Genetic and epigenetic regulation of the organic cation transporter 3, SLC22A3.

Human organic cation transporter 3 (OCT3 and SLC22A3) mediates the uptake of many important endogenous amines and basic drugs in a variety of tissues. OCT3 is identified as one of the important risk loci for prostate cancer, and is markedly underexpressed in aggressive prostate cancers. The goal of this study was to identify genetic and epigenetic factors in the promoter region that influence the expression level of OCT3. Haplotypes that contained the common variants, g.-81G>delGA (rs60515630) (minor allele frequency 11.5% in African American) and g.-2G>A (rs555754) (minor allele frequency>30% in all ethnic groups) showed significant increases in luciferase reporter activities and exhibited stronger transcription factor-binding affinity than the haplotypes that contained the major alleles. Consistent with the reporter assays, OCT3 messenger RNA expression levels were significantly higher in Asian (P<0.001) and Caucasian (P<0.05) liver samples from individuals who were homozygous for g.-2A/A in comparison with those homozygous for the g.-2G/G allele. Studies revealed that the methylation level in the basal promoter region of OCT3 was associated with OCT3 expression level and tumorigenesis capability in various prostate cancer cell lines. The methylation level of the OCT3 promoter was higher in 62% of prostate tumor samples compared with matched normal samples. Our studies demonstrate that genetic polymorphisms in the proximal promoter region of OCT3 alter the transcription rate of the gene and may be associated with altered expression levels of OCT3 in human liver. Aberrant methylation contributes to the reduced expression of OCT3 in prostate cancer.

Aberrant CpG-island methylation has non-random and tumour-type-specific patterns.

CpG islands frequently contain gene promoters or exons and are usually unmethylated in normal cells. Methylation of CpG islands is associated with delayed replication, condensed chromatin and inhibition of transcription initiation. The investigation of aberrant CpG-island methylation in human cancer has primarily taken a candidate gene approach, and has focused on less than 15 of the estimated 45,000 CpG islands in the genome. Here we report a global analysis of the methylation status of 1,184 unselected CpG islands in each of 98 primary human tumours using restriction landmark genomic scanning (RLGS). We estimate that an average of 600 CpG islands (range of 0 to 4,500) of the 45,000 in the genome were aberrantly methylated in the tumours, including early stage tumours. We identified patterns of CpG-island methylation that were shared within each tumour type, together with patterns and targets that displayed distinct tumour-type specificity. The expression of many of these genes was reactivated by experimental demethylation in cultured tumour cells. Thus, the methylation of particular subsets of CpG islands may have consequences for specific tumour types.

Epigenetic inactivation of the miR-34a in hematological malignancies.

miR-34a is a transcriptional target of p53 and implicated in carcinogenesis. We studied the role of miR-34a methylation in a panel of hematological malignancies including acute leukemia [acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL)], chronic leukemia [chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML)], multiple myeloma (MM) and non-Hodgkin's lymphoma (NHL). The methylation status of miR-34a promoter was studied in 12 cell lines and 188 diagnostic samples by methylation-specific polymerase chain reaction. miR-34a promoter was unmethylated in normal controls but methylated in 75% lymphoma and 37% myeloma cell lines. Hypomethylating treatment led to re-expression of pri-miR-34a transcript in lymphoma cells with homozygous miR-34a methylation. In primary samples at diagnosis, miR-34a methylation was detected in 4% CLL, 5.5% MM samples and 18.8% of NHL at diagnosis but none of ALL, AML and CML (P = 0.011). In MM patients with paired samples, miR-34a methylation status remained unchanged at progression. Amongst lymphoid malignancies, miR-34a was preferentially methylated in NHL (P = 0.018), in particular natural killer (NK)/T-cell lymphoma. In conclusion, amongst hematological malignancies, miR-34a methylation is preferentially hypermethylated in NHL, in particular NK/T-cell lymphoma, in a tumor-specific manner, therefore the role of miR-34a in lymphomagenesis warrants further study.

Evaluation of the Airtraq and Macintosh laryngoscopes in patients at increased risk for difficult tracheal intubation.

