Evolutionary dynamics of West Nile virus in Georgia, 2001-2011.

From 1999-2001, West Nile virus (WNV) spread throughout the eastern United States (US) and was first detected in Georgia in 2001. To date, the virus has been detected in over 2,500 dead wild bird and mosquito samples from across Georgia. We sequenced the premembrane (preM) and envelope gene (E) (2004 bp) from 111 isolates collected from 2001 to 2011. To assess viral gene flow from other geographic regions in the US, we combined our data with WNV sequences available at the National Center for Biotechnology Information (NCBI) and performed phylogenetic analysis. We found evidence that WNV isolates detected in Chatham County Georgia most likely originated from the Northeastern United States. These results highlight the growing importance of adequate genetic surveillance for monitoring and controlling viruses of public health concern.

Comparative sequence analysis of full-length genome of FIPV at different tissue passage levels.

Feline infectious peritonitis virus (FIPV), an alpha Coronavirus, is the causative agent of a fatal immune mediated disease in cats. It is currently unclear if this virus circulates in the field or develops in felines that are infected with Feline enteric coronavirus. To better understand the genomic changes associated with viral adaptation, we sequenced the complete genomes of FIPV WSU 79-1146 at different tissue passage levels: passage 1, passage 8, and passage 50 tissue culture. Twenty-one amino acid differences were observed in the polyprotein 1a/ab between the different passages. Only one residue change was observed in the spike glycoprotein, which reverted back on subsequent passages, four changes were observed in the 3c protein, and one change was observed in each 3a, small membrane, nucleocapsid and 7a proteins. The mutation rate was calculated to be 5.08-6.3 × 10(-6) nucleotides/site/passage in tissue culture suggesting a relatively stable virus. Our data show that FIPV has a low mutation rate as it is passed in cell culture but has the capacity for change specifically in nsp 2, 3c, and 7b as it is passed in cell culture.

Changes in nonstructural protein 3 are associated with attenuation in avian coronavirus infectious bronchitis virus.

Full-length genome sequencing of pathogenic and attenuated (for chickens) avian coronavirus infectious bronchitis virus (IBV) strains of the same serotype was conducted to identify genetic differences between the pathotypes. Analysis of the consensus full-length genome for three different IBV serotypes (Ark, GA98, and Mass41) showed that passage in embryonated eggs, to attenuate the viruses for chickens, resulted in 34.75-43.66% of all the amino acid changes occurring in nsp 3 within a virus type, whereas changes in the spike glycoprotein, thought to be the most variable protein in IBV, ranged from 5.8 to 13.4% of all changes. The attenuated viruses did not cause any clinical signs of disease and had lower replication rates than the pathogenic viruses of the same serotype in chickens. However, both attenuated and pathogenic viruses of the same serotype replicated similarly in embryonated eggs, suggesting that mutations in nsp 3, which is involved in replication of the virus, might play an important role in the reduced replication observed in chickens leading to the attenuated phenotype.

Panlobular emphysema is associated with COPD disease severity: A study of emphysema subtype by computed tomography.

BACKGROUND: Computed tomography has the potential to inform COPD prognosis. We sought to determine associations of emphysema phenotype with clinical parameters including lung function, inflammatory markers, and quality of life. METHODS: Participants of this single-center observational cohort (n = 83) were 40-80 years old, had ≥10 pack-year smoking, and a diagnosis of COPD confirmed by spirometry. All participants had available historic chest CT scans which were systematically reviewed by a single expert radiologist and scored for emphysema subtype, extent, and distribution. Associations between radiographic findings and clinical parameters were determined. RESULTS: Median age of participants was 72 years, median smoking 40 pack-years, and median FEV1 59% predicted. 84% of the participants had radiographic emphysema. Of those, 26% had panlobular emphysema (PLE), 68% centrilobular emphysema (CLE), and 6% paraseptal emphysema (PSE). As compared to the participants with no radiographic emphysema, the presence of PLE-dominant emphysema was associated with a lower BMI (P = 0.012) and greater extent of emphysema (P = 0.014). After adjusting for age, sex, and pack-years smoking history, PLE was associated with greater airflow obstruction by FEV1% (48% vs 71%, P = 0.005), greater symptom burden by CAT score (18 vs 9, P = 0.015), worse quality of life by SGRQ score (43 vs 22, P = 0.025), and more systemic inflammation by erythrocyte sedimentation rate (P = 0.001). CLE- or PSE-dominant emphysema were not similarly associated with clinical features or symptom burden. CONCLUSIONS: The presence of PLE-dominant emphysema was associated with greater extent of emphysema, greater airflow obstruction, increased respiratory symptoms, worse quality of life, and systemic inflammation. Further investigation is indicated to explore the pathogenesis of the PLE phenotype and the prognostic and treatment implications of PLE.

