Kurt J. Williams

Equine Multinodular Pulmonary Fibrosis: A Newly Recognized Herpesvirus-Associated Fibrotic Lung Disease:

Pulmonary fibrosis and interstitial lung disease are poorly understood in horses; the causes of such conditions are rarely identified. Equine herpesvirus 5 (EHV-5) is a γ-herpesvirus of horses that has not been associated with disease in horses. Pathologic and virologic findings from 24 horses with progressive nodular fibrotic lung disease associated with EHV-5 infection are described and compared with 23 age-matched control animals. Gross lesions consisted of multiple nodules of fibrosis throughout the lungs. Histologically, there was marked interstitial fibrosis, often with preservation of an “alveolar-like” architecture, lined by cuboidal epithelial cells. The airways contained primarily neutrophils and macrophages. Rare macrophages contained large eosinophilic intranuclear viral inclusion bodies; similar inclusion bodies were also found cytologically. The inclusions were identified as herpesviral-like particles by transmission electron microscopy in a single horse. In situ hybridization was used to detect EHV-5 nucleic acids within occasional macrophage nuclei. With polymerase chain reaction (PCR), the herpesviral DNA polymerase gene was detected in 19/24 (79.2%) of affected horses and 2/23 (8.7%) of the control horses. Virus genera–specific PCR was used to detect EHV-5 in all of the affected horses and none of the control horses. EHV-2 was detected in 8/24 (33.3%) of affected horses and 1/9 (11.1%) of the control horses. This disease has not been reported before, and the authors propose that based upon the characteristic gross and histologic findings, the disease be known as equine multinodular pulmonary fibrosis. Further, we propose that this newly described disease develops in association with infection by the equine γ-herpesvirus, EHV-5.

An Experimental Model of Allergic Asthma in Cats Sensitized to House Dust Mite or Bermuda Grass Allergen

Background: Animal models are used to mimic human asthma, however, not all models replicate the major characteristics of the human disease. Spontaneous development of asthma with hallmark features similar to humans has been documented to occur with relative frequency in only one animal species, the cat. We hypothesized that we could develop an experimental model of feline asthma using clinically relevant aeroallergens identified from cases of naturally developing feline asthma, and characterize immunologic, physiologic, and pathologic changes over 1 year. Methods: House dust mite (HDMA) and Bermuda grass (BGA) allergen were selected by screening 10 privately owned pet cats with spontaneous asthma using a serum allergen-specific IgE ELISA. Parenteral sensitization and aerosol challenges were used to replicate the naturally developing disease in research cats. The asthmatic phenotype was characterized using intradermal skin testing, serum allergen-specific IgE ELISA, serum and bronchoalveolar lavage fluid (BALF) IgG and IgA ELISAs, airway hyperresponsiveness testing, BALF cytology, cytokine profiles using TaqMan PCR, and histopathologic evaluation. Results: Sensitization with HDMA or BGA in cats led to allergen-specific IgE production, allergen-specific serum and BALF IgG and IgA production, airway hyperreactivity, airway eosinophilia, an acute T helper 2 cytokine profile in peripheral blood mononuclear cells and BALF cells, and histologic evidence of airway remodeling. Conclusions: Using clinically relevant aeroallergens to sensitize and challenge the cat provides an additional animal model to study the immunopathophysiologic mechanisms of allergic asthma. Chronic exposure to allergen in the cat leads to a variety of immunologic, physiologic, and pathologic changes that mimic the features seen in human asthma.

