Impact of empagliflozin added on to basal insulin in type 2 diabetes inadequately controlled on basal insulin: a 78-week randomized, double-blind, placebo-controlled trial.

AIMS: To investigate the efficacy and tolerability of empagliflozin added to basal insulin-treated type 2 diabetes. METHODS: Patients inadequately controlled [glycated haemoglobin (HbA1c) >7 to ≤10% (>53 to ≤86 mmol/mol)] on basal insulin (glargine, detemir, NPH) were randomized to empagliflozin 10 mg (n = 169), empagliflozin 25 mg (n = 155) or placebo (n = 170) for 78 weeks. The baseline characteristics were balanced among the groups [mean HbA1c 8.2% (67 mmol/mol), BMI 32.2 kg/m(2) ]. The basal insulin dose was to remain constant for 18 weeks, then could be adjusted at investigator's discretion. The primary endpoint was change from baseline in HbA1c at week 18. Key secondary endpoints were changes from baseline in HbA1c and insulin dose at week 78. RESULTS: At week 18, the adjusted mean ± standard error changes from baseline in HbA1c were 0.0 ± 0.1% (-0.1 ± 0.8 mmol/mol) for placebo, compared with -0.6 ± 0.1% (-6.2 ± 0.8 mmol/mol) and -0.7 ± 0.1% (-7.8 ± 0.8 mmol/mol) for empagliflozin 10 and 25 mg, respectively (both p < 0.001). At week 78, empagliflozin 10 and 25 mg significantly reduced HbA1c, insulin dose and weight vs placebo (all p < 0.01), and empagliflozin 10 mg significantly reduced systolic blood pressure vs placebo (p = 0.004). Similar percentages of patients had confirmed hypoglycaemia in all groups (35-36%). Events consistent with urinary tract infection were reported in 9, 15 and 12% of patients on placebo, empagliflozin 10 and 25 mg, and events consistent with genital infection were reported in 2, 8 and 5%, respectively. CONCLUSIONS: Empagliflozin for 78 weeks added to basal insulin improved glycaemic control and reduced weight with a similar risk of hypoglycaemia to placebo.

Chromatin H3K27me3/H3K4me3 histone marks define gene sets in high-grade serous ovarian cancer that distinguish malignant, tumour-sustaining and chemo-resistant ovarian tumour cells.

In embryonic stem (ES) cells, bivalent chromatin domains containing H3K4me3 and H3K27me3 marks silence developmental genes, while keeping them poised for activation following differentiation. We have identified gene sets associated with H3K27me3 and H3K4me3 marks at transcription start sites in a high-grade ovarian serous tumour and examined their association with epigenetic silencing and malignant progression. This revealed novel silenced bivalent marked genes, not described previously for ES cells, which are significantly enriched for the PI3K (P<10(-7)) and TGF-ß signalling pathways (P<10(-5)). We matched histone marked gene sets to gene expression sets of eight normal fallopian tubes and 499 high-grade serous malignant ovarian samples. This revealed a significant decrease in gene expression for the H3K27me3 and bivalent gene sets in malignant tissue. We then correlated H3K27me3 and bivalent gene sets to gene expression data of ovarian tumour 'stem cell-like' sustaining cells versus non-sustaining cells. This showed a significantly lower expression for the H3K27me3 and bivalent gene sets in the tumour-sustaining cells. Similarly, comparison of matched chemo-sensitive and chemo-resistant ovarian cell lines showed a significantly lower expression of H3K27me3/bivalent marked genes in the chemo-resistant compared with the chemo-sensitive cell line. Our analysis supports the hypothesis that bivalent marks are associated with epigenetic silencing in ovarian cancer. However it also suggests that additional tumour specific bivalent marks, to those known in ES cells, are present in tumours and may potentially influence the subsequent development of drug resistance and tumour progression.

Candidate DNA methylation drivers of acquired cisplatin resistance in ovarian cancer identified by methylome and expression profiling.