The Airtraq, a novel single use indirect laryngoscope, has demonstrated promise in the normal and simulated difficult airway. We compared the ease of intubation using the Airtraq with the Macintosh laryngoscope, in patients at increased risk for difficult tracheal intubation, in a randomised, controlled clinical trial. Forty consenting patients presenting for surgery requiring tracheal intubation, who were deemed to possess at least three characteristics indicating an increased risk for difficulty in tracheal intubation, were randomly assigned to undergo tracheal intubation using a Macintosh (n = 20) or Airtraq (n = 20) laryngoscope. All patients were intubated by one of three anaesthetists experienced in the use of both laryngoscopes. Four patients were not successfully intubated with the Macintosh laryngoscope, but were intubated successfully with the Airtraq. The Airtraq reduced the duration of intubation attempts (mean (SD); 13.4 (6.3) vs 47.7 (8.5) s), the need for additional manoeuvres, and the intubation difficulty score (0.4 (0.8) vs 7.7 (3.0)). Tracheal intubation with the Airtraq also reduced the degree of haemodynamic stimulation and minor trauma compared to the Macintosh laryngoscope.

Methylation-related chromatin structure is associated with exclusion of transcription factors from and suppressed expression of the O-6-methylguanine DNA methyltransferase gene in human glioma cell lines.

There is considerable interest in identifying factors responsible for expression of the O-6-methylguanine DNA methyltransferase (MGMT) gene, as MGMT is a major determinant in the response of glioma cells to the chemotherapeutic agent 1,3 bis(2-chloroethyl)-1-nitrosourea. Recently we have shown that MGMT expression is correlated in a direct, graded fashion with methylation in the body of the MGMT gene and in an inverse, graded fashion with promoter methylation in human glioma cell lines. To determine if promoter methylation is an important component of MGMT expression, this study addressed the complex interactions between methylation, chromatin structure, and in vivo transcription factor occupancy in the MGMT promoter of glioma cell lines with different levels of MGMT expression. Our results show that the basal promoter in MGMT-expressing glioma cell lines, which is 100% unmethylated, was very accessible to restriction enzymes at all sites tested, suggesting that this region may be nucleosome free. The basal promoter in glioma cells with minimal MGMT expression, however, which is 75% unmethylated, was much less accessible, and the basal promoter in nonexpressing cells, which is 50% unmethylated, was entirely inaccessible to restriction enzymes. Despite the presence of the relevant transcription factors in all cell lines examined, in vivo footprinting showed DNA-protein interactions at six Sp1 binding sites and one novel binding site in MGMT-expressing cell lines but no such interactions in nonexpressors. We conclude that in contrast to findings of previous in vitro studies, Sp1 is an important component of MGMT transcription. These correlations also strongly suggest that methylation and chromatin structure, by determining whether Sp1 and other transcription factors can access the MGMT promoter, set the transcriptional state of the MGMT gene.

Methylation of discrete regions of the O6-methylguanine DNA methyltransferase (MGMT) CpG island is associated with heterochromatinization of the MGMT transcription start site and silencing of the gene.

O6-Methylguanine DNA methyltransferase (MGMT) repairs the mutagenic and cytotoxic O6-alkylguanine lesions produced by environmental carcinogens and the chemotherapeutic nitrosoureas. As such, MGMT-mediated repair of O6-alkylguanine lesions constitutes a major form of resistance to nitrosourea chemotherapy and makes control of MGMT expression of clinical interest. The variability of expression in cell lines and tissues, along with the ease with which the MGMT phenotype reverts under various conditions, suggests that MGMT is under epigenetic control. One such epigenetic mechanism, 5-methylation of cytosines, has been linked to MGMT expression. We have used an isogenic human multiple myeloma tumor cell line model composed of an MGMT-positive parent cell line, RPMI 8226/S, and its MGMT-negative variant, termed 8226/V, to study the control of MGMT expression. The loss of MGMT activity in 8226/V was found to be due to the loss of detectable MGMT gene expression. Bisulfite sequencing of the MGMT CpG island promoter revealed large increases in the levels of CpG methylation within discrete regions of the 8226/V MGMT CpG island compared to those in 8226/S. These changes in CpG methylation are associated with local heterochromatinization of the 8226/V MGMT transcription start site and provide a likely mechanism for the loss of MGMT transcription in 8226/V.

Silencing of p16/CDKN2 expression in human gliomas by methylation and chromatin condensation.

