Risk of exposure to Coxiella burnetii from ruminant livestock exhibited at Iowa agricultural fairs.

Coxiella burnetii is a zoonotic pathogen typically associated with clinical and asymptomatic infection in ruminant livestock. A re-emerging pathogen of significant public health importance, C. burnetii has caused recent epidemics in the United States and Europe, and public livestock exhibitions are increasingly scrutinized as a potential source of C. burnetii exposure. Although C. burnetii prevalence data among North American domestic ruminants are extremely limited, contemporary studies suggest that this pathogen is both geographically widespread and highly prevalent on a herd basis, especially in dairy cattle and goat populations. We utilized a real-time PCR assay to detect C. burnetii faecal shedding by clinically normal, non-periparturient beef cattle, meat goats and sheep exhibited at Iowa agricultural fairs. Individual faecal samples were collected from beef cattle, meat goats and sheep exhibited at twelve Iowa county fairs during the summer of 2009. The sample pool was blocked by species and fair, and ten samples from each block were randomly selected for the diagnostic assay; this test pool is considered sufficient to identify with 95% confidence a shedding animal in a population prevalence of 2.85% (cattle and sheep) and 6.25% (goats). Detection of C. burnetii DNA was determined through use of a real-time PCR assay validated for use in bovine, ovine and caprine faeces; threshold of detection is one DNA copy per PCR (sensitivity 95.8%, specificity 100%). All tested samples were negative for C. burnetii DNA. We conclude that non-dairy, non-periparturient ruminants exhibited at Iowa fairs are unlikely to shed C. burnetii in their faeces and that this population should not be considered to be a significant exposure risk to other livestock or fair attendees.

Shortfalls of the use of HbA1C-derived eAG in older adults with diabetes.

AIMS: The hemoglobin HbA1C (HbA1C) value, translated into estimated average glucose concentration (eAG), is commonly used to assess glycaemic control and manage treatment regimens in people with diabetes. However, the relationships among HbA1C-derived eAG, and mean glucose concentration derived from continuous glucose monitoring (CGM) in different populations have not been well studied. We examined this relationship in older people with diabetes and compared the results to those currently used in clinical practice. METHODS: Data from three studies evaluating CGM in older adults (≥70 years of age), with stable glycaemic control were analyzed retrospectively. Mean glucose and mean amplitude of glucose excursion (MAGE) were calculated from CGM data and correlated with HbA1C and HbA1C-derived eAG using the ADAG study formula. RESULTS: HbA1C and CGM data were analyzed from 90 patients with mean age 76±5 years, HbA1C 7.9±1.2% (63±13 mmol/mol) and 77% with Type 2 diabetes. The HbA1C and HbA1C-derived eAG correlated significantly with CGM-measured mean glucose (r(2)=0.30, p<0.0001) and MAGE (r(2)=0.16, p=0.00013) in this population and all its subgroups, but the slopes of the relationship between HbA1C and eAG or CGM-measured mean glucose were significantly different. CONCLUSIONS: HbA1C-derived eAG values may not accurately reflect CGM-measured mean glucose or MAGE in older adults with diabetes. Wide glucose excursions should be considered and HbA1C should be interpreted cautiously when making treatment changes based on HbA1C.

Drosophila male courtship behavior is modulated by ecdysteroids.

Temperature-dependent induction of ecdysteroid deficiency in the ecdysoneless mutant ecd(1) adult Drosophila melanogaster results in altered courtship behavior in males. Ecdysteroid deficiency brings about significantly elevated male-male courtship behavior including song production resembling that directed toward females. Supplementation with dietary 20-hydroxyecdysone reduces male-male attraction, but does not change motor activity, courtship patterns or attraction to females. These observations support the hypothesis that reduced levels of ecdysteroids increase the probability that male fruit flies will display courtship behaviors to male stimuli.

Considerations in developing an integrated pest management programme for control of sea lice on farmed salmon in Pacific Canada.

In the development of integrated pest management (IPM) plans for the control of sea lice there are some components that are common to many areas. However, effective plans must be tailored to regionally varying environmental and biological factors affecting the severity of sea lice infections. This paper describes factors that would be involved in the development of an IPM plan for sea lice in the Broughton Archipelago, British Columbia. Temperature, salinity and currents affect the production, dispersion and competence of larvae of sea lice, Lepeophtheirus salmonis (Krøyer), as they develop to the infective copepodid stage. This information can be coupled with oceanographic conditions in the Broughton Archipelago and emerging computer models to define zones of infection where infections of new hosts are most likely. Salinity and temperature depend, in part, on river discharge in estuarine systems. River discharge depends on precipitation, snow pack and ambient temperatures, which can be monitored to help forecast the intensity of sea lice infections associated with both farmed and wild hosts. One of the goals of IPM planning is to reduce reliance on pesticides to avoid development of resistance in targeted parasites and to minimize environmental residues. Recommendations for developing an IPM plan specific to the Broughton Archipelago are provided along with a discussion of the additional information needed to refine IPM plans in this and other areas.

