Dirofilariasis mouse models for heartworm preclinical research.

Introduction: Dirofilariasis, including heartworm disease, is a major emergent veterinary parasitic infection and a human zoonosis. Currently, experimental infections of cats and dogs are used in veterinary heartworm preclinical drug research. Methods: As a refined alternative in vivo heartworm preventative drug screen, we assessed lymphopenic mouse strains with ablation of the interleukin-2/7 common gamma chain (γc) as susceptible to the larval development phase of Dirofilaria immitis. Results: Non-obese diabetic (NOD) severe combined immunodeficiency (SCID)γc-/- (NSG and NXG) and recombination-activating gene (RAG)2-/-γc-/- mouse strains yielded viable D. immitis larvae at 2-4 weeks post-infection, including the use of different batches of D. immitis infectious larvae, different D. immitis isolates, and at different laboratories. Mice did not display any clinical signs associated with infection for up to 4 weeks. Developing larvae were found in subcutaneous and muscle fascia tissues, which is the natural site of this stage of heartworm in dogs. Compared with in vitro-propagated larvae at day 14, in vivo-derived larvae had completed the L4 molt, were significantly larger, and contained expanded Wolbachia endobacteria titres. We established an ex vivo L4 paralytic screening system whereby assays with moxidectin or levamisole highlighted discrepancies in relative drug sensitivities in comparison with in vitro-reared L4 D. immitis. We demonstrated effective depletion of Wolbachia by 70%-90% in D. immitis L4 following 2- to 7-day oral in vivo exposures of NSG- or NXG-infected mice with doxycycline or the rapid-acting investigational drug, AWZ1066S. We validated NSG and NXG D. immitis mouse models as a filaricide screen by in vivo treatments with single injections of moxidectin, which mediated a 60%-88% reduction in L4 larvae at 14-28 days. Discussion: Future adoption of these mouse models will benefit end-user laboratories conducting research and development of novel heartworm preventatives via increased access, rapid turnaround, and reduced costs and may simultaneously decrease the need for experimental cat or dog use.

Pharmacokinetic differences in exposure to camphor after intraruminal dosing in selectively bred lines of goats.

A pharmacokinetic dosing study with camphor was used to determine whether selection lines of high-juniper-consuming goats (HJC, n = 12) and low-juniper-consuming goats (LJC, n = 12) differed in their respective disposition kinetics. Postdosing plasma camphor concentrations were used to examine whether a timed single blood sample collected after intraruminal administration of camphor would be a useful screening test to aid in the identification of HJC. Yearling female Boer x Spanish goats (n = 24) received a single intraruminal dose of monoterpene cocktail (0.270 g/kg of BW) containing 4 different monoterpenes that represented their composition previously reported for Ashe juniper (Juniperus ashei). Camphor, the predominant monoterpene in Ashe juniper, was 49.6% of the mix and was the monoterpene analyzed for this study. Blood samples were taken at 15 time points from 0 to 8 h after dosing. Concentrations of camphor were measured in plasma using solid phase extraction and gas chromatography/flame-ionization detection analysis. Maximal plasma concentration of camphor was greater for LJC than HJC (P = 0.01), and area under the curve extrapolated to infinity was greater for LJC than HJC (P < 0.01). Total systemic exposure (area under the curve) to camphor was 5 times less in HJC goats. We conclude that 1) HJC goats possess internal mechanisms to reduce the bioavailability of camphor, and 2) a blood sample taken at 45 min or at 60 min after intraruminal administration of camphor may be useful for identifying HJC individual animals from within large populations of goats.

Development of predictive models for airflow obstruction in alpha-1-antitrypsin deficiency.

Alpha-1-antitrypsin deficiency is a genetic condition associated with severe, early-onset chronic obstructive pulmonary disease (COPD). However, there is significant variability in lung function impairment among persons with the protease inhibitor ZZ genotype. Early identification of persons at highest risk of developing lung disease could be beneficial in guiding monitoring and treatment decisions. Using a multicenter, family-based study sample (2002-2005) of 372 persons with the protease inhibitor ZZ genotype, the authors developed prediction models for forced expiratory volume in 1 second (FEV(1)) and the presence of severe COPD using demographic, clinical, and genetic variables. Half of the data sample was used for model development, and the other half was used for model validation. In the training sample, variables found to be predictive of both FEV(1) and severe COPD were age, sex, pack-years of smoking, bronchodilator responsiveness, chronic bronchitis symptoms, and index case status. In the validation sample, the predictive model for FEV(1) explained 50% of the variance in FEV(1), and the model for severe COPD exhibited excellent discrimination (c statistic = 0.88).

