DNA methylation of Th2 lineage determination genes at birth is associated with allergic outcomes in childhood.

BACKGROUND: There is now increasing evidence that asthma and atopy originate in part in utero, with disease risk being associated with the altered epigenetic regulation of genes. OBJECTIVE AND METHODS: To determine the relationship between variations in DNA methylation at birth and the development of allergic disease, we examined the methylation status of CpG loci within the promoter regions of Th1/2 lineage commitment genes (GATA3, IL-4, IL-4R, STAT4 and TBET) in umbilical cord DNA at birth in a cohort of infants from the Southampton Women's Survey (n = 696) who were later assessed for asthma, atopic eczema and atopy. RESULTS: We found that higher methylation of GATA3 CpGs -2211/-2209 at birth was associated with a reduced risk of asthma at ages 3 (median ratio [median methylation in asthma group/median methylation in non-asthma group] = 0.74, P = .006) and 6-7 (median ratio 0.90, P = .048) years. Furthermore, we demonstrated that the GATA3 CpG loci associated with later risk of asthma lie within a NF-κB binding site and that methylation here blocks transcription factor binding to the GATA3 promoter in the human Jurkat T-cell line. Associations between umbilical cord methylation of CpG loci within IL-4R with atopic eczema at 12 months (median ratio 1.02, P = .028), and TBET with atopy (median ratio 0.98, P = .017) at 6-7 years of age were also observed. CONCLUSIONS AND CLINICAL RELEVANCE: Our findings provide further evidence of a developmental contribution to the risk of later allergic disorders and suggest that involvement of epigenetic mechanisms in childhood asthma is already demonstrable at birth.

Synthesis of fruity ethyl esters by acyl coenzyme A: alcohol acyltransferase and reverse esterase activities in Oenococcus oeni and Lactobacillus plantarum.

AIMS: To assess the abilities of commercial wine lactic acid bacteria (LAB) to synthesize potentially flavour active fatty acid ethyl esters and determine mechanisms involved in their production. METHODS AND RESULTS: Oenococcus oeni AWRI B551 produced significant levels of ethyl hexanoate and ethyl octanoate following growth in an ethanolic test medium, and ester formation generally increased with increasing pH (4.5 > 3.5), anaerobiosis and precursor supplementation. Cell-free extracts of commercial O. oeni strains and Lactobacillus plantarum AWRI B740 were also tested for ester-synthesizing capabilities in a phosphate buffer via: (i) acyl coenzyme A: alcohol acyltransferase (AcoAAAT) activity and (ii) reverse esterase activity. For both ester-synthesizing activities, strain-dependent variation was observed, with AcoAAAT activity generally greater than reverse esterase. Reverse esterase in O. oeni AWRI B551 also esterified 1-propanol to produce propyl octanoate, and deuterated substrates ([(2)H(6)]ethanol and [(2)H(15)]octanoic acid) to produce the fully deuterated ester, [(2)H(5)]ethyl [(2)H(15)]octanoate. CONCLUSIONS: Wine LAB exhibit ethyl ester-synthesizing capability and possess two different ester-synthesizing activities, one of which is associated with an acyl coenzyme A: alcohol acyltransferase. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that wine LAB exhibit enzyme activities that can augment the ethyl ester content of wine. This knowledge will facilitate greater control over the impacts of malolactic fermentation on the fruity sensory properties and quality of wine.

The GTPase rho controls a p53-dependent survival checkpoint during thymopoiesis.

During the early stages of thymopoiesis, cell survival is controlled by cytokines that regulate the expression of antiapoptotic proteins such as Bcl-2. At the pre-T cell stage, a critical checkpoint for beta chain selection is monitored by the tumor suppressor p53: pre-T cells can survive and differentiate when p53 is removed genetically or when its proapoptotic function is inactivated physiologically as a consequence of signaling through the pre-T cell receptor complex. Previous work has shown that the guanine nucleotide binding protein Rho controls cell survival in T cell progenitors. Here we define the survival pathways controlled by Rho in pre-T cells and show that this GTPase is a pivotal regulator of the p53-mediated checkpoint operating at the time of beta selection: loss of Rho function results in apoptosis in pre-T cells, but this cell death is prevented by loss of p53. The prevention of cell death by loss of p53 restored numbers of early T cell progenitors but did not fully restore thymic cellularity. Further analysis revealed that loss of Rho function caused survival defects in CD4/8 double-positive thymocytes that is independent of p53 but can be prevented by ectopic expression of Bcl-2. These studies highlight that the GTPase Rho is a crucial component of survival signaling pathways in at least two different thymocyte subpopulations: Rho controls the p53 survival checkpoint in pre-T cells and is also crucial for a p53 independent survival signaling pathway in CD4/8 double positives.

