Evaluation of multiplex ligation-dependent probe amplification as a method for the detection of copy number abnormalities in B-cell precursor acute lymphoblastic leukemia.

Recent genomic studies have shown that copy number abnormalities (CNA) of genes involved in lymphoid differentiation and cell cycle control are common in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). We have evaluated Multiplex Ligation-dependent Probe Amplification (MLPA) on 43 BCP-ALL patients for the detection of the most common deletions among these genes and compared the results to those obtained by fluorescence in situ hybridization (FISH) and genomic quantitative PCR (qPCR). There was good correlation between methods for CDKN2A/B, IKZF1, and PAX5 deletions in the majority of cases and MLPA confirmed the presence of deletions within the PAR1 region in two of three cases identified by FISH. Small intragenic aberrations detected by MLPA, which were below the resolution of FISH for CDKN2A/B (n = 7), IKZF1 (n = 3), and PAX5 (n = 3) were confirmed by qPCR. MLPA and qPCR were unable to detect populations present at a low level (<20%) by FISH. In addition, although MLPA identified the presence of a deletion, it was unable to discern the presence of mixed cell populations which had been identified by FISH: CDKN2A/B (n = 3), IKZF1 (n = 1), PAX5 (n = 2), and PAR1 deletion (n = 1). Nevertheless, this study has demonstrated that MLPA is a robust technique for the reliable detection of CNA involving multiple targets in a single test and thus is ideal for rapid high throughput testing of large cohorts with a view to establishing incidence and prognostic significance.

What defines airflow obstruction in asthma?

Asthma guidelines from the Global Initiative for Asthma (GINA) and from the National Heart, Lung, and Blood Institute provide conflicting definitions of airflow obstruction, suggesting a fixed forced expiratory volume in 1 s (FEV(1))/forced vital capacity (FVC) cut-off point and the lower limit of normality (LLN), respectively. The LLN was recommended by the recent American Thoracic Society/European Respiratory Society guidelines on lung function testing. The problem in using fixed cut-off points is that they are set regardless of age and sex in an attempt to simplify diagnosis at the expense of misclassification. The sensitivity and specificity of fixed FEV(1)/FVC ratios of 0.70, 0.75 and 0.80 versus the LLN were evaluated in 815 subjects (aged 20-44 yrs) with a diagnosis of asthma within the framework of the European Community Respiratory Health Survey. In males, the 0.70 ratio showed 76.5% sensitivity and 100.0% specificity, the 0.75 ratio 100.0% sensitivity and 92.4% specificity, and the 0.80 ratio 100.0% sensitivity but 58.1% specificity. In females, the 0.70 ratio showed 57.3% sensitivity and 100.0% specificity, the 0.75 ratio 91.5% sensitivity and 95.9% specificity, and the 0.80 ratio 100.0% sensitivity but 72.9% specificity. The fixed cut-off points cause a lot of misidentification of airflow obstruction in young adults, with overestimation with the 0.80 ratio and underestimation with the 0.70 ratio. In conclusion, the GINA guidelines should change their criteria for defining airflow obstruction.

Underestimation of airflow obstruction among young adults using FEV1/FVC <70% as a fixed cut-off: a longitudinal evaluation of clinical and functional outcomes.

