Lineage-specific RUNX3 hypomethylation marks the preneoplastic immune component of gastric cancer.

Runt domain transcription factor 3 (RUNX3) is widely regarded as a tumour-suppressor gene inactivated by DNA hypermethylation of its canonical CpG (cytidine-phosphate-guanidine) island (CGI) promoter in gastric cancer (GC). Absence of RUNX3 expression from normal gastric epithelial cells (GECs), the progenitors to GC, coupled with frequent RUNX3 overexpression in GC progression, challenge this longstanding paradigm. However, epigenetic models to better describe RUNX3 deregulation in GC have not emerged. Here, we identify lineage-specific DNA methylation at an alternate, non-CGI promoter (P1) as a new mechanism of RUNX3 epigenetic control. In normal GECs, P1 was hypermethylated and repressed, whereas in immune lineages P1 was hypomethylated and widely expressed. In human GC development, we detected aberrant P1 hypomethylation signatures associated with the early inflammatory, preneoplastic and tumour stages. Aberrant P1 hypomethylation was fully recapitulated in mouse models of gastric inflammation and tumorigenesis. Cell sorting showed that P1 hypomethylation reflects altered cell-type composition of the gastric epithelium/tumour microenvironment caused by immune cell recruitment, not methylation loss. Finally, via long-term culture of gastric tumour epithelium, we revealed that de novo methylation of the RUNX3 canonical CGI promoter is a bystander effect of oncogenic immortalization and not likely causal in GC pathogenesis as previously argued. We propose a new model of RUNX3 epigenetic control in cancer, based on immune-specific, non-CGI promoter hypomethylation. This novel epigenetic signature may have utility in early detection of GC and possibly other epithelial cancers with premalignant immune involvement.

Exploring transcriptional conservation between Ancylostoma caninum and Haemonchus contortus by oligonucleotide microarray and bioinformatic analyses.

In this study, we identified, using an established oligonucleotide microarray platform for the parasitic nematode Haemonchus contortus, transcripts that are 'conserved' between serum-activated and non-activated L3s of Ancylostoma caninum (aL3 and L3, respectively) and H. contortus by cross-species hybridization (CSH) at high stringency and conducted extensive bioinformatic analyses of the cross-hybridizing expressed sequence tags (ESTs). The microarray analysis revealed significant differential hybridization between aL3 and L3 for 32 molecules from A. caninum, of which 29 were shown to have homologues/orthologues in the free-living nematode Caenorhabditis elegans and/or A. caninum and the other three molecules had no homologues in current gene databases. 'Non-wildtype' RNAi phenotypes were recorded for 13 of the C. elegans homologues. A subset of 16 C. elegans homologues/orthologues (i.e. genes abce-1, act-2, C08H9.2, C55F2.1, calu-1, col-181, cpr-6, elo-2, asp-1, K07E3.4, rpn-2, sel-9, T28C12.4, hsb-1, Y57G11C.15 and ZK593.1) were predicted to interact genetically with a total of 156 (range 1-88) other genes. Gene ontology (GO) analysis of the interacting genes revealed that the most common subcategories were signal transduction (7%), intracellular protein transport and glycolysis (6.2%) within 'biological process'; nuclear (25.7%) and intracellular (19.8%) within 'cellular component'; and ATP-binding (14.4%) and protein-binding (8.4%) within 'molecular function'. The potential roles of key molecules in the two blood-feeding parasitic nematodes are discussed in relation to the known roles of their homologues/orthologues in C. elegans. The CSH approach used may provide a tool for the screening of genes conserved across a range of different taxa of parasites for which DNA microarray platforms are not available.

Do levels of airborne grass pollen influence asthma hospital admissions?

