Tatjana Lazic

Adenovirus-Mediated Gene Therapy Enhances Parainfluenza Virus 3 Infection in Neonatal Lambs

ABSTRACT Parainfluenza viruses are a common cause of seasonal respiratory disease, but in high-risk individuals (e.g., young children) these viruses can cause severe clinical manifestations that require hospitalization. Beta-defensins are a subclass of antimicrobial peptides with antiviral activity. Use of adenovirus-mediated beta-defensin gene expression has been proposed as therapy for chronic bacterial infections commonly seen in cystic fibrosis patients; however, its use during parainfluenza virus 3 (PIV3) infection has not been evaluated. The hypothesis in this experiment was that adenovirus expression of human beta-defensin 6 (HBD6) would diminish concurrent PIV3 infection in neonatal lambs. The group infected with adenovirus HBD6 and PIV3 had increased levels of pulmonary neutrophil recruitment compared to those for the group infected with PIV3 or PIV3 and adenovirus, with an increased respiration rate and body temperature late in the course of the PIV3-adenovirus HBD6 infection. Interestingly, the adenovirus-treated groups had higher levels of immunohistochemical staining for PIV3 and syncytial cell formation than the group infected with PIV3, suggesting that treatment with the adenovirus vector, regardless of whether it was carrying a target gene, exacerbated the PIV3 infection. The levels of expression of mRNA for antimicrobial surfactant proteins A and D and sheep beta-defensin 1 were increased by PIV3 and adenovirus treatment, and the increased levels of expression roughly corresponded to the degree of inflammation. While pulmonary administration of a high-dose adenovirus vector has been associated with undesirable inflammation, this is the first study to show that it can exacerbate concurrent viral infection, a concern that needs to be addressed for future studies of adenovirus in the lung. Additionally, this study showed that adenovirus-mediated HBD6 expression increases neutrophil recruitment, a recently described attribute of beta-defensins, with mild accentuation of PIV3 activity and inflammation.

Exposure to ethanol during the last trimester of pregnancy alters the maturation and immunity of the fetal lung.

The effects of ethanol exposure on fetal lungs remain under investigation. Previously, we demonstrated that lambs exposed to ethanol during gestation had impaired expression of pulmonary surfactant protein A, a crucial component of lung immunity. In this study, we investigated the effects of in utero exposure to ethanol on maturation and immunity of the fetal lung. Pregnant ewes were surgically implanted with an abomasal cannula and administered 1g ethanol/kg (n=8) or water (n=8) during the last trimester of pregnancy. Lambs were delivered prematurely or naturally. Neonatal lungs were assessed for maturation markers (hypoxia-inducible factor-1α [HIF-1α], HIF-2α, HIF-3α, vascular endothelial growth factor-A [VEGF-A], VEGFR-1, VEGFR-2, glycogen, and lung protein levels) and immunity (cytokines and chemokines). Preterm animals exposed to ethanol had significantly reduced VEGF-A mRNA (P=.066) and protein levels, HIF-1α (P=.055), HIF-2α (P=.019), VEGFR-1 (P=.088), and VEGFR-2 (P=.067) mRNA levels but no changes in HIF-3α mRNA. No significant changes occurred in full-term animals exposed to ethanol. Glycogen levels were significantly higher in preterm animals exposed to ethanol (P=.006) but not in full-term animals. Ethanol exposure was associated with significantly lower lung protein levels in preterm (P=.03) but not full-term animals. Preterm animals exposed to ethanol had significantly reduced TNF-α (P=.05), IL-10 (P=.03), chemokine (C-C motif) ligand 5 (CCL5) (P=.017), and monocyte chemotactic protein-1 (MCP-1) (P=.0004) mRNA. In full-term animals exposed to ethanol, the immune alterations were either sustained (TNF-α, P=.009; IL-10, P=.03) or returned to near baseline levels (CCL5 and MCP-1). The ethanol-mediated alterations in fetal lung maturation and immunity may explain the increased incidence of respiratory infections in neonates exposed to ethanol in utero.