BACKGROUND: Early detection of airflow obstruction is particularly important among young adults because they are more likely to benefit from intervention. Using the forced expiratory volume in 1 s (FEV(1)) to forced vital capacity (FVC) (FEV(1)/FVC) <70% fixed ratio, airflow obstruction may be underdiagnosed. The lower limit of normal (LLN), which is statistically defined by the lower fifth percentile of a reference population, is physiologically appropriate but it still needs a clinical validation. METHODS: To evaluate the characteristics and longitudinal outcomes of subjects misidentified as normal by the fixed ratio with respect to the LLN, 6249 participants (aged 20-44 years) in the European Community Respiratory Health Survey were examined and divided into three groups (absence of airflow obstruction by the LLN and the fixed ratio; presence of airflow obstruction only by the LLN; presence of airflow obstruction by the two criteria) for 1991-1993. LLN equations were obtained from normal non-smoking participants. A set of clinical and functional outcomes was evaluated in 1999-2002. RESULTS: The misidentified subjects were 318 (5.1%); only 45.6% of the subjects with airflow obstruction by the LLN were also identified by the fixed cut-off. At baseline, FEV(1) (107%, 97%, 85%) progressively decreased and bronchial hyperresponsiveness (slope 7.84, 6.32, 5.57) progressively increased across the three groups. During follow-up, misidentified subjects had a significantly higher risk of developing chronic obstructive pulmonary disease and a significantly higher use of health resources (medicines, emergency department visits/hospital admissions) because of breathing problems than subjects without airflow obstruction (p<0.001). CONCLUSIONS: Our findings show the importance of using statistically derived spirometric criteria to identify airflow obstruction.

Using the lower limit of normal for the FEV1/FVC ratio reduces the misclassification of airway obstruction.

AIM: The prevalence of airway obstruction varies widely with the definition used. OBJECTIVES: To study differences in the prevalence of airway obstruction when applying four international guidelines to three population samples using four regression equations. METHODS: We collected predicted values for forced expiratory volume in 1 s/forced vital capacity (FEV(1)/FVC) and its lower limit of normal (LLN) from the literature. FEV(1)/FVC from 40 646 adults (including 13 136 asymptomatic never smokers) aged 17-90+years were available from American, English and Dutch population based surveys. The prevalence of airway obstruction was determined by the LLN for FEV(1)/FVC, and by using the Global Initiative for Chronic Obstructive Lung Disease (GOLD), American Thoracic Society/European Respiratory Society (ATS/ERS) or British Thoracic Society (BTS) guidelines, initially in the healthy subgroup and then in the entire population. RESULTS: The LLN for FEV(1)/FVC varied between prediction equations (57 available for men and 55 for women), and demonstrated marked negative age dependency. Median age at which the LLN fell below 0.70 in healthy subjects was 42 and 48 years in men and women, respectively. When applying the reference equations (Health Survey for England 1995-1996, National Health and Nutrition Examination Survey (NHANES) III, European Community for Coal and Steel (ECCS)/ERS and a Dutch population study) to the selected population samples, the prevalence of airway obstruction in healthy never smokers aged over 60 years varied for each guideline: 17-45% of men and 7-26% of women for GOLD; 0-18% of men and 0-16% of women for ATS/ERS; and 0-9% of men and 0-11% of women for BTS. GOLD guidelines caused false positive rates of up to 60% when applied to entire populations. CONCLUSIONS: Airway obstruction should be defined by FEV(1)/FVC and FEV(1) being below the LLN using appropriate reference equations.

Fibromuscular differentiation in deeply infiltrating endometriosis is a reaction of resident fibroblasts to the presence of ectopic endometrium.

BACKGROUND: In this study, we characterized the fibromuscular (FM) tissue, typical of deeply infiltrating endometriosis, investigated which cells are responsible for the FM reaction and evaluated whether transforming growth factor-beta (TGF-beta) signaling is involved in this process. METHODS: FM differentiation and TGF-beta signaling were assessed in deeply infiltrating endometriosis lesions (n = 20) and a nude mouse model of endometriosis 1, 2, 3 and 4 weeks post-transplantation. The FM reaction was evaluated by immunohistochemistry using different markers of FM and smooth muscle cell differentiation (vimentin, desmin, alpha-smooth muscle actin, smooth muscle myosin heavy chain). TGF-beta signaling was assessed by immunostaining for its receptors and phosphorylated Smad. RESULTS: Deeply infiltrating endometriosis lesions contain myofibroblast-like cells that express multiple markers of FM differentiation. Expression of TGF-beta receptors and phospho-Smad was more pronounced in the endometrial component of the lesions than in the FM component. In the nude mouse model, alpha-smooth muscle actin expression was observed in murine fibroblasts surrounding the lesion, but not in human endometrial stroma. CONCLUSIONS: FM differentiation in deeply infiltrating endometriosis is the result of a reaction of the local environment to the presence of ectopic endometrium. It shares characteristics with pathological wound healing, but cannot be explained by TGF-beta signaling alone.