Heritability of lung function in severe alpha-1 antitrypsin deficiency.

Severe alpha-1 antitrypsin (AAT) deficiency is a proven genetic risk factor for COPD, but there is marked variation in the development of COPD among AAT deficient subjects. To investigate familial aggregation of lung function in subjects with AAT deficiency, we estimated heritability for forced expiratory volume in 1 s (FEV1) and FEV1/forced vital capacity (FVC) in 378 AAT deficient subjects from 167 families in the AAT Genetic Modifiers Study; all subjects were verified homozygous for the Z AAT deficiency allele. Heritability was evaluated for models that included and excluded an ascertainment correction, as well as for models that excluded, included and were stratified by a cigarette smoking covariate. In models without an ascertainment correction, and in all models without a covariate for smoking, no evidence for familial aggregation of lung function was observed. In models conditioned on the index proband with covariates for smoking, post-bronchodilator FEV1/FVC demonstrated significant heritability (0.26 +/- 0.14, p = 0.03). When we limited the analysis to subjects with a smoking history, post-bronchodilator FEV1 demonstrated significant heritability (0.47 +/- 0.21, p = 0.02). Severity rate phenotypes were also assessed as potential phenotypes for genetic modifier studies. Significant heritability was found with all age-of-onset threshold models that included smoking and ascertainment adjustments. Using the t-distribution, the heritability estimates ranged from 0.43 to 0.64, depending on the age-of-onset of FEV1 decline used for the severity rate calculation. Correction for ascertainment and consideration of gene-by-smoking interactions will be crucial for the identification of genes that may modify susceptibility for COPD in families with AAT deficiency.

Phosphorylated c-Src in the nucleus is associated with improved patient outcome in ER-positive breast cancer.

Elevated c-Src protein expression has been shown in breast cancer and in vitro evidence suggests a role in endocrine resistance. To investigate whether c-Src is involved in endocrine resistance, we examined the expression of both total and activated c-Src in human breast cancer specimens from a cohort of oestrogen receptor (ER)-positive tamoxifen-treated breast cancer patients. Tissue microarray technology was employed to analyse 262 tumour specimens taken before tamoxifen treatment. Immunohistochemistry using total c-Src and activated c-Src antibodies was performed. Kaplan-Meier survival curves were constructed and log-rank test were performed. High level of nuclear activated Src was significantly associated with improved overall survival (P=0.047) and lower recurrence rates on tamoxifen (P=0.02). Improved patient outcome was only seen with activated Src in the nucleus. Nuclear activated Src expression was significantly associated with node-negative disease and a lower NPI (P<0.05). On subgroup analysis, only ER-positive/progesterone receptor (PgR)-positive tumours were associated with improved survival (P=0.004). This shows that c-Src activity is increased in breast cancer and that activated Src within the nucleus of ER-positive tumours predicts an improved outcome. In ER/PgR-positive disease, activated Src kinase does not appear to be involved in de novo endocrine resistance. Further study is required in ER-negative breast cancer as this may represent a cohort in which it is associated with poor outcome.

A survey of current surgical treatment of acute gallstone disease in the west of Scotland.

