Efficacy and safety of oxitropium bromide, theophylline and their combination in COPD patients: a double-blind, randomized, multicentre study (BREATH Trial).

We compare the efficacy including spirometry, peak expiratory flow (PEFR) and quality of life and safety of an 8-week treatment with inhaled oxitropium, theophylline or their combination in patients with mild-to-severe chronic obstructive pulmonary disease (COPD). We conducted a multicentre, double-blind, double-dummy randomized, parallel-group study at 29 Italian outpatients clinics. A group of 236 patients with mild-to-severe COPD (baseline FEV1 < or = 70% of predicted value) were recruited. Treatments were as follows: Inhaled oxitropium bromide 200 microg (N=75), sustained-release oral theophylline 300 mg (N=81) or their combination (N=80), taken twice daily. Spirometry (FEV1 and FVC) was evaluated every 4 weeks, and morning and evening PEFR (before and 2-4 h after drug intake) was measured daily. Symptoms, cough and dysponea, were recorded daily. Health status was evaluated at baseline and week 8 using the disease specific St George' Respiratory Questionnaire (SGRQ). Any adverse event occurring during the treatment period was recorded on a diary card. FEV1 and FVC improved in all the groups at 4 and 8 weeks, but the difference between treatment groups did not reach statistically significant levels. Differences between groups in pre-dosing morning and evening PEFR were not significant. Post-dosing morning and evening PEFR were increased and the largest increase was seen in patients treated with both drugs. However, differences between groups was significant only for evening values (P=0.008). The proportion of patients who experienced a decrease in symptoms was high in all groups but no differences among groups were observed. SGRQ total scores decreased in all treatment groups after 8 weeks, particularly in the oxitropium and combination groups. Clinically significant change (> or = 4 units) was only observed in patients treated with oxitropium bromide whether with or without theophylline. Adverse events related to treatments were higher in the group treated with theophylline alone (P < 0.02). We conclude that inhaled oxitropium bromide alone was associated with an improvement in FEV1, PEFR and symptoms in patients with COPD that was not statistically different from that of oral theophylline alone or of the combination of both drugs. Oxitropium bromide in combination with theophylline provided a greater improvement in evening post-dosing PEFR. Oxitropium bromide alone or in combination with theophylline improved the quality of life better than theophylline alone.

Thymic hyperplasia and lung carcinomas in a line of mice transgenic for keratin 5-driven HPV16 E6/E7 oncogenes.

Human Papillomavirus type 16 (HPV-16) is the cause of both benign lesions and ano-genital cancers. In HPV-associated cancers the transforming properties of the expressed viral E6 and E7 proteins have been revealed by a number of different assays. We have generated transgenic mice expressing HPV-16 E6/E7 genes under the control of the murine keratin 5 gene promoter, which should confer cell-type specific expression in the basal cells of squamous stratified epithelia. Transgenic mice developed thymic hyperplasia and lung neoplasia with 100% frequency, the thymus showing a size increase at 2 months and reaching the maximum dimension at 6 months, when lung carcinomas appeared. After this time the size of hyperplastic thymi decreased, while malignant formations invaded the mediastinal area. Hepatic metastasis could be also observed in some of the animals at the autopsy and death invariably occurred around 10-11 months of age.

Evaluation of hepatitis C virus glycoprotein E2 for vaccine design: an endoplasmic reticulum-retained recombinant protein is superior to secreted recombinant protein and DNA-based vaccine candidates.

Hepatitis C virus (HCV) is the leading causative agent of blood-borne chronic hepatitis and is the target of intensive vaccine research. The virus genome encodes a number of structural and nonstructural antigens which could be used in a subunit vaccine. The HCV envelope glycoprotein E2 has recently been shown to bind CD81 on human cells and therefore is a prime candidate for inclusion in any such vaccine. The experiments presented here assessed the optimal form of HCV E2 antigen from the perspective of antibody generation. The quality of recombinant E2 protein was evaluated by both the capacity to bind its putative receptor CD81 on human cells and the ability to elicit antibodies that inhibited this binding (NOB antibodies). We show that truncated E2 proteins expressed in mammalian cells bind with high efficiency to human cells and elicit NOB antibodies in guinea pigs only when purified from the core-glycosylated intracellular fraction, whereas the complex-glycosylated secreted fraction does not bind and elicits no NOB antibodies. We also show that carbohydrate moieties are not necessary for E2 binding to human cells and that only the monomeric nonaggregated fraction can bind to CD81. Moreover, comparing recombinant intracellular E2 protein to several E2-encoding DNA vaccines in mice, we found that protein immunization is superior to DNA in both the quantity and quality of the antibody response elicited. Together, our data suggest that to elicit antibodies aimed at blocking HCV binding to CD81 on human cells, the antigen of choice is a mammalian cell-expressed, monomeric E2 protein purified from the intracellular fraction.

