William L. Castleman

Enhanced pulmonary pathology associated with the use of formalin-inactivated respiratory syncytial virus vaccine in cotton rats is not a unique viral phenomenon☆

The specificity of viral antigens in the formalin-inactivated, alum-precipitated respiratory syncytial virus (FI-RSV) vaccine in augmenting the pulmonary inflammatory response was evaluated. Cotton rats were immunized with a FI-RSV vaccine derived from Vero cells, a monkey cell line, or HEp-2 cells, a human cell line. The FI-RSV/Vero and the FI-RSV/HEp-2 vaccines were prepared similarly to the original Lot-100 FI-RSV vaccine that was associated with enhanced disease in the mid-1960s field trials. Each vaccine was administered intramuscularly at various doses and intervals. At 1, 4 or 7 weeks after the last vaccine dose, cotton rats were challenged with 10(6) plaque-forming units of live RSV grown in HEp-2 cells. For controls, FI-parainfluenza, FI-HEp-2 and alum vaccines, and live RSV primary infection were used. For measuring virus replication and histopathology, lungs were harvested at 4 and 8 days postchallenge. A dose-response relationship to vaccine dose was observed for ELISA, neutralizing and antifusion antibodies. All animals given three doses or two of the higher doses of FI-RSV/Vero vaccine developed significant neutralizing antibody, were protected against pulmonary virus replication and had similar low levels of histopathology compared with live RSV and controls. Two immunizations of the lowest dose of FI-RSV/Vero vaccine did not induce neutralizing antibody, did not provide protection of the lung against RSV and did not enhance the pulmonary cellular response. However, FI-RSV/HEp-2 vaccine was associated with significant enhanced pulmonary histopathology despite inducing high titres of neutralizing antibody and protecting the lungs against RSV infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental Infection of Neonatal Foals with Rhodococcus equi Triggers Adult-Like Gamma Interferon Induction

ABSTRACT Rhodococcus equi is a facultative intracellular pathogen that causes pneumonia in young foals but does not induce disease in immunocompetent adult horses. Clearance of R. equi depends mainly on gamma interferon (IFN-γ) production by T lymphocytes, whereas the predominance of interleukin 4 (IL-4) is detrimental. Young foals, like neonates of many other species, are generally deficient in the ability to produce IFN-γ. The objective of this study was to compare the cytokine profiles, as well as cell-mediated and antibody responses, of young foals to those of adult horses following intrabronchial challenge with R. equi. The lymphoproliferative responses of bronchial lymph node (BLN) cells to concanavalin A were significantly higher in foals than in adult horses. In contrast, adult horses had significantly higher lymphoproliferative responses to R. equi antigens than did foals. Infected foals had significantly lower IL-4 mRNA expression but significantly higher IFN-γ expression and IFN-γ/IL-4 ratio in R. equi-stimulated BLN lymphocytes than did infected adults. Infection with R. equi in foals resulted in a significant increase in the percentage of T lymphocytes and CD4+ T lymphocytes in bronchoalveolar lavage fluid in association with a significant decrease in the percentage of these cell populations in BLNs. Infection of foals also resulted in a marked increase in serum immunoglobulin Ga (IgGa) and IgGb levels, resulting in concentrations in serum that were significantly higher than those of adult horses. This study demonstrates that the immune response to R. equi in foals is not biased toward IL-4 and is characterized by the predominant induction of IFN-γ.

Lack of detectable enhanced pulmonary histopathology in cotton rats immunized with purified F glycoprotein of respiratory syncytial virus (RSV) when challenged at 3–6 months after immunization

The cotton rat model has been used to evaluate the potential for immunogens to induce respiratory syncytial virus (RSV)-enhanced pulmonary histopathology. A recent study evaluated purified F protein in this model when animals were challenged intranasally with RSV 3 or 6 months after immunization. The authors concluded that the purified F protein was associated with the same level of histopathological changes as observed with the positive control, a formalin-inactivated RSV immunogen. Three pathologists have independently evaluated the lung sections from the animals of this study and the results are reported in this article. In contrast to the previously published data, we have found that F protein was associated with a substantially milder and qualitatively different response to that observed with the formalin-inactivated RSV vaccine. We concluded that the minimal histological changes observed and lack of clinical disease make it very difficult to assess the issue of enhanced pulmonary RSV disease with the cotton rat model.