Response of an estuarine benthic community to application of the pesticide carbaryl and cultivation of Pacific oysters (Crassostrea gigas) in Willapa Bay, Washington.

Oyster culture operations on the West coast of North America have developed into complete farming operations for the introduced Japanese oyster, Crassostrea gigas, which now covers vast areas of the intertidal landscape, particularly in Washington State where the pesticide carbaryl has also been used to control burrowing thalassinid shrimp for more than 30 years. Field experiments were conducted to examine the effects of these habitat modifications on the benthic community in Willapa, Bay Washington (124 degrees 06'W,46 degrees 24'N) where 50% of the state's oyster production occurs. Results indicated that the primary long-term effect of carbaryl application was removal of the two species of thalassinid shrimp (Neotrypaea californiensis and Upogebia pugettensis), which dominated the community at the start of the experiment and clearly influenced community composition themselves. Small peracarid crustaceans like the amphipods Corophium acherusicum and Eohaustorius estuarius experienced the most significant short-term mortalities, but generally recruited back to treated sites within 3 months, and were often more abundant on treated than untreated sites 1 year after carbaryl application. Results for molluscs were mixed, with no significant effect on Macoma spp, but a significant effect on the commensal clam Crytomya californica and mixed results for the cockle Clinocardium nutalli. Polychaetes were the least susceptible to carbaryl and with the exception of a short-term effect on oligochaetes, no significant negative effects were observed. The addition of oysters did not affect the infaunal community in this study, however greater abundance of epifaunal organisms like mussels, scaleworms, and the amphipod Amphithoe valida, which builds tubes in algae attached to shells, was observed. Carbaryl, which is currently applied to roughly 242 ha (< 6% of the intertidal) in Willapa Bay on an annual basis, has a variable but relatively short-term effect on the benthic community, which should be viewed in the context of other oyster culture operations like the addition of oysters themselves to a community often dominated by burrowing thalassinid shrimp which clearly control its dynamics.

RNA: a method to specifically inhibit PCR amplification of known members of a multigene family by degenerate primers.

The polymerase chain reaction (PCR) is a versatile method to amplify specific DNA with oligonucleotide primers. By designing degenerate PCR primers based on amino acid sequences that are highly conserved among all known gene family members, new members of a multigene family can be identified. The inherent weakness of this approach is that the degenerate primers will amplify previously identified, in addition to new, family members. To specifically address this problem, we synthesized a specific RNA for each known family member so that it hybridized to one strand of the template, adjacent to the 3'-end of the primer, allowing the degenerate primer to bind yet preventing extension by DNA polymerase. To test our strategy, we used known members of the soluble, nitric oxide-sensitive guanylyl cyclase family as our templates and degenerate primers that discriminate this family from other guanylyl cyclases. We demonstrate that amplification of known members of this family is effectively and specifically inhibited by the corresponding RNAs, alone or in combination. This robust method can be adapted to any application where multiple PCR products are amplified, as long as the sequence of the desired and the undesired PCR product(s) is sufficiently distinct between the primers.

A clinical trial to selectively change dietary fat and/or energy intake in women: the Women's Diet Study.

Dietary fat and energy intake have been implicated in breast cancer etiology. To examine the relative importance of these dietary factors on markers of cancer risk in women, we designed an intervention trial to selectively decrease fat and/or energy intake in free-living, premenopausal women who were somewhat overweight. The study used a 2 x 2 factorial design to evaluate the independent and interactive effects of dietary fat and energy. The diets were nonintervention, low fat (15% of energy from fat, maintenance of energy intake), low energy (25% energy reduction), and combination low fat and low energy. We utilized an individualized counseling approach with self-selection of foods. Women on the low-fat and combination diets were asked to meet given daily goals for fat grams and food group exchanges, while women on the low-energy diet used only food group exchanges. Of the 113 premenopausal women randomized who were eligible for analysis, 43% were African-American. A total of 88 women completed the 12-week program, and adherence to the dietary goals was similar in both racial groups. Women on the low-fat diet were able to reduce dietary fat intake to 19% of energy by 4 weeks and to 17% by 12 weeks with a slight decrease in energy intake. Women on the low-energy diet met their energy reduction goals by four weeks while maintaining percentage of energy from fat. Women on the combination diet largely met their goals by four weeks as well. These data indicate that it is possible to selectively manipulate dietary fat and energy intake in women over a short period of time, which makes clinical studies on the relative effects of these two dietary variables on cancer risk biomarkers readily feasible.

