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1.  Overexpression of eCLCA1 in Small Airways of Horses with Recurrent Airway Obstruction 
SUMMARY
The human hCLCA1 and murine mCLCA3 (chloride channels, calcium-activated) have recently been identified as promising therapeutic targets in asthma. Recurrent airway obstruction in horses is an important animal model of human asthma. Here, we have cloned and characterized the first equine CLCA family member, eCLCA1. The 913 amino acids eCLCA1 polypeptide forms a 120-kDa transmembrane glycoprotein that is processed to an 80-kDa protein in vivo. Three single nucleotide polymorphisms were detected in the eCLCA1 coding region in 14 horses, resulting in two amino acid changes (485H/R and 490V/L). However, no functional differences were recorded between the channel properties of the two variants in transfected HEK293 cells. The eCLCA1 protein was detected immunohistochemically in mucin-producing cells in the respiratory and intestinal tracts, cutaneous sweat glands, and renal mucous glands. Strong overexpression of eCLCA1 was observed in the airways of horses with recurrent airway obstruction using Northern blot hybridization, Western blotting, immunohistochemistry, and real-time quantitative RT-PCR. The results suggest that spontaneous or experimental recurrent airway obstruction in horses may serve as a model to study the role of CLCA homologs in chronic airway disease with overproduction of mucins.
doi:10.1369/jhc.4A6599.2005
PMCID: PMC1383431  PMID: 15879574
asthma; chronic obstructive pulmonary disease; calcium-activated chloride channels; goblet cells; mucus overproduction
2.  The Novel Human Influenza A(H7N9) Virus Is Naturally Adapted to Efficient Growth in Human Lung Tissue 
mBio  2013;4(5):e00601-13.
ABSTRACT
A novel influenza A virus (IAV) of the H7N9 subtype has been isolated from severely diseased patients with pneumonia and acute respiratory distress syndrome and, apparently, from healthy poultry in March 2013 in Eastern China. We evaluated replication, tropism, and cytokine induction of the A/Anhui/1/2013 (H7N9) virus isolated from a fatal human infection and two low-pathogenic avian H7 subtype viruses in a human lung organ culture system mimicking infection of the lower respiratory tract. The A(H7N9) patient isolate replicated similarly well as a seasonal IAV in explanted human lung tissue, whereas avian H7 subtype viruses propagated poorly. Interestingly, the avian H7 strains provoked a strong antiviral type I interferon (IFN-I) response, whereas the A(H7N9) virus induced only low IFN levels. Nevertheless, all viruses analyzed were detected predominantly in type II pneumocytes, indicating that the A(H7N9) virus does not differ in its cellular tropism from other avian or human influenza viruses. Tissue culture-based studies suggested that the low induction of the IFN-β promoter correlated with an efficient suppression by the viral NS1 protein. These findings demonstrate that the zoonotic A(H7N9) virus is unusually well adapted to efficient propagation in human alveolar tissue, which most likely contributes to the severity of lower respiratory tract disease seen in many patients.
IMPORTANCE
Humans are usually not infected by avian influenza A viruses (IAV), but this large group of viruses contributes to the emergence of human pandemic strains. Transmission of virulent avian IAV to humans is therefore an alarming event that requires assessment of the biology as well as pathogenic and pandemic potentials of the viruses in clinically relevant models. Here, we demonstrate that an early virus isolate from the recent A(H7N9) outbreak in Eastern China replicated as efficiently as human-adapted IAV in explanted human lung tissue, whereas avian H7 subtype viruses were unable to propagate. Robust replication of the H7N9 strain correlated with a low induction of antiviral beta interferon (IFN-β), and cell-based studies indicated that this is due to efficient suppression of the IFN response by the viral NS1 protein. Thus, explanted human lung tissue appears to be a useful experimental model to explore the determinants facilitating cross-species transmission of the H7N9 virus to humans.
doi:10.1128/mBio.00601-13
PMCID: PMC3791893  PMID: 24105764
3.  Modulation of respiratory dendritic cells during Klebsiella pneumonia infection 
Respiratory Research  2013;14(1):91.