A critical role for Syk in signal transduction and phagocytosis mediated by Fcgamma receptors on macrophages.

Receptors on macrophages for the Fc region of IgG (FcgammaR) mediate a number of responses important for host immunity. Signaling events necessary for these responses are likely initiated by the activation of Src-family and Syk-family tyrosine kinases after FcgammaR cross-linking. Macrophages derived from Syk-deficient (Syk-) mice were defective in phagocytosis of particles bound by FcgammaRs, as well as in many FcgammaR-induced signaling events, including tyrosine phosphorylation of a number of cellular substrates and activation of MAP kinases. In contrast, Syk- macrophages exhibited normal responses to another potent macrophage stimulus, lipopolysaccharide. Phagocytosis of latex beads and Escherichia coli bacteria was also not affected. Syk- macrophages exhibited formation of polymerized actin structures opposing particles bound to the cells by FcgammaRs (actin cups), but failed to proceed to internalization. Interestingly, inhibitors of phosphatidylinositol 3-kinase also blocked FcgammaR-mediated phagocytosis at this stage. Thus, PI 3-kinase may participate in a Syk-dependent signaling pathway critical for FcgammaR-mediated phagocytosis. Macrophages derived from mice deficient for the three members of the Src-family of kinases expressed in these cells, Hck, Fgr, and Lyn, exhibited poor Syk activation upon FcgammaR engagement, accompanied by a delay in FcgammaR-mediated phagocytosis. These observations demonstrate that Syk is critical for FcgammaR-mediated phagocytosis, as well as for signal transduction in macrophages. Additionally, our findings provide evidence to support a model of sequential tyrosine kinase activation by FcgammaR's analogous to models of signaling by the B and T cell antigen receptors.

[Coronary CT angiography using prospective ECG triggering: high diagnostic accuracy with low radiation dose]. / CT-Angiographie der Koronarien mit prospektivem EKG-Triggering: Hohe diagnostische Genauigkeit bei niedriger Strahlendosis.

BACKGROUND: The purpose of this study was to evaluate the diagnostic performance of coronary CT angiography (coronary CTA) using prospective ECG triggering (PT) for the detection of significant coronary artery stenosis compared to invasive coronary angiography (ICA). METHODS: A total of 20 patients underwent coronary CTA with PT using a 128-slice CT scanner (Definition AS+, Siemens) and ICA. All coronary CTA studies were evaluated for significant coronary artery stenoses (>or=50% luminal narrowing) by 2 observers in consensus using the AHA-15-segment model. Findings in CTA were compared to those in ICA. RESULTS: Coronary CTA using PT had 88% sensitivity in comparison to 100% with ICA, 95% to 88% specificity, 80% to 92% positive predictive value and 97% to 100% negative predictive value for diagnosing significant coronary artery stenosis on per segment per patient analysis, respectively. Mean effective radiation dose-equivalent of CTA was 2.6+/-1 mSv. CONCLUSION: Coronary CTA using PT enables non-invasive diagnosis of significant coronary artery stenosis with high diagnostic accuracy in comparison to ICA and is associated with comparably low radiation exposure.

Iodinated contrast media: effect of osmolarity and injection temperature on erythrocyte morphology in vitro.

