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Speciation leads to adaptive changes in organ cellular physiology and creates challenges for studying rare cell-type functions that diverge between humans and mice. Rare cystic fibrosis transmembrane conductance regulator (CFTR)-rich pulmonary ionocytes exist throughout the cartilaginous airways of humans1,2, but limited presence and divergent biology in the proximal trachea of mice has prevented the use of traditional transgenic models to elucidate ionocyte functions in the airway. Here we describe the creation and use of conditional genetic ferret models to dissect pulmonary ionocyte biology and function by enabling ionocyte lineage tracing (FOXI1-CreERT2::ROSA-TG), ionocyte ablation (FOXI1-KO) and ionocyte-specific deletion of CFTR(FOXI1-CreERT2::CFTRL/L). By comparing these models with cystic fibrosis ferrets3,4, we demonstrate that ionocytes control airway surface liquid absorption, secretion, pH and mucus viscosity—leading to reduced airway surface liquid volume and impaired mucociliary clearance in cystic fibrosis, FOXI1-KO and FOXI1-CreERT2::CFTRL/Lferrets. These processes are regulated by CFTR-dependent ionocyte transport of Cl−and HCO3−. Single-cell transcriptomics and in vivo lineage tracing revealed three subtypes of pulmonary ionocytes and a FOXI1-lineage common rare cell progenitor for ionocytes, tuft cells and neuroendocrine cells during airway development. Thus, rare pulmonary ionocytes perform critical CFTR-dependent functions in the proximal airway that are hallmark features of cystic fibrosis airway disease. These studies provide a road map for using conditional genetics in the first non-rodent mammal to address gene function, cell biology and disease processes that have greater evolutionary conservation between humans and ferrets.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.amjsurg.2008.07.009 Byline: David K. Meyerholz (a), Deborah E. Williard (b), Ana-Maria Grittmann (b), Isaac Samuel (b)(c) Keywords: Acute pancreatitis; Mouse; Acute lung injury; Pancreatic duct; Morphology; Acinar cell; Stress kinases Abstract: Acute lung injury is a major determinant of outcomes in acute pancreatitis. We evaluated acute lung injury and stress kinase activation in ligation-induced acute pancreatitis in mice. Author Affiliation: (a) Department of Pathology, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA, USA (b) Department of Surgery, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA, USA (c) Department of Surgery, Veterans Affairs Medical Center, Iowa City, IA, USA Article History: Received 20 May 2008; Revised 21 July 2008 Article Note: (footnote) Dr. I. Samuel was supported for this work by a National Institutes of Health NIDDK Career Development Award (Grant No. K08-DK062805) and a VA Merit Review Award. Dr. D.K. Meyerholz was supported in part by the Department of Pathology, University of Iowa Carver College of Medicine.

Coronavirus disease 2019 (COVID-19) is especially severe in aged populations1. Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are highly effective, but vaccine efficacy is partly compromised by the emergence of SARS-CoV-2 variants with enhanced transmissibility2. The emergence of these variants emphasizes the need for further development of anti-SARS-CoV-2 therapies, especially for aged populations. Here we describe the isolation of highly virulent mouse-adapted viruses and use them to test a new therapeutic drug in infected aged animals. Many of the alterations observed in SARS-CoV-2 during mouse adaptation (positions 417, 484, 493, 498 and 501 of the spike protein) also arise in humans in variants of concern2. Their appearance during mouse adaptation indicates that immune pressure is not required for selection. For murine SARS, for which severity is also age dependent, elevated levels of an eicosanoid (prostaglandin D2(PGD2)) and a phospholipase (phospholipase A2 group 2D (PLA2G2D)) contributed to poor outcomes in aged mice3,4. mRNA expression of PLA2G2D and prostaglandin D2receptor (PTGDR), and production of PGD2also increase with ageing and after SARS-CoV-2 infection in dendritic cells derived from human peripheral blood mononuclear cells. Using our mouse-adapted SARS-CoV-2, we show that middle-aged mice lacking expression of PTGDR or PLA2G2D are protected from severe disease. Furthermore, treatment with a PTGDR antagonist, asapiprant, protected aged mice from lethal infection. PTGDR antagonism is one of the first interventions in SARS-CoV-2-infected animals that specifically protects aged animals, suggesting that the PLA2G2D–PGD2/PTGDR pathway is a useful target for therapeutic interventions.

Research in bone tissue engineering aims to design materials that are effective at generating bone without causing significant side effects. The osteogenic potential of combining matrices and protein growth factors has been well documented, however, improvements are necessary to achieve optimal therapeutic benefits upon clinical translation. In this article, rat calvarial defects were treated with gene-activated matrices (GAMs). The GAMs used were collagen sponges mineralized with a simulated body fluid (SBF) containing a nonviral gene delivery system. Both in vitroand in vivostudies were performed to determine the optimal mode of gene delivery. After 6 weeks, the defects were extracted to assess bone formation and tissue quality through histological and microcomputed tomography analyses. The optimal GAM consisted of a collagen sponge with polyethylenimine plasmid DNA (PEI-pDNA) complexes embedded in a calcium phosphate coating produced by SBF, which increased total bone formation by 39% compared with 19% for control samples. A follow-up in vivostudy was performed to optimize the ratio of growth factors included in the GAM. The optimal ratio for supporting bone formation after 6 weeks of implantation was five parts of pBMP-2 to three parts pFGF-2. These studies demonstrated that collagen matrices biomimetically mineralized and activated with plasmids encoding fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2) can optimally improve bone regeneration outcomes.Impact statementBone tissue engineering has explored both nonviral gene delivery and the concept of biomimetic mineralization. In this study, we combined these two concepts to further enhance bone regeneration outcomes. We demonstrated that embedding polyethylenimine (PEI)-based gene delivery within a mineral layer formed from simulated body fluid (SBF) immersion can increase bone formation rates. We also demonstrated that the ratio of growth factors utilized for matrix fabrication can impact the amount of bone formed in the defect site. This research highlights a combined approach using SBF and nonviral gene delivery both in vitroand in vivoand prepares the way for future optimization of synthetic gene activated matrices.