Detection of a novel familial deletion of four genes between BP1 and BP2 of the Prader-Willi/Angelman syndrome critical region by oligo-array CGH in a child with neurological disorder and speech impairment.

Two common classes of deletions are described in the literature in individuals with Prader-Willi/Angelman syndrome (PWS/AS): one between breakpoint 1 (BP1) to BP3 and the other between BP2 to BP3 of the PWS/AS critical region on chromosome 15q11-->q13. We present here a novel observation of an approximately 253-kb deletion between BP1 and BP2 on 15q11.2, in a 3(1/2)-year-old boy, who was referred to us with a clinical suspicion of having Angelman syndrome and presenting with mental retardation, neurological disorder, developmental delay and speech impairment. Karyotype and FISH results were found to be normal. The microdeletion between BP1 and BP2 includes four genes - NIPA1, NIPA2, CYFIP1 and TUBGCP5 which was detected by a high-resolution oligonucleotide array-CGH that was further validated by a Multiplex Ligation-dependent Probe Amplification (MLPA) assay. The same deletion was observed in the father who presented with similar but relatively milder clinical features as compared to the affected son. Methylation studies by methylation-specific MLPA (MS-MLPA) of the SNRPN imprinting center (IC) showed a normal imprinting pattern, both in the patient and the father. To our knowledge a microdeletion limited only to the BP1-BP2 region has not yet been reported. The familial genetic alteration together with the striking clinical presentation in this study are interesting, but from our single case study it is difficult to suggest if the deletion is causative of some of the abnormal features or if it is a normal variant. The study however further strengthens the fact that genome-wide analysis by array CGH in individuals with developmental delay and mental retardation is very useful in detecting such hidden interstitial chromosomal rearrangements.

Effects of 4 months of smoking in mice with ovalbumin-induced airway inflammation.

BACKGROUND: The effects of smoking on asthma pathogenesis are complex and not well studied. We have shown recently that 3 weeks of smoking attenuates ovalbumin (OVA)-induced airway inflammation in mice and that 4-6 months of smoking induces emphysema in mice without airway inflammation. Effects of combined long-term smoking and OVA exposure have not been investigated so far. OBJECTIVE: To study whether long-term smoking affects progression of allergic airway inflammation and/or enhances the development of emphysema in mice. METHODS: Mice were sensitized to OVA and challenged with saline or OVA aerosols for 6 months. From 2 months onwards, mice were also exposed to air or smoke. Lung tissue was analysed for extent of inflammation, emphysema, remodelling and for cytokine levels, and serum for OVA-specific IgE levels. RESULTS: Chronic OVA exposure of 6 months resulted in a T helper type 2 (Th2)-type inflammation with increased levels of IL-4, IL-5, IL-6 and infiltration of eosinophils, CD4(+) T cells, macrophages and plasma cells. Smoking induced a Th17-type of airway inflammation, characterized by neutrophils, macrophages, B cells and increased levels of IL-17, IL-6, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor and monocyte chemoattractant protein-1. Concomittant smoking and OVA exposure resulted in inflammation similar to OVA exposure alone. OVA exposure increased IgE levels compared with saline exposure, and smoking did not further increase these levels. CONCLUSION: We did not find evidence for increased inflammation, IgE levels or emphysema in mice with allergic airway inflammation after 4 months of smoking compared with non-smoking. However, a 4-month exposure to smoke alone did enhance neutrophilic airway inflammation characterized by high pulmonary IL-17 levels. A Th2 inflammatory environment due to OVA exposure may be one explanation as to why no further detrimental effects of smoking on allergic airway inflammation were found.

Preventive intervention possibilities in radiotherapy- and chemotherapy-induced oral mucositis: results of meta-analyses.