BACKGROUND: Some side effects of intravenously injected iodinated contrast media are thought to be linked to the biological properties of the various agents and their effect on blood components. PURPOSE: To assess the effect of osmolarity and injection temperature of iodinated contrast media on erythrocyte (RBC) morphology in vitro. MATERIAL AND METHODS: Blood from 20 volunteers was incubated with three different contrast media (320 mg I/ml iso-osmolar iodixanol, 300 mg I/ml low-osmolar iopromide, 300 mg I/ml low-osmolar iopamidol) injected at 37 degrees C, 43 degrees C, and 48 degrees C, and in two different volumes corresponding to the estimated concentration at the site of venous injection and after systemic distribution. After 10 min incubation, aliquots were removed for complete blood count analysis and blood smears. Two hematologists blindedly and independently reviewed all smears, and determined the grade of morphological RBC changes compared to a blank sample. RESULTS: There was excellent (kappa = 0.98) inter-reader correlation for grading RBC changes. At systemic concentration at 37 degrees C, the grade of RBC changes was significantly (P<0.05) less in blood samples exposed to iso-osmolar iodixanol (mean 0.21) as compared to low-osmolar iopromide (mean 0.26) and low-osmolar iopamidol (mean 0.58). These differences became more significant at higher volumes, corresponding to concentrations at the site of injection and higher injection temperatures. CONCLUSION: In vitro, RBC morphology is less affected by iso-osmolar as compared to low-osmolar contrast media. These differences become more significant at higher injection temperatures that are proposed to improve flow dynamics for high-speed injection.

A new in vitro assay to test UVR protection of dermal extracellular matrix components by a flat spectrum sunscreen.

The efficacy of topical sunscreens is currently assessed by crude, costly and time consuming in vivo assays. We have previously demonstrated that components of the dermal extracellular matrix (ECM), rich in UV-absorbing amino acids, are susceptible to damage by solar simulated radiation (SSR) in vitro. Here we developed an in vitro method to test the ability of sunscreens to protect fibrillin-rich microfibrils (FRM) and fibronectin, key components of the dermal ECM from UV-induced damage. Solutions of FRM or fibronectin were irradiated without protection, in the presence of a vehicle or a commercially-available flat-spectrum sunscreen. The effect of SSR on molecular structure was determined by atomic force microscopy (FRM) and SDS-PAGE (fibronectin). Following irradiation, FRM periodicity became bi-modally distributed (peaks: 40nm & 59nm) compared to the unimodal distribution in unexposed controls (peak: 50nm). Irradiation in the presence of flat-spectrum sunscreen protected against this change, maintaining the unimodal distribution. SSR induced significant aggregation of fibronectin (p=0.005), which was abrogated by sunscreen. These results demonstrate that this in vitro assay system is sufficiently sensitive to act as an initial/additional screen of sunscreen efficacy. We conclude that sunscreen can reduce UV-mediated damage of key dermal ECM in vitro and thereby prevent remodelling associated with photoageing.

Frequent loss of imprinting of PEG1/MEST in invasive breast cancer.

The human PEG1 gene is a newly identified imprinted gene on 7q32. Genetic aberrations of this chromosomal region are often detected in invasive breast carcinomas. In this study, we show monoallelic PEG1 expression in normal breast tissue, indicating the presence of a functional imprint, and more importantly, we demonstrate loss of imprinting (LOI) in all of seven informative invasive breast carcinomas. In contrast to this, in one case of atypical ductal hyperplasia (ADH) found in residual breast, imprinting was maintained. This raises the possibility that aberrant imprinting of PEG1 may be involved in the progression from hyperplasia to invasive breast cancer.

Loss of Rho function in the thymus is accompanied by the development of thymic lymphoma.

In vitro studies in model cell lines have implicated the GTPase Rho in the control of diverse cellular responses including the control of the actin cytoskeleton and the regulation of cell cycle progression. It is also reported that the transformation of fibroblasts via oncogenic Ras requires intact Rho signalling. An invaluable tool used to investigate Rho function is the bacterial toxin C3 transferase derived from Clostridium botulinum. C3 transferase ribosylates Rho in its effector domain thereby abolishing interaction with downstream effectors. We have previously reported the use of C3 transferase under the control of the thymocyte specific lck promoter to explore the role of Rho in T cell biology. Strikingly, lck-C3 mice develop aggressive malignant thymic lymphoblastic lymphomas between 4 and 8 months of age. These studies reveal that loss of Rho function is associated with prediposition to lymphoid cell transformation. Inhibition of Rho function has been suggested as a therapeutic strategy for treatment of Ras-transformed tumours. The development of lymphomas in mice devoid of functional Rho in their T cell compartment shows that such a strategy would need to be used with caution.

Inhibition of integrin linked kinase (ILK) suppresses beta-catenin-Lef/Tcf-dependent transcription and expression of the E-cadherin repressor, snail, in APC-/- human colon carcinoma cells.