Multiple DNA methylation changes in the cancer methylome are associated with the acquisition of drug resistance; however it remains uncertain how many represent critical DNA methylation drivers of chemoresistance. Using isogenic, cisplatin-sensitive/resistant ovarian cancer cell lines and inducing resensitizaton with demethylating agents, we aimed to identify consistent methylation and expression changes associated with chemoresistance. Using genome-wide DNA methylation profiling across 27 578 CpG sites, we identified loci at 4092 genes becoming hypermethylated in chemoresistant A2780/cp70 compared with the parental-sensitive A2780 cell line. Hypermethylation at gene promoter regions is often associated with transcriptional silencing; however, expression of only 245 of these hypermethylated genes becomes downregulated in A2780/cp70 as measured by microarray expression profiling. Treatment of A2780/cp70 with the demethylating agent 2-deoxy-5'-azacytidine induces resensitization to cisplatin and re-expression of 41 of the downregulated genes. A total of 13/41 genes were consistently hypermethylated in further independent cisplatin-resistant A2780 cell derivatives. CpG sites at 9 of the 13 genes (ARHGDIB, ARMCX2, COL1A, FLNA, FLNC, MEST, MLH1, NTS and PSMB9) acquired methylation in ovarian tumours at relapse following chemotherapy or chemoresistant cell lines derived at the time of patient relapse. Furthermore, 5/13 genes (ARMCX2, COL1A1, MDK, MEST and MLH1) acquired methylation in drug-resistant ovarian cancer-sustaining (side population) cells. MLH1 has a direct role in conferring cisplatin sensitivity when reintroduced into cells in vitro. This combined genomics approach has identified further potential key drivers of chemoresistance whose expression is silenced by DNA methylation that should be further evaluated as clinical biomarkers of drug resistance.

CpG-island methylation study of liver fluke-related cholangiocarcinoma.

BACKGROUND: Genetic changes have been widely reported in association with cholangiocarcinoma (CCA), while epigenetic changes are poorly characterised. We aimed to further evaluate CpG-island hypermethylation in CCA at candidate loci, which may have potential as diagnostic or prognostic biomarkers. METHODS: We analysed methylation of 26 CpG-islands in 102 liver fluke related-CCA and 29 adjacent normal samples using methylation-specific PCR (MSP). Methylation of interest loci was confirmed using pyrosequencing and/or combined bisulfite restriction analysis, and protein expression by immunohistochemistry. RESULTS: A number of CpG-islands (OPCML, SFRP1, HIC1, PTEN and DcR1) showed frequency of hypermethylation in >28% of CCA, but not adjacent normal tissues. The results showed that 91% of CCA were methylated in at least one CpG-island. The OPCML was the most frequently methylated locus (72.5%) and was more frequently methylated in less differentiated CCA. Patients with methylated DcR1 had significantly longer overall survival (Median; 41.7 vs 21.7 weeks, P=0.027). Low-protein expression was found in >70% of CCA with methylation of OPCML or DcR1. CONCLUSION: Aberrant hypermethylation of certain loci is a common event in liver fluke-related CCA and may potentially contribute to cholangiocarcinogenesis. The OPCML and DcR1 might serve as methylation biomarkers in CCA that can be readily examined by MSP.

Protein inhibitor of nitric oxide synthase (NOS) and the N-methyl-D-aspartate receptor are expressed in the rat and mouse penile nerves and colocalize with penile neuronal NOS.

Nitrergic neurotransmission triggering penile erection is mediated by nitric oxide (NO) synthesized in the cavernosal nerves of the penis by penile neuronal NO synthase (PnNOS). In the central nervous system, nNOS is activated by the N-methyl-D-aspartate receptor (NMDAR) and, presumably, is inhibited by the protein inhibitor of NOS (PIN). The PnNOS and NMDAR are expressed in the penis, and PnNOS has been localized in penile nerves. Both proteins colocalize with PIN in the hypothalamus and the spinal cord involved in the control of erection. The present study aimed to elucidate the relationship between PnNOS, PIN, and NMDAR in the penis. It was found that in the rat, PIN was expressed in the pelvic ganglion and the cavernosal nerve, and penile PIN cDNA was cloned, sequenced, and expressed. Immunohistochemistry localized PIN to the cavernosal and dorsal nerve of the penis, whereas NMDAR was not detected in the latter. Dual-fluorescence labeling showed that PnNOS colocalized with PIN in both nerves but with NMDAR only in the cavernosal nerve. Aging did not affect the mRNA levels of PnNOS, nNOS, NMDAR, and PIN. Both PIN and NMDAR were detected in penile nerves of the wild-type and nNOS(-/-) mouse. The PIN protein did not inhibit or bind NOS in penile extracts, and in vivo, PIN cDNA reduced the erectile response to electrical field stimulation. In conclusion, PIN and NMDAR colocalize with PnNOS in penile nerves, but the functional significance of these protein interactions for penile erection remains to be elucidated.