The product of the p16/CDKN2 locus, p16ink4, negatively regulates the cell cycle through binding and inactivation of cyclin-dependent kinases (CDKs) 4 and 6. This locus is frequently targeted for deletion in cell lines and primary tumor tissues. In gliomas, although up to 50% do not have detectable expression of p16/CDKN2 protein or mRNA, often the gene is wild type in sequence. Here, we tested the hypothesis that transcriptional repression of p16/CDKN2 in gliomas may be mediated by aberrant methylation of the CpG island, which is in the 5' region of the locus. Partial rather than complete p16/CDKN2 methylation was detected in 24% (10 of 42) of the gliomas, regardless of tumor grade, but was not observed in normal brain (0 of 10). We tested whether this partial methylation could inhibit expression in a human tumor cell line in which suppressed p16/CDKN2 expression was associated with both methylation and tightly compacted chromatin around the p16/CDKN2 promoter. Exposure of these cells to 5-aza-2-deoxycytidine resulted in a dramatic increase in promoter accessibility and induction of p16/CDKN2 expression, indicating that chromatin structure, CpG island methylation, and p16/CDKN2 expression are intimately associated. Taken together, these data suggest that methylation occurs in only a subset of cells within gliomas and that the methylation-associated inactivation of p16/CDKN2 expression observed in many common human cancers may mechanistically result from structural changes in the chromatin containing the p16/CDKN2 locus.

Cyclin-dependent kinase 6 (CDK6) amplification in human gliomas identified using two-dimensional separation of genomic DNA.

DNA amplification is a common mechanism invoked by many human tumors to elicit overexpression of genes whose products are involved in drug resistance or cell proliferation. Although amplified regions in tumor DNA may exceed several megabases in size, segments of amplicons with a high probability of containing gene sequences may be amenable to detection by restriction landmark genomic scanning (RLGS), a high-resolution DNA analysis that separates labeled NotI fragments in two dimensions. Here, we tested this by applying RLGS to matched samples of glioma and normal brain DNA and found tumor-specific amplification of the gene encoding cyclin-dependent kinase 6 (CDK6), an observation not previously reported in human tumors. The CDK6 gene has been localized to chromosome 7q21-22, but in the gliomas studied here, it was not coamplified with either the syntenic MET (7q31) or epidermal growth factor receptor (7p11-p12) genes, suggesting that this may be part of a novel amplicon in gliomas. We then corroborated this finding by identifying both amplification-associated and amplification-independent increases in CDK6 protein levels in gliomas relative to matched normal brain samples. These data implicate the CDK6 gene in genomic amplification and illustrate the potential of RLGS for the more general identification and cloning of novel genes that are amplified in human cancer.

Methylation-associated silencing of the tissue inhibitor of metalloproteinase-3 gene suggest a suppressor role in kidney, brain, and other human cancers.

Tissue inhibitor of metalloproteinase-3 (TIMP-3) antagonizes matrix metalloproteinase activity and can suppress tumor growth, angiogenesis, invasion, and metastasis. Loss of TIMP-3 has been related to the acquisition of tumorigenesis. Herein, we show that TIMP-3 is silenced in association with aberrant promoter-region methylation in cell lines derived from human cancers. TIMP-3 expression was restored after 5-aza-2'deoxycytidine-mediated demethylation of the TIMP-3 proximal promoter region. Genomic bisulfite sequencing revealed that TIMP-3 silencing was related to the overall density of methylation and that discrete regions within the TIMP-3 CpG island may be important for the silencing of this gene. Aberrant methylation of TIMP-3 occurred in primary cancers of the kidney, brain, colon, breast, and lung, but not in any of 41 normal tissue samples. The most frequent TIMP-3 methylation was found in renal cancers, which originate in the tissue that normally expresses the highest TIMP-3 levels. This methylation correlated with a lack of detectable TIMP-3 protein in these tumors. Together, these data show that methylation-associated inactivation of TIMP-3 is frequent in many human tumors.

Selective airway responsiveness in asthma.

Hyperresponsiveness of airway smooth muscle accounts for the susceptibility of asthmatic subjects to diverse bronchoconstrictor agents. It is widely presumed that hyperresponsiveness is not spasmogen selective. Hence, inhalation of methacholine is used routinely for clinical assessment of asthma and for evaluation of anti-asthma drugs. Comparative studies employing multiple spasmogens have revealed hyperresponsiveness to be markedly spasmogen selective. Because of this pronounced heterogeneity of hyperresponsiveness, sensitivity to methacholine cannot provide a reliable index of responsiveness. Development of exceptional hyperresponsiveness to bradykinin and to peptidoleukotrienes during allergic and other reactions could warrant the development of specific antagonists for asthma therapy. These issues are discussed here by Brian O'Connor, Simon Crowther, John Costello and John Morley.