Loss of functional adenomatous polyposis coli (APC) protein results in the stabilization of cytosolic beta-catenin and activation of genes that are responsive to Lef/Tcf family transcription factors. We have recently shown that an independent cell adhesion and integrin linked kinase (ILK)-dependent pathway can also activate beta-catenin/LEF mediated gene transcription and downregulate E-cadherin expression. In addition, ILK activity and expression are elevated in adenomatous polyposis and colon carcinomas. To examine the role of this pathway in the background of APC mutations we inhibited ILK activity in APC-/- human colon carcinoma cell lines. In all cases, inhibition of ILK resulted in substantial inhibition of TCF mediated gene transcription and inhibition of transcription and expression of the TCF regulated gene, cyclin D1. Inhibition of ILK resulted in decreased nuclear beta-catenin expression, and in the inhibition of phosphorylation of GSK-3 and stimulation of its activity, leading to accelerated degradation of beta-catenin. In addition, inhibition of ILK suppressed cell growth in culture as well as growth of human colon carcinoma cells in SCID mice. Strikingly, inhibition of ILK also resulted in the transcriptional stimulation of E-cadherin expression and correlated with the inhibition of gene transcription of snail, a repressor of E-cadherin gene expression. Overexpression of ILK caused a stimulation of expression of snail, but snail expression was found not to be regulated by beta-catenin/Tcf. These data demonstrate that ILK can regulate beta-catenin/TCF and snail transcription factors by distinct pathways. We propose that inhibition of ILK may be a useful strategy in the control of progression of colon as well as other carcinomas. Oncogene (2001) 20, 133 - 140.

The integrin linked kinase (ILK) induces an invasive phenotype via AP-1 transcription factor-dependent upregulation of matrix metalloproteinase 9 (MMP-9).

Overexpression of Integrin Linked Kinase (ILK) in intestinal and mammary epithelial cells results in a highly invasive phenotype, associated with increased levels of expression of the matrix metalloproteinase MMP-9. This increase was at the transcriptional level as determined by MMP-9 promoter-CAT reporter assays. Mutations in the two AP-1 binding sites within the MMP-9 promoter completely inhibited the reporter activity. We have previously shown that ILK inhibits glycogen synthase kinase-3 (GSK-3) activity. Transient transfection of wild-type GSK-3beta in ILK-overexpressing cells decreased MMP-9 promoter activity and AP-1 activity, indicating that ILK can stimulate MMP-9 expression via GSK-3beta and AP-1 transcription factor. A small molecule inhibitor of the ILK kinase reduced the in vitro invasiveness of ILK-overexpressing cells as well as the invasiveness of several human brain tumor cell lines. Furthermore, both MMP-9 promoter and AP-1 activities were inhibited by the ILK inhibitor. Invasiveness of ILK-overexpressing cells was also reduced by inhibition of MMP-9. These data demonstrate that ILK can induce an invasive phenotype via AP-1-dependent upregulation of MMP-9.

Critical role for the tyrosine kinase Syk in signalling through the high affinity IgE receptor of mast cells.

Activation of the high affinity IgE receptor (Fc epsilon RI) of mast cells, a member of the antigen receptor family, leads to the release of allergic mediators, a critical event in the onset of immediate hypersensitivity. Stimulation of Fc epsilon RI results in the rapid association and activation of the Syk tyrosine kinase. Using Syk-deficient mast cells we show that they fail to degranulate, synthesize leukotrienes and secrete cytokines when stimulated through Fc epsilon RI, conclusively demonstrating an essential role for Syk in Fc epsilon RI signalling. Furthermore, our data strongly supports a model of Fc epsilon RI engagement leading to the sequential activation of the tyrosine kinases Lyn and then Syk. A similar mechanism is likely to apply to signal transduction through all members of the antigen receptor family.

Divalent cation effects on the binding of human anti-phospholipid antibodies.

Anti-phospholipid antibodies from sera of subjects with documented syphilis were measured as a result of their individual interactions with cardiolipin, phosphatidylserine and phosphatidic acid, each impregnated in nitrocellulose paper from chloroform solution, followed by reaction with a labelled second antibody. Binding curves generated by increasing the cardiolipin concentration over a standard area of nitrocellulose paper showed saturation. The presence of Ca2+ or Mg2+ during the antibody binding step resulted in a complex pattern of binding as a function of the cation concentration. When the extent of binding was normalized to percent of maximum bound, virtually super-impossible patterns were seen with different sera. These patterns were distinctive for both the phospholipid and the cation. The speculation is presented, albeit without any direct evidence, that the extent of antibody binding is sensitive to a variety of intermediate phospholipid phase structures which may be initiated by the presence of the specific divalent cation at a particular concentration.