[Technical realization of an LCD-based vision test device]. / Technische Realisierung eines LCD-basierten Sehtestgerätes.

We describe a computer-aided self-test vision screener for testing near and far visual acuity. The device generates images separately for each eye on a LCD. Acuity can be measured in the range between 0.1 and 1.6 for any distance between 0.34 m and 6 m. In addition, it also enables testing for colour deficiency, phoria, stereopsis and contrast sensitivity. The device is fully automatic and enables self-testing of the above mentioned functions. Initial practical application in an industrial environment has demonstrated the practicability of the device.

Total body water reference values and prediction equations for adults.

BACKGROUND: The clinical interpretation of total body water (TBW) necessitates the availability of timely comparative reference data. The prediction of TBW volume in renal disease is critical in order to prescribe and monitor the dose of dialysis in the determination of Kt/V. In clinical practice, urea distribution (V) is commonly predicted from anthropometric equations that are several decades old and for white patients only. This article presents new reference values and prediction equations for TBW from anthropometry for white and black adults. METHODS: The study sample included four data sets, two from Ohio and one each from New Mexico and New York, for a total of 604 white men, 128 black men, 772 white women, and 191 black women who were 18 to 90 years of age. The TBW concentration was measured by the deuterium or tritium oxide dilution method, and body composition was measured with a Lunar DXA machine. An all-possible-subsets of regression was used to predict TBW. The accuracy of the selected equations was confirmed by cross-validation. RESULTS: Blacks had larger TBW means than whites at all age groups. The 75th TBW percentile for whites approximated the TBW median for blacks at most ages. The white men and black men and women had the largest TBW means ever reported for healthy individuals. The race- and sex-specific TBW prediction equations included age, weight, and stature, with body mass index (BMI) substituted for weight in the white men. The root mean square errors (RMSEs) and standard errors for the individual (SEIs) ranged from approximately 3.8 to 5.0 L for the men and from 3.3 to 3.6 L for the women. In both men and women, high values of TBW were associated with high levels of total body fat (TBF) and fat-free mass (FFM). CONCLUSION: : TBW in these healthy adults is relatively stable through a large portion of adulthood. There are significant race and sex differences in TBW. These accurate and precise equations for TBW provide a useful tool for the clinical prediction of TBW in renal disease for white and black adults. These are the first TBW prediction equations that are specific for blacks.

ET gets HIT--thrombocytotic heparin-induced thrombocytopenia (HIT) in a patient with essential thrombocythemia (ET).

Immune-mediated heparin-induced thrombocytopenia (HIT) is a relatively common complication in patients receiving heparin, and represents a strong risk factor for thromboembolic disease associated with high morbidity and mortality. The condition is commonly defined as a platelet count fall of greater than 30-50% to values below 150 x 10(9)/l. Despite this, several cases with platelet count nadirs in the normal range have been reported. This report describes a patient with status post-carotid thrombendarterectomy presenting with neurological symptoms and thrombocytosis (1235 x 10(9)/l), which in due course was diagnosed to be caused by essential thrombocythemia (ET). Heparin therapy was established and symptoms resolved markedly. After 5 days of standard heparin therapy, serologically confirmed HIT with new neurological symptoms occurred. The platelet count nadir attributed to HIT was far above normal (633 x 10(9)/l), which nevertheless represented a substantial relative platelet count fall (49%). This is the first reported case of serologically confirmed HIT in a patient with ET. Furthermore, it represents the first published case of HIT being present in thrombocytosis. Clinicians should be aware that atypical HIT can even be present in thrombocytosis and, because of nomenclature, HIT with normal and elevated platelet count nadirs is likely to be largely underdiagnosed.

Expression of penile neuronal nitric oxide synthase variants in the rat and mouse penile nerves.