Hyperinsulinemic Hypoglycemia Syndrome in 2 Dogs with Bartonellosis

A 6-month-old male castrated, 21-kg Weimaraner developed seizures after surgical castration and umbilical hernia repair. Two weeks earlier, presurgical laboratory abnormalities included lymphocytosis (7,800/lL), eosinophilia (1,700/lL), monocytosis (1,200/lL), basophilia (170/lL), thrombocytosis (459,000/lL), and an elevated ALP activity (135 IU/ L). Blood glucose was 111 mg/dL. Surgery and recovery were uncomplicated, until 2 hours postoperatively when generalized tonic clonic seizures developed. Seizures continued despite 2 doses of diazepam (1.5 mL IV), so propofol (10 mg) was administered IV. Hypoglycemia was not suspected and blood glucose was not measured. The dog was transported to an emergency clinic, where laboratory abnormalities included neutrophilia (16,210/lL) and hyperphosphatemia (7.7 mg/ dL), with a normal blood glucose concentration (106 mg/dL). When transferred to a specialty hospital the following morning, the dog was dysphoric, nonambulatory, and had absent menace reflexes bilaterally. No other cranial nerve deficits or anisocoria were noted. Serum chemistry and bile acid values before (1.3 lmol/L) and after feeding (1.8 lmol/L) were within reference intervals. After intravenous administration of mannitol and phenobarbital, no additional seizures were observed, but the dog remained stuporous, with absent menace reflexes until discharged 2 days later. Phenobarbital was continued (2 mg/kg PO q12). The following morning, the dog was found salivating, minimally responsive, and recumbent. Laboratory abnormalities included hypoglycemia (glucose 25 mg/ dL), neutrophilia (12,030/lL), and monocytosis (3,290/ lL). A blood ammonia concentration (30 lmol/L) was within reference intervals. Enrofloxacin (5 mg/kg IV) and a 5% dextrose infusion were administered. The dog was fed every 2 hours and periodically determined glucose values ranged from 40 to 75 mg/dL. After transfer to a specialty hospital, hypoglycemia (59, 48, 22, 41, 23, and 40 mg/dL at 10:00 PM, 1:00, 3:00, 4:00, 5:00, and 6:00 AM, respectively) persisted, despite frequent small feedings and continuous administration of 10% dextrose solution with periodic boluses. Dexamethasone sodium phosphate (0.15 mg/kg IV) was administered, after which the dog was referred to NCSU-CVM-VTH for further evaluation. Historically purchased from a breeder in Virginia, the dog had been healthy before surgery 6 days earlier. Littermates were reportedly healthy, vaccinations current, heartworm, flea, and tick preventive medications were used routinely. The owner denied exposure of the dog to toxins such as xylitol or oral hypoglycemic drugs. At NCSU-CVM-VTH, the dog was laterally recumbent, pupils fixed, menace reflexes absent bilaterally, and blood glucose was 20 mg/dL. Hematologic abnormalities included mild microcytic, normochromic, nonregenerative anemia (PCV 32%, reticulocytes 0.44%), and monocytosis (2,129/lL). Normoglycemia (129 mg/dL) was documented after an intravenous bolus of dextrose. Serum biochemical abnormalities included hypoalbuminemia (2.4 g/dL), low SUN (4 mg/dL) and creatinine (0.3 mg/dL), hypomagnesemia (1.7 mg/dL) and hypokalemia (3.7 mmol/L), all potentially related to fluid administration, and hemodilution. Blood ammonia (11 lmol/L) and coagulation measurements were normal, except for a mildly From the Intracellular Pathogens Research Laboratory, Center for Comparative Medicine and Translational Research, College of Veterinary Medicine, North Carolina State University, Raleigh, NC (Breitschwerdt, Mascarelli); the University of Florida College of Veterinary Medicine, Gainesville, FL (Goldkamp, Castleman, Schaer); and the Veterinary Health Complex, College of Veterinary Medicine, North Carolina State University, Raleigh, NC (Cullen, Thalhem). Corresponding author: Dr E.B. Breitschwerdt, College of Veterinary Medicine, North Carolina State University, 1060 William Moore Drive, Raleigh, NC 27607; e-mail: ed_breitschwerdt@ncsu. edu. Submitted September 9, 2013; Revised January 23, 2014; Accepted April 22, 2014. Copyright