CXCR2 antagonists for the treatment of pulmonary disease.

Chemokines have long been implicated in the initiation and amplification of inflammatory responses by virtue of their role in leukocyte chemotaxis. The expression of one of the receptors for these chemokines, CXCR2, on a variety of cell types and tissues suggests that these receptors may have a broad functional role under both constitutive conditions and in the pathophysiology of a number of acute and chronic diseases. With the development of several pharmacological, immunological and genetic tools to study CXCR2 function, an important role for this CXC chemokine receptor subtype has been identified in chronic obstructive pulmonary disease (COPD), asthma and fibrotic pulmonary disorders. Interference with CXCR2 receptor function has demonstrated different effects in the lungs including inhibition of pulmonary damage induced by neutrophils (PMNs), antigen or irritant-induced goblet cell hyperplasia and angiogenesis/collagen deposition caused by lung injury. Many of these features are common to inflammatory and fibrotic disorders of the lung. Clinical trials evaluating small molecule CXCR2 antagonists in COPD, asthma and cystic fibrosis are currently underway. These studies hold considerable promise for identifying novel and efficacious treatments of pulmonary disorders.

High temporal resolution gastric emptying breath tests in mice.

BACKGROUND: Gastric emptying is a complex physiological process regulating the division of a meal into smaller partitions for the small intestine. Disrupted gastric emptying contributes to digestive disease, yet current measures may not reflect different mechanisms by which the process can be altered. METHODS: We have developed high temporal resolution solid and liquid gastric emptying breath tests in mice using [13 C]-octanoic acid and off axis- integrated cavity output spectroscopy (OA-ICOS). Stretched gamma variate and 2-component stretched gamma variate models fit measured breath excretion data. KEY RESULTS: These assays detect acceleration and delay using pharmacological (7.5 mg/kg atropine) or physiological (nutrients, cold exposure stress, diabetes) manipulations and remain stable over time. High temporal resolution resolved complex excretion curves with 2 components, which was more prevalent in mice with delayed gastric emptying following streptozotocin-induced diabetes. There were differences in the gastric emptying of Balb/c vs C57Bl6 mice, with slower gastric emptying and a greater occurrence of two-phase gastric emptying curves in the latter strain. Gastric emptying of C57Bl6 could be accelerated by halving the meal size, but with no effect on the occurrence of two-phase gastric emptying curves. A greater proportion of two-phase gastric emptying was induced in Balb/c mice with the administration of PYY (8-80 nmol) 60 min following meal ingestion. CONCLUSIONS AND INFERENCES: Collectively, these results demonstrate the utility of high temporal resolution gastric emptying assays. Two-phase gastric emptying is more prevalent than previously reported, likely involves intestinal feedback, but contributes little to the overall rate of gastric emptying.

Inhaled efficacious dose translation from rodent to human: A retrospective analysis of clinical standards for respiratory diseases.

Clinical pharmacologists and toxicologists are often faced with predicting equivalent dosages for humans from biological observations in laboratory animals. Allometric scaling has been used extensively as the basis for extrapolation of drug dosage that might be expected to produce the equivalent biological effects. Allometry is the study of size and its consequences and it is based on the anatomical, physiological, and biochemical similarities between animals. In this review, retrospective analyses have been performed based on data reported in the literature in an attempt to determine the utility of allometric scaling for human dose projections from pre-clinical data for compounds that are delivered by inhalation. The limited pre-clinical efficacy data available on inhaled drugs that are also used clinically supports the current method of scaling using a fixed allometric exponent of 0.67. An example of the utility of the human inhaled dose projections for planning inhaled toxicology studies is also presented.