Monocytic/Macrophagic Pneumonitis after Intrabronchial Deposition of Vascular Endothelial Growth Factor in Neonatal Lambs

Preterm and young neonates are prone to inadequate surfactant production and are susceptible to respiratory distress syndrome characterized by alveolar damage and hyaline-membrane formation. Glucocorticoid therapy is commonly used in preterm and young infants to enhance lung maturation and surfactant synthesis. Recently, vascular endothelial growth factor (VEGF) was suggested to be a novel therapeutic agent for lung maturation that lacked adverse effects in mice. The purpose of this study was to assess the safety of incremental concentration (0.0005, 0.005, and 0.05 mg/ml) and duration (16, 24, and 32 hours) of recombinant human VEGF after bronchoscopic instillation (10 ml) in neonatal lambs. High-dose VEGF caused locally extensive plum-red consolidation that was microscopically characterized by interstitial and alveolar infiltrates of cells that were morphologically and phenotypically (CD68+) consistent with monocytes/macrophages. T cells (CD3+) and B cells (CD79+) were located primarily in bronchus/bronchiole-associated lymphoid tissue and were not consistently altered by treatment with VEGF. The dose of VEGF had significant effects on both gross lesions (P < .0047) and microscopic monocyte/macrophage recruitment scores (P < .0001). Thus, the VEGF dose instilled into the lung greatly influenced cellular recruitment and lesion development. The post-dosing interval of VEGF in this study had minor impact (no statistical significance) on cellular recruitment. This study showed that airway deposition of VEGF in the neonatal lamb induces monocyte/macrophage recruitment to the lung and high doses can cause severe lesions. The cellular recruitment suggests further research is needed to define dosages that are efficacious in enhancing lung maturation while minimizing potential adverse effects.

Effects of nicotine on pulmonary surfactant proteins A and D in ovine lung epithelia.

Maternal smoking during pregnancy increases the incidence and severity of respiratory infections in neonates. Surfactant proteins A and D (SP‐A and SP‐D, respectively) are components of pulmonary innate immunity and have an important role in defense against inhaled pathogens. The purpose of this study was to determine if nicotine exposure during the third trimester of pregnancy alters the expression of SP‐A and SP‐D of fetal lung epithelia. Pregnant ewes were assigned to four groups; a nicotine‐exposed full‐term and pre‐term group, and control full‐term and pre‐term group. Lung tissue was collected for Western blot and IHC analysis of SP‐A level, Western blot analysis of SP‐D level and qPCR analysis of SP‐A and SP‐D mRNA expression. Exposure to nicotine significantly decreased SP‐A gene expression (P = 0.01) and SP‐A protein level in pre‐term lambs. This finding suggests that maternal nicotine exposure during the last trimester of pregnancy alters a key component of lung innate immunity in offspring. Pediatr Pulmonol. 2010; 45:255–262.

Regulation of surfactant protein and defensin mRNA expression in cultured ovine type II pneumocytes by all-trans retinoic acid and VEGF

Beta‐defensins and surfactant proteins are components of the pulmonary innate immune system. Their gene expression is regulated by development, hormones, growth and immunoregulatory factors. It was our hypothesis that growth and differentiation factors such as all‐trans retinoic acid (RA) and vascular endothelial growth factor (VEGF) may affect expression of selected innate immune genes by respiratory epithelial cells. Ovine JS7 cells (alveolar type II pneumocytes) were incubated in serum‐free Dulbeccos modified Eagles medium (DMEM) complete media that contained: no treatment (negative control), RA (500 nM), or VEGF (100 ng/ml) for 6, 12 or 24 h incubation. Total RNA was isolated, cDNA synthesized, and relative mRNA levels of surfactant protein A (SP‐A) and SP‐D, and sheep beta‐defensin‐1 (SBD‐1) were determined by real‐time reverse transcriptase‐polymerase chain reaction (RT‐PCR). Cells had significantly increased expression of SP‐D mRNA at 6 h and 24 h, decreased expression of SP‐A mRNA at 12 h, and unchanged levels of SBD‐1 mRNA after the treatment with RA compared with their respective negative controls. VEGF did not alter the expression of the three innate immune genes. These findings suggest that SP‐A and SP‐D have different transcription regulation pathways, and that expression of SBD‐1 is not inducible by RA similar to its human homolog HBD‐1. The lack of changes induced by VEGF treatment suggests that VEGF does not have a direct effect on epithelial cells, but may affect gene expression indirectly.

