Effects of estrogen and progesterone on spatial memory consolidation in aged females

Interpretation of data illustrating that estrogen, with or without progestin, is detrimental to memory in post-menopausal women is complicated by the fact that little is known about the effects of progestins on memory. The present study examined if estrogen, alone or with progesterone, affects spatial memory consolidation in ovariectomized aged female mice. Mice received eight training trials in a spatial Morris water maze followed immediately by injection of water-soluble 17beta-estradiol (E(2); 0.2 mg/kg) or vehicle. Mice were re-tested 24 h later. All mice learned to find the platform on Day 1. On Day 2, the performance of control, but not E(2) mice, deteriorated, suggesting that E(2) enhanced memory for the platform location. In a second experiment, mice were injected with E(2) and 10 or 20 mg/kg water-soluble progesterone. The 10 mg/kg dose of progesterone did not affect estrogen's ability to enhance spatial memory consolidation, but 20 mg/kg blocked this effect. These data indicate that estrogen can improve spatial memory consolidation in aged females and that this effect can be attenuated by progesterone.     

Separation of adeno-associated virus type 2 empty particles from genome containing vectors by anion-exchange column chromatography

Adeno-associated virus (AAV) empty capsids typically co-purify with genome containing AAV2 vectors purified by column chromatography. This study describes a method to remove empty capsids from genome containing vector particles by anion exchange chromatography. The separation is based on the slightly less anionic character of empty particles compared to vectors. Detailed methods to achieve AAV2 vector purification and particle separation using cation exchange resin POROS 50HS followed by anion exchange resin Q-Sepharose^xl are described. Chromatographic separation of AAV2 particles was achieved using gradients based on sodium acetate and ammonium acetate, and was optimal at pH 8.5. Efficient removal of particle surface nucleic acid impurities was found to be important to achieve good particle separation. In a large scale experiment performed using partially purified vector containing a mixture of 1.56 × 10¹⁴ vg and 2.52 × 10¹⁵ empty capsids as a starting material, the optimized anion exchange chromatography method resulted in a vector peak of 1.15 × 10¹⁴ vg containing 0.25 × 10¹⁴ empty capsids, corresponding to 74% vector yield and 86-fold reduction in empty capsids in the vector product.     

Characterization of a Trypanosoma cruzi acetyltransferase: cellular location, activity and structure

Trypanosomatids are widespread parasites that cause three major tropical diseases. In trypanosomatids, as in most other organisms, acetylation is a common protein modification that is important in multiple, diverse processes. This paper describes a new member of the Trypanosoma cruzi acetyltransferase family. The gene is single copy and orthologs are also present in the other two sequenced trypanosomatids, Trypanosoma brucei and Leishmania major. This protein (TcAT-1) has the essential motifs present in members of the GCN5-related acetyltransferase (GNAT) family, as well as an additional motif also found in some enzymes from plant and animal species. The protein is evolutionarily more closely related to this group of enzymes than to histone acetyltransferases. The native protein has a cytosolic cellular location and is present in all three life-cycle stages of the parasite. The recombinant protein was shown to have autoacetylation enzymatic activity.     

Components of airway hyperresponsiveness and their associations with inflammation and remodeling in mice

BACKGROUND: Pathologic changes, including inflammation and remodeling, occur in the asthmatic airway. However, their relative contribution to the components of airway hyperresponsiveness (AHR) remains unclear. OBJECTIVE: Attempting to delineate AHR into discrete immune-mediated and structural remodeling components, we performed a detailed time course of the development, progression, and persistence of maximal respiratory system resistance, airway reactivity, and airway sensitivity. METHODS: Mice exposed to increasing durations of persistent allergen were assessed for airway function, morphometry, and inflammation. RESULTS: Allergen exposure resulted in increases for all indices of AHR that persisted for at least 4 weeks after chronic allergen exposure (P < .01 for all values). Early increases in AHR were associated with increases in immune-mediated events, including airway eosinophils (P < .01), whereas sustained AHR was associated with structural remodeling events. Increased maximal respiratory system resistance, evident by 6 weeks postallergen and persisting for at least 4 weeks after 8 weeks of chronic exposure, was associated with an increase in collagen deposition (P < .01). Increased airway reactivity and sensitivity, each evident by 1 week after allergen and persisting for at least 4 weeks after 8 weeks of chronic exposure, were associated with an increase in airway smooth muscle area (P < .01). CONCLUSION: Our novel observation of distinct temporal relationships in the development, progression, and persistence of the individual indices of AHR supports our hypothesis that multiple underlying factors contribute to airway dysfunction. CLINICAL IMPLICATIONS: These findings illustrate the importance of clearly addressing specific components of airway dysfunction to provide greater insight into specific pathophysiologic mechanisms in airway disease.     