The aim of these meta-analyses was to evaluate the effectiveness of interventions for the prevention of oral mucositis in cancer patients treated with head and neck radiotherapy and/or chemotherapy, with a focus on randomized clinical trials. A literature search was performed for reports of randomized controlled clinical studies, published between 1966 and 2004, the aim of which was the prevention of mucositis in cancer patients undergoing head and neck radiation, chemotherapy, or chemoradiation. The control group consisted of a placebo, no intervention, or another intervention group. Mucositis was scored by either the WHO, the National Cancer Institute-Common Toxicity Criteria (NCI-CTC) score, or the absence or presence of ulcerations, or the presence or absence of grades 3 and 4 mucositis. The meta-analyses included 45 studies fulfilling the inclusion criteria, in which 8 different interventions were evaluated: i.e., local application of chlorhexidine; iseganan; PTA (polymyxin E, tobramycine, and amphotericin B); granulocyte macrophage-colony-stimulating factor/granulocyte colony-stimulating factor (GM-CSF/G-CSF); oral cooling; sucralfate and glutamine; and systemic administration of amifostine and GM-CSF/G-CSF. Four interventions showed a significant preventive effect on the development or severity of oral mucositis: PTA with an odds ratio (OR) = 0.61 (95% confidence interval [CI], 0.39-0.96); GM-CSF, OR = 0.53 (CI: 0.33-0.87); oral cooling, OR = 0.3 (CI: 0.16-0.56); and amifostine, OR = 0.37 (CI: 0.15-0.89). To date, no single intervention completely prevents oral mucositis, so combined preventive therapy strategies seem to be required to ensure more successful outcomes.

Predictors of lung function and its decline in mild to moderate COPD in association with gender: results from the Euroscop study.

BACKGROUND: There is increasing appreciation of gender differences in COPD but scant data whether risk factors for low lung function differ in men and women. We analysed data from 3 years follow-up in 178 women and 464 men with COPD, participants in the Euroscop Study who were smokers unexposed to inhaled corticosteroids. METHODS: Explanatory variables of gender, age, starting age and pack-years smoking, respiratory symptoms, FEV(1)%FVC and FEV(1)%IVC (clinically important measures of airway obstruction), body mass index (BMI), and change in smoking were included in multiple linear regression models with baseline and change in post-bronchodilator FEV(1) as dependent variables. RESULTS: Reduced baseline FEV(1) was associated with respiratory symptoms in men only. Annual decline in FEV(1) was not associated with respiratory symptoms in either men or women, and was 55 ml less in obese men (BMI 30 kg/m(2)) than men having normal BMI, an effect not seen in women. It was 32 ml faster in women with FEV(1)%FVC

Partial NSD1 deletions cause 5% of Sotos syndrome and are readily identifiable by multiplex ligation dependent probe amplification.

BACKGROUND: Most cases of Sotos syndrome are caused by intragenic NSD1 mutations or 5q35 microdeletions. It is uncertain whether allelic or genetic heterogeneity underlies the residual cases and it has been proposed that other mechanisms, such as 11p15 defects, might be responsible for Sotos cases without NSD1 mutations or 5q35 microdeletions. OBJECTIVE: To develop a multiplex ligation dependent probe amplification (MLPA) assay to screen NSD1 for exonic deletions/duplications. METHODS: Analysis was undertaken of 18 classic Sotos syndrome cases in which NSD1 mutations and 5q35 microdeletions were excluded. Long range polymerase chain reaction (PCR) was used to characterise the mechanism of generation of the partial NSD1 deletions. RESULTS: Eight unique partial NSD1 deletions were identified: exons 1-2 (n = 4), exons 3-5, exons 9-13, exons 19-21, and exon 22. Using long range PCR six of the deletions were confirmed and the precise breakpoints in five cases characterised. This showed that three had arisen through Alu-Alu recombination and two from non-homologous end joining. CONCLUSIONS: MLPA is a robust, inexpensive, simple technique that reliably detects both 5q35 microdeletions and partial NSD1 deletions that together account for approximately 15% of Sotos syndrome.