Cholesterol effects on the interaction of cardiolipin with anti-cardiolipin antibody.

Human antibodies to cardiolipin, phosphatidic acid and phosphatidylserine were assessed by binding to nitrocellulose paper and subsequent reaction with an enzyme-linked or radioactively labelled second antibody to human IgG. The addition of cholesterol to constant amounts of cardiolipin impregnated in the nitrocellulose paper resulted in a profound fall in antibody binding beginning at a 0.5 to 1 molar ratio of cholesterol to cardiolipin and stabilizing at about 15% of the original level. Antibody binding to phosphatidic acid and phosphatidylserine also showed extensive cholesterol-induced inhibition beginning at a slightly lower molar ratio of cholesterol to phospholipid. The structural array of neither the cardiolipin alone impregnated in nitrocellulose nor the phospholipid together with cholesterol is known. It is possible that the specific cardiolipin phase structure required for human antibody recognition was disrupted by cholesterol.

Stereospecificity of the hydroxyeicosatetraenoic and hydroxyoctadecadienoic acids produced by cultured bovine endothelial cells.

Characterization of the stereospecificity of the derivatives of arachidonic acid and linoleic acid produced by endothelial cells is needed to define the enzymatic origin of these compounds and their role in vascular physiology. In studies utilizing two bovine endothelial cell lines (CPAE and AG04762), both free 15-hydroxyeicosatetraenoic acid (15-HETE) and 11-hydroxyeicosatetraenoic acid (11-HETE) were generated during incubations with exogenous arachidonic acid and both free 9-hydroxyoctadecadienoic acid (9-HODE) and 13-hydroxyoctadecadienoic acid (13-HODE) were generated during incubations with exogenous linoleic acid. Esterification of 15-HETE, 9-HODE and 13-HODE during these incubations was demonstrated. The analyses included reversed-phase high performance liquid chromatography of the free acid and its methyl ester and chiral separation of the methyl ester on straight phase chiral columns. The ratio of 9-HODE/13-HODE averaged 2.7 in the chromatographic analyses of the extracts of the incubations with linoleic acid. The combined production of 13-HODE and 9-HODE from linoleic acid was four times greater than that of 15-HETE and 11-HETE from arachidonic acid. With regard to the products of the CPAE endothelial cell line, the S/R ratio of the stereoisomers averaged 1.5 for free 15-HETE, 5.7 for free 13-HODE and 0.2 for free 9-HODE. The 11-HETE had strict (R) stereospecificity. The products from the AG04762 endothelial cell line had similar stereochemistry. All these stereochemical findings point to the activity of a cyclooxygenase rather than that of a lipoxygenase.

Integrin-linked kinase, a promising cancer therapeutic target: biochemical and biological properties.

Integrin-linked kinase (ILK) is an ankyrin repeat-containing Ser/Thr kinase that interacts with the cytoplasmic domains of beta(1) and beta(3) integrins. ILK is widely expressed in tissues throughout the body, and, as might be expected, appears to mediate a diversity of functions relating to its role in coupling integrins and growth factor receptors to downstream signaling pathways. Through its downstream targets protein kinase B/Akt and glycogen synthase kinase-3beta, ILK appears to be involved in several oncogenesis-related events, including suppression of apoptosis and promotion of cell survival, as well as cell migration and invasion. Over-expression of ILK in epithelial cells results in anchorage-independent cell growth with increased cell cycle progression. Inoculation of nude mice with ILK over-expressing cells leads to tumor formation. Furthermore, increased ILK expression and activity have been correlated with malignancy in several human tumor types, including breast, prostate, brain, and colon carcinomas. Based on these findings, ILK represents an excellent therapeutic target for the prevention of tumor progression. Here, we provide an overview of the physical and biochemical properties of ILK, and present data describing the impact of small-molecule ILK inhibitors on several ILK-mediated cellular functions.

The developmental environment, epigenetic biomarkers and long-term health.