BACKGROUND: The effects of environmental factors and ambient concentrations of grass pollen on allergic asthma are yet to be established. OBJECTIVE: We sought to estimate the independent effects of grass pollen concentrations in the air over Melbourne on asthma hospital admissions for the 1992-1993 pollen season. METHODS: Daily grass pollen concentrations were monitored over a 24-h period at three stations in Melbourne. The outcome variable was defined as all-age asthma hospital admissions with ICD9-493 codes. The ambient air pollutants were average daily measures of ozone, nitrogen dioxide and sulphur dioxide, and the airborne particle index representing fine particulate pollution. Semi-parametric Poisson regression models were used to estimate these effects, adjusted for air temperature, humidity, wind speed, rainfall, day-of-the-week effects and seasonal variation. RESULTS: Grass pollen was a strong independent non-linear predictor of asthma hospital admissions in a multi-pollutant model (P=0.01). Our data suggest that grass pollen had an increasing effect on asthma hospital admissions up to a threshold of 30 grains/m3, and that the effect remains stable thereafter. CONCLUSION: Our findings suggest that grass pollen levels influence asthma hospital admissions. High grass pollen days, currently defined as more than 50 grains/m3, are days when most sensitive individuals will experience allergic symptoms. However, some asthmatic patients may be at a significant risk even when airborne grass pollen levels are below this level. Patients with pollen allergies and asthma would be advised to take additional preventive medication at lower ambient concentrations.

Mechanical devices for urinary incontinence in women.

BACKGROUND: Incontinence can have a devastating effect on the lives of sufferers with significant economic implications. Non-surgical treatments such as pelvic floor muscle training and the use of mechanical devices are usually the first line of management. The latter more so when a person did not want surgery or when considered unfit for surgery. Mechanical devices are inexpensive and do not compromise future surgical treatment. OBJECTIVES: To determine the effects of mechanical devices in the management of adult female urinary incontinence. SEARCH STRATEGY: We searched the Cochrane Incontinence Group Specialised Trials Register (7 December 2005). The register contains trials identified from MEDLINE, the Cochrane Central Register of Controlled Trials (CENTRAL), CINAHL and handsearching of journals and conference proceedings. SELECTION CRITERIA: All randomised or quasi-randomised controlled trials of mechanical devices in the management of adult female urinary incontinence determined either by symptom classification or by urodynamic diagnosis. DATA COLLECTION AND ANALYSIS: Three reviewers assessed the identified studies for eligibility and methodological quality and independently extracted data from the included studies. Data analysis was performed using RevMan software (version 4.2). MAIN RESULTS: There were six trials involving a total of 286 women. Two small trials compared a mechanical device with no treatment and although they suggested that use of a mechanical device might be better than no treatment, the evidence for this was inconclusive. Five trials compared one mechanical device with another. Quantitative synthesis of data from these trials was not possible because different mechanical devices were compared in each trial using different outcome measures. Data from the individual trials showed no clear difference between devices, but with wide confidence intervals. There were no trials comparing a mechanical device with another type of treatment. AUTHORS' CONCLUSIONS: The place of mechanical devices in the management of urinary incontinence remains in question. Currently there is little evidence from controlled trials on which to judge whether their use is better than no treatment and a large well-conducted trial is required for clarification. There was also insufficient evidence in favour of one device over another and no evidence to compare mechanical devices with other forms of treatment.

Mechanical devices for urinary incontinence in women.

BACKGROUND: Incontinence can have a devastating effect on the lives of sufferers with significant economic implications. Non-surgical treatments such as pelvic floor muscle training and the use of mechanical devices are usually the first line of management. The latter more so when a person did not want surgery or when considered unfit for surgery. Mechanical devices are inexpensive and do not compromise future surgical treatment. OBJECTIVES: To determine the effects of mechanical devices in the management of adult female urinary incontinence. SEARCH STRATEGY: We searched the Cochrane Incontinence Group Specialised Trials Register (7 December 2005). The register contains trials identified from MEDLINE, the Cochrane Central Register of Controlled Trials (CENTRAL), CINAHL and handsearching of journals and conference proceedings. SELECTION CRITERIA: All randomised or quasi-randomised controlled trials of mechanical devices in the management of adult female urinary incontinence determined either by symptom classification or by urodynamic diagnosis. DATA COLLECTION AND ANALYSIS: Three reviewers assessed the identified studies for eligibility and methodological quality and independently extracted data from the included studies. Data analysis was performed using RevMan software (version 4.2). MAIN RESULTS: There were six trials involving a total of 286 women. Two small trials compared a mechanical device with no treatment and although they suggested that use of a mechanical device might be better than no treatment, the evidence for this was inconclusive. Five trials compared one mechanical device with another. Quantitative synthesis of data from these trials was not possible because different mechanical devices were compared in each trial using different outcome measures. Data from the individual trials showed no clear difference between devices, but with wide confidence intervals. There were no trials comparing a mechanical device with another type of treatment. AUTHORS' CONCLUSIONS: The place of mechanical devices in the management of urinary incontinence remains in question. Currently there is little evidence from controlled trials on which to judge whether their use is better than no treatment and a large well-conducted trial is required for clarification. There was also insufficient evidence in favour of one device over another and no evidence to compare mechanical devices with other forms of treatment.