Penile erection is mediated by nitric oxide (NO) synthesized by the neuronal nitric oxide synthase (nNOS). In the rat penis, the main nNOS mRNA variant, PnNOS, differs from cerebellar nNOS (CnNOS) by a 102 base pair insert encoding a 34-amino acid sequence. In the mouse, two nNOS mRNAs have been identified: nNOSalpha, encoding a 155-kDa protein, and an exon 2-deletion variant, nNOSbeta, encoding a 135-kDa protein that lacks a domain where a protein inhibitor of nNOS (PIN) binds. We wished to determine whether PnNOSalpha and beta are expressed in the rat penis and are located in the nerves and whether the beta form persists in the potent nNOS knock-out mouse (nNOS( big up tri, open big up tri, open)). A PnNOS antibody against the insert common to both PnNOSalpha and beta detected the expected 155-kDa protein in PnNOSalpha-transfected cells. This antibody, and the one common to PnNOS/CnNOS, showed (on Western blots) the 155- and 135-kDa nNOS variants in rat penile tissue during development and aging. PnNOSalpha mRNA and its subvariants were found as the main nNOS in the penile corpora, the cavernosal nerve, and the pelvic ganglia, with lower levels of PnNOSbeta mRNA. In tissue sections, PnNOS protein was immunodetected in the penile nerve endings in the rat and in the nNOS wild-type and nNOS( big up tri, open big up tri, open) mice. An antibody against the sequence encoded by exon 2 did not react (on Western blots) with the 135-kDa band, which confirms that this protein is the beta form. In conclusion, both PnNOSalpha and beta are expressed in the rat penis at all ages and are located in the nerves. The beta form may allow nitric oxide synthesis during erection to be partially insensitive to PIN. The residual expression of PnNOS, and possibly CnNOS, in the penis of the nNOS( big up tri, open big up tri, open) mouse occurs through transcription of the beta mRNA, and this may explain the retention of erectile function when the expression of nNOSalpha is disrupted.

Aging, body composition, and lifestyle: the Fels Longitudinal Study.

BACKGROUND: Changes in body composition in men and women occur with age, but these changes are affected by numerous covariate factors. OBJECTIVE: The study examined patterns of change in body composition and determined the effects of long-term patterns of change in physical activity in older men and women and in menopausal status and estrogen use in women. DESIGN: Serial measures of height, weight, body mass index (BMI), total body fat (BF), percentage BF, and fat-free mass (FFM) from underwater weighing of 102 men and 108 women enrolled in the Fels Longitudinal Study were analyzed. Physical activity levels and menopausal status were included as covariates. RESULTS: There were significant age-related decreases in FFM and height and increases in total BF, percentage BF, weight, and BMI. Physical activity was associated with decreases in total BF, percentage BF, weight, and BMI in men and were associated with increases in FFM and decreases in total BF and percentage BF in women. Postmenopausal women had significantly higher total BF and percentage BF than did pre- and perimenopausal women. The longer the time since menopause the greater were the increases in weight, BMI, total BF, and percentage BF; however, estrogen use attenuated these increases. CONCLUSIONS: Low FFM can be improved by increased physical activity. The effects of an intervention program on body composition can be masked if only body weight or BMI is measured. The effects of physical activity were more profound in postmenopausal than in premenopausal women, and estrogen use had beneficial effects on body composition.

Total body water data for white adults 18 to 64 years of age: the Fels Longitudinal Study.

BACKGROUND: Total body water (TBW) volume is reported to decrease with age, but much of the published data are 20 to almost 50 years old and are cross-sectional. Proper interpretation of clinical levels of TBW and trends with age necessitates the availability of current longitudinal data from healthy individuals. METHODS: Mixed longitudinal data for TBW of 274 white men and 292 white women (18 to 64 years of age) in the Fels Longitudinal Study were collected on a regular schedule over a recent eight-year period. The concentration of deuterium was measured by deuterium nuclear magnetic resonance spectroscopy. Body composition estimates were made with dual-energy x-ray absorptiometry, and random effect models were used to determine the patterns of change over time with and without covariates. RESULTS: The mean TBW data for the Fels men are either similar to or approximately 2 to as much as 6 liters greater than that reported by most other investigators 20 to 50 years ago. For Fels women, the mean TBW ranges from approximately 2 to as much as 5 liters less than that reported previously. These comparisons with much earlier studies reflect cohort effects and the secular changes in overall body size that have occurred during the past 60 to 70 years. These findings are reinforced by the fact that some early data sets included individuals born almost 140 years ago. After adjusting for the covariate effects of total body fat (TBF) and fat-free mass (FFM) with age, there were no significant age or age-squared effects on TBW in the men. In the women, after adjusting for the covariate associations of TBF and FFM with age, there was a small, but significant, negative linear association of TBW with age. In the men and women, the mean ratio of TBW to weight declined with age as a function of an increase in body fatness and more so for the men than the women. CONCLUSION: The findings from these mixed longitudinal data indicate that TBW volume, on average, maintains a reasonable degree of stability in men and women through a large portion of adulthood. These TBW data are recommended as current reference data for healthy adults.

