Control of bovine tuberculosis in a farmed red deer herd in England.

This report describes how Mycobacterium bovis infection was controlled and eventually eradicated in a farmed red deer herd in the north of England, following sustained tuberculin skin testing supplemented with serological (antibody) tests over a period of approximately two years. By taking advantage of the anamnestic antibody response produced by the skin test to detect skin test-negative, antibody-positive infected individuals, a total of 35 additional animals were identified, including 2 with gross visible lesions typical of bovine tuberculosis (BTB). Without detection and removal, these animals would have posed a continued risk of BTB persistence within the herd and potentially contributed to the spread of infection from deer into wildlife and surrounding cattle farms in an area of low BTB incidence. This case supports the use of ancillary diagnostic serological tests to speed up the resolution of incidents of BTB caused by M bovis in captive deer herds.

A polymorphism in the thyroid hormone receptor gene is associated with bronchodilator response in asthmatics.

A pro-asthmatic culture milieu and ß2-agonist (isoproterenol) were previously shown to regulate the expression of select transcription factors (TFs) within human airway epithelial and smooth muscle cells. This study tests 1116 single-nucleotide polymorphisms (SNPs) across 98 of these TF genes for association with bronchodilator response (BDR) in asthma patients. Genotyping was conducted using the Illumina HumanHap550v3 Beadchip in 403 non-Hispanic White asthmatic children and their parents. SNPs were evaluated for association with BDR using family and population-based analyses. Forty-two SNPs providing P-values <0.1 in both analyses were then genotyped in three adult asthma trials. One SNP 5' of the thyroid hormone receptor-ß gene was associated with BDR in the childhood population and two adult populations (P-value=0.0012). This investigation identified a novel locus for inter-individual variability in BDR and represents a translation of a cellular drug-response study to potential personalization of clinical asthma management.

Immunological and pathological responses of red deer resistant or susceptible genotypes, to experimental challenge with Mycobacterium avium subsp. paratuberculosis.

This study aimed to monitor the clinical, immunological and pathological changes in red deer for 49 weeks after experimental oral challenge with Mycobacterium avium subsp. paratuberculosis (MAP) and to assess the heritability of resistance in the offspring of two red stags. Eighteen young deer, which were bred from unselected hinds and sired by two stags resistant (R) or susceptible (S) to paratuberculosis, were challenged with MAP and monitored for 49 weeks. Biopsy samples of the jejunal lymph node were collected at Weeks 4 and 13 and at necropsy after euthanasia of clinically affected animals or when electively killed at Week 49. Three animals (two S and one R) developed clinical disease and were euthanised. The nine S offspring had significantly more severe lesions than the nine R offspring (Mantel-Haenszel Chi-square P=0.017). The average Lesion Severity Score (LSS) of R offspring was 5.9 (mild), and 7/9 had no or very mild lesions. In contrast, the LSS of S offspring averaged 11.7 (severe), and 7/9 had severe lesions. Most of the resistant, but not the susceptible, animals showed evidence of resolving lesions and a reduction in the number of MAP between 13 and 49 weeks after challenge. One R offspring appeared to completely cure itself, and progressed from mild culture-positive paratuberculosis lesions at Week 13 to having no signs of disease or infection 36 weeks later. This study showed significant heritable resistance/susceptibility to paratuberculosis and key differences in immunological responses in the first 3 months after challenge, indicating different paths to relative success or failure to control MAP. In general, R deer had higher IFN-γ levels, low antibody titres and fewer MAP, while S deer had lower IFN-γ levels, higher antibody and more MAP.

Cardiovascular pharmacogenomics of adrenergic receptor signaling: clinical implications and future directions.

G-protein-coupled receptors (GPCRs) are the targets for many drugs, and genetic variation in coding and noncoding regions is apparent in many such receptors. In this superfamily, adrenergic receptors (ARs) were among the first in which single-nucleotide polymorphisms (SNPs) were discovered, and studies including in vitro mutagenesis, genetically modified mouse models, human ex vivo and in vitro studies and pharmacogenetic association studies were conducted. The signal transduction in these receptors includes amplification steps, desensitization, crosstalk, and redundancies, enabling potential mitigation of the size of the clinical effect for a single variant in a single gene. Nevertheless, convincing evidence has emerged that several variants have an impact on therapy, with certain caveats as to how the results are to be interpreted. Here we review these results for selected ARs and associated regulatory kinases relative to the pharmacogenomics of ß-blocker treatment for hypertension and heart failure. We emphasize the linking of clinical results to molecular mechanisms, discuss study design limitations, and offer some recommendations for future directions.