Characterization of structure and function of the mouse retina using pattern electroretinography, pupil light reflex, and optical coherence tomography

OBJECTIVE To perform in vivo analysis of retinal functional and structural parameters in healthy mouse eyes. ANIMAL STUDIED Adult C57BL/6 male mice (n = 37). PROCEDURES Retinal function was evaluated using pattern electroretinography (pERG) and the chromatic pupil light reflex (cPLR). Structural properties of the retina and nerve fiber layer (NFL) were evaluated using spectral-domain optical coherence tomography (SD-OCT). RESULTS The average pERG amplitudes were found to be 11.2 ± 0.7 μV (P50-N95, mean ± SEM), with an implicit time for P50-N95 interval of 90.4 ± 5.4 ms. Total retinal thickness was 229.5 ± 1.7 μm (mean ± SEM) in the area centralis region. The thickness of the retinal nerve fiber layer (mean ± SEM) using a circular peripapillary retinal scan centered on the optic nerve was 46.7 ± 0.9 μm (temporal), 46.1 ± 0.9 μm (superior), 45.8 ± 0.9 μm (nasal), and 48.4 ± 1 μm (inferior). The baseline pupil diameter was 2.1 ± 0.05 mm in darkness, and 1.1 ± 0.05 and 0.56 ± 0.03 mm after stimulation with red (630 nm, luminance 200 kcd/m(2)) or blue (480 nm, luminance 200 kcd/m(2)) light illumination, respectively. CONCLUSIONS Pattern electroretinography, cPLR and SD-OCT analysis are reproducible techniques, which can provide important information about retinal and optic nerve function and structure in mice.

Rapid diagnosis of retina and optic nerve abnormalities in canine patients with and without cataracts using chromatic pupil light reflex testing

OBJECTIVE To develop fast and reliable testing routines for diagnosing retina and optic nerve diseases in canine cataract patients based on chromatic properties of the pupillary light reflex response. PROCEDURES Seventy-seven canine patients with a history of cataract and decreased vision (43 patients with cataracts and no evidence of retina or optic nerve disease, 21 patients with cataracts and retinal degeneration [RD], 13 patients with cataracts and retinal detachment [RDT]), 11 canine patients with optic neuritis (ON) and 23 healthy dogs were examined using chromatic pupillary light reflex (cPLR) analysis with red and blue light and electroretinography. RESULTS Electroretinography analysis showed statistically significant deficits in a- and b-wave amplitudes in dogs with cataracts and RD, or cataracts and RDT, when compared to dogs with cataracts without evidence of retinal abnormalities. Evaluation of b-wave amplitudes showed that presence of 78.5-μV (or lower) amplitudes had high sensitivity of 100% (95% CI: 87.2-100%) and high specificity of 96.7% (95% CI: 88.4-100%) in RD and RDT. Evaluation of cPLR responses using red light showed that presence of the pupil end constriction diameter of 5.5 mm (or higher) had moderately high sensitivity of 76.5% (95% CI: 50.1-93.2%) and high specificity of 100% (95% CI: 91.2-100%) in detecting RD and RDT. Optic neuritis patients had absent cPLR responses, regardless of the visual status. CONCLUSIONS AND CLINICAL RELEVANCE Chromatic evaluation of the pupillary light reflex is a rapid and accurate test for diagnosing retina and optic nerve diseases in canine patients.