BACKGROUND: National guidelines exist for the treatment of acute gallstone pancreatitis, but not for the management of acute cholecystitis (AC). AIMS: To establish the preferred management of uncomplicated AC and adherence to guidelines for the management of mild gallstone pancreatitis in the west of Scotland. METHODS: A postal survey of all 100 consultant general surgeons in the west of Scotland. RESULTS: 67 of 71 responses received were suitable for analysis. For uncomplicated AC, 24 (36%) perform urgent laparoscopic cholecystectomy (LC), 16 (24%) perform same admission LC after clinical improvement. 23 (34%) perform interval LC after discharge. Within this group, 9 surgeons (13%) manage AC conservatively due to insufficient operating time or equipment when on call. In mild gallstone pancreatitis, 33 (49%) perform same admission LC, 13 (19%) perform sphincterotomy, 3 (4.5%) perform one of these depending on the patient and 6 (9.5%) refer to a colleague with an interest in upper gastrointestinal surgery. CONCLUSIONS: The majority of surgeons (over 60%) manage AC with same admission LC. Of those who do not, more than a third report lack of resources as being the reason. The majority of surgeons in the West of Scotland manage mild gallstone pancreatitis in accordance with current guidelines.

Screening for alpha1-Pi deficiency in patients with lung diseases.

In patients with pulmonary emphysema, studies have reported 2-3% of individuals with severe alpha1-Pi deficiency. The aims of this study were to evaluate the accuracy of a new method for quantifying alpha1-Pi through phenotyping from dried blood spots (DBS) and to test the hypothesis that the screening of a population at risk increases the detection rate for severe alpha1-Pi deficiency. The accuracy of phenotyping results from DBS was compared to conventional methods in a total of 555 individuals. In a prospective study 1,060 patients with chronic lung disease were screened for alpha1-Pi deficiency using DBS. The validation of the phenotyping method from DBS showed an accuracy of 100%. Out of 1,060 tested patients, none had a severe PiZ deficiency and only 3 had PiSZ, whilst 36 (3.34%) individuals were identified as heterozygous for PiMS and 39 (3.68%) for PiMZ. No patients with severe alpha1-Pi deficiency could be detected in this population and the frequency of PiMS or PiMZ detected was similar to that of the normal population. Thus, the screening of an unselected population of chronic obstructive pulmonary disease and asthma patients may not detect a large number of individuals with severe alpha1-Pi deficiency.

Linkage analysis of alpha 1-antitrypsin deficiency: lessons for complex diseases.

OBJECTIVES: Severe alpha 1-antitrypsin (A1AT) deficiency is the one proven genetic risk factor for chronic obstructive pulmonary disease (COPD). Familial aggregation has been demonstrated for COPD among individuals who do not have A1AT deficiency, but linkage analysis of COPD has not been reported. To investigate the optimal phenotype definitions and analytical methods for the linkage analysis of COPD, we examined a set of 28 A1AT- deficient families containing 155 individuals. We have used the protease inhibitor (PI) type as a genetic marker rather than a disease gene, and we have performed linkage analysis between PI type and serum A1AT level and spirometry-related phenotypes. METHODS: Linkage analysis was performed on the quantitative phenotypes forced expiratory volume at 1 s (FEV(1) as % predicted), the ratio of FEV(1) to forced vital capacity (FEV(1)/FVC as % predicted), and serum A1AT level using the variance component approach in SOLAR, the generalized estimating equation approach in RELPAL, and the model-based classical lod score method in LINKAGE. Linkage analysis with qualitative A1AT and spirometry phenotypes was performed using a model-based method (LINKAGE) and a model-free method (GENEHUNTER). Adjustments for smoking effects were investigated under each method. RESULTS: All of the methods demonstrated linkage of PI type to serum A1AT level. Interestingly, however, the other quantitative phenotypes provided only weak evidence for linkage of PI type to lung disease. Better evidence for linkage of lung disease to PI type was found using a moderate or a mild threshold for the definition of airflow obstruction. CONCLUSIONS: For linkage analysis of spirometry phenotypes in A1AT deficiency, qualitative phenotypes provided stronger evidence for linkage than quantitative phenotypes. Possible contributors to the stronger evidence for linkage to qualitative spirometry phenotypes include the ascertainment scheme and the nonnormality of the pulmonary function data in PI Z subjects. This study provides guidelines for studies of the genetics of COPD unrelated to A1AT deficiency.

Multum in parvo: explorations with a small bag of carbon dioxide.