Evidence of Mycobacterium avium subsp. paratuberculosis infection in Dutch farmed red deer.

Paratuberculosis is a chronic disease in ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Most economic losses due to MAP occur in the dairy industry. However, the infection is not restricted to cattle, but also occurs in other ruminants, such as sheep, goat, and deer. Although deer are of minimal economic importance in The Netherlands, they may constitute a source of infection for the dairy industry. This pilot study was conducted to estimate the prevalence of Johne's disease in farmed red deer in The Netherlands. Serum and faecal samples were collected from 140 animals, originating from 8 different farms. Four of the farms had animals that tested positive for Johne's disease. The within-herd MAP seroprevalence varied between 4.8% and 21.2%. In conclusion, this pilot study provides evidence of MAP infection in the Dutch farmed deer population, and thus there might be a risk of MAP transmission between farmed red deer and dairy cattle.

Pharmacogenetics of beta1-adrenergic receptors in heart failure and hypertension.

Currently it is generally accepted that an individual's genetic makeup can modify the efficacy of drug treatment or the risk of adverse reactions. Although not a new concept, the availability of human genome sequence and rapid genotyping at variable loci in drug targets or metabolizing genes has provided new opportunities for the field termed "pharmacogenetics". Somewhat surprisingly, multiple studies have shown the existence of common variants (polymorphisms) in members of the G-protein coupled receptor superfamily, which constitute around 50% of all the targets of currently prescribed drugs. The beta1-adrenergic receptors (beta1ARs) are interesting candidates for pharmacogenetic studies in two complex cardiovascular disease, heart failure and hypertension, since they mediate the effects of catecholamines in the sympathetic nervous system. These receptors are involved in the progression and treatment (beta-blockers therapy) of both diseases, and have polymorphisms that show altered function or regulation as compared to their allelic counterparts in recombinant expression systems and genetically modified mice. These results have prompted prospective and retrospective clinical studies examining whether polymorphisms of these genes are risk factors, disease modifiers, or predictors of b-blocker response in heart failure and hypertension. To date, it appears that beta1AR variants are very likely one genetic component that defines responsiveness to beta-blockers in heart failure and hypertension. Altogether, results are promising, but discrepancies between studies require resolution before these polymorphisms can be utilized in practice. With the goal of personalizing therapy based on an individual's genetic makeup, additional adequately powered, multiethnic, multi-drug studies will be needed.

Overview of the pharmacogenetics of asthma treatment.

Asthma affects approximately 300 million individuals worldwide. Medications comprise a substantial portion of asthma expenditures. Despite the availability of three primary therapeutic classes of medications, there are a significant number of nonresponders to therapy. Available data, as well as previous pharmacogenetic studies, suggest that genetics may contribute as much as 60-80% to the interindividual variability in treatment response. In this methodologic review, after providing a broad overview of the asthma pharmacogenetics literature to date, we describe the application of a novel family-based screening algorithm to the analysis of pharmacogenetic data and highlight our approach to identifying and verifying loci influencing asthma treatment response. This approach seeks to address issues related to multiple comparisons, statistical power, population stratification, and failure to replicate from which previous population-based or case-control pharmacogenetic association studies may suffer. Identification of such replicable loci is the next step towards the goal of 'individualized therapy' for asthma.

Tuberculosis in ruminants: characteristics of intra-tonsilar Mycobacterium bovis infection models in cattle and deer.