Evidence from both human and animal studies has shown that the prenatal and early postnatal environments influence susceptibility to chronic disease in later life and suggests that epigenetic processes are an important mechanism by which the environment alters long-term disease risk. Epigenetic processes, including DNA methylation, histone modification and non-coding RNAs, play a central role in regulating gene expression. The epigenome is highly sensitive to environmental factors in early life, such as nutrition, stress, endocrine disruption and pollution, and changes in the epigenome can induce long-term changes in gene expression and phenotype. In this review we focus on how the early life nutritional environment can alter the epigenome leading to an altered susceptibility to disease in later life.

Astrocytoma adhesion to extracellular matrix: functional significance of integrin and focal adhesion kinase expression.

Evidence is accumulating implicating a role for integrins in the pathogenesis of cancer, a disease in which alterations in cellular growth, differentiation, and adhesive characteristics are defining features. In the present report we studied a panel of 8 human astrocytoma cell lines for their expression of integrin subunits by RT-PCR, and of integrin heterodimers by immunoprecipitation analyses. The functionality of integrin heterodimers was assessed using cell attachment assays to plastic or single matrix substrates. Downstream effects of integrin activation were studied by western blot analyses of FAK expression in human astrocytoma cell lines growing on plastic and on a fibronectin matrix, and in 13 primary human brain tumor specimens of varying histopathological grade. Furthermore, we studied tyrosine phosphorylation of FAK in astrocytoma cells growing on plastic versus fibronectin. Finally, we analyzed the effects of intermediate filament gene transfer on FAK phosphorylation in SF-126 astrocytoma cells. Our data show that astrocytoma cell lines express various integrin subunits by RT-PCR, and heterodimers by immunoprecipitation analyses. The beta1 and alphav integrin subunits were expressed by all astrocytoma cell lines. The alpha3 subunit was expressed by all cell lines except SF-188. By immunoprecipitation, the fibronectin receptor (alpha5beta1 integrin heterodimer) and the vitronectin receptor (alphavbeta3) were identified in several cell lines. Astrocytoma cell attachment studies to human matrix proteins suggested that these integrin heterodimers were functional. Using confocal laser microscopy, we showed that FAK was colocalized to actin stress fibers at sites of focal adhesion complexes. By western blot, FAK was variably but quite ubiquitously expressed in human astrocytoma cell lines, and in several primary human astrocytoma specimens. When U373 and U87 MG astrocytoma cells bind to a fibronectin matrix, FAK is phosphorylated. GFAP-transfected SF-126 human astrocytoma cells were shown to overexpress the phosphorylated form of FAK only when these cells were placed on a fibronectin matrix. This result is of interest because it suggests that manipulations of the astrocytoma cytoskeleton per se can bring about potential signaling changes that channel through integrins and focal adhesion sites leading to activation of key kinases such as FAK.

Dose-dependent inhibition of theophylline metabolism by disulfiram in recovering alcoholics.

The influence of disulfiram on theophylline metabolism was studied in 20 recovering alcoholics. Ten of the patients, who were selected at random, received 250 mg of disulfiram daily. The other 10 patients received 500 mg of disulfiram daily. Two single-dose studies of theophylline kinetics were performed--one as a baseline control and the other after 1 week of treatment with disulfiram. With disulfiram pretreatment, the plasma clearance of theophylline was decreased from 105.7 +/- 10.2 (mean +/- SEM) to 83.1 +/- 8.1 ml/kg per hour (p less than 0.001) in the 250 mg group and from 94.3 +/- 13.3 to 65.4 +/- 10.7 ml/mg per hour (p less than 0.001) in the 500 mg group. The elimination half-life was prolonged significantly in both groups. The percent reduction in theophylline clearance was greater in the 500 mg group (32.5 +/- 3.1; range, 21.6 to 49.6) than it was in the 250 mg group (21.2 +/- 1.7; range, 14.6 to 29.6; p less than 0.01). Disulfiram decreased the formation of all theophylline metabolites in smokers in both treatment groups. In each group, the hydroxylation pathway was affected more than the demethylation pathway. These data indicate that at therapeutic doses disulfiram exerts a dose-dependent inhibitory effect on theophylline metabolism. Depending on the dose of disulfiram, a dose reduction of theophylline by as much as 50% may be necessary to minimize the risk of toxicity.