A collection of 12 papers published between 1957 and 1972 are revisited. The papers had a common theme of the use of rebreathing carbon dioxide and explored a variety of topics in respiratory physiology. The first study established a method for the noninvasive and indirect estimation of arterial carbon dioxide pressure that was suitable for the routine clinical monitoring of respiratory failure and whose clinical utility remains to this day, but which also provided observations that were the stimulus for the studies that followed. The rate of rise in the partial pressure of carbon dioxide (PCO(2)) during rebreathing led to an analysis of body carbon dioxide storage capacity. Knowledge of carbon dioxide storage led to a method for quantifying lactate production in exercise without the need for blood sampling. The changes in ventilation that accompanied the increase in PCO(2) provided the basis for a rapid method for measuring aspects of breathing control (Read's method), which was later modified to measure the ventilatory response to hypoxia. The physiology of breath-holding was explored through observations of the fall in breath-holding time as PCO(2) climbed. Rebreathing also allowed increases in voluntary ventilation to be achieved without the development of alkalosis, leading to studies of maximal voluntary ventilation and respiratory muscle fatigue. Equilibration of PCO(2) during rebreathing was used to measure mixed venous PCO(2) during exercise and develop an integrated approach to the physiology of exercise in health and disease; alveolar-arterial disequilibrium in PCO(2) during exercise was uncovered. Equilibration of PCO(2), as well as PO(2), during rebreathing of carbon dioxide and nitrogen gas mixtures showed different time courses of venous gases at the onset of exercise. Starting with the rebreathing of carbon dioxide in oxygen mixtures in a small rubber bag, an astonishing range of topics in respiratory physiology was explored, with observations that remain valid, but in some respects unresolved, to the present day.

Catalytic Meerwein-Pondorf-Verley reduction by simple aluminum complexes.

[reaction: see text] Catalytic MPV reduction was successfully carried out using simple aluminum precatalysts. Alkylaluminum reagents were converted to a low-aggregation aluminum alkoxide that was highly active for the MPV reduction of several carbonyl substrates in high yield (50-99%) using (i)PrOH as the reducing agent. A high degree of cis/trans selectivity was achieved in the reduction of 2-methylcyclohexanone (cis/trans = 20/80) by (i)PrOH. When chiral hydride sources were utilized in the reduction of 2-chloroacetophenone, high enantioselectivity (68-80% ee) was observed.

Imidazolinium salts as catalysts for the ring-opening alkylation of meso epoxides by alkylaluminum complexes.

[reaction: see text] Imidazolinium salts and their N-heterocyclic carbene (NHC) derivatives catalyze the alkylation of a variety of meso epoxides in the presence of triethylaluminum (yield = 70-90%), under mild conditions. Imidazolinium salts are better catalysts than their NHC derivatives but can lead to dimerization side reactions under extended reaction time. Preformed NHC.AlEt(3) complexes and Wanzlick-type olefins, which are dimers of free NHCs, are also catalysts for this reaction.

Alpha-1-antitrypsin genotyping with mouthwash specimens.

Alpha1-antitrypsin (alpha1-AT) deficiency is diagnosed as a two-stage procedure (concentration and phenotype). However the latter does not provide clues to the presence of null genes without family studies and obtaining blood from patients at a distance often proves difficult. The aim of the study was to assess the feasibility of genotyping alpha1-AT using buccal cells. Mouthwash specimens were sent by 84 patients (with a variety of phenotypes of alpha1-antitrypsin) through the post. Deoxyribonucleic acid (DNA) was isolated from buccal cells in each sample and subjected to polymerase chain reaction (PCR) using a genotyping kit to detect the S and Z alleles. Eighty-three of 84 samples received were suitable for amplification. The specific primers successfully identified the S and Z alleles in each case. However, five of the 35 samples obtained from patients thought to be Z allele homozygotes were found to be heterozygotes for another severe deficiency allele. These data confirm the feasibility of "at distance" testing for alpha1-antitrypsin deficiency alleles using buccal cells from mouthwash samples. The results raise the possibility that other deficiency alleles are more common than has previously been suspected.

Assessment of airway neutrophils by sputum colour: correlation with airways inflammation.