Mycobacterium bovis infection produces tubercular lymphadenitis in the head lymphatics of cattle and deer, in addition to pulmonary disease. A low-dose intra-tonsilar infection model that establishes tuberculosis (Tb) lymphadenitis in cattle and deer is characterised in this study. Intra-tonsilar infection of red deer (500 cfus of M. bovis) was monitored longitudinally at 6-week intervals over a period of 23 weeks. Lesion characteristics, bacteriological and immunological parameters were assessed, and compared against those observed in cattle at 20 weeks post-infection, where the latter were infected with 500 or 5000 cfus of M. bovis. Intra-tonsilar inoculation of M. bovis established infection in >90% of deer and cattle, with lesion frequencies at the draining sentinel lymphatic site (left medial retropharyngeal node) of 68-86% and tissue bacterial burdens >3.5 logs/g of tissue, the tonsil being a major site of M. bovis persistence in deer only. Mineralisation occurred at lesion sites in both species in the later stages (18-23 weeks) of infection, with extensive coarse mineralisation observed mainly in cattle. The severity of infection or disease in cattle that received the higher or lower dose of M. bovis did not differ markedly. Pathogen-induced cellular immune response (lymphocyte transformation) and humoral responses (IgG and IgG(1) anti-mycobacterial antibodies) were recorded in both species, and the magnitude of these was noticeably amplified by skin tuberculin testing. IgG(1) antibodies were detectable within 6 weeks post-inoculation in deer and could be associated with early detection of lymphadenitis. Deer and cattle show similar levels of susceptibility to M. bovis infection.

Serine 232 of the alpha(2A)-adrenergic receptor is a protein kinase C-sensitive effector coupling switch.

alpha(2)-adrenergic receptors (alpha(2)AR) couple to multiple effectors including adenylyl cyclase and phospholipase C. We hypothesized that signaling selectivity to these effectors is dynamically directed by kinase-sensitive domains within the third intracellular loop of the receptor. Substitution of Ala for Ser232, which is in the N-terminal region of this loop in the alpha(2A)AR, resulted in a receptor that was markedly uncoupled ( approximately 82% impairment) from stimulation of inositol phosphate accumulation while the capacity to inhibit adenylyl cyclase remained relatively intact. In S232A alpha(2A)AR transfected cell membranes, agonist-promoted [(35)S]GTPgammaS binding was reduced by approximately 50%. Coexpression of modified G proteins rendered insensitive to pertussis toxin revealed that the S232A receptor was uncoupled from both G(i) and G(o). S232 is a potential PKC phosphorylation site, and whole cell phosphorylation studies showed that the mutant had depressed phosphorylation compared to wild type (1.3- vs 2.1-fold/basal). Consistent with S232 directing coupling to phospholipase C, PMA exposure resulted in approximately 67% desensitization of agonist-promoted inositol phosphate accumulation without significantly affecting inhibition of adenylyl cyclase. The dominant effect of mutation or phosphorylation at this site on inositol phosphate as compared to cAMP signaling was found to most likely be due to the low efficiency of signal transduction via phospholipase C vs adenylyl cyclase. Taken together, these results indicate that S232 acts as a selective, PKC-sensitive, modulator of effector coupling of the alpha(2A)AR to inositol phosphate stimulation. This represents one mechanism by which cells route stimuli directed to multifunctional receptors to selected effectors so as to attain finely targeted signaling.

Identification and functional characterization of alpha(2)-adrenoceptor polymorphisms.

For each alpha(2)-adrenoceptor subtype (alpha(2A), alpha(2B) and alpha(2C)), sequence variations within the coding region of each gene have been identified in humans. These result in substitutions or deletions of amino acids in the third intracellular loops of each receptor. This article summarizes the genetics and molecular biology of alpha(2)-adrenoceptor polymorphisms, including the consequences of each polymorphism on receptor signaling, as determined in transfected cells. These effects include alterations in G-protein coupling, desensitization and G-protein receptor kinase-mediated phosphorylation. Studies so far provide the mechanistic basis for future studies to investigate genetic risk factors and pharmacogenetics in pathophysiological conditions linked to alpha(2)-adrenoceptor function.

Targeted transgenic expression of beta(2)-adrenergic receptors to type II cells increases alveolar fluid clearance.

Clearance of edema fluid from the alveolar space can be enhanced by endogenous and exogenous beta-agonists. To selectively delineate the effects of alveolar type II (ATII) cell beta(2)-adrenergic receptors (beta(2)-ARs) on alveolar fluid clearance (AFC), we generated transgenic (TG) mice that overexpressed the human beta(2)-AR under control of the rat surfactant protein C promoter. In situ hybridization showed that transgene expression was consistent with the distribution of ATII cells. TG mice expressed 4.8-fold greater beta(2)-ARs than nontransgenic (NTG) mice (939 +/- 113 vs. 194 +/- 18 fmol/mg protein; P < 0.001). Basal AFC in TG mice was approximately 40% greater than that in untreated NTG mice (15 +/- 1.4 vs. 10.9 +/- 0.6%; P < 0.005) and approached that of NTG mice treated with the beta-agonist formoterol (19.8 +/- 2.2%; P = not significant). Adrenalectomy decreased basal AFC in TG mice to 9.7 +/- 0.5% but had no effect on NTG mice (11.5 +/- 1.0%). Na(+)-K(+)-ATPase alpha(1)-isoform expression was unchanged, whereas alpha(2)-isoform expression was approximately 80% greater in the TG mice. These findings show that beta(2)-AR overexpression can be an effective means to increase AFC in the absence of exogenous agonists and that AFC can be stimulated by activation of beta(2)-ARs specifically expressed on ATII cells.