The ongoing coronavirus disease 2019 (COVID-19) pandemic is associated with substantial morbidity and mortality. Although much has been learned in the first few months of the pandemic, many features of COVID-19 pathogenesis remain to be determined. For example, anosmia is a common presentation, and many patients with anosmia show no or only minor respiratory symptoms1. Studies in animals infected experimentally with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of COVID-19, provide opportunities to study aspects of the disease that are not easily investigated in human patients. Although the severity of COVID-19 ranges from asymptomatic to lethal2, most experimental infections provide insights into mild disease3. Here, using K18-hACE2 transgenic mice that were originally developed for SARS studies4, we show that infection with SARS-CoV-2 causes severe disease in the lung and, in some mice, the brain. Evidence of thrombosis and vasculitis was detected in mice with severe pneumonia. Furthermore, we show that infusion of convalescent plasma from a recovered patient with COVID-19 protected against lethal disease. Mice developed anosmia at early time points after infection. Notably, although pre-treatment with convalescent plasma prevented most signs of clinical disease, it did not prevent anosmia. Thus, K18-hACE2 mice provide a useful model for studying the pathological basis of both mild and lethal COVID-19 and for assessing therapeutic interventions.

Animals are valuable resources in biomedical research in investigations of biological processes, disease pathogenesis, therapeutic interventions, safety, toxicity, and carcinogenicity. Interpretation of data from animals requires knowledge not only of the processes or diseases (pathophysiology) under study but also recognition of spontaneous conditions and background lesions (pathology) that can influence or confound the study results. Species, strain/stock, sex, age, anatomy, physiology, spontaneous diseases (noninfectious and infectious), and neoplasia impact experimental results and interpretation as well as animal welfare. This review and the references selected aim to provide a pathology resource for researchers, pathologists, and veterinary personnel who strive to achieve research rigor and validity and must understand the spectrum of “normal” and expected conditions to accurately identify research-relevant experimental phenotypes as well as unusual illness, pathology, or other conditions that can compromise studies involving laboratory mice, rats, gerbils, guinea pigs, hamsters, naked mole rats, and rabbits.

Animal models provide a valuable tool and resource for biomedical researchers as they investigate biological processes, disease pathogenesis, novel therapies, and toxicologic studies. Interpretation of animal model data requires knowledge not only of the processes/diseases being studied but also awareness of spontaneous conditions and background lesions in the model that can influence or even confound the study results. Species, breed/stock, sex, age, anatomy, physiology, diseases (noninfectious and infectious), and neoplastic processes are model features that can impact the results as well as study interpretation. Here, we review these features in several common laboratory animal species, including ferret, dog (beagle), pig, sheep, and goats.

Hepatobiliary disease causes significant morbidity in people with cystic fibrosis (CF), yet this problem remains understudied. We previously found that newborn CF pigs have microgallbladders with significant luminal obstruction in the absence of infection and consistent inflammation. In this study, we sought to better understand the early pathogenesis of CF pig gallbladder disease. We hypothesized that loss of CFTR would impair gallbladder epithelium anion/liquid secretion and increase mucin production. CFTR was expressed apically in non-CF pig gallbladder epithelium but was absent in CF. CF pig gallbladders lacked cAMP-stimulated anion transport. Using a novel gallbladder epithelial organoid model, we found that Cl−or HCO3−was sufficient for non-CF organoid swelling. This response was absent for non-CF organoids in Cl−/HCO3−-free conditions and in CF. Single-cell RNA-sequencing revealed a single epithelial cell type in non-CF gallbladders that coexpressed CFTR, MUC5AC, and MUC5B. Despite CF gallbladders having increased luminal MUC5AC and MUC5B accumulation, there was no significant difference in the epithelial expression of gel-forming mucins between non-CF and CF pig gallbladders. In conclusion, these data suggest that loss of CFTR-mediated anion transport and fluid secretion contribute to microgallbladder development and luminal mucus accumulation in CF.

Despite being a leading cause of severe respiratory disease, there remains no licensed respiratory syncytial virus (RSV) vaccine. Neutralizing antibodies reduce the severity of RSV-associated disease, but are not sufficient for preventing reinfection. In contrast, the role of memory CD8 T cells in protecting against a secondary RSV infection is less established. We recently demonstrated that high-magnitude memory CD8 T cells efficiently reduced lung viral titers following RSV infection, but induced fatal immunopathology that was mediated by IFN-γ. To evaluate the ability of RSV-specific neutralizing antibodies to prevent memory CD8 T cell-mediated immunopathology, mice with high-magnitude memory CD8 T cell responses were treated with neutralizing antibodies prior to RSV challenge. Neutralizing antibody treatment significantly reduced morbidity and prevented mortality following RSV challenge compared with IgG-treated controls. Neutralizing antibody treatment restricted early virus replication, which caused a substantial reduction in memory CD8 T cell activation and IFN-γ production, directly resulting in survival. In contrast, therapeutic neutralizing antibody administration did not impact morbidity, mortality, or IFN-γ levels, despite significantly reducing lung viral titers. Therefore, only pre-existing neutralizing antibodies prevent memory CD8 T cell-mediated immunopathology following RSV infection. Overall, our results have important implications for the development of future RSV vaccines.

Inflammatory bowel disease (IBD) is a heterogeneous group of inflammation‐mediated pathologies that include Crohn's disease and ulcerative colitis and primarily affects the colon and small intestine. Previous studies have shown that a disintegrin and metalloprotease (ADAM) 17, a membrane‐bound sheddase, capable of cleaving the proinflammatory cytokine TNF and epidermal growth factor receptor ligands, plays a critical role in maintaining gut homeostasis and modulating intestinal inflammation during IBD. Rhomboid 5 homolog 2 (RHBDF2), a catalytically inactive member of the rhomboid family of intramembrane serine proteases, was recently identified as a crucial regulator of ADAM17. Here, we assessed the role of RHBDF2 in the development of colitis in the context of IL10 deficiency. Il10−/−/Rhbdf2−/−mice developed spontaneous colitis and experienced severe weight loss starting at 8 wk of age, without the need for exogenous triggers. Severity of disease pathology in Il10−/−/Rhbdf2−/−mice correlated with a dysbiotic gut microbiota and elevated Th1‐associated immune responses with increased interferon gamma and IL2 production. In addition, Il10−/−/Rhbdf2−/−mice failed to maintain their epithelial cell homeostasis, although the intestinal epithelial barrier of Rhbdf2−/−mice is intact and loss of Rhbdf2did not significantly exacerbate sensitivity to dextran sulfate sodium‐induced colitis, suggesting differences in the underlying disease pathway of intestinal inflammation in this model. Taken together, our results demonstrate a critical regulatory role for RHBDF2 in the maintenance of the unique homeostasis between intestinal microbiota and host immune responses in the gut that is dysregulated during the pathogenesis of IBD. RHBDF2 participates in maintaining the critical balance between intestinal microbiota and host immune responses during the pathogenesis of colitis in Il10‐deficient mice.