DNA copy number changes at 8q11-24 in metastasized colorectal cancer.

BACKGROUND: C-Myc, a well-known oncogene located on 8q24.12-q24.23, is often amplified and over-expressed in both primary and metastasizing colorectal cancer. In addition, PRL-3 (also known as PTP4A3), a tyrosine phosphatase located on 8q24.3, is amplified in colorectal cancer metastasis. Beside PRL-3 and c-myc, other oncogenes located on the 8q23-24 region might be involved in this process. Therefore, the present study aims to correlate DNA copy number status of a series of genes at 8q23-24 in colorectal cancer at high resolution in correlation to metastatic disease. MATERIALS AND METHODS: Thirty-two cases of colorectal cancer, 10 stage B1, 10 B2 and 12 D (Astler-Coller) with their corresponding liver metastasis and one colorectal cell line (colo205, previously analyzed by array-CGH), were included in this study. A chromosome 8 specific MLPA probe mixture was used to analyze the presence of DNA copy number changes. The probe mixture contained 29 probes covering 25 genes on chromosome 8, as well as 6 control probes on other chromosomes. RESULTS AND DISCUSSION: MLPA results obtained of the colo205 colorectal cell line were comparable with previous array-CGH results, thus validating the MLPA probe mixture. Astler-Coller B1 and B2 colorectal cancers differed significantly in DNA copy number of the genes, MOS (p=0.04), MYC (p=0.007), DDEF1 (p=0.004), PTK2 (p=0.02) and PTP4A3 (p=0.04). When comparing these with Astler-Coller D primary tumors, significant differences were seen for several genes as well (MYC (p<0.000), DDEF1 (p<0.000), SLA (p<0.000), PTK2 (p<0.000), PTP4A3 (p=0.002), and RECQL4 (p=0.01)). When comparing primary Astler-Coller D tumors and their corresponding liver metastases, a similar pattern of gains and losses was observed. Most of the liver metastases showed higher DNA copy number ratios than the corresponding primary tumors, but this difference was only significant for TPD52 (p=0.02) and EIF3S6 (p=0.007). CONCLUSION: In addition to c-myc, multiple genes on chromosome 8 differed significantly between primary colorectal cancers with and without liver metastases. This observation is consistent with the concept that clinical behaviour, like risk of liver metastasis, is determined by the genomic profile that is already present in the primary tumor.

Gene expression profiling of minimal residual disease in acute myeloid leukaemia by novel multiplex-PCR-based method.

In acute myeloid leukaemia (AML), alterations in apoptotic pathways are crucial for treatment outcome, resulting either in refractoriness or in minimal residual disease (MRD). The apoptosis characteristics of MRD cells may differ from those at diagnosis and thereby determine the adequacy of further treatment. Such characteristics are largely unknown, since studies hereto are hampered by minimal cell availability. This study explores the applicability of the recently described RT-Multiplex Ligation-dependent Probe Amplification (RT-MLPA) for gene expression analysis of small amounts of RNA obtained from MRD cells. Reproducibility and dilution experiments showed that the relative expression of 37 apoptosis-related genes starting with only 1000 cells could be measured with 12% variation; for 100 cells, 31/37 genes could still be quantified, though expression variation increased. In practice 100-1000 leukemic cells can be obtained from bone marrow samples with clinically relevant MRD percentages of 0.01-0.1. Procedures often necessary to obtain AML blasts, that is, FACS-sorting, freeze-thawing or combinations are possible, provided that selected viable nonapoptotic cells are used. Concluding, RT-MLPA allows accurate gene expression profiling of MRD cells. This method will help to gain insight into the processes of MRD emergence and persistence in AML, which may ultimately guide new therapeutic strategies in AML.

Host factors and environmental determinants associated with skin test reactivity and eosinophilia in a community-based population study.