The incidence of deep prosthetic infections in a specialist orthopaedic hospital: a 15-year prospective survey.

The Control of Infection Committee at a specialist orthopaedic hospital prospectively collected data on all episodes of bacteriologically-proven deep infection arising after primary hip and knee replacements over a 15-year period from 1987 to 2001. There were 10 735 patients who underwent primary hip or knee replacement. In 34 of 5947 hip replacements (0.57%) and 41 of 4788 knee replacements (0.86%) a deep infection developed. The most common infecting micro-organism was coagulase-negative staphylococcus, followed by Staphylococcus aureus, enterococci and streptococci. Of the infecting organisms, 72% were sensitive to routine prophylactic antimicrobial agents. Of the infections, 29% (22) arose in the first three months following surgery, 35% between three months and one year (26), and 36% (27) after one year. Most cases were detected early and treated aggressively, with eradication of the infection in 96% (72). There was no significant change in the infection rate or type of infecting micro-organism over the course of this study. These results set a benchmark, and importantly emphasise that only 64% of peri-prosthetic infections arise within one year of surgery. These results also illustrate the advantages of conducting joint replacement surgery in the isolation of a specialist hospital.

Basolateral K channels in an insect epithelium. Channel density, conductance, and block by barium.

K channels in the basolateral membrane of insect hindgut were studied using current fluctuation analysis and microelectrodes. Locust recta were mounted in Ussing-type chambers containing Cl-free saline and cyclic AMP (cAMP). A transepithelial K current was induced by raising serosal [K] under short-circuit conditions. Adding Ba to the mucosal (luminal) side under these conditions had no effect; however, serosal Ba reversibly inhibited the short-circuit current (Isc), increased transepithelial resistance (Rt), and added a Lorentzian component to power density spectra of the Isc. A nonlinear relationship between corner frequency and serosal [Ba] was observed, which suggests that the rate constant for Ba association with basolateral channels increased as [Ba] was elevated. Microelectrode experiments revealed that the basolateral membrane hyperpolarized when Ba was added: this change in membrane potential could explain the nonlinearity of the 2 pi fc vs. [Ba] relationship if external Ba sensed about three-quarters of the basolateral membrane field. Conventional microelectrodes were used to determine the correspondence between transepithelially measured current noise and basolateral membrane conductance fluctuations, and ion-sensitive microelectrodes were used to measure intracellular K activity (acK). From the relationship between the net electrochemical potential for K across the basolateral membrane and the single channel current calculated from noise analysis, we estimate that the conductance of basolateral K channels is approximately 60 pS, and that there are approximately 180 million channels per square centimeter of tissue area.

Fibrillin secretion and microfibril assembly by Marfan dermal fibroblasts.

The Marfan syndrome has been linked to the FBN1 gene encoding the microfibrillar glycoprotein fibrillin. To date, there have been no descriptions of microfibrillar abnormalities characteristic of this connective tissue disorder, although biochemical analyses have highlighted apparent abnormalities in fibrillin synthesis, secretion and processing. We have conducted a biochemical and ultrastructural investigation of fibrillin expression and assembly by a panel of dermal fibroblast lines from patients with Marfan syndrome and related diseases. The study has highlighted marked differences between cells in terms of secretion and aggregation of newly-synthesised fibrillin. In addition, electron microscopic visualization of fibrillin assemblies has clearly demonstrated for the first time the plethora of microfibrillar abnormalities that underlie this heterogeneous disorder. These data emphasize the molecular complexity that is a feature of the diverse clinical phenotypes exhibited by Marfan patients.

Fibrin stimulates platelets to increase factor VIIIa binding site expression.

