Increasing the Potency of an Alhydrogel-Formulated Anthrax Vaccine by Minimizing Antigen-Adjuvant Interactions

Aluminum salts are the most widely used vaccine adjuvants, and phosphate is known to modulate antigen-adjuvant interactions. Here we report an unexpected role for phosphate buffer in an anthrax vaccine (SparVax) containing recombinant protective antigen (rPA) and aluminum oxyhydroxide (AlOH) adjuvant (Alhydrogel). Phosphate ions bind to AlOH to produce an aluminum phosphate surface with a reduced rPA adsorption coefficient and binding capacity. However, these effects continued to increase as the free phosphate concentration increased, and the binding of rPA changed from endothermic to exothermic. Crucially, phosphate restored the thermostability of bound rPA so that it resembled the soluble form, even though it remained tightly bound to the surface. Batches of vaccine with either 0.25 mM (subsaturated) or 4 mM (saturated) phosphate were tested in a disease model at batch release, which showed that the latter was significantly more potent. Both formulations retained their potency for 3 years. The strongest aluminum adjuvant effects are thus likely to be via weakly attached or easily released native-state antigen proteins.     

Seroprevalence and geographic distribution of Dirofilaria immitis and tick-borne infections (Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, and Ehrlichia canis) in dogs from Romania

Tick-borne diseases are of great concern worldwide. Despite this, in Romania there is only limited information regarding the prevalence of vector-borne pathogens in dogs. In all, 1146 serum samples were tested by SNAP(?) 4Dx(?) (IDEXX Laboratories, Inc., Westbrook, ME) for Anaplasma phagocytophilum, Borrelia burgdorferi, and Ehrlichia canis antibodies, and for Dirofilaria immitis antigen. The correlation between positive cases and their geographic distribution, as well as potential risk factors (age, sex, breed, type of dog, habitat, and prophylactic treatments) were evaluated. Overall, 129 dogs (11.3%) were serologically-positive to one or more of the tested pathogens. The seroprevalence for the four infectious agents were: A. phagocytophilum 5.5% (63/1146), D. immitis 3.3% (38/1146), E. canis 2.1% (24/1146), and B. burgdorferi 0.5% (6/1146). Co-infection with E. canis and A. phagocytophilum was registered in 2 dogs (0.2%). The geographical distribution of the seropositive cases suggests clustered foci in southern regions and in the western part of the country for D. immitis, and in the southeastern region (Constan?a County) for E. canis. A. phagocytophilum and B. burgdorferi showed a homogenous distribution, with a tendency for Lyme-positive samples to concentrate in central Romania. For D. immitis, A. phagocytophilum, and E. canis, administering prophylactic treatments was a risk factor associated with infection. Another associated risk factor was the type of dog (stray dogs were at risk being positive for D. immitis, shelter dogs for E. canis, and hunting dogs for B. burgdorferi). The prevalence of D. immitis was significantly higher in males and in dogs older than 2 years. This survey represents the first data detailing A. phagocytophilum and E. canis seroprevalence in Romanian dogs, and the most comprehensive epidemiological study on vector-borne infections in dogs from this country.     

Sclerostin antibody stimulates bone regeneration after experimental periodontitis

The reconstruction of large osseous defects due to periodontitis is a challenge in regenerative therapy. Sclerostin, secreted by osteocytes, is a key physiological inhibitor of osteogenesis. Pharmacologic inhibition of sclerostin using sclerostin‐neutralizing monoclonal antibody (Scl‐Ab) thus increases bone formation, bone mass and bone strength in models of osteopenia and fracture repair. This study assessed the therapeutic potential of Scl‐Ab to stimulate alveolar bone regeneration following experimental periodontitis (EP). Ligature‐induced EP was induced in rats to generate localized alveolar bone defects. Following 4 weeks of disease induction, Scl‐Ab (+EP) or vehicle (+/? EP) were systemically delivered, twice weekly for up to 6 wks to determine the ability of Scl‐Ab to regenerate bone around tooth‐supporting osseous defects. 3 and 6 wks after the initiation of Scl‐Ab or vehicle treatment, femur and maxillary jawbones were harvested for histology, histomorphometry, and micro‐computed tomography (micro‐CT) of linear alveolar bone loss (ABL) and volumetric measures of bone support, including bone volume fraction (BVF) and tissue mineral density (TMD). Serum was analyzed to examine bone turnover markers during disease and regenerative therapy. Vehicle + EP animals exhibited maxillary bone loss (BVF, TMD and ABL) at ligature removal and thereafter. 6 weeks of Scl‐Ab significantly improved maxillary bone healing, as measured by BVF, TMD and ABL, when compared to vehicle + EP. After 6 weeks of treatment, BVF and TMD values in the Scl‐Ab + EP group were similar to those of healthy controls. Serum analysis demonstrated higher levels of bone formation markers osteocalcin and PINP in Scl‐Ab treatment groups. Scl‐Ab restored alveolar bone mass following experimental periodontitis. These findings warrant further exploration of Scl‐Ab therapy in this and other oral bone defect disease scenarios. © 2013 American Society for Bone and Mineral Research.     