We examined the relationship of host factors (age, gender) and environmental determinants (smoking status, area of residence) to indicators of allergy (skin test reactivity and eosinophil count) in a random population sample in the Netherlands. Positive skin test reactivity was associated with age (decreasing with increasing age), with male gender (versus female gender), and with urban residence (versus rural residence). Positive skin test reactivity was not associated with smoking. Elevated eosinophil counts were associated with male gender (versus female gender), with urban residence (versus rural residence), and with current smoking (versus never smoking). Elevated eosinophil counts were not clearly associated with age (if adjusted for the age-related effects of skin test reactivity). Additionally, this study specifically demonstrates that skin test reactivity increases with increasing eosinophil count and vice versa, indicating that the two traits are interrelated. Furthermore, this interrelationship was demonstrated to be age-dependent (decreasing with increasing age).

Comparing FEV1 in adults in two community-based studies.

Data collected in two large-scale longitudinal respiratory studies were analyzed to estimate age-related changes in FEV1 and to determine the between-study differences in change in FEV1 among nonsmoking participants. Two adult cohorts in the Netherlands were followed up for 21 years at 3-year intervals and a cohort of adults in 6 cities in the eastern United States was followed up for 6 years. The age distributions and follow-up rate differed for the two studies. The mean rates of decline in FEV1 for 50-year old male and female never smokers were 27.9 +/- 2.8 mL/yr and 22.1 +/- 0.9 mL/yr, respectively, in the Netherlands cohort. The estimates for men and women in the Six Cities cohort were 33% and 32% greater, respectively, than those for the Netherlands cohort (p < 0.01). The estimated rate of decline in FEV1 associated with each 1-year increase in age was 0.50 +/- 0.12 mL/yr for men and 0.61 +/- 0.08 mL/yr for women in the Netherlands cohort. This estimate was comparable for the Six Cities women and the Netherlands women but significantly different for the two cohorts of men. Inclusion of survey (period) effect in the regression model had only a trivial influence on accelerated rate of decline in FEV1 and did not alter the more rapid acceleration of decline in FEV1 among the Six Cities men. The present study also showed that use of a "prediction equation" for age adjustment to compare across population samples may produce a biased conclusion if the estimated rate of decline in FEV1 associated with age is different and the age distributions are different for the two populations. In addition, height adjustment for FEV1 was found to have a negligible influence on the longitudinal estimates of FEV1 decline in both study samples. In conclusion, substantial between study variation in decline in FEV1 occurs despite use of similar data analysis techniques. Differences in the initial age distributions and loss to follow-up appeared to be important factors in this analysis.

Effects of long-term treatment with corticosteroids in COPD.

STUDY OBJECTIVE: To determine the effectiveness of treatment with corticosteroids in patients with COPD. METHODS: In this study, we investigated the effect of a 2-year treatment with corticosteroids on clinical symptoms and the decline of lung function in 58 nonallergic patients with COPD. Subjects were treated in a double-blind, randomized, placebo-controlled, parallel way with inhaled budesonide (bud), 1,600 micrograms/d; inhaled budesonide, 1,600 micrograms/d, plus oral prednisolone, 5 micrograms/d (bud + pred); or placebo (plac). Clinical assessment (history, physical examination, and spirometry) was carried out every 2 months. The rate of decline in FEV1 was assessed by calculating individual regression co-efficients from linear regression of FEV1 on time for each subject. RESULTS: Eleven patients dropped out. The number of withdrawals due to pulmonary problems was significantly higher in the plac group (n = 5 out of 18) than in the actively treated groups (n = 2 out of 40). Treatment with corticosteroids significantly reduced pulmonary symptoms. Median decline of FEV1 was 60 mL/yr in the plac group, 40 mL/yr in the bud + pred group, and 30 mL/yr in the bud group. Variation was large and differences were not statistically significant. No treatment effect was found on frequency or duration of exacerbations, possibly because of the high number of withdrawals due to pulmonary deterioration in the plac group. Treatment with a combination of inhaled plus oral corticosteroids was not more effective than inhaled corticosteroids alone. Morning plasma cortisol levels remained within the normal range in all three groups. CONCLUSIONS: Our study shows beneficial effects of long-term daily treatment with inhaled corticosteroids in patients with COPD with regard to symptoms and drop out due to pulmonary problems. Lung function decline tends to decrease during treatment with inhaled corticosteroids. The observed effects are limited but warrant further studies on the effectiveness of corticosteroids in larger numbers of patients with COPD.