Selection of aspirin dosages for aspirin desensitization treatment in patients with aspirin-exacerbated respiratory disease

BACKGROUND: Aspirin desensitization followed by daily aspirin therapy is effective add-on treatment for patients with aspirin-exacerbated respiratory disease. Prior studies used 650 mg of aspirin twice daily, but studies at lower dosages were inconclusive. OBJECTIVE: We sought to determine the optimal daily dosage of aspirin treatment. METHODS: We studied 137 patients who had undergone successful aspirin desensitization and randomized them into 2 groups, 650 mg twice daily versus 325 mg twice daily. After 1 month, patients either increased or decreased their dosage based on their symptom control and continued that dosage for the remainder of the year. RESULTS: Patients taking either 650 mg twice daily or 325 mg twice daily showed significant improvements in number of sinus infections, sinus operations, and hospitalizations for asthma (all P < .0001). Anosmia, nasal/sinus symptoms, and asthma symptoms also improved in both groups (all P < .03). Systemic corticosteroid dosages decreased by 3- and 4-fold in the 325 mg twice daily and 650 mg twice daily groups, respectively. Of the 137 patients, 32 had adverse effects from or discontinued aspirin therapy: 14 (44%) of 32 from the group randomized to taking 650 mg twice daily and 18 (56%) of 32 from the group randomized to 325 mg twice daily. The most common adverse effect was dyspepsia. CONCLUSION: Both dosages were efficacious, and side effects occurred in both groups at similar frequencies. Some patients initially taking 325 mg twice daily required an increase to 650 mg twice daily for optimal symptom control. CLINICAL IMPLICATIONS: We recommend that patients begin daily aspirin therapy with 650 mg twice daily and subsequently decrease to the lowest effective dosage (usually 325 mg twice daily).     

Pediatric patients with eosinophilic esophagitis: an 8-year follow-up

BACKGROUND: Eosinophilic esophagitis (EE) is a gastrointestinal disorder that is increasingly diagnosed in pediatric patients. OBJECTIVE: We aimed to define, in pediatric patients with EE, their demographic and atopic characteristics, the histopathology of all segments of the gastrointestinal tract, and the effect of therapeutic interventions on the natural history. METHODS: We conducted a retrospective analysis of a database of pediatric patients with EE followed over a period of 8 years. RESULTS: In 89 pediatric patients with EE, male sex (78.6%), white race (94.4%), young age at diagnosis, mean +/- SD, 6.2 +/- 4.8 years, and atopy with sensitization to environmental and food allergens in 79% and 75%, respectively, were prevalent. Patients had EE of the proximal and distal esophagus, and 77% had in addition either mucosal eosinophilia or noneosinophilic histopathology in the stomach, duodenum, and colon. EE was chronic, with a duration of mean +/- SD, 0.91 +/- 0.84 years, until first resolution, and was recurrent; of 66% of the patients who had resolution, 79% later relapsed. CONCLUSION: Eosinophilic esophagitis in the pediatric population is a chronic and relapsing condition, associated with atopy and sometimes with subsequent histopathology in segments of the gastrointestinal tract other than the esophagus. CLINICAL IMPLICATIONS: Physicians evaluating pediatric patients with chronic gastrointestinal symptoms should consider the diagnosis of EE, particularly in young white male patients with atopy. Once diagnosed and treated, the physicians should follow the patients over a period of several years because the course of the disease is protracted, other gastrointestinal segments may be affected, and relapses are common.     

Comparative genomics of Canadian epidemic lineages of methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogen that has disseminated throughout Canadian hospitals and communities. Pulsed-field gel electrophoresis of over 9,300 MRSA isolates obtained from the Canadian Nosocomial Infection Surveillance Program has identified 10 epidemic strain types in Canada (CMRSA1 to CMRSA10). In an attempt to determine specific genetic factors that have contributed to their high prevalence in community and/or hospital settings, the genomic content of representative isolates for each of the 10 Canadian epidemic types was compared using comparative genomic hybridizations. Comparison of the community-associated Canadian epidemic isolates (CMRSA7 and CMRSA10) with the hospital-associated Canadian epidemic isolates revealed one open reading frame (ORF) (SACOL0046) encoding a putative protein belonging to a metallo-beta-lactamase family, which was present only in the community-associated Canadian epidemic isolates. A more restricted comparison involving only the most common hospital-associated Canadian epidemic isolates (CMRSA1 and CMRSA2) with the community-associated Canadian epidemic isolates did reveal additional factors that might be contributing to their prevalence in the community and hospital settings, which included ORFs encoding potential virulence factors involved in capsular biosynthesis, serine proteases, epidermin, adhesion factors, regulatory functions, leukotoxins, and exotoxins.     