Methylation matters.

DNA methylation is not just for basic scientists any more. There is a growing awareness in the medical field that having the correct pattern of genomic methylation is essential for healthy cells and organs. If methylation patterns are not properly established or maintained, disorders as diverse as mental retardation, immune deficiency, and sporadic or inherited cancers may follow. Through inappropriate silencing of growth regulating genes and simultaneous destabilisation of whole chromosomes, methylation defects help create a chaotic state from which cancer cells evolve. Methylation defects are present in cells before the onset of obvious malignancy and therefore cannot be explained simply as a consequence of a deregulated cancer cell. Researchers are now able to detect with exquisite sensitivity the cells harbouring methylation defects, sometimes months or years before the time when cancer is clinically detectable. Furthermore, aberrant methylation of specific genes has been directly linked with the tumour response to chemotherapy and patient survival. Advances in our ability to observe the methylation status of the entire cancer cell genome have led us to the unmistakable conclusion that methylation abnormalities are far more prevalent than expected. This methylomics approach permits the integration of an ever growing repertoire of methylation defects with the genetic alterations catalogued from tumours over the past two decades. Here we discuss the current knowledge of DNA methylation in normal cells and disease states, and how this relates directly to our current understanding of the mechanisms by which tumours arise.

Gene amplification in PNETs/medulloblastomas: mapping of a novel amplified gene within the MYCN amplicon.

OBJECTIVES: The pathological entity of primitive neuroectodermal tumour/medulloblastoma (PNET/MB) comprises a very heterogeneous group of neoplasms on a clinical as well as on a molecular level. We evaluated the importance of DNA amplification in medulloblastomas and other primitive neuroectodermal tumours (PNETs) of the CNS. METHOD: Restriction landmark genomic scanning (RLGS), a method that allows the detection of low level amplification, was used. RLGS provides direct access to DNA sequences circumventing positional cloning efforts. Furthermore, we analysed several samples by CGH. DESIGN: Twenty primary medulloblastomas, five supratentorial PNETs, and five medulloblastoma cell lines were studied. RESULTS: Although our analysis confirms that gene amplification is generally a rare event in childhood PNET/MB, we found a total of 17 DNA fragments that were amplified in seven different tumours. Cloning and sequencing of several of these fragments confirmed the previous finding of MYC amplification in the cell line D341 Med and identified novel DNA sequences amplified in PNET/MB. We describe for the first time amplification of the novel gene, NAG, in a subset of PNET/MB. Despite genomic amplification, NAG was not overexpressed in the tumours studied. We have determined that NAG maps less than 50 kb 5' of DDX1 and approximately 400 kb telomeric of MYCN on chromosome 2p24. CONCLUSION: We found a similar but slightly higher frequency of amplification than previously reported. We present several DNA fragments that may belong to the CpG islands of novel genes amplified in a small subset of PNET/MB. As an example we describe for the first time the amplification of NAG in the MYCN amplicon in PNET/MB.

Cold air as a bronchial provocation technique. Reproducibility and comparison with histamine and methacholine inhalation.

Bronchial provocation testing with cold air was carried out on 36 asthmatic and 13 normal subjects in order to assess the reproducibility and clinical relevance of the technique as a test of airways reactivity. Sixteen subjects underwent repeat testing after an interval of two to three weeks. Using a least squares linear regression analysis, the technique was highly reproducible, with a correlation of r = 0.93 (p less than 0.001). The 21 asthmatic subjects who had exercise-provoked symptoms required a significantly lower level of ventilation of cold air to produce a 35 percent drop in specific airways conductance (PD35) than did those who had no exercise-induced asthma (33.9 L min-1 vs 45.8 L min-1; p less than 0.02). Subjects requiring no regular treatment for their asthma had a geometric mean PD35 of 62.6 L min-1, significantly higher than those requiring inhaled therapy (44.9 L min-1; p less than 0.005). Subjects requiring oral in addition to inhaled treatment had the lowest PD35 (23.6 L min-1; p less than 0.02). Atopic status did not appear to influence the response. There was a strong correlation between the PD35 to cold air and to histamine (r = 0.92; p less than 0.001) and between the PD35 to cold air and to methacholine (r = 0.86; p less than 0.001). The three techniques of assessing bronchial reactivity were equally successful in separating the normal and asthmatic groups. The results indicate that cold air provocation may be reliably and reproducibly used to assess bronchial reactivity. The use of a naturally-occurring stimulus of asthma in all subjects has great potential as an investigational technique.