Exploring Raman spectroscopy for the evaluation of glaucomatous retinal changes

Glaucoma is a chronic neurodegenerative disease characterized by apoptosis of retinal ganglion cells and subsequent loss of visual function. Early detection of glaucoma is critical for the prevention of permanent structural damage and irreversible vision loss. Raman spectroscopy is a technique that provides rapid biochemical characterization of tissues in a nondestructive and noninvasive fashion. In this study, we explored the potential of using Raman spectroscopy for detection of glaucomatous changes in vitro. Raman spectroscopic imaging was conducted on retinal tissues of dogs with hereditary glaucoma and healthy control dogs. The Raman spectra were subjected to multivariate discriminant analysis with a support vector machine algorithm, and a classification model was developed to differentiate disease tissues versus healthy tissues. Spectroscopic analysis of 105 retinal ganglion cells (RGCs) from glaucomatous dogs and 267 RGCs from healthy dogs revealed spectroscopic markers that differentiated glaucomatous specimens from healthy controls. Furthermore, the multivariate discriminant model differentiated healthy samples and glaucomatous samples with good accuracy [healthy 89.5% and glaucomatous 97.6% for the same breed (Basset Hounds); and healthy 85.0% and glaucomatous 85.5% for different breeds (Beagles versus Basset Hounds)]. Raman spectroscopic screening can be used for in vitro detection of glaucomatous changes in retinal tissue with a high specificity.

Respiratory herpesvirus infection in two Indian Ringneck parakeets

A flock of Indian Ringneck parakeets (Psittacula krameri manillensis) was imported to the United States from Australia. Soon after, 1 parakeet suddenly died, and a second parakeet died after a 2-day course of illness, which consisted of anorexia, lethargy, emaciation, and dyspnea. At necropsy, the affected birds had diffuse consolidation and red discoloration of the lungs, as well as thickened, congested air sacs. The microscopic examination revealed multifocal, necrotizing bronchitis, parabronchitis, and interstitial pneumonia. The lumen of the affected airways contained numerous, large syncytial cells with up to 15 nuclei. The nuclei of these syncytial cells often contained large, eosinophilic inclusion bodies, consistent with herpesvirus. The epithelium of the trachea and air sacs was hypertrophied and contained syncytial cells with intranuclear inclusion bodies similar to the bronchi. In addition, a few intranuclear inclusion bodies were also present in the epithelial cells that line the air capillaries. On ultrastructural examination, the nuclei of degenerating epithelial cells contained clusters of viral nucleocapsid proteins and unenveloped, icosahedral, viral particles that were approximately 90 nm in diameter. In addition, some epithelial cells contained clusters of enveloped viral particles approximately 105 nm in diameter, within the cytocavitary network. These lesions are characteristic of those caused by respiratory herpesvirus of parakeets.

Detection and characterization of glaucoma-like canine retinal tissues using Raman spectroscopy

Abstract. Early detection of pathological changes and progression in glaucoma and other neuroretinal diseases remains a great challenge and is critical to reduce permanent structural and functional retina and optic nerve damage. Raman spectroscopy is a sensitive technique that provides rapid biochemical characterization of tissues in a nondestructive and noninvasive fashion. In this study, spectroscopic analysis was conducted on the retinal tissues of seven beagles with acute elevation of intraocular pressure (AEIOP), six beagles with compressive optic neuropathy (CON), and five healthy beagles. Spectroscopic markers were identified associated with the different neuropathic conditions. Furthermore, the Raman spectra were subjected to multivariate discriminate analysis to classify independent tissue samples into diseased/healthy categories. The multivariate discriminant model yielded an average optimal classification accuracy of 72.6% for AEIOP and 63.4% for CON with 20 principal components being used that accounted for 87% of the total variance in the data set. A strong correlation (R2>0.92) was observed between pattern electroretinography characteristics of AEIOP dogs and Raman separation distance that measures the separation of spectra of diseased tissues from normal tissues; however, the underlining mechanism of this correlation remains to be understood. Since AEIOP mimics the pathological symptoms of acute/early-stage glaucoma, it was demonstrated that Raman spectroscopic screening has the potential to become a powerful tool for the detection and characterization of early-stage disease.