The Ile164 beta(2)-adrenoceptor polymorphism alters salmeterol exosite binding and conventional agonist coupling to G(s).

beta(2)-adrenoceptors (beta(2)AR) are polymorphic at amino acid 164 (Thr or Ile) of the fourth transmembrane domain. In transfected fibroblasts, six agonists commonly used in the treatment of bronchospasm were studied. Isoproterenol, albuterol, metaproterenol, terbutaline, formoterol, and salmeterol displayed decreased binding affinities (K(i)s were 1.2-3.0-fold higher) and a significant degree of impaired maximal stimulation of adenylyl cyclase ( approximately 40%), was observed with all agonists for the Ile164 receptor. The ratios of signal transduction efficiencies (Tau function, Ile164/Thr164) varied from a low of 0.17 for terbutaline to 0.49 for salmeterol. In addition, Ile164 bound salmeterol at the exosite, as delineated in perfusion washout studies, at a decreased level (31+/-4.8% vs. 49+/-4.4% retained salmeterol, respectively, P=0.02). In cAMP production studies under perfusion conditions, this decreased exosite binding caused a approximately 50% decrease in the duration of action of salmeterol at Ile164 (t(1/2)=21.0+/-3.6 vs. 46.8+/-4.1 min for Thr164, P=0.001). The durations of action for isoproterenol and formoterol under similar perfusion conditions were not different between the two receptors. These in vitro results indicate the Ile164 polymorphic receptor represents a pharmacogenetic locus for the most commonly utilized agonists in the treatment of asthma with a unique phenotype for salmeterol.

Modification of the beta 2-adrenergic receptor to engineer a receptor-effector complex for gene therapy.

Depressed G-protein-coupled receptor (GPCR) signaling has been implicated as a component of the pathophysiology of a number of complex diseases including heart failure and asthma, and augmentation or restoration of signaling by various means has been shown to improve organ function. Because some properties of native GPCRs are disadvantageous for ectopic therapeutic expression, we utilized the beta(2)-adrenergic receptor (beta(2)AR) as a scaffold to construct a highly modified therapeutic receptor-effector complex (TREC) suitable for gene therapy. Altogether, 19 modifications were made to the receptor. The ligand-binding site was re-engineered in TM-3 so that a beta-hydroxylmethyl side chain acts as a proton donor for the binding of a novel ligand. In addition, sites critical for agonist-promoted down-regulation in the amino terminus and for phosphorylation by GPCR kinases, and protein kinases A and C, in the third intracellular loop and the carboxyl terminus of the receptor were altered. These modifications of the receptor resulted in depressed agonist-stimulated adenylyl cyclase activity (26.8 +/- 2.1 versus 41.4 +/- 8 pmol/min/mg for wild-type beta(2)AR). This was fully restored by fusing the carboxyl terminus of the modified receptor to G alpha(s) (43.3 +/- 2.7 pmol/min/mg). The fully modified fused receptor was not activated by beta-agonists but rather by a nonbiogenic amine agonist that itself failed to activate the wild-type beta(2)AR. This two-way selectivity thus provides targeted activation based on physiologic status. Furthermore, the TREC did not display tachyphylaxis to prolonged agonist exposure (desensitization was 1 +/- 5% versus 55 +/- 4% for wild-type beta(2)AR). Thus, despite extensive alterations in regions of conformational lability, the beta(2)AR can be tailored to have optimal signaling characteristics for gene therapy. As a general paradigm, TRECs for enhancement of other G-protein signaling appear to be feasible for modification of other pathologic states.

Effect of polymorphism of the beta(2)-adrenergic receptor on response to regular use of albuterol in asthma.