Bombesin immunoreactive neurons in the hypothalamic paraventricular nucleus innervate the dorsal vagal complex in the rat.

Bombesin-like immunoreactivity has been localized within neuronal cell bodies of the hypothalamus and nerve terminals within the dorsal vagal complex. The possibility that the hypothalamus is a source for bombesin-like immunoreactive terminals within the dorsal vagal complex was examined using the combined retrograde tracing and immunohistochemical technique. After injections of retrograde tracer were made into the dorsal vagal complex, cells in the hypothalamus labeled with both retrograde tracer and bombesin immunoreactivity were localized in the parvocellular part of the paraventricular nucleus. In the paraventricular nucleus most of the vagal projecting bombesin immunoreactive neurons were located within the medial parvocellular subdivision. Approximately 30% of the bombesin immunoreactive neurons in this subnucleus projected to the dorsal vagal complex. The results suggest that the paraventricular hypothalamic nucleus is a major source of bombesin terminals within the dorsal vagal complex. This pathway may mediate some of the autonomic nervous system changes that are observed when bombesin is injected within the central nervous system. Additionally, this data adds to a growing amount of evidence supporting the role of bombesin as a peptide neurotransmitter.

Aberrant methylation of genes in low-grade astrocytomas.

The underlying basis of the malignant progression of astrocytomas is a specific and cumulative series of genetic alterations, most of which are confined to high-grade tumors. In contrast, a proportion of low-grade astrocytomas have a relatively normal-appearing genome when examined with standard genetic screening methods. These methods do not detect epigenetic events such as aberrant methylation of CpG island, which result in transcriptional silencing of important cancer genes. To determine if aberrant methylation is involved in the early stages of astrocytoma development, we assessed the methylation status of 1,184 genes in each of 14 low-grade astrocytomas using restriction landmark genome scanning (RLGS). The results showed nonrandom and astrocytoma-specific patterns of aberrantly methylated genes. We estimate that an average of 1,544 CpG island-associated genes (range, 38 to 3,731) of the approximately 45,000 in the genome are aberrantly methylated in each tumor. Expression of a significant proportion of the genes could be reactivated by 5-aza-2-deoxycytidine-induced demethylation in cultured glioma cell lines. The data suggest that aberrant methylation of genes is more prevalent than genetic alterations and may have consequences for the development of low-grade astrocytomas.

Sputum cysteinyl-leukotriene levels correlate with the severity of pulmonary disease in children with cystic fibrosis.

Sputum samples from 13 children with cystic fibrosis (CF) were analyzed for leukotrienes (LTs) LTB4, LTC4, LTD4, and LTE4. Distribution of LTB4 appeared to be normal, and of cysteinyl-LTs log normal. Total cysteinyl-LT levels, of which on average 75% was LTE4, were nearly 10 times higher than in earlier studies. Log LTE4 and total cysteinyl-LT levels correlated with the overall severity of pulmonary disease assessed by Chrispin-Norman chest radiograph score (Log LTE4: r = 0.701, r2 = 49.1%, P = 0.008. Log total cysteinyl-LTs: r = 0.715, r2 = 51.1%, P = 0.006). There was no apparent relationship between LTB4 levels and Chrispin-Norman chest radiograph score, nor between the level of any of the LTs and age or organism cultured from sputum. These findings suggest that the cysteinyl-LTs may be involved in the pathophysiology of pulmonary disease in CF.

Bronchial responsiveness is not always increased after allergen challenge.

