We describe a new method for relative quantification of 40 different DNA sequences in an easy to perform reaction requiring only 20 ng of human DNA. Applications shown of this multiplex ligation-dependent probe amplification (MLPA) technique include the detection of exon deletions and duplications in the human BRCA1, MSH2 and MLH1 genes, detection of trisomies such as Down's syndrome, characterisation of chromosomal aberrations in cell lines and tumour samples and SNP/mutation detection. Relative quantification of mRNAs by MLPA will be described elsewhere. In MLPA, not sample nucleic acids but probes added to the samples are amplified and quantified. Amplification of probes by PCR depends on the presence of probe target sequences in the sample. Each probe consists of two oligonucleotides, one synthetic and one M13 derived, that hybridise to adjacent sites of the target sequence. Such hybridised probe oligonucleotides are ligated, permitting subsequent amplification. All ligated probes have identical end sequences, permitting simultaneous PCR amplification using only one primer pair. Each probe gives rise to an amplification product of unique size between 130 and 480 bp. Probe target sequences are small (50-70 nt). The prerequisite of a ligation reaction provides the opportunity to discriminate single nucleotide differences.

In patients with mild COPD who continue smoking, the use of inhaled budesonide is associated with a small one-time improvement in lung function but does not appreciably affect the long-term progressive decline.

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.Chronic obstructive pulmonary disease (COPD) is a major public health concern as a cause of chronic morbidity and mortality.

We have studied in vivo responses of ''spontaneous'' Brca1-and p53-deficient mammary tumors arising in conditional mouse mutants to treatment with doxorubicin, docetaxel, or cisplatin. Like human tumors, the response of individual mouse tumors varies, but eventually they all become resistant to the maximum tolerable dose of doxorubicin or docetaxel. The tumors also respond well to cisplatin but do not become resistant, even after multiple treatments in which tumors appear to regrow from a small fraction of surviving cells. Classical biochemical resistance mechanisms, such as up-regulated drug transporters, appear to be responsible for doxorubicin resistance, rather than alterations in drug-damage effector pathways. Our results underline the promise of these mouse tumors for the study of tumor-initiating cells and of drug therapy of human cancer.multidrug resistance ͉ P-glycoprotein ͉ cancer stem cells