Multinodular pulmonary fibrosis in five horses

CASE DESCRIPTION 5 horses were evaluated because of decreased appetite, weight loss, fever, cough, tachypnea, and respiratory distress. CLINICAL FINDINGS Tachycardia, tachypnea, increased respiratory effort, lethargy, fever, poor body condition, and nasal discharge were detected in various combinations on initial physical examination. Evaluation of the lower portion of the respiratory tract via radiography and ultrasonography revealed a severe nodular interstitial pattern. Histologic examination of lung tissue revealed interstitial expansion of alveolar parenchyma with collagen, intraluminal accumulation of neutrophils and macrophages within the alveoli, and occasional intranuclear inclusion bodies within alveolar macrophages. Equine herpesvirus type 5 was detected in samples of lung tissue, bronchoalveolar lavage fluid, or both via polymerase chain reaction assay in all cases. A diagnosis of equine multinodular pulmonary fibrosis (EMPF) was established. TREATMENT AND OUTCOME Horses were provided supportive treatment and were administered a variety of medications including corticosteroids and acyclovir. Two horses survived and returned to their previous level of activity. Three horses were euthanized because of either deterioration of clinical condition (n=2) or failure to improve within 4 weeks of initiation of treatment (1). CLINICAL RELEVANCE EMPF should be considered as a differential diagnosis for adult horses with interstitial pneumonia and should be suspected on the basis of characteristic radiographic, ultrasonographic, and histopathologic findings. Equine herpesvirus type 5 is found in association with EMPF; although the exact pathogenic role this virus plays in EMPF is unknown, equine herpesvirus type 5 may be an etiologic agent or cofactor in the development of EMPF.

Regional Pulmonary Veno-occlusion: A Newly Identified Lesion of Equine Exercise-induced Pulmonary Hemorrhage

Exercise-induced pulmonary hemorrhage (EIPH) is common in horses following intense exertion, occurring in up to 75% of racing Thoroughbreds and Standardbreds. In spite of this, the pathogenesis of EIPH is poorly understood. In 7 racing Thoroughbred horses with EIPH, 6 sections were collected from the left and right lung, representing the cranial, middle, and caudal region of the dorsal and ventral lung (84 sites total). Grossly, both right and left lungs had numerous dark brown to blue-black foci along the caudodorsal visceral pleura. Tissue sections were stained with hematoxylineosin, Massons trichrome, and Prussian blue. Verhoeff Van Gieson and immunohistochemistry for α-smooth muscle actin were used to assess the pulmonary vasculature. Histologic scores (HS = 0–3) were assigned to each region/slide for the presence and severity of 5 findings: interstitial fibrosis, hemosiderin accumulation, pleural/interlobular septal thickness, arterial and venous wall thickness, and evidence of angiogenesis (maximum cumulative HS = 15). Thirty-nine of the 84 (46%) sections were histologically normal (HS = 0); 33/84 (39%) were mildly to moderately affected, with small amounts of hemosiderin and fibrosis (HS = 1–9) while 12/84 (14%), primarily from the dorsocaudal lung, had severe vascular remodeling, fibrosis, and hemosiderin accumulation (HS = 10–15). In the latter, veno-occlusive remodeling of the intralobular veins colocalized with hemosiderosis, fibrosis, hypertrophy of vessels within the pleura, and interlobular septa and bronchial neovascularization. We propose that regional veno-occlusive remodeling, especially within the caudodorsal lung fields, contributes to the pathogenesis of EIPH, with the venous remodeling leading to regional vascular congestion and hemorrhage, hemosiderin accumulation, fibrosis, and bronchial angiogenesis.

Modulation of Airway Responses to Influenza A/PR/8/34 by Δ9-Tetrahydrocannabinol in C57BL/6 Mice

Δ9-Tetrahydrocannabinol (Δ9-THC) has been widely established as a modulator of host immune responses. Accordingly, the objective of the present study was to examine the effects of Δ9-THC on the immune response within the lungs and associated changes in the morphology of the bronchiolar epithelium after one challenge with a nonlethal dose of the influenza virus A/PR/8 (PR8). C57BL/6 mice were treated by oral gavage with Δ9-THC and/or vehicle (corn oil) for 5 consecutive days. On day 3, mice were instilled intranasally with 50 plaque-forming units of PR8 and/or vehicle (saline) 4 h before Δ9-THC exposure. Mice were subsequently killed 7 and 10 days postinfection (dpi). Viral hemagglutinin 1 (H1) mRNA levels in the lungs were increased in a dose-dependent manner with Δ9-THC treatment. Enumeration of inflammatory cell types in bronchoalveolar lavage fluid showed an attenuation of macrophages and CD4+ and CD8+ T cells in Δ9-THC-treated mice compared with controls. Likewise, the magnitude of inflammation and virus-induced mucous cell metaplasia, as assessed by histopathology, was reduced in Δ9-THC-treated mice by 10 dpi. Collectively, these results suggest that Δ9-THC treatment increased viral load, as assessed by H1 mRNA levels, through a decrease in recruitment of macrophages and lymphocytes, particularly CD4+ and CD8+ T cells, to the lung.