Increased bronchial responsiveness has been reported at various time points following allergen challenge (AC), and may be related to the magnitude of the late response (LAR). We have studied 20 mild asthmatics, who were known to develop a late asthmatic response to inhalation of house dust mite extract (fall of > 15% from post-diluent baseline FEV1 from 2 to 7h after AC). The provocation concentration of methacholine causing a 20% fall in FEV1 (PC20 FEV1) was measured before and 24 h after challenge with house dust mite extract (HDM). The mean (SEM) change in log(PC20) was 0.08 (0.09) mg ml-1, and was not significant (P = 0.38; paired t-test). The change in PC20 for each subject was not significantly correlated with the size of LAR (r = -0.33; P > 0.05), but was significantly correlated with the absolute change from baseline FEV1 at 24 h (r = 0.67; P < 0.01). Our subjects had a high baseline responsiveness, when compared with previous studies. We suggest they may have been approaching a maximally responsive state prior to study, and allergen challenge may have had little effect in further increasing responsiveness. Exposure to allergen in late responders is not necessarily followed by an increase in non-specific bronchial responsiveness.

O6-methylguanine-DNA methyltransferase in tumors and cells of the oligodendrocyte lineage.

BACKGROUND: Oligodendrogliomas respond to nitrosourea-based chemotherapy and are induced in rats following transplacental exposure to ethylnitrosourea, observations suggesting that neoplastic and normal cells of the oligodendrocyte lineage are "sensitive" to nitrosoureas. Nitrosoureas alkylate DNA at O6-guanine with repair mediated by O6-methylguanine-DNA methyltransferase (MGMT). The cytotoxic and carcinogenic properties of the nitrosoureas appear related to MGMT activity. METHODS: To explore why oligodendrogliomas respond to chemotherapy, we measured MGMT activity in five chemosensitive human oligodendrogliomas and in rat oligodendrocyte lineage cells. We also measured MGMT activity in rat astrocytes and compared the cytotoxic effects of carmustine (BCNU) on oligodendrocyte lineage cells and astrocytes. RESULTS: Low levels of MGMT activity were found in five of five human oligodendrogliomas. Cultures of neonatal rat glia enriched for oligodendrocyte lineage cells also had low levels of MGMT activity, approximately one-third that found in astrocytes (p < 0.02), and oligodendrocyte lineage cells were more sensitive to BCNU than astrocytes. CONCLUSIONS: Low MGMT activity may contribute to the chemosensitivity of some human oligodendrogliomas and rat oligodendrocyte lineage cells also have low levels. If drug resistance mechanisms in tumors reflect the biochemical properties of their cells of origin, then normal glia may serve as a laboratory substitute for human glioma.

Leukotriene B4 and oral cancer.

The quantitation of leukotriene B4 (LTB4) in induced squamous cell carcinoma (SCC) of the Syrian hamster cheek pouch and histologically proven human oral SCC was investigated by a combination of reverse phase-high performance liquid chromatography (RP-HPLC) and radioimmunoassay (RIA). Healthy tissue obtained from these same patients and animals treated with vehicle alone were used as controls. From both animal and human studies our results show a 10 to 30 fold increase in the levels of LTB4 found in tumour compared to control tissue. Furthermore, this dihydroxy acid was not detected in the mucosal tissue of normal subjects undergoing routine surgery. Since LTB4 is a potent inflammatory mediator and modulator of immune responses, its presence at biologically active concentrations in human squamous cell carcinoma suggests a possible role in the pathogenesis of head and neck cancer.

Relationship between bronchial airway responsiveness and clinical severity of asthma.

The minimum medication required to control symptoms was individually established in a systematic manner in 10 asthmatics. A scoring system for the severity of asthma was designed using the indices of initial airway calibre (a reflection of the degree of airway obstruction) and the minimum medication requirement; all the subjects were so scored. Bronchial airway responsiveness to histamine, methacholine and isocapnic hyperventilation of cold air was then measured in these subjects. The relationship between the level of bronchial responsiveness and the asthma severity score was examined. The mean airway responsiveness to histamine or methacholine for the subjects who required a combination of drugs was not significantly greater than that for those who required single medication intermittently or daily, while the airway responsiveness to cold air was significantly different between the subjects in the two treatment subgroups. Similarly, there was no correlation between the asthma severity score and airway responsiveness to methacholine and histamine (r = -0.38 and -0.48; P greater than 0.1) while a significant correlation was found with responsiveness to cold air (r = 0.72; P less than 0.02). The results suggest that there is a qualitative difference between the bronchoconstriction induced in asthmatic subjects by pharmacological constrictor substances and natural physical stimuli such as cold air.