The comparison of the effect of LTR72 and MF59 adjuvants on mouse humoral response to intranasal immunisation with human papillomavirus type 6b (HPV-6b) virus-like particles.

Infections with genital human papillomaviruses (HPV) are likely to be neutralised more efficiently if a mucosal immune response can be elicited at the viral entry site. Local IgA antibodies are highly induced when antigens are co-administered with mucosal adjuvants, such as cholera toxin (CT) and Escherichia coli heat labile enterotoxin (LT) which, however, are not expected to have wide application because of their pronounced toxicity. We have immunised mice intranasally with HPV-6b virus-like particles (VLPs) and a genetically modified LT-derived molecule with only residual toxicity, LTR72, and compared the humoral responses with those obtained following systemic immunisation with VLPs and the MF59 adjuvant. Titration of anti-HPV antibodies in sera and vaginal secretions established that LTR72 was able to elicit higher serum and mucosal IgA titers, in addition to IgG serum levels, comparable to those obtained by parenteral immunisation. These results confirm the potential of toxin-derived adjuvants and extend their use in combination with HPV antigens.

Intravenous itasetron: establishing the effective dose range for the prophylactic control of acute emesis in cancer patients undergoing high-dose cisplatin chemotherapy.

Nausea and vomiting induced by chemotherapy are a major cause of distress to patients and reduce compliance with potentially beneficial treatment. Itasetron hydrochloride is a new 5-hydroxytryptamine3 (5-HT3) antagonist with potent antiemetic properties. It is more potent than ondansetron in animal models and in early clinical studies it demonstrates a long half-life and does not undergo hepatic biotransformation before elimination. The aim of this open, uncontrolled study was to establish the effective dose range of itasetron hydrochloride given intravenously (i.v.) to patients due to receive high-dose cisplatin chemotherapy (50-120 mg/m2) for the first time. Thirty-nine patients were enrolled in the trial and received a single i.v. infusion of itasetron hydrochloride at a dose of 17-280 microg/kg body weight before commencing the cisplatin infusion (median dose 90-110 mg/m2). Antiemetic protection was demonstrated by doses in the range of 35-280 microg/kg. The 17 microg/kg dose was not effective. Treatment failure (>5 emetic episodes/24 hours) was reported in only six (16%) of the 38 evaluable patients over all treatment groups. Adverse events were generally mild or moderate and of a similar type and incidence to those of current 5-HT3 antagonists. Physicians' and patients' assessments of efficacy and tolerability of itasetron hydrochloride were similar, the majority rating the treatment as 'good' or 'very good'. In conclusion, itasetron hydrochloride is effective in the dose range 35-280 microg/kg in preventing cisplatin-induced emesis. Taken together with results from a larger dose-finding study, a dose corresponding to 35 microg/kg (equivalent to 2.5 mg itasetron, calculated as free base) has been pursued in Phase III studies with the i.v. formulation.

Twenty-four-hour intragastric acidity following early evening or bedtime administration of roxatidine in duodenal ulcer patients.

AIM: To assess the usefulness of early evening administration of roxatidine 150 mg as an alternative to the traditional bedtime regimen. METHODS: Twenty-four patients with healed duodenal ulcer were dosed according to a balanced incomplete-block design, with two of the following regimens: placebo, roxatidine 150 mg at 07.30 h (early evening) or roxatidine 150 mg at 22.00 h (bedtime). Twenty-four-hour intragastric pH-metry was started at 18.00 h on the first day of dosing. Median pH was determined for the 24-h period, and for the following time intervals: 20.00-00.00 h, 00.00-08.00 h and 08.00-18.00 h. Percentage time in the 24-h period with pH greater than 4.0 was also calculated. RESULTS: The two roxatidine regimens proved significantly superior to the placebo, decreasing 24-h acidity for all the time intervals, except the 20.00-00.00 h period, when mean intragastric pH with the early evening regimen (4.5 +/- 1.1) proved significantly higher than after placebo (2.2 +/- 1.0) or when roxatidine was taken at bedtime (2.4 +/- 1.1). CONCLUSION: Early evening administration of roxatidine may afford satisfactory control of 24-h acidity, offering a useful alternative to conventional bedtime administration.