Constructing "sound science" and "good epidemiology": tobacco, lawyers, and public relations firms.

The tobacco industry has attacked "junk science" to discredit the evidence that secondhand smoke-among other environmental toxins-causes disease. Philip Morris used public relations firms and lawyers to develop a "sound science" program in the United States and Europe that involved recruiting other industries and issues to obscure the tobacco industry's role. The European "sound science" plans included a version of "good epidemiological practices" that would make it impossible to conclude that secondhand smoke-and thus other environmental toxins-caused diseases. Public health professionals need to be aware that the "sound science" movement is not an indigenous effort from within the profession to improve the quality of scientific discourse, but reflects sophisticated public relations campaigns controlled by industry executives and lawyers whose aim is to manipulate the standards of scientific proof to serve the corporate interests of their clients.

A human monoclonal IgE antibody defines a highly allergenic fragment of the major timothy grass pollen allergen, Phl p 5: molecular, immunological, and structural characterization of the epitope-containing domain.

Almost 90% of grass pollen-allergic patients are sensitized against group 5 grass pollen allergens. We isolated a monoclonal human IgE Fab out of a combinatorial library prepared from lymphocytes of a grass pollen-allergic patient and studied its interaction with group 5 allergens. The IgE Fab cross-reacted with group 5A isoallergens from several grass and corn species. By allergen gene fragmentation we mapped the binding site of the IgE Fab to a 11.2-kDa N-terminal fragment of the major timothy grass pollen allergen Phl p 5A. The IgE Fab-defined Phl p 5A fragment was expressed in Escherichia coli and purified to homogeneity. Circular dichroism analysis revealed that the rPhl p 5A domain, as well as complete rPhl p 5A, assumed a folded conformation consisting predominantly of an alpha helical secondary structure, and exhibited a remarkable refolding capacity. It reacted with serum IgE from 76% of grass pollen-allergic patients and revealed an extremely high allergenic activity in basophil histamine release as well as skin test experiments. Thus, the rPhl p 5A domain represents an important allergen domain containing several IgE epitopes in a configuration optimal for efficient effector cell activation. We suggest the rPhl p 5A fragment and the corresponding IgE Fab as paradigmatic tools to explore the structural requirements for highly efficient effector cell activation and, perhaps later, for the development of generally applicable allergen-specific therapy strategies.

Tobacco industry efforts subverting International Agency for Research on Cancer's second-hand smoke study.

Scientific reports on second-hand smoke have stimulated legislation on clean indoor air in the USA, but less so in Europe. Recently, the largest European study, by the International Agency for Research on Cancer (IARC), demonstrated a 16% increase in the point estimate of risk in lung cancer for nonsmokers, a result consistent with earlier studies. However, the study was described by newspapers and the tobacco industry as demonstrating no increase in risk. To understand the tobacco industry's strategy on the IARC study we analysed industry documents released in US litigation and interviewed IARC investigators. The Philip Morris tobacco company feared that the study (and a possible IARC monograph on second-hand smoke) would lead to increased restrictions in Europe so they spearheaded an inter-industry, three-prong strategy to subvert IARC's work. The scientific strategy attempted to undercut IARC's research and to develop industry-directed research to counter the anticipated findings. The communications strategy planned to shape opinion by manipulating the media and the public. The government strategy sought to prevent increased smoking restrictions. The IARC study cost $2 million over ten years; Philip Morris planned to spend $2 million in one year alone and up to $4 million on research. The documents and interviews suggest that the tobacco industry continues to conduct a sophisticated campaign against conclusions that second-hand smoke causes lung cancer and other diseases, subverting normal scientific processes.