Factor (F)VIII functions as an enzymatic cofactor on the membranes of stimulated platelets. However, thrombin stimulates platelets to express only a small number of binding sites for FVIII. We wished to determine whether molecules that are likely to be present in a developing thrombus stimulate platelets to up-regulate FVIII binding site expression. Flow cytometry was utilized to measure binding of fluorescein-labeled FVIIIa to activated platelets and a FXase assay was utilized to measure platelet-dependent function. Various agonists as well as normal and mutant fibrinogens and fibrin were evaluated as co-stimuli. Thrombin-stimulated platelets expressed 214 +/- 67 binding sites for thrombin-activated FVIII (FVIIIa) and none of the established soluble agonists enhanced binding site exposure. However, the presence of 5 micro g mL(-1) fibrin increased the number of FVIIIa binding sites/platelet three- to eight-fold (1470 +/- 130, range 600-1800) with a parallel increase in platelet-based FXase assay. Binding site up-regulation was not stimulated by fibrinogen and was blocked by inhibitors of GPIIbIIIa. Mutant fibrin lacking the gamma-chain C-terminal four residues was ineffective while fibrin with altered RGD sequences did stimulate expression of FVIIIa binding sites indicating that co-stimulation is mediated by the fibrin gamma-chain termini. Fibrin-enhanced expression of FVIIIa binding sites was not supported by D364H fibrin, which does not aggregate normally, and was blocked by the GPRP peptide, which inhibits fibrin polymerization. Polymerized fibrin can function as a platelet co-stimulus, up-regulating expression of binding sites for FVIIIa.

7-Ketocholesterol forms crystalline domains in model membranes and murine aortic smooth muscle cells.

7-Ketocholesterol (7-keto) is one of the major oxygenated products found in oxidized low-density lipoproteins (LDL) and in atherosclerotic plaque, where it is believed to play a role in arterial pathology. We hypothesize that direct membrane effects independent of receptor binding may mediate its biological activity. To test this, small-angle x-ray diffraction approaches were used to examine the interactions of 7-keto with other membrane components in well-defined lipid vesicles and in murine aortic smooth muscle cell membranes. These data were compared with the interactions of 25-hydroxycholesterol (25-OHC) and cholesterol. Replacement of cholesterol with 7-keto in lipid vesicles produced distinct changes in membrane structure, including a marked increase in molecular volume associated with the hydrocarbon core (+/-0-8 A from the bilayer center). Additionally, there was an increase in electron density associated with the upper acyl chain region (+/-9-21 A), corresponding to the bilayer location of the steroid nucleus of 7-keto. In contrast, 25-OHC did not appear to intercalate into the membrane hydrocarbon core and did not form separate domains. Cells grown in the presence of the 7-keto developed extracellular crystals concomitant with the formation of membrane domains having a unit cell periodicity of 35.4 or 1.4 A greater than measured with cholesterol. Domains were formed within 4 h and persisted up to 72 h, after which cells showed signs of declining viability. We conclude that 7-keto is found in a membrane location distinct from cholesterol, does not condense phospholipids as efficiently as cholesterol and is able to self-associate into discrete intrabilayer domains. While these domains may decrease its cytotoxicity by inducing the formation of sterol crystals in smooth muscle cells, they may, in a broader capacity, contribute to the sterol crystals found in advanced atherosclerotic lesions.

Biological actions of synthetic locust ion transport peptide (ITP).

Locust Ion Transport Peptide (ITP) a member of the arthropod neuropeptide family which includes hyperglycemic, vitellogenesis-inhibiting, and moult-inhibiting hormones (CHH, VIH, MIH, respectively) was synthesized as proposed by Meredith et al. (1996) with terminal amidation of amino acid residue 72 and with 3 disulphide bridges. This is the first member of this family to be synthesized. Biological activities of synthetic ITP (synITP) were very similar to those previously reported for ITP purified from Schistocerca corpora cardiaca (ScgITP) and partially sequenced by Audsley et al. (1992a, b). Dose-response curves for both synITP and ScgITP on ileal transport of Cl- (measured as increased short-circuit current, delta Isc), were similar with a EC50 of 1-2 nM. The Isc time course and maximum delta Isc across ileal epithelia at different dosages of synITP and ScgITP had similar patterns as did changes in transepithelial open-circuit potential (Vt) and resistance (Rt), reflecting changes in salt transport which drives fluid absorption. Disulphide bridges were shown to be required for biological activity of synITP, which caused the same 4-fold increase in ileal fluid transport rate (Jv) as previously reported for ScgITP. Both synITP and ScgITP caused only partial stimulation of rectal Isc and had no significant effect on rectal Jv. These results indicate that the structure of ITP predicted earlier from cDNA is correct.