Background
Klebsiella pneumoniae is a leading cause of severe hospital-acquired respiratory tract infections and death but little is known regarding the modulation of respiratory dendritic cell (DC) subsets. Plasmacytoid DC (pDC) are specialized type 1 interferon producing cells and considered to be classical mediators of antiviral immunity.
Method
By using multiparameter flow cytometry analysis we have analysed the modulation of respiratory DC subsets after intratracheal Klebsiella pneumonia infection.
Results
Data indicate that pDCs and MoDC were markedly elevated in the post acute pneumonia phase when compared to mock-infected controls. Analysis of draining mediastinal lymph nodes revealed a rapid increase of activated CD103+ DC, CD11b+ DC and MoDC within 48 h post infection. Lung pDC identification during bacterial pneumonia was confirmed by extended phenotyping for 120G8, mPDCA-1 and Siglec-H expression and by demonstration of high Interferon-alpha producing capacity after cell sorting. Cytokine expression analysis of ex vivo-sorted respiratory DC subpopulations from infected animals revealed elevated Interferon-alpha in pDC, elevated IFN-gamma, IL-4 and IL-13 in CD103+ DC and IL-19 and IL-12p35 in CD11b+ DC subsets in comparison to CD11c+ MHC-class IIlow cells indicating distinct functional roles. Antigen-specific naive CD4+ T cell stimulatory capacity of purified respiratory DC subsets was analysed in a model system with purified ovalbumin T cell receptor transgenic naive CD4+ responder T cells and respiratory DC subsets, pulsed with ovalbumin and matured with Klebsiella pneumoniae lysate. CD103+ DC and CD11b+ DC subsets represented the most potent naive CD4+ T helper cell activators.
Conclusion
These results provide novel insight into the activation of respiratory DC subsets during Klebsiella pneumonia infection. The detection of increased respiratory pDC numbers in bacterial pneumonia may indicate possible novel pDC functions with respect to lung repair and regeneration.
doi:10.1186/1465-9921-14-91
PMCID: PMC3848864  PMID: 24044871
Klebsiella pneumonia; Pneumonia; Plasmacytoid dendritic cells
4.  Impaired Autoproteolytic Cleavage of mCLCA6, a Murine Integral Membrane Protein Expressed in Enterocytes, Leads to Cleavage at the Plasma Membrane Instead of the Endoplasmic Reticulum 
Molecules and Cells  2012;33(3):251-257.
CLCA proteins (calcium-activated chloride channel regulators) have been linked to diseases involving secretory disorders, including cystic fibrosis (CF) and asthma. They have been shown to modulate endogenous chloride conductance, possibly by acting as metalloproteases. Based on the differential processing of the subunits after post-translational cleavage, two subgroups of CLCA proteins can be distinguished. In one subgroup, both subunits are secreted, in the other group, the carboxy-terminal subunit possesses a transmembrane segment, resulting in shedding of only the amino-terminal subunit. Recent data on the post-translational cleavage and proteolytic activity of CLCA are limited to secreted CLCA. In this study, we characterized the cleavage of mCLCA6, a murine CLCA possessing a transmembrane segment. As for secreted CLCA, the cleavage in the endoplasmic reticulum was not observed for a protein with the E157Q mutation in the HEXXH motif of mCLCA6, suggesting that this mutant protein and secreted CLCA family members share a similar autoproteolytic cleavage mechanism. In contrast to secreted CLCA proteins with the E157Q mutation, the uncleaved precursor of the mCLCA6E157Q mutant reached the plasma membrane, where it was cleaved and the amino-terminal subunit was shed into the supernatant. Using crude membrane fractions, we showed that cleavage of the mCLCA6E157Q protein is zinc-dependent and sensitive to metalloprotease inhibitors, suggesting secondary cleavage by a metalloprotease. Interestingly, anchorage of mCLCA6E157Q to the plasma membrane is not essential for its secondary cleavage, because the mCLCA6Δ™E157Q mutant still underwent cleavage. Our data suggest that the processing of CLCA proteins is more complex than previously recognized.