Linkage analysis narrows the critical region for oculodentodigital dysplasia to chromosome 6q22-q23.

Oculodentodigital dysplasia (ODDD) is an autosomal dominant condition with high penetrance and variable expressivity. The anomalies of the craniofacial region, eyes, teeth, and limbs indicate abnormal morphogenesis during early fetal development. Neurologic abnormalities occur later in life and appear to be secondary to white matter degeneration and basal ganglia changes. In familial cases, the dysmorphic and/or neurodegenerative components of the phenotype can be more severe and/or present at a younger age in subsequent generations, suggesting genetic anticipation. These clinical features suggest that the ODDD gene is pleiotropic with important functions throughout pre- and postnatal development. We have performed two-point linkage analysis with seven ODDD families and 19 microsatellite markers on chromosome 6q spanning a genetic distance of approximately 11 cM in males and 20 cM in females. We have refined the location of the ODDD gene between DNA markers D6S266/D6S261 (centromeric) and D6S1639 (telomeric), an interval of 1.01 (male) to 2.87 (female) cM. The strongest linkage was to DNA marker D6S433 (Zmax = 8.96, thetamax = 0.001). Families show significant linkage to chromosome 6q22-q23 and no evidence for genetic heterogeneity.

Human skeletal muscle HSP70 response to training in highly trained rowers.

Previous studies have demonstrated exercise-induced heat shock protein 70 (HSP70) in animals. The purpose of this study was to investigate human skeletal muscle HSP70 response to rowing training. Ten male rowers trained for 4 wk with different forms, durations, and intensities of exercise. Biopsy was performed in the right musculus vastus lateralis before training and at the end of each week. HSP70 in 5 microg of total protein from the muscle sample was determined by using Western blot and immunodetection with chemiluminescence technique, by means of laser densitometer referring to a series of known standard HSP70. Compared with pretraining (100%), HSP70 increased during training (181, 405, 456, and 363% from the first to fourth training week, respectively) with the maximum HSP70 production at the end of second training week. Thus HSP70 is induced in highly trained human muscle by long-term training.

Total-body and regional bone mineral content and areal bone mineral density in children aged 8-18 y: the Fels Longitudinal Study.

BACKGROUND: Techniques for cross-calibration of bone mineral content (BMC) and bone mineral density (BMD) between manufacturers of dual-energy X-ray absorptiometry (DXA) instruments are currently inadequate for total body measurements. Therefore, manufacturer-specific data for BMC and BMD in children are needed. OBJECTIVE: We provided age- and sex-specific means and SDs for total-body and regional BMC and areal BMD in 8-18-y-old white children. DESIGN: BMC and BMD of the head, arms, legs, pelvis, spine, and total body were determined by DXA. Data include 465 annual measurements from 148 healthy children with body weights between 30 and 100 kg and statures <190 cm. RESULTS: There were significant sex differences in BMC at ages 15-18 y for the total body and legs, at ages 12 and 15-18 y for arms and pelvis, at ages 11-13 and 16-18 y for the spine, and at ages 10-11 y for the head. There were significant sex differences in BMD at ages 16-18 y for total body, arms, and legs; at ages 12-13 and 16-18 y for the pelvis; at ages 12-14 and 18 y for the spine; and at ages 13-18 y for the head. CONCLUSIONS: Data presented in this investigation can be used to compare the BMC and BMD of 8-18-y-old white children (with statures <190 cm and body weights between 30 and 100 kg) using DXA.

Detection and isolation of lectin-transfected COS cells based on cell adhesion to immobilized glycosphingolipids.