BACKGROUND: Airway inflammation, with recruitment of neutrophils to the airway lumen, results in purulent secretions and a variety of potential adverse consequences for patients with chronic bronchitis and bronchiectasis. We hypothesised that gradations of sputum colour would correlate directly with the myeloperoxidase content of sputum and with various other indicators of the activity and consequences of bronchial diseases. METHODS: To test this hypothesis, we quantified sputum colour by reference to a sensitive nine point colour chart and correlated this assessment with indices of a number of inflammatory mediators in sputum. RESULTS: The results indicate that standardised visual measurements of sputum colour correlated strongly with myeloperoxidase, interleukin 8, leucocyte elastase (both activity and total quantity), sputum volume, protein leak, and secretory leucocyte proteinase inhibitor (p<0.001 for all). In addition, there was a strong direct correlation between leucocyte elastase and both myeloperoxidase (p<0.003) and sputum volume (p<0.001), but a strong negative correlation with secretory leucocyte proteinase inhibitor (p<0.001). CONCLUSIONS: These results indicate that sputum colour graded visually relates to the activity of the underlying markers of bronchial inflammation. The results of this simple visual analysis of sputum provides guidance concerning underlying inflammation and its damaging potential. It also provides a useful scientific tool for improving the monitoring of chronic airways diseases and response to treatment.

Gender-related differences in severe, early-onset chronic obstructive pulmonary disease.

Men have higher prevalence rates of chronic obstructive pulmonary disease (COPD) than women, which has been attributed to the historically higher rates of cigarette smoking in males. However, the increased rates of cigarette smoking in females within the last several decades have been associated with steadily increasing rates of COPD in women. As part of a study of the genetics of severe, early-onset COPD, we assembled a group of 84 probands with severe, early-onset COPD (without severe alpha(1)-antitrypsin deficiency) and 348 of their first-degree relatives. We found a markedly elevated prevalence (71.4%) of females among the early-onset COPD probands. Among the entire group of first-degree relatives of early-onset COPD probands, univariate analysis demonstrated similar spirometric values and bronchodilator responsiveness in males and females; however, among current or ex-smokers, female first-degree relatives had significantly lower FEV(1)/ FVC (81.4 +/- 17.2% in females versus 87.0 +/- 12.9% in males, p = 0.009) and significantly greater bronchodilator responsiveness (expressed as percentage of baseline FEV(1)) (7.7 +/- 9.4% pred in females versus 4.1 +/- 6.4% pred in males, p = 0.002). Female smoking first-degree relatives were significantly more likely to demonstrate profound reductions in FEV(1) (< 40% pred) than male smoking first-degree relatives (p = 0. 03). Multivariate analysis, performed with generalized estimating equations, demonstrated that current or ex-smoking female first-degree relatives had significantly greater risk of FEV(1) < 80% pred (OR 1.91, 95% CI 1.03- 3.54), FEV(1) < 40% pred (OR 3.56, 95% CI 1.08-11.71), and bronchodilator response greater than 10% of baseline FEV(1) (OR 4.74, 95% CI 1.91-11.75). These results suggest that women may be more susceptible to the development of severe COPD.

Association between airway bacterial load and markers of airway inflammation in patients with stable chronic bronchitis.