Tcf1 and Lef1 have versatile functions in regulating T cell development and differentiation, but intrinsic requirements for these factors in regulatory T (T reg) cells remain to be unequivocally defined. Specific ablation of Tcf1 and Lef1 in T reg cells resulted in spontaneous multi-organ autoimmunity that became more evident with age. Tcf1/Lef1-deficient T regs showed reduced protection against experimentally induced colitis, indicative of diminished immuno-suppressive capacity. Transcriptomic analysis revealed that Tcf1 and Lef1 were responsible for positive regulation of a subset of T reg–overrepresented signature genes such as Ikzf4 and Izumo1r. Unexpectedly, Tcf1 and Lef1 were necessary for restraining expression of cytotoxic CD8+ effector T cell–associated genes in T reg cells, including Prdm1 and Ifng. Tcf1 ChIP-seq revealed substantial overlap between Tcf1 and Foxp3 binding peaks in the T reg cell genome, with Tcf1-Foxp3 cooccupancy observed at key T reg signature and cytotoxic effector genes. Our data collectively indicate that Tcf1 and Lef1 are critical for sustaining T reg suppressive functions and preventing loss of self-tolerance.

Failure to reproduce results from some scientific studies has raised awareness of the critical need for reproducibility in translational studies. Macroscopic and microscopic examination is a common approach to determine changes in tissues, but text descriptions and visual images have limitations for group comparisons. Semiquantitative scoring is a way of transforming qualitative tissue data into numerical data that allow more robust group comparisons. Semiquantitative scoring has broad uses in preclinical and clinical studies for evaluation of tissue lesions. Reproducibility can be improved by constraining bias through appropriate experimental design, randomization of tissues, effective use of multidisciplinary collaborations, and valid masking procedures. Scoring can be applied to tissue lesions (eg, size, distribution, characteristics) and also to tissues through evaluation of staining distribution and intensity. Semiquantitative scores should be validated to demonstrate relevance to biological data and to demonstrate observer reproducibility. Statistical analysis should make use of appropriate tests to give robust confidence in the results and interpretations. Following key principles of semiquantitative scoring will not only enhance descriptive tissue evaluation but also improve quality, reproducibility, and rigor of tissue studies.

Evaluation of tissues is a common and important aspect of translational research studies. Labeling techniques such as immunohistochemistry can stain cells/tissues to enhance identification of specific cell types, cellular activation states, and protein expression. While qualitative evaluation of labeled tissues has merit, use of semiquantitative and quantitative scoring approaches can greatly enhance the rigor of the tissue data. Adhering to key principles for reproducible scoring can enhance the quality and reproducibility of the tissue data so as to maximize its biological relevance and scientific impact.

Loss of cystic fibrosis transmembrane conductance regulator (CFTR) function causes cystic fibrosis (CF), predisposing the lungs to chronic infection and inflammation. In young infants with CF, structural airway defects are increasingly recognized before the onset of significant lung disease, which suggests a developmental origin and a possible role in lung disease pathogenesis. The role(s) of CFTR in lung development is unclear and developmental studies in humans with CF are not feasible. Young CF pigs have structural airway changes and develop spontaneous postnatal lung disease similar to humans; therefore, we studied lung development in the pig model (non-CF and CF). CF trachea and proximal airways had structural lesions detectable as early as pseudoglandular development. At this early developmental stage, budding CF airways had smaller, hypo-distended lumens compared to non-CF airways. Non-CF lung explants exhibited airway lumen distension in response to forskolin/IBMX as well as to fibroblast growth factor (FGF)-10, consistent with CFTR-dependent anion transport/secretion, but this was lacking in CF airways. We studied primary pig airway epithelial cell cultures and found that FGF10 increased cellular proliferation (non-CF and CF) and CFTR expression/function (in non-CF only). In pseudoglandular stage lung tissue, CFTR protein was exclusively localized to the leading edges of budding airways in non-CF (but not CF) lungs. This discreet microanatomic localization of CFTR is consistent with the site, during branching morphogenesis, where airway epithelia are responsive to FGF10 regulation. In summary, our results suggest that the CF proximal airway defects originate during branching morphogenesis and that the lack of CFTR-dependent anion transport/liquid secretion likely contributes to these hypo-distended airways.

Neurofibromatosis type 1 (NF1) is a common, cancer-predisposing disease caused by mutations in the NF1tumor gene. Patients with NF1 have an increased risk for benign and malignant tumors of the nervous system (e.g., neurofibromas, malignant peripheral nerve sheath tumors, gliomas) and other tissues (e.g., leukemias, rhabdomyosarcoma, etc.) as well as increased susceptibility to learning disabilities, chronic pain/migraines, hypertension, pigmentary changes, and developmental lesions (e.g., tibial pseudoarthrosis). Pigs are an attractive and upcoming animal model for future NF1 studies, but a potential limitation to porcine model research has been the lack of validated reagents for direct translational study to humans. To address that issue, we used formalin-fixed tissues (human and pigs) to evaluate select immunohistochemical markers (activated caspase-3, allograft inflammatory factor-1, beta-tubulin III, calbindin D, CD13, CD20, desmin, epithelial membrane antigen, glial fibrillary acidic protein, glucose transporter-1, laminin, myelin basic protein, myoglobin, proliferating cell nuclear antigen, S100, vimentin, and von Willebrand factor). The markers were validated by comparing known expression and localization in human and pig tissues. Validation of these markers on fixed tissues will facilitate prospective immunohistochemical studies of NF1 pigs, as well as other pig models, in a more efficient, reproducible, and translationally relevant manner.

This issue of ILAR Journal focusses on pathology and pathologists in biomedical research, more specifically in preclinical translational research involving (nonhuman) animals, emphasizing academic settings. Considerations in study design and planning to maximize benefit from pathologists and pathology resources are reviewed. Adjunctive technologies including molecular techniques, digital pathology, and imaging are highlighted. Additional considerations regarding safety and regulatory concerns, and veterinary clinical trials are reviewed as well. Pathology has been fundamental to understanding clinical disease, remains fundamental to diagnosing disease, and is required in drug and device development. Broader integration of pathology expertise and well-designed pathology investigations have much to offer research rigor and reproducibility, and successful translation from biomedical research.