doi:10.1007/s10059-012-2217-1
PMCID: PMC3887709  PMID: 22350745
CLCA protein; HEXXH zinc-binding amino-acid motif; metalloprotease
5.  Macrophage-expressed IFN-β Contributes to Apoptotic Alveolar Epithelial Cell Injury in Severe Influenza Virus Pneumonia 
PLoS Pathogens  2013;9(2):e1003188.
Influenza viruses (IV) cause pneumonia in humans with progression to lung failure and fatal outcome. Dysregulated release of cytokines including type I interferons (IFNs) has been attributed a crucial role in immune-mediated pulmonary injury during severe IV infection. Using ex vivo and in vivo IV infection models, we demonstrate that alveolar macrophage (AM)-expressed IFN-β significantly contributes to IV-induced alveolar epithelial cell (AEC) injury by autocrine induction of the pro-apoptotic factor TNF-related apoptosis-inducing ligand (TRAIL). Of note, TRAIL was highly upregulated in and released from AM of patients with pandemic H1N1 IV-induced acute lung injury. Elucidating the cell-specific underlying signalling pathways revealed that IV infection induced IFN-β release in AM in a protein kinase R- (PKR-) and NF-κB-dependent way. Bone marrow chimeric mice lacking these signalling mediators in resident and lung-recruited AM and mice subjected to alveolar neutralization of IFN-β and TRAIL displayed reduced alveolar epithelial cell apoptosis and attenuated lung injury during severe IV pneumonia. Together, we demonstrate that macrophage-released type I IFNs, apart from their well-known anti-viral properties, contribute to IV-induced AEC damage and lung injury by autocrine induction of the pro-apoptotic factor TRAIL. Our data suggest that therapeutic targeting of the macrophage IFN-β-TRAIL axis might represent a promising strategy to attenuate IV-induced acute lung injury.
Author Summary
Acute lung injury induced by influenza virus (IV) infection has been linked to an unbalanced release of pro-inflammatory cytokines including type I interferons (IFN) causing immune-mediated organ damage. Using ex vivo and in vivo IV infection models, we demonstrate that alveolar macrophage-expressed IFN-β induces alveolar epithelial cell injury by autocrine induction of the pro-apoptotic TNF-related apoptosis-inducing ligand (TRAIL). Elucidating the cell-specific underlying signalling pathways revealed that IV-induced IFN-β release from alveolar macrophages (AM) strictly depended on protein kinase R- (PKR-) and NF-κB-signalling. Autocrine activation via the macrophage type I IFN receptor (IFNAR) resulted in increased expression and release of TRAIL which caused apoptosis of IV-infected and non-infected alveolar epithelial cells and promoted alveolar barrier dysfunction as demonstrated in ex vivo co-cultures and in bone marrow chimeric mouse models in vivo. Importantly, we found TRAIL highly upregulated in and released from AM of hospitalized patients with pandemic H1N1-induced lung failure. Therapeutic targeting of the macrophage IFN-β-TRAIL axis might therefore represent a promising strategy to attenuate IV-induced acute lung injury.
doi:10.1371/journal.ppat.1003188
PMCID: PMC3585175  PMID: 23468627
6.  Modulation of the host Th1 immune response in pigeon protozoal encephalitis caused by Sarcocystis calchasi 
Veterinary Research  2013;44(1):10.
