The safety and efficacy of inhaled dry powder mannitol as a bronchial provocation test for airway hyperresponsiveness: a phase 3 comparison study with hypertonic (4.5%) saline

Background

Inhaled mannitol is a new bronchial provocation test (BPT) developed to improve portability and standardisation of osmotic challenge testing. Osmotic challenge tests have an advantage over the traditional methods of measuring airway hyperresponsiveness using methacholine as they demonstrate higher specificity to identify asthma and thus the need for treatment with inhaled corticosteroids (ICS). The safety and the efficacy of mannitol (M) as a BPT to measure airway hyperresponsiveness were compared to hypertonic (4.5%) saline (HS) in people both with and without signs and symptoms of asthma.

Methods

A phase III, multi-centre, open label, operator-blinded, crossover design, randomised trial, with follow-up. Asthmatics and non-asthmatics (6–83 yr) were recruited and 592 subjects completed the study. Mannitol was delivered using a low resistance dry powder inhaler and HS was delivered using an ultrasonic nebuliser. The FEV₁ was measured 60 seconds after each dose of mannitol (5,10,20,40,80,160,160,160 mg) and after each exposure to HS (0.5,1.0,2.0,4.0,8.0 minutes). A 15% fall in FEV₁ defined a positive test. Adverse events were monitored and diaries kept for 7 days following the tests.

Results

Mean pre-test FEV1 (mean ± SD) was 95.5 ± 14% predicted. 296 were positive to mannitol (M+) and 322 positive to HS (HS+). A post study physician conducted clinical assessment identified 82.3% asthmatic (44% classified mild) and 17.7% non-asthmatic. Of those M+, 70.1% were taking ICS and of those mannitol negative (M-), 81.1 % were taking ICS. The % fall in FEV1 for mannitol in asthmatics was 21.0% ± 5.7 and for the non-asthmatics, 5.5% ± 4.8. The median PD15 M was 148 mg and PD15 HS 6.2 ml. The sensitivity of M to identify HS+ was 80.7% and the specificity 86.7%. The sensitivity of M compared with the clinical assessment was 59.8% and specificity 95.2% and increased to 88.7% and 95.0% respectively when the M- subjects taking ICS were excluded. Cough was common during testing. There were no serious adverse events. The diarised events were similar for mannitol and HS, the most common being headache (17.2%M, 19%HS), pharyngolaryngeal pain (5.1%M, 3%HS), nausea (4.3%M, 3%HS), and cough (2.2%M, 2.4%HS).

Conclusion

The efficacy and safety of mannitol was demonstrated in non-asthmatic and clinically diagnosed asthmatic adults and children.     

Aberrant E-cadherin staining patterns in invasive mammary carcinoma

Background

Advances in our understanding of the molecular biology of colorectal cancer have fuelled the search for novel molecular prognostic markers to complement existing staging systems. Markers assessed in combination may perform better than those considered individually. Using high-throughput tissue microarray technology, we describe the prognostic value of combined p53 / Bcl-2 status in colorectal cancer.

Patients and methods

Tumour samples from 462 patients who underwent elective surgery to resect a primary colorectal cancer between 1994 and 2000 (mean follow-up of 75 months) were assembled in tissue microarray format. Clinico-pathological data including tumour grade, stage, vascular invasion status along with disease specific survival data has been collected prospectively. Immunohistochemical analysis of p53 and Bcl-2 expression was performed using antibodies DO-7 (p53) and 124 (Bcl-2), and results correlated with known clinico-pathological variables and outcomes.

Results

Abnormal nuclear p53 accumulation and Bcl-2 overexpression were detected in 221/445 (49.6%) and199/437 (45.5%) tumours respectively, with a significant inverse correlation between the two markers (p = 0.023). On univariate analysis no correlations were found between either marker and standard clinico-pathological variables, however nuclear p53 expression was associated with a significantly reduced survival (p = 0.024). Combined analysis of the two markers indicated that 112/432 (24.2%) cases displayed a p53(-)/Bcl-2(+) phenotype, this occurring more frequently in earlier stage tumours. Kaplan-Meier analysis revealed a significant survival advantage in these p53(-)/Bcl-2(+) tumours compared with the remaining cases (p = 0.0032). On multivariate analysis using the Cox proportional hazards model, neither p53 expression nor Bcl-2 expression alone were of independent prognostic significance, however the combined p53(-)/Bcl-2(+) phenotype was significantly associated with a good prognosis in this series (HR 0.659, 95%CI 0.452–0.959, p = 0.029).