Bile Acid at Low pH Reduces Squamous Differentiation and Activates EGFR Signaling in Esophageal Squamous Cells in 3-D Culture

Background

Barrett's esophagus is a preneoplastic metaplasia in which the normal squamous epithelium of the esophagus changes to an intestinal, columnar phenotype due to long-term gastro-esophageal reflux. The major components of this reflux are bile and stomach acid. Previous in vitro studies on the effect of bile and acid on esophageal cells have predominantly relied on transformed esophageal squamous cells or cancer cells grown in monolayer culture.

Discussion

In this study, we expanded our previous work using an immortalized primary esophageal squamous cell line (EPC1). We demonstrate that EPC1 cells form a multi-layer, stratified epithelium when grown on polyester transwell filters in media supplemented with calcium. When exposed to short pulses of bile and pH 5, but not either condition alone, EPC1 cells demonstrate a reduction in stratification layers and reduced expression of squamous epithelium-specific genes. Bile at pH 5 also causes activation of epidermal growth factor receptor and down-stream pathways. Blocking epidermal growth factor receptor activation partially attenuates the effects of bile acid and pH 5. These results suggest that bile at low pH, but not bile or low pH alone, promotes loss of differentiation status of stratified squamous esophageal epithelium in vitro, possibly by initiating a mucosal repair response through epidermal growth factor activation.     

Systemic Naloxone Infusion May Trigger Spasticity in Patients With Spinal Cord Injury: Case Series

Abstract

Background/Objective: Three patients with spinal cord injury (SCI) and 3 able-bodied (AB) patients were infused with naloxone during a study to examine their neuroendocrine function. An unanticipated side effect occurred during the naloxone infusion. All 3 patients with SCI, but none of the AB patients, experienced profoundly increased spasticity during the naloxone infusion. Our report describes this side effect, which has potential implications for the clinical treatment or scientific evaluation of individuals with SCI.

Methods: All patients were in good general health and medication free for 11 days or longer before the study. Each patient was placed on a 30-hour protocol to analyze pulsatile release of gonadotropins. Physiologic saline was intravenously infused on day 1 to serve as a control period for naloxone infusion on day 2.

Results: AB patients experienced no muscle spasm activity or any other side effects at any time during the study. In contrast, all 3 patients with SCI experienced a profoundly increased frequency and duration of spasticity in muscles innervated by the nerve roots caudal to their level of injury. In all 3 patients with SCI, spasticity increased only during the period of naloxone infusion. Within 1 hour of stopping naloxone, spasticity returned to baseline levels.

Conclusions: Naloxone infusion produced a differential effect on the muscle activity of men with SCI compared to AB men with intact spinal circuits. Consistent with previous studies, the results of this study indicate a relationship between opioid neuromodulation and spasticity after SCI.     

Probing the supramolecular features via π–π interaction of a di-iminopyrene-di-benzo-18-crown-6-ether compound: experimental and theoretical study

A novel DPyDB-C N-18C6 compound was synthesised by linking a pyrene moiety to each phenyl group of dibenzo-18-crown-6-ether, the crown ether, through –HC N– bonds and characterized by FTIR, ¹H-NMR, ¹³C-NMR, TGA, and DSC techniques. The quantitative ¹³C-NMR analysis revealed the presence of two position isomers. The electronic structure of the DPyDB-C N-18C6 molecule was characterized by UV-vis and fluorescence spectroscopies in four solvents with different polarities to observe particular behavior of isomers, as well as to demonstrate a possible non-bonding chemical association (such as ground- and excited-state associations, namely, to probe if there were forming dimers/excimers). The interpretation of the electronic structure was realized through QM calculations. The TD-CAM-B3LYP functional, at the 6-311+G(d,p) basis set, indicated the presence of predominant π → π* and mixed π → π* + n → π* transitions, in line with the UV-vis experimental data. Even though DPyDB-C N-18C6 computational studies revealed a π-extended conjugation effect with predominantly π → π* transitions, thorough fluorescence analysis was observed a weak emission, as an effect of PET and ACQ. In particular, the WAXD analysis of powder and thin films obtained from n-hexane, 1,2-dichloroethane, and ethanol indicated an amorphous organization, whereas from toluene a smectic ordering was obtained. These results were correlated with MD simulation, and it was observed that the molecular geometry of DPyDB-C N-18C6 molecule played a defining role in the pyrene stacking arrangement.

Herein, we report the formation of a potential supramolecular arrangement mediated by inter- and intra-molecular interactions between di-iminopyrene-dibenzo-18-crown-6-ether molecules.