Gray foxes (Urocyon cinereoargenteus) as a potential reservoir of a Bartonella clarridgeiae-like bacterium and domestic dogs as part of a sentinel system for surveillance of zoonotic arthropod-borne pathogens in northern California

Two species of Bartonella, a novel Bartonella clarridgeiae-like bacterium and B. vinsonii subsp. berkhoffii, were isolated from rural dogs and gray foxes in northern California. A novel B. clarridgeiae-like species was isolated from 3 (1.7%) of 182 dogs and 22 (42%) of 53 gray foxes, while B. vinsonii subsp. berkhoffii was isolated from 1 dog (0.5%) and 5 gray foxes (9.4%). PCR and DNA sequence analyses of the citrate synthase (gltA) gene and the 16S-23S intergenic spacer region suggested that strains infecting dogs and gray foxes were identical. Fifty-four dogs (29%) and 48 gray foxes (89%) had reciprocal titers of antibodies against Bartonella spp. of > or =64. The high prevalence of bacteremia and seroreactivity to Bartonella spp. in gray foxes suggests that they may act as a reservoir species for the B. clarridgeiae-like species in this region. Domestic dogs were also tested for other arthropod-borne infectious agents. Fifty-one dogs (28%) were positive for Dirofilaria immitis antigen, seventy-four (40%) were seroreactive to Anaplasma phagocytophilum, and five (2.7%) were seropositive for Yersinia pestis. Fourteen dogs (7.6%) were PCR positive for A. phagocytophilum. Polytomous logistic regression models were used to assess the association of Bartonella antibody titer categories with potential risk factors and the presence of other vector-borne agents in domestic dogs. Older dogs were more likely to be seroreactive to Bartonella spp. There was no association between the exposure of dogs to Bartonella and the exposure of dogs to A. phagocytophilum in this study.     

Fibronectin terminated multilayer films: protein adsorption and cell attachment studies

Electrostatically driven layer-by-layer (LbL) assembly is a simple and robust method for producing structurally tailored thin film biomaterials, of thickness ca. 10nm, containing biofunctional ligands. We investigate the LbL formation of multilayer films composed of polymers of biological origin (poly(L-lysine) (PLL) and dextran sulfate (DS)), the adsorption of fibronectin (Fn)--a matrix protein known to promote cell adhesion--onto these films, and the subsequent spreading behavior of human umbilical vein endothelial cells (HUVEC). We employ optical waveguide lightmode spectroscopy (OWLS) and quartz crystal microgravimetry with dissipation (QCMD) to characterize multilayer assembly in situ, and find adsorbed Fn mass on PLL-terminated films to exceed that on DS terminated films by 40%, correlating with the positive charge and lower degree of hydration of PLL terminated films. The extent and initial rate of Fn adsorption to both PLL and DS-terminated films exceed those onto the bare substrate, indicating the important role of electrostatic complexation between negatively charged protein and positively charged PLL at or near the film surface. We use phase-contrast optical microscopy to investigate the time-dependent morphological changes of HUVEC as a function of layer number, charge of terminal layer, and the presence of Fn. We observe HUVEC to attach, spread, and lose circularity on all surfaces. Positively charged PLL-terminated films exhibit a greater extent of cell spreading than do (negatively charged) DS-terminated films, and spreading is enhanced while circularity loss is suppressed by the presence of adsorbed Fn. The number of layers plays a significant role only for DS-terminated films with Fn, where spreading on a bilayer greatly exceeds that on a multilayer, and PLL-terminated films without Fn, where initial spreading is significantly higher on a monolayer. We observe initial cell spreading to be followed by retraction (i.e. decreased cell area and circularity with time) for films without Fn, and for DS-terminated films with Fn. Overall, the Fn-coated PLL monolayer and the Fn-coated PLL-terminated multilayer are the best performing films in promoting cell spreading. We conclude the presence of Fn to be an important factor (more so than film charge or layer number) in controlling the interaction between multilayer films and living cells, and thus to represent a promising strategy toward in vivo applications such as tissue engineering.