Combined metformin and insulin therapy for patients with type 2 diabetes mellitus.

OBJECTIVE: This study was undertaken to assess the effects of combined treatment with insulin and metformin in patients with type 2 diabetes mellitus in whom dietary measures, weight control, and oral antihyperglycemic therapy had failed. BACKGROUND: Insulin resistance in peripheral tissues, increased hepatic gluconeogenesis, and impaired insulin secretion are the underlying factors in the development of type 2 diabetes. Metformin is a biguanide antihyperglycemic agent that increases peripheral insulin sensitivity, reduces hepatic gluconeogenesis, and decreases intestinal glucose absorption. METHODS: Thirty-one patients (24 women, 7 men; mean age, 61.8 years; mean body mass index [BMI], 28.0 kg/m2) were enrolled in this randomized, double-blind, 2-way, crossover, placebo-controlled study. Patients with type 2 diabetes who were treated previously with insulin or oral hypoglycemic agents and who had a glycosylated hemoglobin (HbA1c) level >9% or a fasting blood glucose level >8 mmol/L were included. Patients who were being treated with oral agents were switched to insulin therapy and required to maintain stable blood glucose control for 2 months prior to randomization. Patients received insulin plus either metformin 1,700 mg/d or placebo for 5 months, followed by a 2-month washout period, and were then crossed over to the other treatment arm for 5 months of additional treatment (total treatment period: 12 months). RESULTS: Thirty patients completed the study; 1 patient withdrew early because of hypoglycemia. Compared with placebo, metformin produced significant reductions from overall baseline in mean daily insulin dose requirement (-8.69 units (17.2%], P < 0.001), HbA1c level (-0.74 [9.9%], P = 0.005), serum fructosamine level (-44.40 micromol/L, P = 0.026), 24-hour blood glucose profile (P = 0.008), and total cholesterol level (-0.42 mmol/L, P = 0.005). No treatment effects were observed on body weight, blood pressure, serum high-density lipoprotein cholesterol levels, or serum triglyceride levels. There was no correlation between BMI and reduction in HbA1C. No major side effects were reported. CONCLUSIONS: Combination therapy with metformin and insulin improves glycemic control and reduces insulin requirements. with no major side effects, in patients with type 2 diabetes and may improve the risk profile in this patient population.

Short term effects of air pollution on emergency hospital admissions for respiratory disease: results of the APHEA project in two major cities in The Netherlands, 1977-89.