Plant homologue of human excision repair gene ERCC1 points to conservation of DNA repair mechanisms.

Nucleotide excision repair (NER), a highly versatile DNA repair mechanism, is capable of removing various types of DNA damage including those induced by UV radiation and chemical mutagens. NER has been well characterized in yeast and mammalian systems but its presence in plants has not been reported. Here it is reported that a plant gene isolated from male germline cells of lily (Lilium longiflorum) shows a striking amino acid sequence similarity to the DNA excision repair proteins human ERCC1 and yeast RAD10. Homologous genes are also shown to be present in a number of taxonomically diverse plant genera tested, suggesting that this gene may have a conserved function in plants. The protein encoded by this gene is able to correct significantly the sensitivity to the cross-linking agent mitomycin C in ERCC1-deficient Chinese hamster ovary (CHO) cells. These findings suggest that the NER mechanism is conserved in yeast, animals and higher plants.

A study of pollen prevalence in relation to pollen allergy in Malaysian asthmatics.

In this paper we report results of skin prick tests (SPT) using pollen extracts on 200 patients with clinical symptoms of asthma, and results of a parallel study in which pollen was collected and classified over a period of 18 months. The patients were outpatients from the University Hospital in Kuala Lumpur, Malaysia, while the pollen grains were collected with a spore trap placed in the campus of the University of Malaya, approximately one kilometer from the University Hospital. Pollen extracts of 3 grasses (Bahia, Bermuda, rough pigweed) and 2 flowering trees, Acacia and Melaleuca, were used in the SPT. Of the 29.5% asthmatics with positive SPT reactions, 21.5% were to one or more of the grass pollens, 21.5% to Acacia and 7.5% to Melaleuca pollen. Acacia and Bermuda grass extracts were the most allergenic, which agreed with results of the pollen collection which showed grass and Acacia pollen grains to be the two most commonly found pollens.

Efficacy and safety of oral desmopressin in the treatment of primary nocturnal enuresis in Asian children.

OBJECTIVE: To determine the efficacy and safety of oral desmopressin (DDAVP) treatment in Asian children with nocturnal enuresis. METHODOLOGY: This was a multicentre randomized placebo-controlled double-blind cross-over study. Patients were randomized to either active treatment with oral 400 mg DDAVP or placebo, with a 2-week medication-free period between the cross-over. Children with primary monosymptomatic nocturnal enuresis, aged between 7 and 18 years, with a minimum frequency of wetting of 6 nights or more during a 2-week observation period were recruited. Efficacy was measured by reduction in the average number of wet nights per week. RESULTS: Of the 37 children initially recruited, the outcomes for 34 children were included in the final cross-over analysis, as they had complete data for both the treatment periods. Statistical analysis by ANOVA showed that there was no significant difference between the medication-free period and the pretreatment period. However, the average number of wet nights per week for the DDAVP treatment period (2.5+/-2.7) was significantly lower than that of the placebo treatment period (4.5+/-2.1) (P < 0.0001). In terms of the safety profile, there was no significant change in bodyweight, blood pressure, serum sodium, serum osmolality, and urine osmolality following DDAVP treatment. CONCLUSION: Oral DDAVP is a safe and efficacious drug for the short-term treatment of children with primary nocturnal enuresis.

Efficacy of bronchodilators in the treatment of bronchiolitis.

UNLABELLED: Bronchiolitis is a common respiratory infection affecting young children. Much controversy revolves around the efficacy of bronchodilators in the treatment of bronchiolitis. This study was conducted to address this issue. AIM: To determine the efficacy of bronchodilators in the treatment of bronchiolitis. METHOD: All children less than 2 years old with bronchiolitis were randomly assigned to receive nebulisations of Salbutamol, Ipratropium bromide or normal saline. A fourth group given only humidified oxygen without nebulisation were used as a control. RESULTS: Data were obtained for 120 patients. Fifty-one (42%) had respiratory syncytial virus (RSV) isolated from their nasopharyngeal aspirates. The demographic characteristics of the 4 groups were similar. There was no significant difference between the groups in terms of severity score, number of nebulisations required in the nebulised groups and the outcome as measured by the length of hospitalisation. CONCLUSION: The use of bronchodilators did not alter the course of the disease and is therefore not effective in the treatment of bronchiolitis.