It has been shown that inhalation exposure to copper oxide nanoparticles (CuO NPs) results in pulmonary inflammation. However, immunomodulatory consequences after CuO NP inhalation exposure have been less explored. We tested the effect of CuO NP aerosols on immune responses in healthy, house dust mite (HDM) asthmatic, or allergen immunotherapy (AIT)-treated asthmatic mice (BALB/c, females). The AIT consisted of a vaccine comprising HDM allergens and CpG-loaded nanoparticles (CpG NPs). AIT treatment involved mice being immunized (via subcutaneous (sc) injection; 2 doses) while concomitantly being exposed to CuO NP aerosols (over a 2 week period), starting on the day of the first vaccination. Mice were then sensitized twice by sc injection and subsequently challenged with HDM extract 10 times by intranasal instillation. The asthmatic model followed the same timeline except that no immunizations were administered. All mice were necropsied 24 h after the end of the HDM challenge. CuO NP-exposed healthy mice showed a significant decrease in TH1 and TH2 cells, and an elevation in T-bet+Treg cells, even 40 days after the last exposure to CuO NPs. Similarly, the CuO NP-exposed HDM asthma model demonstrated decreased TH2 responses and increased T-bet+Treg cells. Conversely, CuO NP inhalation exposure to AIT-treated asthmatic mice resulted in an increase in TH2 cells. In conclusion, immunomodulatory effects of inhalation exposure to CuO NPs are dependent on immune conditions prior to exposure.

ABSTRACTStaphylococcus aureusis a leading cause of chronic biofilm infections. Hyaluronic acid (HA) is a large glycosaminoglycan abundant in mammalian tissues that has been shown to enhance biofilm formation in multiple Gram-positive pathogens. We observed that HA accumulated in an S. aureusbiofilm infection using a murine implant-associated infection model and that HA levels increased in a mutant strain lacking hyaluronidase (HysA). S. aureussecretes HysA in order to cleave HA during infection. Through in vitrobiofilm studies with HA, the hysAmutant was found to accumulate increased biofilm biomass compared to the wild type, and confocal microscopy showed that HA is incorporated into the biofilm matrix. Exogenous addition of purified HysA enzyme dispersed HA-containing biofilms, while catalytically inactive enzyme had no impact. Additionally, induction of hysAexpression prevented biofilm formation and also dispersed an established biofilm in the presence of HA. These observations were corroborated in the implant model, where there was decreased dissemination from an hysAmutant biofilm infection compared to the S. aureuswild type. Histopathology demonstrated that infection with an hysAmutant caused significantly reduced distribution of tissue inflammation compared to wild-type infection. To extend these studies, the impact of HA and S. aureusHysA on biofilm-like aggregates found in joint infections was examined. We found that HA contributes to the formation of synovial fluid aggregates, and HysA can disrupt aggregate formation. Taken together, these studies demonstrate that HA is a relevant component of the S. aureusbiofilm matrix and HysA is important for dissemination from a biofilm infection.

A 5-year-old pregnant Ardi goat presented with dystocia of 36 h duration. Vaginal examination showed normal fetal presentation; however, a large neck mass prevented vaginal delivery and cesarean section was performed to deliver the stillborn kid. A well-circumscribed, poorly lobulated subcutaneous mass (12.5 cm in diameter) was found on the ventral lateral aspect of the neck. The cut surface was fleshy and tan to pale white in appearance. Histologically, the tumor exhibited moderate to high cellularity and consisted of elongate to ribbon-like myotubes, with many showing differentiation into larger cells having cross-striations. Nuclei were often multiple (2–5+) and peripheral. Neoplastic cells lacked anaplasia and had a low mitotic index (<1 mitosis per 10 fields of 400× magnification). The neoplastic cells had diffuse myoglobin and multifocal desmin immunostaining. The clinical and histopathological features were consistent with a diagnosis of intermediate-type fetal rhabdomyoma, a rare tumor reported in the human literature. This is to our knowledge the first report of intermediate-type fetal rhabdomyoma in the veterinary literature and the first report of dystocia secondary to a congenital, extracardiac rhabdomyoma.

Dipeptidyl peptidase 4 (DPP4, CD26), a type II transmembrane ectopeptidase, is the receptor for the Middle Eastern respiratory syndrome coronavirus (MERS-CoV). MERS emerged in 2012 and has a high mortality associated with severe lung disease. A lack of autopsy studies from MERS fatalities has hindered understanding of MERS-CoV pathogenesis. We investigated the spatial and cellular localization of DPP4 to evaluate an association MERS clinical disease. DPP4 was rarely detected in the surface epithelium from nasal cavity to conducting airways with a slightly increased incidence in distal airways. DPP4 was also found in a subset of mononuclear leukocytes and in serous cells of submucosal glands. In the parenchyma, DPP4 was found principally in type I and II cells and alveolar macrophages and was also detected in vascular endothelium (eg, lymphatics) and pleural mesothelia. Patients with chronic lung disease, such as chronic obstructive pulmonary disease and cystic fibrosis, exhibited increased DPP4 immunostaining in alveolar epithelia (type I and II cells) and alveolar macrophages with similar trends in reactive mesothelia. This finding suggests that preexisting pulmonary disease could increase MERS-CoV receptor abundance and predispose individuals to MERS morbidity and mortality, which is consistent with current clinical observations. We speculate that the preferential spatial localization of DPP4 in alveolar regions may explain why MERS is characterized by lower respiratory tract disease.

Oxidative stress and chronic low-grade inflammation in the lungs are associated with aging and may contribute to age-related immune dysfunction. To maintain lung homeostasis, chronic inflammation is countered by enhanced expression of proresolving/antiinflammatory factors. Here, we show that age-dependent increases of one such factor in the lungs, a phospholipase A2 (PLA2) group IID (PLA2G2D) with antiinflammatory properties, contributed to worse outcomes in mice infected with severe acute respiratory syndrome-coronavirus (SARS-CoV). Strikingly, infection of mice lacking PLA2G2D expression (Pla2g2d−/− mice) converted a uniformly lethal infection to a nonlethal one (>80% survival), subsequent to development of enhanced respiratory DC migration to the draining lymph nodes, augmented antivirus T cell responses, and diminished lung damage. We also observed similar effects in influenza A virus–infected middle-aged Pla2g2d−/− mice. Furthermore, oxidative stress, probably via lipid peroxidation, was found to induce PLA2G2D expression in mice and in human monocyte–derived macrophages. Thus, our results suggest that directed inhibition of a single inducible phospholipase, PLA2G2D, in the lungs of older patients with severe respiratory infections is potentially an attractive therapeutic intervention to restore immune function.