Conclusion

Patient stratification by combined p53 / Bcl-2 phenotype provides stage-independent prognostic information in colorectal cancer. Specifically, that up to a quarter of patients display a good prognosis p53(-)/Bcl-2(+) phenotype. This may indicate a more clinically indolent phenotype and a subset of patients for whom less aggressive adjuvant treatment appropriate.     

Comparative proteomics using 2-D gel electrophoresis and mass spectrometry as tools to dissect stimulons and regulons in bacteria with sequenced or partially sequenced genomes

We propose two-dimensional gel electrophoresis (2-DE) and mass spectrometry to define the protein components of regulons and stimulons in bacteria, including those organisms where genome sequencing is still in progress. The basic 2-DE protocol allows high resolution and reproducibility and enables the direct comparison of hundreds or even thousands of proteins simultaneously. To identify proteins that comprise stimulons and regulons, peptide mass fingerprint (PMF) with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS) analysis is the first option and, if results from this tool are insufficient, complementary data obtained with electrospray ionization tandem-MS (ESI-MS/MS) may permit successful protein identification. ESI-MS/MS and MALDI-TOF-MS provide complementary data sets, and so a more comprehensive coverage of a proteome can be obtained using both techniques with the same sample, especially when few sequenced proteins of a particular organism exist or genome sequencing is still in progress.     

Contribution of epigenetic silencing of tumor necrosis factor-related apoptosis inducing ligand receptor 1 (DR4) to TRAIL resistance and ovarian cancer

Dysregulation of apoptosis may support tumorigenesis by allowing cells to live beyond their normally intended life span. The various receptors for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) are located on chromosome 8p21.2, a region frequently deleted in ovarian cancer. Lack of expression of TRAIL receptor 1 (death receptor 4, DR4) correlates with resistance to TRAIL-induced apoptosis in ovarian cancer cells. Reconstitution of DR4 in the TRAIL-resistant A2780 ovarian cancer cell line was investigated with the demethylating agent 5-aza-2'-deoxycytidine and transient gene transfer. Regulation of other genes in the TRAIL pathway by 5-aza-2'-deoxycytidine was assessed in DNA GeneChip experiments. Primary ovarian cancers were analyzed by methylation-specific PCR and immunohistochemical analysis of a tissue microarray. Regulation of DR4 expression by demethylation or transient transfection is of functional relevance for TRAIL resistance in an ovarian cancer cell line. Hypermethylation of the DR4 promoter could be found in 10 of 36 (27.7%) DNAs isolated from ovarian cancer tissue. In an independent set of 68 ovarian cancer cases, a complete loss or down-regulation of DR4 protein expression was observed 10.3% and 8.8% patients, respectively. A significant (P = 0.019) majority of these patients was below 50 years of age. Our findings show a functional relevance of the level of DR4 expression in ovarian cancer and suggest a substantial contribution of DR4 hypermethylation and consequent loss of DR4 expression to ovarian cancer pathogenesis, particularly in premenopausal patients.     