Pigeon protozoal encephalitis (PPE) is an emerging central-nervous disease of domestic pigeons (Columba livia f. domestica) reported in Germany and the United States. It is caused by the apicomplexan parasite Sarcocystis calchasi which is transmitted by Accipter hawks. In contrast to other members of the Apicomplexa such as Toxoplasma and Plasmodium, the knowledge about the pathophysiology and host manipulation of Sarcocystis is scarce and almost nothing is known about PPE. Here we show by mRNA expression profiling a significant down-modulation of the interleukin (IL)-12/IL-18/interferon (IFN)-γ axis in the brains of experimentally infected pigeons during the schizogonic phase of disease. Concomitantly, no cellular immune response was observed in histopathology while immunohistochemistry and nested PCR detected S. calchasi. In contrast, in the late central-nervous phase, IFN-γ and tumor necrosis factor (TNF) α-related cytokines were significantly up-modulated, which correlated with a prominent MHC-II protein expression in areas of mononuclear cell infiltration and necrosis. The mononuclear cell fraction was mainly composed of T-lymphocytes, fewer macrophages and B-lymphocytes. Surprisingly, the severity and composition of the immune cell response appears unrelated to the infectious dose, although the severity and onset of the central nervous signs clearly was dose-dependent. We identified no or only very few tissue cysts by immunohistochemistry in pigeons with severe encephalitis of which one pigeon repeatedly remained negative by PCR despite severe lesions. Taken together, these observations may suggest an immune evasion strategy of S. calchasi during the early phase and a delayed-type hypersensitivity reaction as cause of the extensive cerebral lesions during the late neurological phase of disease.
doi:10.1186/1297-9716-44-10
PMCID: PMC3598538  PMID: 23398807
7.  The C-Type Lectin Receptor SIGNR3 Binds to Fungi Present in Commensal Microbiota and Influences Immune Regulation in Experimental Colitis 
Inflammatory bowel disease is a condition of acute and chronic inflammation of the gut. An important factor contributing to pathogenesis is a dysregulated mucosal immunity against commensal bacteria and fungi. Host pattern-recognition receptors (PRRs) sense commensals in the gut and are involved in maintaining the balance between controlled responses to pathogens and overwhelming innate immune activation. C-type lectin receptors (CLRs) are PRRs recognizing glycan structures on pathogens and self-antigens. Here we examined the role of the murine CLR specific intracellular adhesion molecule-3 grabbing non-integrin homolog-related 3 (SIGNR3) in the recognition of commensals and its involvement in intestinal immunity. SIGNR3 is the closest murine homolog of the human dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) receptor recognizing similar carbohydrate ligands such as terminal fucose or high-mannose glycans. We discovered that SIGNR3 recognizes fungi present in the commensal microbiota. To analyze whether this interaction impacts the intestinal immunity against microbiota, the dextran sulfate sodium-induced colitis model was employed. SIGNR3−/− mice exhibited an increased weight loss associated with more severe colitis symptoms compared to wild-type control mice. The increased inflammation in SIGNR3−/− mice was accompanied by a higher level of TNF-α in colon. Our findings demonstrate for the first time that SIGNR3 recognizes intestinal fungi and has an immune regulatory role in colitis.
doi:10.3389/fimmu.2013.00196
PMCID: PMC3712271  PMID: 23882266
SIGNR3; C-type lectin receptor; host innate immunity; colitis; carbohydrate recognition; microbiota; fungi
8.  The Murine Goblet Cell Protein mCLCA3 Is a Zinc-Dependent Metalloprotease with Autoproteolytic Activity 
Molecules and Cells  2011;32(6):535-541.