Primary monosymptomatic nocturnal enuresis in Singapore--parental perspectives in an Asian community.

Primary monosymptomatic nocturnal enuresis (PMNE) is often not openly discussed in Asian societies. We report the parental view of PMNE in Singapore, its impact on patients and their families and the traditional beliefs and its influence on subsequent management. A screening questionnaire was used in evaluating 30 children enrolled in a clinical trial on the use of oral Desmopressin for the treatment of PMNE. Primary monosymptomatic nocturnal enuresis was familial in 56.7% of patients. Fifty per cent of them were previously unevaluated. Earlier remedial attempts included bedtime fluid restriction and voiding (100%), incentive measures (43.3%), traditional practices (26.7%), punishment (20%), drugs (16.7%), psychotherapy (100%) and bladder training (3.3%). Perceived causes of PMNE were maturational delay (50%), deep sleep (50%), familial (43.3%), behavioural problems (43.3%) and excessive fluid intake (26.7%). Reasons for seeking treatment included restricted outdoor activities (90%), parental fatigue (86.7%), disrupted sleep for the household (46.7%) and fear of underlying pathology (26.7%). Perceived adverse effects on patients included social stigma (83.3%), disrupted sleep (33.3%) and impaired school performance (13.3%). Primary monosymptomatic nocturnal enuresis can thus be a chronic distressing problem in Asian communities.

Recombinant expression and epitope mapping of grass pollen allergens.

We have studied the expression of recombinant forms of Group 1 allergens from rye-grass and Bermuda grass pollens. Recombinant Lol p 1 expressed in bacteria bound serum IgE from allergic patients. Based on analysis of fragments of the Lol p 1 cDNA clone, the major IgE-reactive epitope has been mapped to the C-terminus. However, although SDS-denatured natural Cyn d 1 (from Bermuda grass) bound IgE, the full or partial recombinant proteins expressed in bacteria did not bind IgE. We have since expressed Cyn d 1 in the yeast Pichia pastoris and restored IgE binding. cDNA clones encoding two isoforms of Lol p 5, Lol p 5A and Lol p 5B, have been expressed in bacteria and resulting polypeptides show IgE-binding. Random fragments of these clones have been generated and when expressed as partial recombinant proteins in bacteria, allowed us to identify the major IgE-binding epitopes. The allergenic epitopes were localised towards the C-terminal half of the molecule. Although both isoforms shared similar IgE-reactive epitopes, Lol p 5B did not recognise the Lol p 5A-specific monoclonal antibody A7. At sequence level, there appear to be several amino acid differences between the antigenic epitopes of these two isoallergens. These results aid in the design of diagnostics and in grass pollen immunotherapy.

Unusual manifestations of Mycoplasma pneumoniae infection in children.

Mycoplasma pneumoniae infection is no longer a benign condition it was originally thought to be. Many extrapulmonary manifestations affecting major organ systems like the central nervous system, cardiovascular system, haematological system, gastrointestinal system, musculoskeletal system and renal system have been described. Early recognition of these manifestations is often difficult and serological diagnosis may not be helpful. Three patients with large pleural effusions, encephalitis, hemiplegia, hepatitis, autoimmune haemolytic anaemia and renal failure are discussed to highlight the many varied presentations associated with this infection.

Mapping of the antigenic and allergenic epitopes of Lol p VB using gene fragmentation.