Expression of Schistocerca gregaria ion transport peptide (ITP) and its homologue (ITP-L) in a baculovirus/insect cell system.

We expressed an N-terminally extended Schistocerca gregaria ion transport peptide (ScgITP) and its homologue (ion transport peptide-like; ITP-L) in insect Sf9 cells using baculovirus expression vectors. Antibodies raised against peptide fragments of ITP and ITP-L were used to detect and characterize the baculovirus expressed peptides (bacITP, bacITP-L). Biological activity of the expressed peptides was assayed using the highly specific bioassay for native ITP, namely the increase in ileal short-circuit current which is a measure of active Cl- transport. BacITP and bacITP-L expression was optimal in Sf9 cells infected at a multiplicity of infection of 1, grown in Grace's medium, and harvested 2-3 days after infection. Western blots showed that bacITP was 2 kDa larger than native or synthetic ITP. This difference was not due to glycosylation and could in part be attributed to post-translational cleavage of the ITP propeptide at a site 11 amino acids upstream of the cleavage site used by S. gregaria to produce native ITP. BacITP stimulated ileal short-circuit current but is significantly less active (270-fold) than synthetic ITP (synITP) possibly as a result of the N-terminal extension. Production of bacITP-L permitted us to show that it is not stimulatory in the bioassay but reduces the synITP response in vitro and thus may have some potential for enhancing the effectiveness of biological control agents such as baculoviruses.

Regulation of vocal fold transepithelial water fluxes.

Vocal fold hydration is critical to phonation. We hypothesized that the vocal fold generates bidirectional water fluxes, which are regulated by activity of the Na(+)-K(+)- ATPase. Western blots and immunohistochemistry demonstrated the presence of the alpha-subunit Na(+)-K(+)-ATPase in the canine vocal fold (n = 11). Luminal cells, basal and adjacent one to two layers of suprabasal cells within stratified squamous epithelium, were immunopositive, as well as basolateral membranes of submucosal seromucous glands underlying transitional epithelia. Canine (n = 6) and ovine (n = 14) vocal fold mucosae exhibited transepithelial potential differences of 8.1 +/- 2.8 and 9.3 +/- 1.3 mV (lumen negative), respectively. The potential difference and short-circuit current (ovine = 31 +/- 4 microA/cm(2); canine = 41 +/- 10 microA/cm(2)) were substantially reduced by luminal administration of 75 microM acetylstrophanthidin (P < 0.05). Ovine (n = 7) transepithelial water fluxes decreased from 5.1 +/- 0.3 to 4.3 +/- 0.3 microl x min(-1) x cm(-2) from the basal to luminal chamber and from 5.2 +/- 0.2 to 3.9 +/- 0.3 microl x min(-1) x cm(-2) from the luminal to basal chamber by luminal acetylstrophanthidin (P < 0.05). The presence of the Na(+)-K(+)-ATPase in the vocal fold epithelium and the electrolyte transport derived from its activity provide the intrinsic mechanisms to regulate cell volume as well as vocal fold hydration.

Measles in pregnancy: a descriptive study of 58 cases.