STUDY OBJECTIVE: To assess the short term relationship between air pollution and the daily number of emergency hospital admissions for respiratory disease. DESIGN: Data were analysed using autoregressive Poisson regression allowing for overdispersion and controlling for possible confounding factors such as seasonal and other chronological variables, meteorological factors, and influenza epidemics. SETTING: The two major cities in The Netherlands-Amsterdam (694,700 inhabitants) and Rotterdam (576,200 inhabitants). PARTICIPANTS AND MEASUREMENTS: Emergency hospital admissions for respiratory diseases, registered on a daily basis by the National Medical Registration, for the period 1977-89 were used. ICD-9 codes included were: respiratory (460-519), chronic obstructive pulmonary disease (490-492, 494, 496), and asthma (493). The mean (range) of the total daily number of admissions for these three classifications were as follows: 6.70 (0-23), 1.74 (0-9) and 1.13 (0-7) respectively in Amsterdam and 4.79 (0-19), 1.57 (0-9), and 0.53 (0-5) in Rotterdam. Air pollution measurements were provided by the National Institute of Public Health and Environmental Protection. In The Netherlands, air pollution is at a low to moderate ("summer type") or a low ("winter type") level. The levels in Amsterdam and Rotterdam did not differ much for the "summer type". For 1977-89 the mean (range) values of ozone (O3), the "summer type" pollutant (O3-8 h), were 86 (0-252) micrograms/m3 in Amsterdam and 82 (0-286) micrograms/m3 in Rotterdam. The mean (range) of the values "winter type", pollutant, sulphur dioxide (SO2-24 h), were 38 (0-381) micrograms/m3 in Amsterdam and 50 (1-379) micrograms/m3 in Rotterdam. For black smoke (BS-24 h), values were 14 (1-84) micrograms/m3 and 28 (1-144) micrograms/m3 respectively (1986-89). MAIN RESULTS: Ozone had a non-significant positive effect on the number of respiratory emergency admissions in summer in people aged > or = 65 years (relative risk for a 100 micrograms/m3 increase in O3-8 h of 1.127 (0.983, 1.292) in Amsterdam and a significant positive effect of 1.344 (1.097, 1.647) in 1977-81 in Rotterdam). Sulphur dioxide did not show any clear effects; in Amsterdam a significant negative effect was even found. The same was true for nitrogen dioxide in Amsterdam; in Rotterdam, however, nitrogen dioxide showed non-significant positive effects (RR 0.965, 1.342). Black smoke did not show any clear effects in Amsterdam; in Rotterdam it was positively but not significantly related to the number of admissions. CONCLUSIONS: The results show that the relation between short term air pollution and emergency hospital admissions is not always consistent at these rather low levels of daily hospital admissions and of air pollution.

Methodological issues in studies of air pollution and daily counts of deaths or hospital admissions.

STUDY OBJECTIVE: To review the issues and methodologies in epidemiologic time series studies of daily counts of mortality and hospital admissions and illustrate some of the methodologies. DESIGN: This is a review paper with an example drawn from hospital admissions of the elderly in Cleveland, Ohio, USA. MAIN RESULTS: The central issue is control for seasonality. Both over and under control are possible, and the use of diagnostics, including plots, is necessary. Weather dependence is probably non-linear, and adequate methods are necessary to adjust for this. In Cleveland, the use of categorical variables for weather and sinusoidal terms for filtering season are illustrated. After control for season, weather, and day of the week effects, hospital admission of persons aged 65 and older in Cleveland for respiratory illness was associated with ozone (RR = 1.09, 95% CI 1.02, 1.16) and particulates (PM10 (RR = 1.12, 95% CI 1.01, 1.24), and marginally associated with sulphur dioxide (SO2) (RR = 1.03, 95% CI = 0.99, 1.06). All of the relative risks are for a 100 micrograms/m3 increase in the pollutant. CONCLUSIONS: Several adequate methods exist to control for weather and seasonality while examining the associations between air pollution and daily counts of mortality and morbidity. In each case, care and judgement are required.

Repeated provocation tests in asthmatic children for testing tachyphylaxis to histamine.

Tachyphylaxis to histamine was investigated in 16 children, aged 7-15 years, with mild asthma. Three consecutive histamine challenges were performed at intervals of 24 hours and 1 hour, respectively. No significant differences in IVC, FEV1, and PC20-histamine values between the three measurements were observed. After a 24 hour interval there was no difference in percentage fall of FEV1, but there was a slight (not significant) decrease in fall of FEV1 after a 1 hour interval. The PC20-histamine values showed good reproducibility with a 24 hour as well as with a 1-hour period between the tests (geometric mean PC20, 2.04 mg/mL +/- 3.50 %SD, 1.96 mg/mL +/- 4.37 %SD, 2.17 mg/mL +/- 4.12 %SD; correlation coefficients for a 24 hour interval, r = 0.87 and for a one-hour interval, r = 0.94 (P less than 0.01]. We conclude that in children there is no strong evidence for tachyphylaxis to histamine. Our results differ from studies on tachyphylaxis in adult asthmatics. Possibly different mechanisms exist in children and in adults.