A comparison of two methods of endoscopic dilation of acute subglottic stenosis using a ferret model

Balloon dilation is accepted as a first line treatment of acute subglottic stenosis, but its effects on the subglottic tissue remain largely unknown. We aimed to develop an animal model of acute subglottic stenosis using endoscopic techniques. Once developed, this model was used to compare the immediate effects of balloon dilation and endotracheal tube dilation on subglottic tissue.

Comparative evaluation of differential laser-induced perturbation spectroscopy as a technique to discriminate emerging skin pathology

Fluorescence spectroscopy has been widely investigated as a technique for identifying pathological tissue; however, unrelated subject-to-subject variations in spectra complicate data analysis and interpretation. We describe and evaluate a new biosensing technique, differential laser-induced perturbation spectroscopy (DLIPS), based on deep ultraviolet (UV) photochemical perturbation in combination with difference spectroscopy. This technique combines sequential fluorescence probing (pre- and post-perturbation) with sub-ablative UV perturbation and difference spectroscopy to provide a new spectral dimension, facilitating two improvements over fluorescence spectroscopy. First, the differential technique eliminates significant variations in absolute fluorescence response within subject populations. Second, UV perturbations alter the extracellular matrix (ECM), directly coupling the DLIPS response to the biological structure. Improved biosensing with DLIPS is demonstrated in vivo in a murine model of chemically induced skin lesion development. Component loading analysis of the data indicates that the DLIPS technique couples to structural proteins in the ECM. Analysis of variance shows that DLIPS has a significant response to emerging pathology as opposed to other population differences. An optimal likelihood ratio classifier for the DLIPS dataset shows that this technique holds promise for improved diagnosis of epithelial pathology. Results further indicate that DLIPS may improve diagnosis of tissue by augmenting fluorescence spectra (i.e. orthogonal sensing).

Ionic conjugates of lidocaine and sweeteners as better tasting local anesthetics for dentistry

Lidocaine is the most widely utilized intraoral injected dental anesthetic, used for more than 500 million dental injections per year. Local anesthesia is essential for pain-free dentistry, yet intraoral injections are often considered painful and a source of anxiety for many patients. Any new anesthetics that will reduce the stress and anxiety of dental injection are expected to be beneficial. A novel chemical approach to taste modulation is proposed, in which the lidocaine cation is coupled with anionic sweeteners such as saccarinate and acesulfamate. The ionic conjugates synthesized using anion exchange techniques, were much less bitter, demonstrated a high local anesthetic potential in animal studies, and were as safe as the original hydrochloride. Based on the currently robust market for lidocaine it is expected that the resulting anesthetics will be in high demand in clinical practices worldwide.

Effects of 4-ipomeanol on bovine alveolar macrophage function

The objective of this study was to determine whether 4-ipomeanol toxicosis in calves impairs alveolar macrophage functions important in pulmonary defense against infectious agents. Male Holstein calves were given either 4-ipomeanol (3 mg kg-1, i.v.) or vehicle (polyethylene glycol 400). Alveolar macrophages were recovered by pulmonary lavage 3 days later, and their capacities to phagocytose and kill E. coli, migrate toward zymosan-activated immune bovine serum, and produce interferon and interleukin-1 activity were evaluated in vitro. Alveolar macrophages recovered from 4-ipomeanol-treated calves had over a 70% decrease (p < 0.01) in chemotactic activity and over a 37% decrease (p < 0.005) in their capacity to phagocytose E. coli as compared to macrophages from control calves. Interleukin-1 activity in macrophages from 4-ipomeanol-treated calves tended to be higher than that from control calves, but the differences were not statistically significant (p = 0.06). 4-ipomeanol did not affect macrophage bactericidal activity or production of interferon. These results indicate that 4-ipomeanol suppresses select functions of alveolar macrophages in cattle that may be important in pulmonary defense against bacterial pathogens.