OBJECTIVE: To describe the effects of measles in pregnancy using a large case series. METHODS: Pregnant women with measles were identified by county health department records, and their hospital and clinic records were reviewed. When available, records for the infants of case patients were also reviewed. RESULTS: Fifty-eight pregnant women with measles were identified. Thirty-five (60%) were hospitalized for measles, 15 (26%) were diagnosed with pneumonia, and two (3%) died of measles complications. Excluding three induced abortions, 18 pregnancies (31%) ended prematurely; five were spontaneous abortions and 13 were preterm deliveries. All but two of the 18 pregnancies that terminated early did so within 14 days of rash onset. Two term infants were born with minor congenital anomalies, but their mothers had measles late in the third trimester. No newborns were diagnosed with congenital measles. CONCLUSIONS: The incidence of death and other complications from measles during pregnancy may be higher than expected for age-comparable, nonpregnant women. Measles in pregnancy may lead to high rates of fetal loss and prematurity, especially in the first 2 weeks after the onset of rash.

Weight gain during adjuvant and neoadjuvant chemotherapy for breast cancer: an audit of 100 women receiving FEC or CMF chemotherapy.

Weight gain is reported as a common finding in patients treated for breast cancer but its aetiology appears to be complex. The aim of this study was to investigate the incidence and degree of weight gain during chemotherapy and to examine possible contributory factors. Data were collected on 100 women treated with CMF or FEC chemotherapy. The mean change in weight was +3.68 kg (P<0.001). 64% of patients gained more than 2 kg in weight, 31% maintained a stable weight (within + or - 2 kg) and 5 patients lost more than 2 kg. Approximately 1/3 of patients (33) gained more than 5 kg and 6 patients gained more than 10 kg in weight. The majority of patients (85%) received steroids as antiemetics but no effect of steroid dose was seen on the level of weight change. No significant differences in weight gain were seen in patients receiving tamoxifen (37%) compared with those not taking it. Similarly, menopausal status did not appear to be a significant factor influencing weight gain. In summary, a high incidence of weight gain was found. The literature on weight gain in breast cancer and possible interventions to avoid weight gain are discussed.

A new technique for posterior cruciate ligament reconstruction.

We describe a new technique for arthroscopically assisted posterior cruciate ligament reconstruction. We use the TransFix (Arthrex, Naples, FL) pin to provide a single and strong tibial attachment point for the 4-stranded semitendinosus and gracilis tendons. The TransFix technique for anterior cruciate ligament reconstruction has been found to give better initial fixation strength than other techniques using semitendinosus and gracilis grafts, and it provides adequate graft length for secure fixation. Strong and secure graft fixation in knee ligament reconstruction is very important for early and fast rehabilitation.

Isolation of staphylococcal L-phase variants from the blood and egg yolks of normal chickens.

The use of special microbiological media and modified techniques has resulted in the stimulation of growth and development of bacterial L forms in samples of blood and egg yolk free from mature bacteria and derived from normal healthy chickens. After prolonged cultivation the majority of these L form cultures reverted to staphylococci.

First report of clinical disease associated with canine distemper virus infection in a wild black bear (Ursus americana).

An approximately 1-yr-old black bear was discovered on the porch of a rural residence in southwestern Pennsylvania on October 26, 2011, where it remained during the day in spite of efforts to frighten it away. The bear exhibited periods of somnolence and sporadic tremors and seizures. It was euthanized by gunshot that evening. Immediately after euthanasia it was observed to have footpads that exuded fluid when compressed. It was submitted for necropsy the next day where roughened footpads were noted. Histologic examination of the brain demonstrated nonsuppurative encephalitis with eosinophilic intranuclear and intracytoplasmic inclusion bodies in neurons. The footpads were thickened and hyperkeratotic. Canine distemper virus (CDV) was detected by immunohistochemistry (IHC) in the brain and footpads, and by reverse transcription polymerase chain reaction (RT-PCR) from the brain tissue. Phylogenetic analysis indicated that the CDV cDNA from the bear had 98.2% nucleotide identity to the Rockborn-Candur vaccine and a canine isolate from 2004 in Missouri, USA, and 97.3% nucleotide identity to a raccoon CDV isolated in 2011 from Tennessee, USA. This represents a first report of CDV as a cause of encephalitis or footpad hyperkeratosis in a wild black bear.