BACKGROUND: Regular use of inhaled beta-adrenergic agonists may have adverse effects in some asthma patients. Polymorphisms of the beta(2)-adrenergic receptor (beta(2)-AR) can affect its regulation; however, results of smaller studies of the effects of such polymorphisms on response to beta-agonist therapy have been inconsistent. METHODS: We examined the possible effects of polymorphisms at codons 16 (beta(2)-AR-16) and 27 (beta(2)-AR-27) on response to albuterol by genotyping 190 asthmatics who had participated in a trial of regular versus as-needed albuterol use. RESULTS: During the 16-week treatment period, patients homozygous for arginine (Arg/Arg) at beta(2)-AR-16 who used albuterol regularly had a small decline in morning peak expiratory flow (AM PEF). This effect was magnified during a 4-week run-out period, when all patients returned to as-needed albuterol only. By the end of the study, Arg/Arg subjects who had used albuterol regularly had an AM PEF 30.5 +/- 12.1 liters/min lower (p = 0.012) than Arg/Arg patients who had used albuterol as needed only. Subjects homozygous for glycine at beta(2)-AR-16 showed no such decline. Evening PEF also declined in the Arg/Arg regular but not in as-need albuterol users. No significant differences between regular and as-needed treatment were associated with polymorphisms at beta(2)-AR-27. CONCLUSIONS: Polymorphisms of the beta(2)-AR may influence airway responses to regular inhaled beta-agonist treatment.

Interactions between phospholamban and beta-adrenergic drive may lead to cardiomyopathy and early mortality.

BACKGROUND: Relieving the inhibition of sarcoplasmic reticular function by phospholamban is a major target of beta-adrenergic stimulation. Chronic beta-adrenergic receptor activity has been suggested to be detrimental, on the basis of transgenic overexpression of the receptor or its signaling effectors. However, it is not known whether physiological levels of sympathetic tone, in the absence of preexisting heart failure, are similarly detrimental. METHODS AND RESULTS: Transgenic mice overexpressing phospholamban at 4-fold normal levels were generated, and at 3 months, they exhibited mildly depressed ventricular contractility without heart failure. As expected, transgenic cardiomyocyte mechanics and calcium kinetics were depressed, but isoproterenol reversed the inhibitory effects of phospholamban on these parameters. In vivo cardiac function was substantially depressed by propranolol administration, suggesting enhanced sympathetic tone. Indeed, plasma norepinephrine levels and the phosphorylation status of phospholamban were elevated, reflecting increased adrenergic drive in transgenic hearts. On aging, the chronic enhancement of adrenergic tone was associated with a desensitization of adenylyl cyclase (which intensified the inhibitory effects of phospholamban), the development of overt heart failure, and a premature mortality. CONCLUSIONS: The unique interaction between phospholamban and increased adrenergic drive, elucidated herein, provides the first evidence that compensatory increases in catecholamine stimulation can, even in the absence of preexisting heart failure, be a primary causative factor in the development of cardiomyopathy and early mortality.

Polymorphic deletion of three intracellular acidic residues of the alpha 2B-adrenergic receptor decreases G protein-coupled receptor kinase-mediated phosphorylation and desensitization.

A polymorphic variant of the human alpha(2B)-adrenergic receptor (alpha(2B)AR), which consists of a deletion of three glutamic acids (residues 301-303) in the third intracellular loop was found to be common in Caucasians (31%) and to a lesser extent in African-Americans (12%). The consequences of this deletion were assessed by expressing wild-type and the Del301-303 receptors in Chinese hamster ovary and COS cells. Ligand binding was not affected, although a small decrease in coupling efficiency to the inhibition of adenylyl cyclase was observed with the mutant. The deletion occurs within a stretch of acidic residues that is thought to establish the milieu for agonist-promoted phosphorylation and desensitization of the receptor by G protein-coupled receptor kinases (GRKs). Agonist-promoted phosphorylation studies carried out in cells coexpressing the alpha(2B)ARs and GRK2 revealed that the Del301-303 receptor displayed approximately 56% of wild-type phosphorylation. Furthermore, the depressed phosphorylation imposed by the deletion was found to result in a complete loss of short term agonist-promoted receptor desensitization. Thus the major phenotype of the Del301-303 alpha(2B)AR is one of impaired phosphorylation and desensitization by GRKs, and thus the polymorphisms renders the receptor incapable of modulation by this key mechanism of dynamic regulation.