Otitis media (inflammation of the middle ear) is one of the most common diseases of early childhood. Susceptibility to otitis is influenced by a number of factors, including the actions of innate immune molecules secreted by the epithelia lining the nasopharynx, middle ear and Eustachian tube. The SPLUNC1 (short palate, lung, nasal epithelial clone 1) protein is a highly abundant secretory product of the mammalian nasal, oral and respiratory mucosa that is thought to play a multifunctional role in host defense. In this study we investigated Splunc1 expression in the ear of the mouse, and examined whether this protein contributes to overall host defense in the middle ear and/or Eustachian tube. We found that Splunc1 is highly expressed in both the surface epithelium and in submucosal glands in these regions in wild-type mice. In mice lacking Splunc1, we noted histologically an increased frequency of otitis media, characterized by the accumulation of leukocytes (neutrophils with scattered macrophages), proteinaceous fluid and mucus in the middle ear lumens. Furthermore, many of these mice had extensive remodeling of the middle ear wall, suggesting a chronic course of disease. From these observations, we conclude that loss of Splunc1 predisposes mice to the development of otitis media. The Splunc1−/− mouse model should help investigators to better understand both the biological role of Splunc1 as well as host defense mechanisms in the middle ear.

Cystic fibrosis (CF) is a multiorgan disease caused by loss of a functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in many epithelia of the body. Here we report the pathology observed in the gastrointestinal organs of juvenile to adult CFTR-knockout ferrets. CF gastrointestinal manifestations included gastric ulceration, intestinal bacterial overgrowth with villous atrophy, and rectal prolapse. Metagenomic phylogenetic analysis of fecal microbiota by deep sequencing revealed considerable genotype-independent microbial diversity between animals, with the majority of taxa overlapping between CF and non-CF pairs. CF hepatic manifestations were variable, but included steatosis, necrosis, biliary hyperplasia, and biliary fibrosis. Gallbladder cystic mucosal hyperplasia was commonly found in 67% of CF animals. The majority of CF animals (85%) had pancreatic abnormalities, including extensive fibrosis, loss of exocrine pancreas, and islet disorganization. Interestingly, 2 of 13 CF animals retained predominantly normal pancreatic histology (84% to 94%) at time of death. Fecal elastase-1 levels from these CF animals were similar to non-CF controls, whereas all other CF animals evaluated were pancreatic insufficient (<2 μg elastase-1 per gram of feces). These findings suggest that genetic factors likely influence the extent of exocrine pancreas disease in CF ferrets and have implications for the etiology of pancreatic sufficiency in CF patients. In summary, these studies demonstrate that the CF ferret model develops gastrointestinal pathology similar to CF patients.

ABSTRACTThe virulence factors mediating Francisellapathogenesis are being investigated, with an emphasis on understanding how the organism evades innate immunity mechanisms. Francisella tularensisproduces a lipopolysaccharide (LPS) that is essentially inert and a polysaccharide capsule that helps the organism to evade detection by components of innate immunity. Using an F. tularensisSchu S4 mutant library, we identified strains that are disrupted for capsule and O-antigen production. These serum-sensitive strains lack both capsule production and O-antigen laddering. Analysis of the predicted protein sequences for the disrupted genes (FTT1236and FTT1238c) revealed similarity to those for waa(rfa) biosynthetic genes in other bacteria. Mass spectrometry further revealed that these proteins are involved in LPS core sugar biosynthesis and the ligation of O antigen to the LPS core sugars. The 50% lethal dose (LD50) values of these strains are increased 100- to 1,000-fold for mice. Histopathology revealed that the immune response to the F. tularensismutant strains was significantly different from that observed with wild-type-infected mice. The lung tissue from mutant-infected mice had widespread necrotic debris, but the spleens lacked necrosis and displayed neutrophilia. In contrast, the lungs of wild-type-infected mice had nominal necrosis, but the spleens had widespread necrosis. These data indicate that murine death caused by wild-type strains occurs by a mechanism different from that by which the mutant strains kill mice. Mice immunized with these mutant strains displayed >10-fold protective effects against virulent type A F. tularensischallenge.

Pancreatic disease has onset in uteroin humans with cystic fibrosis (CF), and progresses over time to complete destruction of the organ. The exact mechanisms leading to pancreatic damage in CF are incompletely understood. Inflammatory cells are present in the pancreas of newborn pigs with CF (CF pigs) and humans, which suggests that inflammation may have a role in the destructive process. We wondered whether tissue inflammation and genes associated with inflammatory pathways were increased in the pancreas of fetal CF pigs [83 to 90 days gestation (normal pig gestation is ∼114 days)] and newborn pigs. Compared with fetal pigs without CF (non-CF pigs), in fetal CF pigs, the pancreas exhibited patchy inflammation and acinar atrophy, with progression in distribution and severity in neonatal CF pigs. Large-scale transcript profiling revealed that the pancreas in fetal and newborn CF pigs exhibited significantly increased expression of proinflammatory, complement cascade, and profibrotic genes when compared with fetal and newborn non-CF pigs. Acinar cells exhibited increased apoptosis in the pancreas of fetal and newborn CF pigs. α-Smooth muscle actin and transforming growth factor β1 were increased in both fetal and newborn CF pig pancreas, suggesting activation of profibrotic pathways. Cell proliferation and mucous cell metaplasia were detected in newborn, but not fetal, CF pigs, indicating that they were not an initiator of pathogenesis but a response. Proinflammatory, complement cascade, proapoptotic, and profibrotic pathways are activated in CF pig pancreas, and likely contribute to the destructive process.