Two methods are described, one for detection and one for isolation of COS cells transiently expressing vertebrate lectins. The methods are based on specific cell adhesion to polystyrene microwells or magnetic beads adsorbed with glycosphingolipids. In the first method, glycolipids were adsorbed to wells of 96-well polystyrene plates. A suspension of lectin-transfected COS cells was added and the plate was incubated to allow cell adhesion to occur. The plate was then immersed in buffer, inverted (while immersed), and placed in a fluid-filled Plexiglas centrifugation chamber which was sealed to avoid introducing an air-liquid interface. The chamber, with the inverted plate enclosed, was centrifuged to remove nonadherent cells. The plate was then removed from the carrier (while immersed) and righted, and adherent cells were quantitated enzymatically or immunochemically using a 96-well plate reader. COS cells transfected with an expression plasmid carrying the gene for the rat Kupffer cell lectin (fucose and N-acetylgalactosamine specific) adhered specifically to globotetraosylceramide. Glycolipid- and lectin-specific cell adhesion was readily detected even when COS cells were transfected with a plasmid mixture containing 0.5% lectin-carrying plasmid and 99.5% irrelevant plasmid. This sensitivity will facilitate screening of plasmid pools to detect and isolate plasmids expressing mammalian lectin genes. To isolate COS cells transiently expressing lectin, glycosphingolipids were adsorbed to carboxylated magnetic polystyrene microspheres, which were mixed with the lectin-transfected COS cells. Adherent cells were collected on a fixed magnet and plasmid recovered for subsequent amplification.

Gangliosides are neuronal ligands for myelin-associated glycoprotein.

Nerve cells depend on specific interactions with glial cells for proper function. Myelinating glial cells are thought to associate with neuronal axons, in part, via the cell-surface adhesion protein, myelin-associated glycoprotein (MAG). MAG is also thought to be a major inhibitor of neurite outgrowth (axon regeneration) in the adult central nervous system. Primary structure and in vitro function place MAG in an immunoglobulin-related family of sialic acid-binding lactins. We report that a limited set of structurally related gangliosides, known to be expressed on myelinated neurons in vivo, are ligands for MAG. When major brain gangliosides were adsorbed as artificial membranes on plastic microwells, only GT1b and GD1a supported cell adhesion of MAG-transfected COS-1 cells. Furthermore, a quantitatively minor ganglioside expressed on cholinergic neurons, GQ1b alpha (also known as Chol-1 alpha-b), was much more potent than GT1b or GD1a in supporting MAG-mediated cell adhesion. Adhesion to either GT1b or GQ1b alpha was abolished by pretreatment of the adsorbed gangliosides with neuraminidase. On the basis of structure-function studies of 19 test glycosphingolipids, an alpha 2,3-N-acetylneuraminic acid residue on the terminal galactose of a gangliotetraose core is necessary for MAG binding, and additional sialic acid residues linked to the other neutral core saccharides [Gal(II) and GalNAc(III)] contribute significantly to binding affinity. MAG-mediated adhesion to gangliosides was blocked by pretreatment of the MAG-transfected COS-1 cells with anti-MAG monoclonal antibody 513, which is known to inhibit oligodendrocyte-neuron binding. These data are consistent with the conclusion that MAG-mediated cell-cell interactions involve MAG-ganglioside recognition and binding.

Bronchial reactivity to isocapnic hyperventilation: results in an unselected population of outpatients with known or suspected asthma.

Isocapnic hyperventilation with dry air is nearly as effective as with dry cold air, and appears to be a valuable screening test for bronchial hyperresponsiveness. However some incidental factors such as prechallenge bronchoconstriction, level of hyperventilation, age and smoking habits have barely been examined or were investigated in small samples of either normals or well-characterised asthmatics. In an inhomogeneous population of 186 outpatients with known asthma, 286 with suspected asthma and 32 normals, a single isocapnic hyperventilation challenge of 6-min duration was performed. There was a weak, but significant correlation between the degree of prechallenge airway function and the bronchial response, assessed by the change in forced expiratory volume in 1 s (r = 0.27, p = 0.000) in known asthmatics, but not in patients with suspected asthma and in normals. No significant relation was found between the level of hyperventilation and the bronchial response when comparing the bronchial response to the single-dose hyperventilation test between the subjects (NS). Increasing age appears to be associated with an attenuation of the response in known asthmatics (r = 0.21, p = 0.004), but not in patients with suspected asthma. Smoking habits did not affect the bronchial response in this study. In conclusion, the main finding is that there is a weak correlation between baseline airway obstruction and the subsequent response to isocapnic hyperventilation, a slow decline in response with age and no increase in responsiveness in smokers. Hence, isocapnic hyperventilation is a relatively robust test for assessing bronchial reactivity in an inhomogeneous population of outpatients like ours.