Progesterone receptor variants found in breast cells repress transcription by wild-type receptors.

Progesterone, through its nuclear receptors (PR), regulates the development and growth of breast cancers. PR also serve as markers of hormone dependence and prognosis in patients with this disease, and functional PR are required to mediate the antiproliferative effects of progestin therapies. We find that normal and malignant breast cells and tissues can express anomalous forms of PR transcripts. We have isolated four variant PR mRNAs that contain precise deletions of exons encoding sections of the DNA- and hormone-binding domains. The transcripts lack exon 2 (PRdelta2), exon 4 (PRdelta4), exon 6 (PRdelta6), or exons 5 and 6 (PRdelta5,6). On immunoblots, PRdelta4, delta6. and delta5, 6 cloned into the background of the PR A-isoform comigrate with similar proteins present in breast tumor extracts; delta6 and delta5, 6 are dominant-negative transcriptional inhibitors of wild-type A- and B-receptors. We propose that expression of variant PR can compromise the accuracy of receptor measurements as markers of hormone-dependent cancers, and can modify the responses of tumors to progestin therapies.

alpha-Adrenergic activity and cardiovascular effects of besipirdine HCl (HP 749) and metabolite P7480 in vitro and in the conscious rat and dog.

Besipirdine displays potent adrenergic activity in a variety of pharmacological and behavioral tests. Based on this property, we evaluated the effects of besipirdine and its N-despropyl metabolite N-despropyl-besipirdine (P7480) on cardiovascular function in rats and dogs. Besipirdine and P7480 bind alpha-2 adrenoceptors (K(I): 380 and 10 nM, respectively) and facilitate the stimulated release of [3H]norepinephrine from rat cortical slices due to presynaptic autoreceptor blockade. In rat aorta rings and the pithed rat, P7480, but not besipirdine, also behaved as a postsynaptic alpha-1 adrenoceptor agonist. In conscious rats, besipirdine (2-10 mg/kg, p.o.) and P7480 (3-10 mg/kg, p.o.) produced dose-related increases in mean arterial pressure. Inhibition of hepatic cytochrome P-450 enzyme activity blocked the pressor effect of besipirdine, but not of P7480; therefore, P7480 mediated besipirdine's pressor effect. The bradycardia after either agent was unaffected. In conscious dogs, besipirdine (0.1-2 mg/kg, p.o.) also produced dose-related hypertension and bradycardia. The hypertension, but not the bradycardia, were sensitive to prazosin (3 mg/kg, p.o.), but not hexamethonium (10 mg/kg, p.o.). Muscarinic and beta-adrenergic receptor blockade studies in anesthetized dogs demonstrated the bradycardia to be due to withdrawal of cardiac sympathetic tone. These findings suggest that besipirdine's peripheral hypertensive effect is primarily mediated by the pressor metabolite P7480, although facilitated norepinephrine release may contribute. Besipirdine's bradycardic action appears to be centrally mediated, because both compounds lacked direct negative chronotropic activity on spontaneously beating guinea pig atria in vitro.

Pharmacological activity and safety profile of P10358, a novel, orally active acetylcholinesterase inhibitor for Alzheimer's disease.

1-[(3-Fluoro-4-pyridinyl)amino]-3-methyl-1(H)-indol-5-yl methyl carbamate (P10358) is a potent, reversible acetylcholinesterase inhibitor that produces central cholinergic stimulation after oral and parental administration in rats and mice. P10358 is a 2.5 times more potent acetylcholinesterase inhibitor than THA in vitro (IC50 = 0.10 +/- 0.02 microM vs. IC50 = 0.25 +/- 0.03 microM). It also inhibits butyrylcholinesterase activity as potently as THA (IC50 = 0.08 +/- 0.05 microM vs. IC50 = 0.07 +/- 0.01 microM). Ex vivo, P10358 (0.2 - 20 mg/kg, p.o.) produced dose-dependent inhibition of brain acetylcholinesterase activity. At 10 and 20 mg/ kg, it produced profound and long-lasting hypothermia in mice. P10358 enhanced performance in rats in a step-down passive avoidance task (0.62 and 1.25 mg/kg) and in a social recognition paradigm (0.32, 0.64 and 1.25 mg/kg) in mice. It reversed scopolamine-induced deficits in the Morris Water maze in rats (1.25 and 2.5 mg/kg) and a higher dose elevated striatal homovanillic acid levels. These behavioral and biochemical effects are consistent with central cholinergic stimulation. Hemodynamic studies in the rat demonstrated a 16-fold separation between behaviorally active doses (1.25 mg/kg) and those that elevated arterial pressure (20 mg/kg). Lethality in rats occurred at an oral dose of 80 mg/kg, but not at lower doses. Chemically, P10358 is an N-aminoindole and may not have the hepatotoxic liability associated with aminoacridine structure of tacrine. P10358 had weak affinity (>10 microM) at a variety of aminergic and peptidergic receptors and uptake carriers. These properties suggest that P10358 may be a safe and promising symptomatic treatment for Alzheimer's disease.