AbstractBackground/Aims:In cystic fibrosis (CF), pancreatic disease begins in utero and progresses over time to complete destruction of the organ. Although inflammatory cells have been detected in the pancreas of humans and pigs with CF, their subtypes have not been characterized. Methods:Using four-color flow cytometry, we analyzed the surface antigens of leukocytes in pancreas, blood, and mesenteric lymph nodes (MLN) of newborn pigs with CF (CFTR–/–and CFTRΔF508/ΔF508) and in those without CF (CFTR+/–, CFTR+/ΔF508, CFTR+/+). Pancreatic histopathology was examined with HE stain. Results:CF pig pancreas had patchy distribution of inflammatory cells with neutrophils/macrophages in dilated acini, and lymphocytes in the interstitium compared to non-CF. B cells, effector (MHC-II+) and cytotoxic (CD2+CD8+) γδ T cells, activated (MHC-II+and/or CD25+) and effector (CD4+CD8+) αβ T helper cells, effector natural killer cells (MHC-II+CD3–CD8+), and monocytes/macrophages and neutrophils were increased in the CF pig pancreas compared to pigs without CF. Blood and MLN leukocyte populations were not different between CF and non-CF pigs. Conclusions:We discovered an activated immune response that was specific to the pancreas of newborn CF pigs; inflammation was not systemic. The presence of both innate and adaptive immune cells suggests that the disease process is complex and extensive.Copyright © 2011 S. Karger AG, Basel and IAP

Identifying the normal cell from which a tumor originates is crucial to understanding the etiology of that cancer. However, retrospective identification of the cell of origin in cancer is challenging because of the accumulation of genetic and epigenetic changes in tumor cells. The biologic state of the cell of origin likely influences the genetic events that drive transformation. We directly tested this hypothesis by performing a Sleeping Beauty transposon mutagenesis screen in which common insertion sites were identified in tumors that were produced by mutagenesis of cells at varying time points throughout the T lineage. Mutation and gene expression data derived from these tumors were then compared with data obtained from a panel of 84 human T-cell acute lymphoblastic leukemia samples, including copy number alterations and gene expression profiles. This revealed that altering the cell of origin produces tumors that model distinct subtypes of human T-cell acute lymphoblastic leukemia, suggesting that even subtle changes in the cell of origin dramatically affect genetic selection in tumors. These findings have broad implications for the genetic analysis of human cancers as well as the production of mouse models of cancer.

AbstractBackground:Suitable experimental models of gallstone pancreatitis with systemic inflammation and mortality are limited. We developed a novel murine model of duct-ligation-induced acute pancreatitis associated with multiorgan dysfunction and severe mortality. Methods:Laparotomy was done on C57/BL6 mice followed by pancreatic duct (PD) ligation, bile duct (BD) ligation without PD ligation, or sham operation. Results:Only mice with PD ligation developed acute pancreatitis and had 100 mortality. Pulmonary compliance was significantly reduced after PD ligation but not BD ligation. Bronchoalveolar lavage fluid neutrophil count and interleukin-1β concentration, and the plasma creatinine level, were significantly elevated with PD ligation but not BD ligation. Pancreatic nuclear factor κB (p65) and activator protein 1 (c-Jun) were activated within 1 h of PD ligation. Conclusion:PD-ligation-induced acute pancreatitis in mice is associated with systemic inflammation, acute lung injury, multiorgan dysfunction and death. The development of this novel model is an exciting and notable advance in the field.Copyright © 2010 S. Karger AG, Basel and IAP

Background: Chronic consumption of ethanol (EtOH) is well recognized to lead to defective innate and adaptive immune responses and increase the severity of pulmonary infections. Our own studies have demonstrated that chronic EtOH consumption decreases CD8 T‐cell immunity to influenza virus infections (IAV) leading to severe infections and mortality. Interestingly, antiviral treatment of IAVs has been shown to be compromised in mice and humans that are immuno‐deficient. It is known that EtOH can alter the pharmacokinetics of antivirals. Therefore, the effectiveness of influenza antiviral therapy during chronic ethanol consumption remains in question.

Cystic fibrosis (CF), which is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), is characterized by multiorgan pathology that begins early in life. To better understand the initial stages of disease, we studied the gastrointestinal pathology of CFTR−/−pigs. By studying newborns, we avoided secondary changes attributable to environmental interactions, infection, or disease progression. Lesions resembling those in humans with CF were detected in intestine, pancreas, liver, gallbladder, and cystic duct. These organs had four common features. First, disease was accelerated compared with that in humans, which could provide a strategy to discover modifying factors. Second, affected organs showed variable hyperplastic, metaplastic, and connective tissue changes, indicating that remodeling was a dynamic component of fetal life. Third, cellular inflammation was often mild to moderate and not always present, which raises new questions as to the role of cellular inflammation in early disease pathogenesis. Fourth, epithelial mucus-producing cells were often increased, producing a striking accumulation of mucus with a layered appearance and resilient structure. Thus, mucus cell hyperplasia and mucus accumulation play prominent roles in early disease. Our findings also have implications for CF lung disease, and they lay the foundation for a better understanding of CF pathogenesis.

The addition of drotrecogin alfa (DA), an anti-inflammatory useful in septic shock, to standard burn shock resuscitation fluids will protect burned, injured skin from further injury.

The collectins surfactant-associated protein A (SP-A) and SP-D are components of innate immunity that are present before birth. Both proteins bind pathogens and assist in clearing infection. The significance of SP-A and SP-D as components of the neonatal immune system has not been investigated. To determine the role of SP-A and SP-D in neonatal immunity, wild-type, SP-A null, and SP-D null mice were bred in a bacterium-laden environment (corn dust bedding) or in a semisterile environment (cellulose fiber bedding). When reared in the corn dust bedding, SP-A null pups had significant mortality (P < 0.001) compared to both wild-type and SP-D null pups exposed to the same environment. The mortality of the SP-A null pups was associated with significant gastrointestinal tract pathology but little lung pathology. Moribund SP-A null newborn mice exhibited Bacillus sp. and Enterococcus sp. peritonitis. When the mother or newborn produced SP-A, newborn survival was significantly improved (P < 0.05) compared to the results when there was a complete absence of SP-A in both the mother and the pup. Significant sources of SP-A likely to protect a newborn include the neonatal lung and gastrointestinal tract but not the lactating mammary tissue of the mother. Furthermore, exogenous SP-A delivered by mouth to newborn SP-A null pups with SP-A null mothers improved newborn survival in the corn dust environment. Therefore, a lack of SP-D did not affect newborn survival, while SP-A produced by either the mother or the pup or oral exogenous SP-A significantly reduced newborn mortality associated with environmentally induced infection in SP-A null newborns.

Virtual microscopy (VM) has been implemented and evaluated in the histology and general and systemic pathology courses at the University of Iowa Carver College of Medicine. Advantages of VM over traditional microscopy include accessibility and efficiency of learning and the ability to integrate VM with computer-assisted interactive learning. Advantages of using VM as opposed to digital photomicrographs include the ability to pan and zoom, explore the slide, and make independent observations. Although VM is used in a case-based format for teaching histopathology to medical students at the University of Iowa, VM may also be effectively implemented in other medical-student teaching models, including integrated and problem-based learning curricula and the classical pathology laboratory. Additional Iowa venues and courses using VM teaching include pathology of human disease for bioscience graduate students, cytology education, a comparative pathology research resource, and histology and histopathology for veterinary medicine. This article reviews the history and evolution of VM in medical pathology and its implementation at Iowa.