The recombinant proteins of Lol p VA and Lol p VB expressed in E. coli reacted with IgE antibodies from sera of allergic patients and mAbs FMC A7 and PpV1. Cross-absorption analyses using these recombinant proteins showed that Lol p VA and Lol p VB possess both similar and unique IgE binding determinants. Gene fragmentation was utilized to localize the antigenic and allergenic determinants of Lol p VB. When full-length cDNA of Lol p VB was digested into three fragments and expressed as the fusions from the glutathione transferase of pGEX vectors, fragments Met1-Val196 and Asp197-Val339 bound IgE while fragment Met1-Pro96 did not. The data suggest that there are at least two IgE binding determinants in Lol p VB. In addition, only fragment Met1-Val196 reacted with mAb PpV1. The localization of these determinants was further resolved using random fragment expression libraries. The mAb PpV1 determinant was near the N-terminal region of Lol p VB molecule. The IgE binding determinants were distributed in the central region: region I (amino acids 111-195) and II (199-254). These IgE binding determinants are conserved in Lol p VA.

Immunological relationships among group I and group V allergens from grass pollen.

Specific IgE antibodies have been affinity-purified from recombinant grass pollen allergens, and used to identify isoforms of the two major allergens of rye-grass pollen, Lol p I and Lol p V and cross-reactive allergens in other grasses. Lol p I-specific IgE (affinity-purified from the recombinant protein expressed by clone 13R which encodes amino acids 96-240 of Lol p I) identified four isoforms of the allergen. The same probe recognized cross-reactive epitopes in pollen proteins from 14 out of 16 grasses. The allergens identified by Lol p V-specific IgE (affinity-purified from the recombinant protein expressed by clones 12R or 19R which encode the full Lol p V protein) varied more in their physicochemical characteristics than the Group I isoforms. At least eight isoforms of Lol p V were identified by the Lol p V-specific IgE. The same probe recognized cross-reactive epitopes in pollen protein from 13 out of 16 grasses. Group I proteins were identified in grasses from two sub-families of the Poaceae, while the Group V allergens were only identified in pollen of grasses from one sub-family, the Pooideae.

Cloning of a cDNA encoding a group-V (group-IX) allergen isoform from rye-grass pollen that demonstrates specific antigenic immunoreactivity.

We have isolated and characterized the cDNA clone, 19R, that encodes an isoform of a major rye-grass pollen allergen, Lol p V [previously referred to as Lol p 1b; Singh et al., Proc. Natl. Acad. Sci. USA 88 (1991) 1384-1388; and Lol p IX; Suphioglu et al., Lancet 339 (1992) 569-572]. Clone 19R was isolated from a rye-grass pollen cDNA expression library using grass pollen-specific immunoglobulin E (IgE) antibodies (Ab) from an allergic serum pool. The nucleotide (nt) sequence of clone 19R potentially encodes a 33.8-kDa protein of 339 amino acids (aa). It possesses a leader peptide essentially identical to the previously characterized isoform of Lol p V (Lol p VA). This indicates a mature processed 31.3-kDa protein of 314 aa, correlating well with the size of the polypeptides revealed by Western analysis of pollen proteins using IgE Ab affinity purified from recombinant fusion protein (reFP) encoded by clone 19R as solid matrix. There is no N-glycosylation motif. The protein encoded by clone 19R, designated Lol p VB, has 66.4% identity and 80.4% similarity with Lol p VA. However, a Lol p VA-specific monoclonal Ab, FMC A7, does not recognize reFP encoded by clone 19R, indicating that Lol p VB does not share this epitope. Cross-reactivity studies using affinity purified IgE Ab showed that both isoforms share similar allergenic epitopes. Immunoblot analysis using sera from a population of 30 patients showed that 80% possess IgE Ab that recognize both Lol p V isoforms. Variation occurred in the signal intensities of IgE binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunodetection methods for grass pollen allergens on western blots.

A comparison is made of eight different methods to detect allergenic proteins in Western blots of rye-grass pollen extracts. Horseradish peroxidase-based enhanced chemiluminescence (ECL) provides a sensitive method for the detection of allergenic proteins. The method has been modified to use more dilute solutions of ECL substrate to reduce the background, can be applied to a standard nitrocellulose membrane, and used with Kodak X-ray film. The assays can be performed rapidly, replacing use of radiolabelled probes. Increased resolution is obtained. This makes the method suitable for detection of cDNA clones on plaque lifts, and for rapid and specific purification of proteins following immunodetection on nitrocellulose membranes.