EGF-dependent phosphorylation of the EGF receptor in plasma membranes isolated from young and senescent WI-38 cells.

Tyrosine-specific phosphorylation of the receptor for epidermal growth factor (EGF) in plasma membranes isolated from WI-38 cells is EGF-dependent and occurs to an equivalent extent and on identical tryptic peptides in preparations from cells of various in vitro ages. There is a marked reduction, however, in phosphorylation of receptor molecules from senescent as compared with young WI-38 cells, if enzyme activity is assayed in an immune complex following solubilization of plasma membranes with Nonidet P-40 (NP-40). Differences in the level of receptor phosphorylation in young vs. senescent NP-40 extracts are not resolved by changing the temperature at which the assay is performed, or the length of incubation. Moreover, addition of NP-40 or chloroform-methanol extracts of young cells to assays measuring receptor phosphorylation in senescent cell NP-40 preparations does not augment the senescent enzyme activity. The immunopurified senescent receptor is, however, capable of catalyzing phosphorylation of exogenous substrates. These results indicate that the loss of receptor autophosphorylation in solubilized preparations may result from a differential sensitivity of the senescent cell receptor to the detergent. This finding provides a marker for senescence and suggests subtle changes in protein structure, conformation, or regulation of the EGF receptor in senescent cells.

SPECT imaging of para-axial neurofibromatosis with technetium-99m DTPA.

Single photon emission computed tomography (SPECT) was used to study uptake of technetium-99m diethylenetriaminepentaacetic acid ([99mTc]DTPA) by para-axial neurofibromas in 13 patients. SPECT imaging led to better resolution of uptake in nine instances and detection of 12 lesions unsuspected on planar imaging (PI). Two false-positive instances and one false-negative instance of uptake are described. The planning of the surgical approach and placement of graft material for spinal fusion was assisted by SPECT in two patients. One patient with disability secondary to recurring abdominal pain had detection of the causative lesion by SPECT and subsequent relief of her symptoms following excision of the tumor. SPECT imaging of soft-tissue tumors of neurofibromatosis appears to have potential use in preplanning surgery on structural or cosmetic lesions, in the detection of occult lesions, and the monitoring of patients with neurofibromatosis at regular intervals.

Effect of nitroglycerin during hemodynamic estimation of valve orifice in patients with mitral stenosis.

In patients with mitral stenosis, valve orifice calculations using pulmonary capillary wedge pressure as a substitute for left atrial pressure may overestimate the severity of disease. Previous studies have shown that mitral valve area determined from transseptal left atrial pressure measurements exceeds that area derived from pulmonary wedge pressure measurements. This is probably due to pulmonary venoconstriction, which is reversed by nitroglycerin. Nitroglycerin, 0.4 mg, was administered sublingually to 20 patients with mitral valve disease during preoperative cardiac catheterization using the pulmonary capillary wedge pressure as the proximal hydraulic variable. At the time of a peak hypotensive effect, 3 to 5 minutes after nitroglycerin administration, the mean pulmonary capillary wedge pressure decreased from 23 +/- 2 (mean +/- SEM) to 19 +/- 2 mm Hg (p less than 0.005). The mean diastolic transmitral pressure gradient (12.6 +/- 1.2 mm Hg before and 11.5 +/- 1.0 mm Hg after nitroglycerin; p = NS) and cardiac output (4.0 +/- 0.3 to 4.1 +/- 0.3 liters/min; p = NS) did not change significantly. Nevertheless, the hemodynamic mitral orifice area, calculated using the Gorlin formula, increased from 0.8 +/- 0.1 to 1.1 +/- 0.2 cm2 (p less than 0.05). In 12 patients with isolated mitral stenosis, without regurgitation, the mitral valve orifice area after nitroglycerin was 0.4 +/- 0.2 cm2 larger than it was before drug administration (p less than 0.05). Administration of nitroglycerin during evaluation of mitral stenosis eliminates pulmonary venoconstriction, which raises the pulmonary capillary wedge pressure above the left atrial pressure in some patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Posttraumatic tibia valga: a case demonstrating asymmetric activity at the proximal growth plate on technetium bone scan.