Preterm infants have increased susceptibility to severe manifestations of respiratory syncytial virus (RSV) infection. The cause(s) for this age-dependent vulnerability is/are not well-defined, but alterations in innate immune products have been implicated. In sheep, RSV disease severity has similar age-dependent characteristics and sheep have several related innate molecules for study during pulmonary infection including surfactant protein A (SP-A), surfactant protein D (SP-D), sheep beta defensin 1 (SBD1), monocyte chemotactic protein 1 (MCP1), and Toll-like receptor 4 (TLR4). However, the in vivo cellular gene expression as a response to RSV infection is poorly understood. In this study, the effect of RSV infection on expression of these innate immune genes was determined for bovine RSV-infected (bRSV+ fluorescence) epithelial cells, adjacent cells lacking bRSV antigen (adjoining cells lacking fluorescence), and control cells from non-infected lung using laser capture microdissection (LCM) and real-time RT-PCR. Control lambs had increased expression of innate immune molecules in full term (term) compared to preterm epithelia with statistical significance in SBD1, SP-D, and TLR4 mRNA. Infected cells (bRSV+ fluorescent cells) had consistently higher mRNA levels of SP-A (preterm and term), MCP1 (preterm and term), and SP-D (preterm). Interestingly, bRSV- cells of infected term lambs had significantly reduced SP-D mRNA expression compared to bRSV+ and control epithelia, suggesting that RSV infected cells may regulate the adjacent epithelial SP-D expression. This study defines specific innate immune components (e.g., SBD1, SP-D, and TLR4) that have differential age-dependent expression in the airway epithelia. Furthermore, cellular bRSV infection enhanced certain innate immune components while suppressing adjacent cellular SP-D expression in term animals. These in vivo gene expression results provide a framework for future studies on age-dependent susceptibility to RSV and RSV pathogenesis.

The purpose of this study was to characterize the incidence and diagnostic features of amyloidosis and other diseases found at necropsy in captive trumpeter swans (Cygnus buccinator). A search of Iowa State University's Department of Veterinary Pathology and Veterinary Diagnostic Laboratory databases yielded 31 trumpeter swan (C. buccinator) necropsy cases from captive swans in protected habitats. Eleven of the 31 birds had amyloid deposition most commonly in the spleen (8 of 11), liver (7 of 11), and kidney (6 of 11) and less often in the pancreas (2 of 11) and adrenal gland (2 of 11). Amyloid deposition effaced normal tissue with adjacent necrosis and hemorrhage in severe cases. Amyloidosis was most often diagnosed in February and March. Other disease diagnoses in the trumpeter swans included aspergillosis (5 of 31, 16%); bacterial infection (5 of 31, 16%); lead toxicosis (3 of 31, 10%); gout (2 of 31, 6%); parasitic infection (2 of 31, 6%); vitamin E deficiency (1 of 31, 3%); trauma (1 of 31, 3%); and ventricular foreign body (1 of 31, 3%). Histopathologic, toxicologic, and microbiologic analyses did not define an etiologic diagnosis in the deaths of 9 trumpeter swans. In these cases, necropsy lesions included emaciation (5 of 9), enteritis (1 of 9), pulmonary hemorrhage (1 of 9), and no lesions (3 of 9). The number of trumpeter swan case submissions was greatest in January and February. This study provides a reference for veterinary diagnosticians concerning incidence and diagnostic features of amyloidosis and other diseases in captive trumpeter swans of the midwestern United States.

ABSTRACTParainfluenza 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.

ABSTRACTDefensins and surfactant protein A (SP-A) and SP-D are antimicrobial components of the pulmonary innate immune system. The purpose of this study was to determine the extent to which parainfluenza type 3 virus infection in neonatal lambs alters expression of sheep beta-defensin 1 (SBD-1), SP-A, and SP-D, all of which are constitutively transcribed by respiratory epithelia. Parainfluenza type 3 viral antigen was detected by immunohistochemistry (IHC) in the bronchioles of all infected lambs 3 days postinoculation and at diminished levels 6 days postinoculation, but it was absent 17 days postinoculation. At all times postinoculation, lung homogenates from parainfluenza type 3 virus-inoculated animals had increased SBD-1, SP-A, and SP-D mRNA levels as detected by fluorogenic real-time reverse transcriptase PCR. Protein levels of SP-A in lung homogenates detected by quantitative-competitive enzyme-linked immunosorbent assay and protein antigen of SP-A detected by IHC were not altered. These studies demonstrate that parainfluenza type 3 virus infection results in enhanced expression of constitutively transcribed innate immune factors expressed by respiratory epithelia and that this increased expression occurs concurrently with decreased viral replication.

Segmented filamentous bacteria (SFB) are found in multiple species and play an important role in the development of mucosal immunity. The mechanism by which the bacteria interact with the immune system has not been well defined. We provide morphologic evidence of direct interaction between SFB and intraepithelial mononuclear cells.

Salmonella entericaserotype Typhimurium phagetype DT104 (DT104) is a multiple antibiotic-resistant pathogen. DT104 infections have been reported in a multitude of hosts including humans, companion animals, livestock and wildlife. Recently, several isolates of DT104 were recovered from veal calves exhibiting abomasitis, a finding that is inconsistent with classic salmonellosis. One of these isolates was used in murine ligated loop experiments where it was observed that multiresistant DT104 can elaborate a putative cytotoxin. Thus it appears that DT104 has the ability to evade pharmacologic interventions, via antibiotic resistance, and elaborate a toxin that can damage cells.

The causative agent of COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), requires host cell surface proteases for membrane fusion and entry into airway epithelia. We tested the hypothesis that inhibitors of these proteases, the serine protease inhibitors camostat and nafamostat, block infection by SARS-CoV-2.

Optimized small-molecule 3C-like protease inhibitors show potency against human coronaviruses in both cell culture and a mouse model.

MERS-CoV causes lethal infection in humans, and there is no vaccine. Our work demonstrates that PIV5 is a promising vector for developing a MERS vaccine. Furthermore, success of PIV5-based MERS vaccine can be employed to develop a vaccine for emerging CoVs such as SARS-CoV-2, which causes COVID-19.