Medical sequencing of candidate genes for nonsyndromic cleft lip and palate

Nonsyndromic or isolated cleft lip with or without cleft palate (CL/P) occurs in wide geographic distribution with an average birth prevalence of 1/700. We used direct sequencing as an approach to study candidate genes for CL/P. We report here the results of sequencing on 20 candidate genes for clefts in 184 cases with CL/P selected with an emphasis on severity and positive family history. Genes were selected based on expression patterns, animal models, and/or role in known human clefting syndromes. For seven genes with identified coding mutations that are potentially etiologic, we performed linkage disequilibrium studies as well in 501 family triads (affected child/mother/father). The recently reported MSX1 P147Q mutation was also studied in an additional 1,098 cleft cases. Selected missense mutations were screened in 1,064 controls from unrelated individuals on the Centre d'Étude du Polymorphisme Humain (CEPH) diversity cell line panel. Our aggregate data suggest that point mutations in these candidate genes are likely to contribute to 6% of isolated clefts, particularly those with more severe phenotypes (bilateral cleft of the lip with cleft palate). Additional cases, possibly due to microdeletions or isodisomy, were also detected and may contribute to clefts as well. Sequence analysis alone suggests that point mutations in FOXE1, GLI2, JAG2, LHX8, MSX1, MSX2, SATB2, SKI, SPRY2, and TBX10 may be rare causes of isolated cleft lip with or without cleft palate, and the linkage disequilibrium data support a larger, as yet unspecified, role for variants in or near MSX2, JAG2, and SKI. This study also illustrates the need to test large numbers of controls to distinguish rare polymorphic variants and prioritize functional studies for rare point mutations.     

Distribution and function of biogenic amines in the heart of <Emphasis Type="Italic">Nautilus pompilius</Emphasis> L. (Cephalopoda,Tetrabranchiata)

Summary Biogenic amines (serotonin and catecholamines), play an important role in the control of the blood flow not only in vertebrates, but also in invertebrates such as cephalopods. In contrast to the well investigated hearts of the ȁ8modern,ȁ9 coleoid cephalopods, the innervation of the heart of the archaic Nautilus pompilius L. has not been studied in detail. In this study the distribution and effects of biogenic amines in the Nautilus heart were investigated. Serotonin and catecholamines were visualised by the glyxoylic acid induced fluorescence. High performance liquid chromatotography analysis was performed to discriminate between the catecholamines, which showed a high content of noradrenaline in the 4 auricles, the aorta and the ventricle, whereas the ventricle showed a high dopamine content. Adrenaline was found at a very low concentration in the ventricle. Serotonin and dopamine were also immunohistochemically localised to larger nerves and throughout the heart, respectively. In organ bath experiments, the auricles showed little spontaneous activity. After adding serotonin, they displayed rhythmical contractions, which were accelerated dose-dependently by noradrenaline. In summary, these data suggest an important role for biogenic amines in the control of the heart of Nautilus pompilius L., with serotonin possibly stimulating excitatory nerve fibres, whereas noradrenaline is likely to influence the muscle contraction itself.     

Protein sequence tags: a novel solution for comparative proteomics

Comparative proteome profiling using stable isotope peptide labelling and mass spectrometry has emerged as a promising strategy. Here, we show the broad potential of our proprietary protein sequence tag (PST) technology. A special feature of PST is its ability to detect a wide variety of proteins including the pharmaceutically relevant membrane and nuclear proteins. This procedure addresses a similar number of proteins, compared to the multidimensional protein identification technology approach, but offers additionally a quantitative analysis with its recently developed quantitative PST version.     

A two-dimensional electrophoretic map of human mitochondrial proteins from immortalized lymphoblastoid cell lines: a prerequisite to study mitochondrial disorders in patients

Mitochondrial diseases may be caused by numerous mutations that alter proteins of the respiratory chain and of other metabolic pathways in the mitochondrium. For clinicians this disease group poses a considerable diagnostic challenge due to ambiguous genotype-phenotype relationships. Until now, only 30% of the mitochondriopathies can be diagnosed at the molecular level. We therefore need a new diagnostic tool that offers a wide view on the mitochondrial proteins. Here, we present a method to generate a high-resolution, large-gel two-dimensional gel electrophoretic (2-DE) map of a purified fraction of mitochondrial proteins from Epstein-Barr virus-immortalized lymphoblastoid cell line (LCL). LCLs can be easily obtained from patients and control subjects in a routine clinical setting. They often express the biochemical phenotype and can be cultured to high cell numbers, sufficient to gain enough purified material for 2-DE. In total we identified 166 mitochondrial proteins. Thirteen proteins were earlier not known to be of mitochondrial origin. Thirty-nine proteins were associated with human diseases ranging from respiratory chain enzyme deficiencies to disorders of beta-oxidation and amino acid metabolism. This 2-DE map is intended to be the first step to diagnose mitochondrial diseases at the proteomic level.