Posttraumatic tibia valga is a well-recognized complication following fracture of the upper tibial metaphysis in young children. We present a case of a child who developed a valgus deformity following fracture of the proximal tibia and fibula in which quantitative bone scintigraphy at 5 months after injury demonstrated increased uptake at the proximal tibial growth plate with proportionally greater uptake on the medial side. This finding suggests that the valgus deformity in this patient was due to a relative increase in vascularity and consequent overgrowth of the medial portion of the proximal tibial physis.

Expression of cell cycle-dependent genes in young and senescent WI-38 fibroblasts.

We studied the expression of 11 cell cycle-dependent genes in senescent WI-38 fibroblasts and compared the results to those obtained in WI-38 cells from early passages (young cells). Every gene we examined is expressed in the senescent cells at levels similar to those in the young cells, including two genes maximally expressed at the G1/S phase boundary--genes for thymidine kinase and histone H3. The results clearly show that senescent, noncycling WI-38 cells are not similar to quiescent cells. Rather, such senescent WI-38 cells may be blocked just prior to the onset of DNA synthesis.

Neurofibromas: location by scanning with Tc-99m DTPA. Work in progress.

The accumulation of technetium-99m diethylenetriamine pentaacetic acid (Tc-99m DTPA) in benign soft-tissue neurofibromatosis tumors is reported. In a series of 16 patients with clinical stigmata of neurofibromatosis, 28 sites of abnormal soft-tissue localization of the isotope observed scintigraphically were documented to be sites of soft-tissue tumor by clinical and/or radiographic (predominantly computed tomographic) correlations. The smallest lesion detected was a 1.5-cm subcutaneous neurofibroma. Normal physiologic nonrenal distribution of the Tc-99m DTPA was established by scintigraphic imaging of a control population.

Cellular senescence: factors modulating cell proliferation in vitro.

In our view, two major areas of the investigation of the aging process have been most fruitful over the past few years: namely, the genetic and hormonal strategies aimed at the understanding of in vitro cellular senescence. The genetic studies have primarily utilized cell fusion techniques and viral probes. Along with cell cycle studies involving the induction of thymidine kinase activity and TTP synthesis, the cell fusion studies are most consistent with a late G1 block in senescent cells. This effect would appear to be distinct from the G0 arrest of density-inhibited or mitogen-restricted cell populations. The hormonal studies which have centered on the regulation of cell proliferation have recently focused on peptide hormones. EGF has been of particular interest since it is so well characterized. This receptor system remains largely unchanged throughout the lifespan with the notable exception of the purified receptor-associated, autocatalytic, tyrosine-specific kinase activity, which decreases with age. The functional significance of this decrease in enzyme activity is unknown, although its growth regulatory importance is implicated in several systems, and may well represent a critical early G0/G1 event which is absent in senescent cells.

Effect of nitroglycerin on the pulmonary venous gradient in patients after mitral valve replacement.

Because the equality of the pulmonary artery wedge pressure and left atrial pressure has been questioned in patients with mitral valve disease and pulmonary hypertension, this study examined how vasomotor activity in the pulmonary capacitance vessels might contribute to a discrepancy between these pressures. The difference between the pulmonary wedge and left atrial pressures (designated as the pulmonary venous gradient) was measured after nitroglycerin administration in nine patients who had pulmonary hypertension (mean pulmonary artery pressure 40 mm Hg) after mitral valve replacement. Five minutes after sublingual nitroglycerin, 0.4 mg, the mean pulmonary wedge pressure decreased from 19 +/- 2 to 13 +/- 2 mm Hg (p less than 0.005), exceeding the decrease in left atrial pressure (15 +/- 2 to 11 +/- 2 mm Hg; p less than 0.005). Pulmonary blood flow increased from 4.6 +/- 0.4 to 4.9 +/- 0.4 liters/min (p less than 0.005). The decrease in mean pulmonary venous gradient from 4.0 +/- 0.8 to 1.7 +/- 0.6 mm Hg (p less than 0.025) was attributed to nitrate-mediated pulmonary venodilation. The ratio of venous gradient to blood flow, an index of pulmonary venous tone, decreased after nitroglycerin from 0.9 +/- 0.2 to 0.4 +/- 0.1 (p less than 0.01). These data indicate that reversible pulmonary vasoconstriction contributes to elevation of the pulmonary wedge pressure above the left atrial pressure in patients with chronic mitral valve disease and pulmonary hypertension and that nitroglycerin may produce pulmonary venodilation decreasing the pulmonary venous gradient.