Inhibition of estrogen-mediated uterine gene expression responses by dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exhibits antiestrogenic properties, including the inhibition of estrogen-induced uterine growth and proliferation. The inhibition of estrogen-mediated gene expression through ER/AhR cross-talk has been proposed as a plausible mechanism; however, only a limited number of inhibited responses have been investigated that are unlikely to fully account for the antiuterotrophic effects of TCDD. Therefore, the effects of TCDD on ethynyl estradiol (EE)-mediated uterine gene expression were investigated using cDNA microarrays with complementary physiological and histological phenotypic anchoring. Mice were gavaged with vehicle, 3 daily doses of 10 μg/kg EE, a single dose of 30 μg/kg TCDD, or a combination of EE plus TCDD and sacrificed after 4, 12, 24, and 72 h. TCDD cotreatment inhibited EE-induced uterine wet weight by 37, 23, and 45% at 12, 24, and 72 h, respectively. TCDD cotreatment also reduced EE-mediated stromal edema, hypertrophy, and hyperplasia and induced marked luminal epithelial cell apoptosis. A 2 × 2 factorial microarray design was used to identify EE- and TCDD-specific differential gene expression responses as well as their interactive effects. Only 133 of the 2753 EE-mediated differentially expressed genes were significantly modulated by TCDD cotreatment, indicating a gene-specific inhibitory response. The EE-mediated induction of many genes, including trefoil factor 1 and keratin 14, were inhibited by greater than 90% by TCDD. Functional annotation of inhibited responses was associated with cell proliferation, water and ion transport, and maintenance of cellular structure and integrity. These inhibited responses correlate with the observed histological alterations and may contribute to the antiuterotrophic effects of TCDD.

Targeted deletion of cannabinoid receptors CB1 and CB2 produced enhanced inflammatory responses to influenza A/PR/8/34 in the absence and presence of Δ9-tetrahydrocannabinol

We have previously reported that Δ‐9‐tetrahydrocannabinol (Δ9‐THC)‐treated mice challenged with influenza virus A/PR/8/34 (PR8) developed increased viral hemagglutinin 1 (H1) mRNA levels and decreased monocyte and lymphocyte recruitment to the pulmonary airways when compared with mice challenged with PR8 alone. The objective of the present study was to examine the role of cannabinoid (CB1/CB2) receptors in mediating the effects of Δ9‐THC on immune and epithelial cell responses to PR8. In the current study, Δ9‐THC‐treated CB1/CB2 receptor null (CB1−/−/CB2−/−) and wild‐type mice infected with PR8 had marked increases in viral H1 mRNA when compared with CB1−/−/CB2−/− and wild‐type mice challenged with PR8 alone. However, the magnitude of the H1 mRNA levels was greatly reduced in CB1−/−/CB2−/− mice as compared with wild‐type mice. In addition, Δ9‐THC‐treated CB1−/−/CB2−/− mice infected with PR8 had increased CD4+ T cells and IFN‐γ in bronchoalveolar lavage fluid with greater pulmonary inflammation when compared with Δ9‐THC‐treated wild‐type mice infected with PR8. Δ9‐THC treatment of CB1−/−/CB2−/− mice in the presence or absence of PR8 challenge also developed greater amounts of mucous cell metaplasia in the affected bronchiolar epithelium. Collectively, the immune and airway epithelial cell responses to PR8 challenge in Δ9‐THC‐treated CB1−/−/CB2−/− and wild‐type mice indicated the involvement of CB1/CB2 receptor‐dependent and ‐independent mechanisms.

Experimental induction of pulmonary fibrosis in horses with the gammaherpesvirus equine herpesvirus 5.