A nuclear protein, synthesized in growth-arrested human hepatoblastoma cells, is a novel member of the short-chain alcohol dehydrogenase family.

We have described a protein (Hep27) [Donadel, G., Garzelli, C., Frank, R. & Gabrielli, F. (1991) Eur. J. Biochem. 195, 723-729] which is synthesized and accumulated in the nucleus of human hepatoblastoma (HepG2) cells, following growth arrest induced by butyrate treatment. The synthesis of Hep27 is inhibited in cells that, released from the butyrate block, have resumed DNA synthesis. This report describes the cloning and the characterization of the cDNA coding for the Hep27 protein. The translation of the Hep27 cDNA predicts an amino acid sequence that can be aligned with those of the known short-chain alcohol dehydrogenase enzymes (SCAD) family. Both the recognition of enzymic functional domains and the similarity with the SCAD family of proteins of several amino acid blocks throughout the molecule, strongly suggest that this protein is a new member of the SCAD family. In agreement with its nuclear localization Hep27 has a region similar to the bipartite nuclear-targeting sequence. The study of Hep27 mRNA expression and protein synthesis suggests the existence of a regulation at the post-transcriptional level. The possible nuclear role of the Hep27 protein is discussed.

Double-blind randomized, multicenter study comparing aluminium phosphate gel with rantitidine in the short-term treatment of duodenal ulcer.

The efficacy and safety of aluminium phosphate and ranitidine in the short-term treatment of duodenal ulcer were compared in a multicenter, randomized, double-blind study. A total of 304 patients with endoscopically proven active duodenal ulcer were recruited: 153 received 11 g of aluminium phosphate gel five times a day (equivalent to a daily acid buffering capacity of 182 mEq/HC1) and 151 rantitidine 300 mg once daily for 6 weeks. At the end of the treatment period, 74 out of 113 patients (65%) treated with aluminium phosphate and 84 out of 105 patients (80%) treated with rantitidine showed ulcer healing at endoscopy (p = 0.02). Ulcer symptoms, identified as frequency and intensity of daytime and nocturnal epigastric pain, were significantly reduced by both treatments, but rantitidine proved more effective than aluminium phosphate in reducing the frequency of daytime pain (p < 0.01) and its severity (p < 0.01); in contrast, no significant differences were found with regard to frequency and severity of nocturnal pain. The incidence of unwanted effects was significantly higher in the aluminium phosphate group. The main adverse event observed was constipation which, however, was hardly ever severe enough to warrant discontinuing treatment.

Characterization of a functional NF-kappa B site in the human interleukin 1 beta promoter: evidence for a positive autoregulatory loop.

The -300 region of the interleukin 1 beta (IL-1 beta) promoter contains a functional NF-kappa B binding site composed of the decamer sequence 5'-GGGAAAATCC-3'. Probes representing the -300 region or the NF-kappa B site alone interacted with NF-kappa B proteins present in phorbol myristate acetate-, lipopolysaccharide-, or Sendai virus-induced myeloid cell extracts as well as recombinant NFKB1 (p50) and RelA (p65); furthermore, NF-kappa B protein-DNA complex formation was dissociated in vitro by the addition of recombinant I kappa B alpha. Mutation of the NF-kappa B site in the context of the IL-1 beta promoter reduced the responsiveness of the IL-1 beta promoter to various inducers, including phorbol ester, Sendai virus, poly(rI-rC), and IL-1 beta. A 4.4-kb IL-1 beta promoter fragment linked to a chloramphenicol acetyltransferase reporter gene was also preferentially inducible by coexpression of individual NF-kappa B subunits compared with a mutated IL-1 beta promoter fragment. When multiple copies of the IL-1 beta NF-kappa B site were linked to an enhancerless simian virus 40 promoter, this element was able to mediate phorbol ester- or lipopolysaccharide-inducible gene expression. In cotransfection experiments, RelA (p65) and c-Rel (p85) were identified as the main subunits responsible for the activation of the IL-1 beta NF-kappa B site; also, combinations of NFKB1 (p50) and RelA (p65) or c-Rel and RelA were strong transcriptional activators of reporter gene activity. The presence of a functional NF-kappa B binding site in the IL-1 beta promoter suggests that IL-1 positively autoregulates its own synthesis, since IL-1 is a strong inducer of NF-kappa B binding activity. Thus, the IL-1 beta gene may be considered as an important additional member of the family of cytokine genes regulated in part by the NF-kappa B/rel family of transcription factors.