A 5-year-old pet rabbit (Oryctolagus cuniculus) died after a 3-day history of anorexia and depression. At necropsy, the stomach was distended with dough-like ingesta and hair consistent with gastric stasis syndrome. The lungs had multifocal, raised red nodules with circumferential hemorrhage. Microscopic examination showed pulmonary hemorrhage with intravascular fibrin thrombi and bacterial colonies, which were present in lesser amounts in the kidney, heart, and liver. Bacterial culture of the lung produced a heavy pure growth of Actinobacillus capsulatus.Acute septicemia is a novel presentation for this pathogen. This is the first documented case of A. capsulatusdisease in the contiguous United States and may represent an underdiagnosed to emerging disease of lagomorphs.

ABSTRACTADP-ribosylation is a common posttranslational modification that may have antiviral properties and impact innate immunity. To regulate this activity, macrodomain proteins enzymatically remove covalently attached ADP-ribose from protein targets. All members of the Coronavirinae, a subfamily of positive-sense RNA viruses, contain a highly conserved macrodomain within nonstructural protein 3 (nsp3). However, its function or targets during infection remain unknown. We identified several macrodomain mutations that greatly reduced nsp3’s de-ADP-ribosylation activity in vitro. Next, we created recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) strains with these mutations. These mutations led to virus attenuation and a modest reduction of viral loads in infected mice, despite normal replication in cell culture. Further, macrodomain mutant virus elicited an early, enhanced interferon (IFN), interferon-stimulated gene (ISG), and proinflammatory cytokine response in mice and in a human bronchial epithelial cell line. Using a coinfection assay, we found that inclusion of mutant virus in the inoculum protected mice from an otherwise lethal SARS-CoV infection without reducing virus loads, indicating that the changes in innate immune response were physiologically significant. In conclusion, we have established a novel function for the SARS-CoV macrodomain that implicates ADP-ribose in the regulation of the innate immune response and helps to demonstrate why this domain is conserved in CoVs.IMPORTANCEThe macrodomain is a ubiquitous structural domain that removes ADP-ribose from proteins, reversing the activity of ADP-ribosyltransferases. All coronaviruses contain a macrodomain, suggesting that ADP-ribosylation impacts coronavirus infection. However, its function during infection remains unknown. Here, we found that the macrodomain is an important virulence factor for a highly pathogenic human CoV, SARS-CoV. Viruses with macrodomain mutations that abrogate its ability to remove ADP-ribose from protein were unable to cause lethal disease in mice. Importantly, the SARS-CoV macrodomain suppressed the innate immune response during infection. Our data suggest that an early innate immune response can protect mice from lethal disease. Understanding the mechanism used by this enzyme to promote disease will open up novel avenues for coronavirus therapies and give further insight into the role of macrodomains in viral pathogenesis.

Ca2+/calmodulin-dependent protein kinase (CaMKII) transduces oxidative stress into asthma-related diseases.

A common mutation in human cystic fibrosis, CFTR-ΔF508, results in misprocessed CFTR and a cystic fibrosis–like clinical phenotype in pigs.

Our laboratory recently generated a cystic fibrosis transmembrane conductance regulator gene (CFTR) knockout (KO) ferret model of cystic fibrosis (CF) using viral gene-targeting and somatic cell nuclear transfer (SCNT). Here we describe the phenotype of CFTR null ferrets and methods for genetic complementation of disease using tissue-specific transgenesis in CFTR-KO fibroblasts by SCNT. Neonatal CFTR-KO ferrets demonstrated many of the characteristics of human CF disease, including defective airway chloride transport, variably penetrant meconium ileus (MI), pancreatic and liver disease. 75% of all CFTR-KO kits die from meconium ileus with in 36-48 hrs after birth. Severe malabsorption by the gastrointestinal tract was the primary cause of death in the 25% of CF kits that escaped MI. Since a major reason for generating the CFTR-KO ferret model was to study the progression of lung disease in adult animals, methods to overcome the limitations imposed by the severe gut phenotype in this model were needed. To this end, we cloned a gut-corrected CFTR-KO ferret by somatic cell nuclear transfer using transgenic CFTR-KO fibroblasts expressing fCFTR under the direction of the intestine-specific fatty acid protein (FABPi) promoter. A transgenic construct containing the rat FABPi promoter driving the fCFTR cDNA and a floxed PGK promoter driven zeocin resistant gene cassette was engineered and transfected into ferret fibroblasts generated from a 28 day CFTR-KO embryo. The transfected cells were selected in zeocin and the pooled cells (i.e., non-clonal) were used for SCNT. Three clones were born, but only one survived the early post-natal period and passed meconium. The two additional clones born had to be euthanized within 36 hrs after birth due to severe MI with microcolon. The surviving clone was euthanized to determine the expression patterns of recombinant fCFTR and this clone had a grossly normal intestine lacking any signs of MI. Fibroblasts, liver, lung, and intestine were harvested from this MI-complemented founder clone for analysis of recombinant fCFTR tissue expression patterns prior to nuclear transfer recloning and expansion of the line. Results from analysis of this MI-complemented FABPi-fCFTR/CFTR-KO clone demonstrated the presence of the transgene cassette in the genomic DNA and recombinant fCFTR protein expression in the intestine but not in the lung and liver (endogenous fCFTR protein was expressed in all these organs from wild type ferrets but not CFTR-KO ferrets). In a separate set of experiments, we cloned non-transgenic CFTR-KO kits with fibroblasts derived from a CFTR-KO kit that suffered from severe MI with microcolon; all four clones born from this experiment suffered from MI with varying degrees of microcolon. Thus, the intestinal phenotype of cloned CFTR-KO kits appears to closely reflect that of the CFTR-KO founder. Cumulatively, these findings demonstrate the successful generation of a transgenic FABPi-fCFTR/CFTR-KO founder for which tissue-specific expression of recombinant fCFTR in the intestine can correct the MI phenotype. Currently, nuclear transfer is being performed to expand the FABPi-fCFTR/CFTR-KO line with and without CRE-excision of the PGK-Zeocin cassette. This first demonstration of generating a transgenic ferret proves the feasibility of promoter-directed complementation in the ferret by SCNT, and may significantly enhance the utility of the CF ferret model for research.(poster)

The lungs of just-born piglets with cystic fibrosis fail to efficiently eliminate bacteria, suggesting that lung problems in cystic fibrosis patients may be secondary to impaired antibacterial defense mechanisms.