PURPOSE: Viable bacteria are often isolated from airway secretions in clinically stable patients with chronic bronchitis. We hypothesized that the number of organisms and bacterial species might be important modulators of airway inflammation. SUBJECTS AND METHODS: We performed quantitative sputum cultures in 160 stable patients [55 with chronic obstructive pulmonary disease (COPD) and normal serum alpha(1)-antitrypsin levels, 62 with COPD and severe alpha(1)-antitrypsin deficiency (PiZ), and 43 with idiopathic bronchiectasis]. The results were related to several indicators of the mechanisms and severity of airway inflammation. RESULTS: Airway bacterial load correlated with sputum myeloperoxidase level, an indirect measure of neutrophil activation and number (r = 0.50, P<0. 001); sputum neutrophil chemoattractants [interleukin-8 level (r = 0. 68, P<0.001) and leukotriene B4 level (r = 0.53, P<0.001)]; sputum leukocyte elastase activity (r = 0.55, P<0.001); and albumin leakage from serum to sputum (r = 0.26, P<0.01). Markers of inflammation increased at bacterial loads of 10(6) to 10(7) colony-forming units per milliliter, and increased progressively with increasing bacterial load. For example, the median (interquartile range) sputum myeloperoxidase level was 0.3 U/mL (0.1 to 0.5 U/mL) for patients who were not colonized or who had mixed normal oropharyngeal flora alone; 0.5 U/mL (0.2 to 0.7 U/mL) for patients with 10(5) to 10(6) colony-forming units per milliliter (P = 0.07); 0.5 U/mL (0.3 to 1.2 U/mL) for patients with 10(6) to 10(7) colony-forming units per milliliter (P<0.01); 0.7 U/mL (0.3 to 1.2 U/mL) for patients with 10(7) to 10(8) colony-forming units per milliliter (P <0.005); and 2.4 U/mL (0.7 to 4.8 U/mL) for patients with 10(8) or greater colony-forming units per milliliter (P<0.0001). The bacterial species influenced airway inflammation; for example, sputum myeloperoxidase activity was greater (P<0.005) in patients colonized with Pseudomonas aeruginosa [median 32 U/mL (interquartile range, 20 to 65 U/mL)] than those colonized with nontypeable Hemophilus influenzae [4 U/mL (2 to 31 U/mL)], which in turn was greater (P = 0.01) than among those colonized with Moraxella catarrhalis [1.1 U/mL (0.6 to 1.8 U/mL)]. We did not find a relation between bacterial load and lung function. CONCLUSIONS: The bacterial load and species contribute to airway inflammation in patients with stable chronic bronchitis. Further studies are required to determine the consequences of bacterial colonization on patient morbidity and decline in lung function.

Bioactive proteinase 3 on the cell surface of human neutrophils: quantification, catalytic activity, and susceptibility to inhibition.

Although proteinase 3 (PR3) is known to have the potential to promote inflammation and injure tissues, the biologic forms and function of PR3 in polymorphonuclear neutrophils (PMN) from healthy donors have received little attention. In this paper, we show that PMN contain 3.24 +/- SD 0.24 pg of PR3 per cell, and that the mean concentration of PR3 in azurophil granules of PMN is 13.4 mM. Low levels of PR3 are detectable on the cell surface of unstimulated PMN. Exposure of PMN to cytokines or chemoattractants alone induces modest (1.5- to 2.5-fold) increases in cell surface-bound PR3. In contrast, brief priming of PMN with cytokines, followed by activation with a chemoattractant, induces rapid and persistent, 5- to 6-fold increases in cell surface expression of PR3, while causing minimal free release of PR3. Membrane-bound PR3 on PMN is catalytically active against Boc-Alanine-Alanine-Norvaline-thiobenzyl ester and fibronectin, but in marked contrast to soluble PR3, membrane-bound PR3 is resistant to inhibition by physiologic proteinase inhibitors. PR3 appears to bind to the cell surface of PMN via a charge-dependent mechanism because exposure of fixed, activated PMN to solutions having increasing ionic strength results in elution of PR3, HLE, and CG, and there is a direct relationship between their order of elution and their isoelectric points. These data indicate that rapidly inducible PR3 expressed on the cell surface of PMN is an important bioactive form of the proteinase. If PR3 expression on the cell surface of PMN is dysregulated, it is well equipped to amplify tissue injury directly, and also indirectly via the generation of autoantibodies.

Alpha1-antitrypsin deficiency: incidence and detection program.

Most individuals with AAT deficiency have escaped detection by healthcare systems worldwide. The large number of undiagnosed individuals has made it difficult to define the natural history of the clinical disease accurately, and severe ascertainment bias has colored the clinical descriptions of the disease. Most importantly, undetected individuals lose opportunities for important lifestyle changes and preventive therapies. To address this problem, the World Health Organization has recommended that all patients with chronic obstructive lung disease, and all adults and adolescents with asthma, be tested for AAT deficiency. Historically, the AAT Deficiency Detection Center has tested more than 30 000 individuals for the disease, and we have identified more than 1000 cases of AAT deficiency (approximately 30% of the known cases in the United States). Currently, we are implementing methods for determining AAT concentration (level), phenotype, and genotype in specimens of whole blood dried onto filter paper. This full spectrum of robust tests is performed on samples that are easily obtained and shipped to a central laboratory for processing. Wide application of these procedures may help to bring large numbers of presently undiagnosed patients to medical attention.