Loop substitution as a tool to identify active sites of interleukin-1 beta.

By computer analysis of the amino acid sequence of human interleukin-1 beta (IL-1 beta) and of the human type I IL-1 receptor (IL-1RI), we have identified two hydropathically complementary peptides (Fassina, G., Roller, P. P., Olson, A. D., Thorgeirsson, S. S., and Omichinski, J. G. (1989) J. Biol. Chem. 264, 11252-11257) capable of binding to each other. The sequence of the IL-1 beta peptide corresponds to that of residues 88-99 (loop 7 of the crystal structure of mature IL-1 beta) of mature IL-1 beta, one of the exposed and highly charged regions of the molecule. The substitution of this loop with an amino acid sequence of the same length but different hydropathic profile generates a mutant with drastically reduced binding activity to IL-1RI. In contrast, the binding affinity to the type II IL-1R (IL-1RII) is the same as that of wild type IL-1 beta. The results show that 1) loop 7 is part of the binding site of IL-1 beta to IL-1RI, but not to IL-1RII. 2) The structure of the mutant protein is not grossly altered except locally at the position of the substituted loop. 3) The substitution of amino acids by site-directed mutagenesis of the loop 7 region generates mutants with binding affinity constants slightly lower than that of wild type IL-1 beta and not comparable to that of the loop substitution analogue. 4. All mutants analyzed, including the loop substitutions, are biologically active, confirming the structural integrity of the proteins. We propose a binding site in which the cooperation of several low energy bonds extended over a wide area results in a high affinity complex between IL-1 and the type I receptor.

Operative arthroscopy of the ankle.

The long-term results of 30 operative arthroscopies of the ankle performed from 1983 to 1989 are the basis of this study. The most frequent lesions are synovitis and osteochondral defects of the talus. The treatment consists of lavage, synovial debridement, osteochondral debridement, and the removal of loose bodies. Of the 30 cases, 86.7% obtained excellent or good results and 88% of the athletes returned to their sport.

An inducible enhancer controls the expression of the human interleukin 1 beta gene.

This paper describes regulatory sequences responsible for the control of human interleukin 1 beta gene expression in cells of the monocytic lineage. Hybrid plasmids containing different regions of the interleukin 1 beta gene and the bacterial chloramphenicol acetyltransferase gene were transfected into human monocytic THP-1 or U937 cells. After treatment with 12-O-tetradecanoylphorbol-13-acetate, which triggers cell differentiation, we have identified an enhancer sequence which mediates the induction of gene activity by 12-O-tetradecanoylphorbol-13-acetate. The enhancer, approximately 180 base pairs long, is located between positions -2982 and -2795 upstream from the transcriptional start site. Further analysis has shown that 132 base pairs immediately upstream from the start site of transcription are sufficient to promote constitutive expression of the gene. Additional promoter elements, located within the first intron, maximize the level of gene expression. Although activated monocytes and macrophages are the main producers of interleukin 1 beta, both the enhancer and the constitutive promoter can function in monocytic cells as well as in nonmonocytic HeLa cells.

Longterm treatment of irritable bowel syndrome with cimetropium bromide: a double blind placebo controlled clinical trial.

The aim of this study was to evaluate the efficacy of cimetropium bromide, a new antimuscarinic compound, in relieving symptoms of patients with irritable bowel syndrome over a three month period. Seventy consecutive outpatients were given cimetropium (50 mg tid) or placebo according to a double blind, randomised, parallel groups design. Symptoms were evaluated initially and at monthly intervals up to the end of the study period. One patient receiving placebo withdrew because of treatment failure. Pain score decreased by 40, 66, 85% in the cimetropium group, at the end of the first, second and third months respectively, compared with 26, 32 and 52% reductions among controls (p = 0.0005). At the end of treatment there was a 86% reduction in the number of abdominal pain episodes per day in the cimetropium group compared with 50% in the placebo group (p = 0.001). Constipation and diarrhoea scores decreased by 59 and 49% in the cimetropium treated patients, compared with 37 and 39% in controls, the differences between being not significant. At the end of the study 89% of the patients treated with cimetropium considered themselves as globally improved as opposed to 69% in the placebo group (p = 0.039). The corresponding 95% confidence intervals for the differences between the proportion of improved patients in the two groups were from 11% to 29%. Six patients taking cimetropium complained of slight dry mouth. The results of this study showed that cimetropium bromide is effective in relieving pain in patients with irritable bowel syndrome.