Gammaherpesviruses (γHV) are implicated in the pathogenesis of pulmonary fibrosis in humans and murine models of lung fibrosis, however there is little direct experimental evidence that such viruses induce lung fibrosis in the natural host. The equine γHV EHV 5 is associated with equine multinodular pulmonary fibrosis (EMPF), a progressive fibrosing lung disease in its natural host, the horse. Experimental reproduction of EMPF has not been attempted to date. We hypothesized that inoculation of EHV 5 isolated from cases of EMPF into the lungs of clinically normal horses would induce lung fibrosis similar to EMPF. Neutralizing antibody titers were measured in the horses before and after inoculation with EHV 5. PCR and virus isolation was used to detect EHV 5 in antemortem blood and BAL samples, and in tissues collected postmortem. Nodular pulmonary fibrosis and induction of myofibroblasts occurred in EHV 5 inoculated horses. Mean lung collagen in EHV 5 inoculated horses (80 µg/mg) was significantly increased compared to control horses (26 µg/mg) (p < 0.5), as was interstitial collagen (32.6% ± 1.2% vs 23% ± 1.4%) (mean ± SEM; p < 0.001). Virus was difficult to detect in infected horses throughout the experiment, although EHV 5 antigen was detected in the lung by immunohistochemistry. We conclude that the γHV EHV 5 can induce lung fibrosis in the horse, and hypothesize that induction of fibrosis occurs while the virus is latent within the lung. This is the first example of a γHV inducing lung fibrosis in the natural host.

Comparative temporal and dose-dependent morphological and transcriptional uterine effects elicited by tamoxifen and ethynylestradiol in immature, ovariectomized mice

BackgroundUterine temporal and dose-dependent histopathologic, morphometric and gene expression responses to the selective estrogen receptor modulator tamoxifen (TAM) were comprehensively examined to further elucidate its estrogen receptor-mediated effects. These results were systematically compared to the effects elicited by the potent estrogen receptor ligand 17α-ethynylestradiol (EE) to identify pathways similarly and uniquely modified by each compound.ResultsThree daily doses of 100 μg/kg TAM elicited a dose-dependent increase in uterine wet weight (UWW) in immature, ovariectomized C57BL/6 mice at 72 hrs with concurrent increases in luminal epithelial cell height (LECH), luminal circumference and glandular epithelial tubule number. Significant UWW and LECH increases were detected at 24 hrs after a single dose of 100 μg/kg TAM. cDNA microarray analysis identified 2235 differentially expressed genes following a single dose of 100 μg/kg TAM at 2, 4, 8, 12, 18 and 24 hrs, and at 72 hrs after three daily doses (3 × 24 hrs). Functional annotation of differentially expressed genes was associated with cell growth and proliferation, cytoskeletal organization, extracellular matrix modification, nucleotide synthesis, DNA replication, protein synthesis and turnover, lipid metabolism, glycolysis and immunological responses as is expected from the uterotrophic response. Comparative analysis of TAM and EE treatments identified 1209 common, differentially expressed genes, the majority of which exhibited similar profiles despite a temporal delay in TAM elicited responses. However, several conserved and treatment specific responses were identified that are consistent with proliferation (Fos, Cdkn1a, Anapc1), and water imbibition (Slc30a3, Slc30a5) responses elicited by EE.ConclusionOverall, TAM and EE share similar gene expression profiles. However, TAM responses exhibit lower efficacy, while responses unique to EE are consistent with the physiological differences elicited between compounds.

Multinodular pulmonary fibrosis, pancytopenia and equine herpesvirus-5 infection in a Thoroughbred gelding

Summary A 21-year-old Thoroughbred gelding was examined for intermittent fever, lethargy, inappetance and weight loss. Initial diagnostic evaluation revealed pancytopenia (anaemia, thrombocytopenia and neutropenia), hyperfibrinogenaemia and multifocal pulmonary nodules. Bacterial and fungal culture and viral isolation on transtracheal aspirate samples were negative, consistent with either pulmonary neoplasia, idiopathic granulomatous pneumonia or equine multinodular pulmonary fibrosis (EMPF). Bone marrow evaluation was consistent with pancytopenia due to a combination of peripheral consumption/destruction and suppression/destruction of progenitor cells at the level of the marrow. Pancytopenia resolved and clinical signs improved transiently with immunosuppressive corticosteroid therapy, but the horse deteriorated rapidly one month after presentation and was subjected to euthanasia. Necropsy findings were consistent with EMPF, and equine herpesvirus-5 (EHV-5) DNA was found in both the lung and bone marrow. The specific role of EHV-5 in the development of the pulmonary pathology in EMPF and the pancytopenia in this horse is unclear, but an aberrant host immune/inflammatory response to EHV-5 infection may be important.