia quantitation of hematopoietic growth factors might allow to predict the deterioration of in utero life conditions.

Sixty-minute alteplase protocol: a new accelerated recombinant tissue-type plasminogen activator regimen for thrombolysis in acute myocardial infarction.

OBJECTIVES: Our aim was to design and evaluate a new and easily administered recombinant tissue-type plasminogen activator (rt-PA) regimen for thrombolysis in acute myocardial infarction (AMI) based on established pharmacokinetic data that improve the reperfusion success rate. BACKGROUND: Rapid restoration of Thrombolysis in Myocardial Infarction (TIMI) grade 3 flow is a primary predictor of mortality after thrombolysis in AMI. However, TIMI grade 3 patency rates 90 min into thrombolysis of only 50% to 60% indicate an obvious need for improved thrombolytic regimens. METHODS: Pharmacokinetic simulations were performed to design a new rt-PA regimen. We aimed for a plateau tissue-type plasminogen activator (t-PA) plasma level similar to that of the first plateau of the Neuhaus regimen. These aims were achieved with a 20-mg rt-PA intravenous (i.v.) bolus followed by an 80-mg i.v. infusion over 60 min (regimen A). This regimen was tested in a consecutive comparative trial in 80 patients versus 2.25 10(6) IU of streptokinase/60 min (B), and 70 mg (C) or 100 mg (D) of rt-PA over 90 min. Subsequently, a confirmation trial of regimen A in 254 consecutive patients was performed with angiographic assessment by independent investigators of patency at 90 min. RESULTS: The comparative phase of the trial yielded, respectively, TIMI grade 3 and total patency (TIMI grades 2 and 3) of 80% and 85% (regimen A), 35% and 50% (B), 50% and 55% (C) and 60% and 70% (D). In the confirmation phase of the trial, regimen A yielded 81.1% TIMI grade 3 and 87.0% total patency. At follow-up angiography 7 (4.1%) of 169 vessels had reoccluded. In-hospital mortality rate was 1.2%. Nadir levels of fibrinogen, plasminogen and alpha2-antiplasmin were 3.6 +/- 0.8 mg/ml, 60 +/- 21% and 42 +/- 16%, respectively (mean +/- SD). Fifty-seven patients (22.4%) suffered from bleeding; 3.5% needed blood transfusions. CONCLUSIONS: The 60-min alteplase thrombolysis in AMI protocol achieved a TIMI grade 3 patency rate of 81.1% at 90 min with no indication of an increased bleeding hazard; it was associated with a 1.2% overall mortality rate. These results are substantially better than those reported from all currently utilized regimens. Head to head comparison with established thrombolytic regimens in a large-scale randomized trial is warranted.