Treatment of chronic erosive gastritis: a double-blind trial of pirenzepine and cimetidine.

In a double-blind study, 59 patients with chronic erosive gastritis received 50 mg of pirenzepine twice daily and 55 patients received 400 mg of cimetidine twice daily for six weeks. In both groups, days of pain, of heartburn, and of nausea per week were significantly reduced during treatment (P less than 0.01). After six weeks, 64% of the pirenzepine group and 62% of the cimetidine group were free of symptoms and endoscopy revealed healing of lesions in 78% and 80%, respectively. Differences between groups were not significant.

[Effect of cimetropium bromide in the treatment of colic in the first 3 months of life. Randomized study]. / Effetto di cimetropio bromuro nel trattamento delle coliche del primo trimestre. Studio randomizzato.

UNLABELLED: 20% of unweaned children aged below three months suffer from so-called gaseous colic. The effectiveness and tolerance of an antimuscarinic drug with high spasmolytic activity on the smooth visceral musculature, cimetropium bromide, has been evaluated, excluding cases of intolerance to cow's milk and other pathologies. 40 random patients of both sexes of average age 4.4 weeks (symptoms lasting for one week with crying fits lasting for more than 90 minutes consecutively, on at least 3 days a week) split into two groups of 20 patients each were studied. TREATMENT: A) 1.2 mg/kg 1 hour before bottle; B) 2.0 mg/kg 1 hour before bottle. Speedy reduction in the number of crying episodes (Group A: from 2.8 +/- 0.3 to 0.1 +/- 0.1; Group B: from 2.8 +/- 0.4 to 0.6 +/- 0.4; differences n.s.) and in their duration (Group A: 99 min +/- 10 min to 5 min +/- 3 min; Group B: from 121 min +/- 11 min to 15 min +/- 9 min; differences n.s.). The pharmacological treatment was considered: Group A: very good in 75% of cases; Group B: very good in 70% of cases. In Group B 4 episodes of stypsis occurred and these resolved immediately upon suspension of the drug. Given the equal effectiveness and better tolerance, the use of cimetropium bromide is recommended at the lower dosage.

Hepatic lipid peroxidation and aldehyde dehydrogenase activity in alcoholic and non alcoholic liver disease.

It has been suggested that lipid peroxidation plays a role in the pathogenesis of chronic alcoholic liver disease (CALD). However, whether or not CALD differs from chronic non alcoholic liver disease (CLD) in lipid peroxidation, is still questionable. Thirty-eight patients affected by CALD and CLD who were matched for age, sex, nutrition and liver function tests (LFTs) and 17 controls (C) took part in this study. The following tests were performed: serum and liver malondialdehyde (MDA) determination by the TBA test, liver total glutathione (GSH) estimate, mitochondrial (ALDH2) and cytosolic (ALDH1) aldehyde dehydrogenase activity determinations. Patients who showed signs of malnutrition were excluded from this study. Serum and hepatic TBA-reactive substances resulted in a slight increase in chronic liver patients compared to controls but did not show any difference between CALD and CLD groups. Liver total glutathione did not show any change. Hepatic ALDH2 activity was significantly (P less than 0.01) higher in CALD than in CLD and control patients whereas ALDH1 did not show any difference. These results suggest that the increased lipid peroxidation in CALD and in CLD is probably secondary to liver damage rather than being the pathogenic factor.

Human interleukin-1 beta gene.

We report the nucleotide sequence of the human chromosomal gene which encodes the interleukin-1 beta protein (IL-1 beta). The gene spans a region of 7.5 kb and the coding part is divided into seven exons. Comparison with the homologous mouse gene reveals that the structural organization is conserved through evolution. In addition to this, human and murine IL-1 beta genes show extensive sequence homology within the intervening sequences.

The genome of Bacillus subtilis phage SPP1: structure of an early promoter.

The strongest of five 'early' promoters of Bacillus subtilis phage SPP1 was localized in a DNA restriction fragment by analysis of RNA polymerase binding and R-loop formation. The nucleotide sequence of the promoter region was established. The signal structures identified were similar to those recognized by the sigma 55 RNA polymerase of B. subtilis. The promoter precedes an open reading frame with 51 codons. A protein with the Mr predicted from the nucleotide sequence was identified in minicells.