Several members of the CLCA family of proteins, originally named chloride channels, calcium-activated, have been shown to modulate chloride conductance in various cell types via an unknown mechanism. Moreover, the human (h) hCLCA1 is thought to modulate the severity of disease in asthma and cystic fibrosis (CF) patients. All CLCA proteins are post-translationally cleaved into two subunits, and recently, a conserved HEXXH zinc-binding amino acid motif has been identified, suggesting a role for CLCA proteins as metalloproteases. Here, we have characterized the cleavage and autoproteolytic activity of the murine model protein mCLCA3, which represents the murine orthologue of human hCLCA1. Using crude membrane fractions from transfected HEK293 cells, we demonstrate that mCLCA3 cleavage is zinc-dependent and exclusively inhibited by cation-chelating metalloprotease inhibitors. Cellular transport and secretion were not affected in response to a cleavage defect that was introduced by the insertion of an E157Q mutation within the HEXXH motif of mCLCA3. Interspecies conservation of these key results was further confirmed with the porcine (p) orthologue of hCLCA1 and mCLCA3, pCLCA1. Importantly, the mCLCA3E157Q mutant was cleaved after co-transfection with the wild-type mCLCA3 in HEK293 cells, suggesting that an intermolecular autoproteolytic event takes place. Edman degradation and MALDI-TOF-MS of the protein fragments identified a single cleavage site in mCLCA3 between amino acids 695 and 696. The data strongly suggest that secreted CLCA proteins have zinc-dependent autoproteolytic activity and that they may cleave additional proteins.
doi:10.1007/s10059-011-0158-8
PMCID: PMC3887686  PMID: 22080371
CLCA; cystic fibrosis; HEXXH motif; intermolecular auto-proteolysis; metalloprotease
9.  Molecular characterization of the feline T-cell receptor γ alternate reading frame protein (TARP) ortholog 
Journal of Veterinary Science  2012;13(4):345-353.
T-cell receptor γ alternate reading frame protein (TARP) is expressed by human prostate epithelial, prostate cancer, and mammary cancer cells, but is not found in normal mammary tissue. To date, this protein has only been described in humans. Additionally, no animal model has been established to investigate the potential merits of TARP as tumor marker or a target for adoptive tumor immunotherapy. In this study conducted to characterize feline T-cell receptor γ sequences, constructs very similar to human TARP transcripts were obtained by RACE from the spleen and prostate gland of cats. Transcription of TARP in normal, hyperplastic, and neoplastic feline mammary tissues was evaluated by conventional RT-PCR. In felines similarly to the situation reported in humans, a C-region encoding two open reading frames is spliced to a J-region gene. In contrast to humans, the feline J-region gene was found to be a pseudogene containing a deletion within its recombination signal sequence. Our findings demonstrated that the feline TARP ortholog is transcribed in the prostate gland and mammary tumors but not normal mammary tissues as is the case with human TARP.
doi:10.4142/jvs.2012.13.4.345
PMCID: PMC3539119  PMID: 23271175
cat; C-region; J-region; T-cell receptor γ alternate reading frame protein (TARP); tumor marker
10.  Transcriptome and Proteome Research in Veterinary Science: What Is Possible and What Questions Can Be Asked? 
The Scientific World Journal  2012;2012:254962.
In recent years several technologies for the complete analysis of the transcriptome and proteome have reached a technological level which allows their routine application as scientific tools. The principle of these methods is the identification and quantification of up to ten thousands of RNA and proteins species in a tissue, in contrast to the sequential analysis of conventional methods such as PCR and Western blotting. Due to their technical progress transcriptome and proteome analyses are becoming increasingly relevant in all fields of biological research. They are mainly used for the explorative identification of disease associated complex gene expression patterns and thereby set the stage for hypothesis-driven studies. This review gives an overview on the methods currently available for transcriptome analysis, that is, microarrays, Ref-Seq, quantitative PCR arrays and discusses their potentials and limitations. Second, the most powerful current approaches to proteome analysis are introduced, that is, 2D-gel electrophoresis, shotgun proteomics, MudPIT and the diverse technological concepts are reviewed. Finally, experimental strategies for biomarker discovery, experimental settings for the identification of prognostic gene sets and explorative versus hypothesis driven approaches for the elucidation of diseases associated genes and molecular pathways are described and their potential for studies in veterinary research is highlighted.
doi:10.1100/2012/254962
PMCID: PMC3259802  PMID: 22262952
11.  Avian Malaria Deaths in Parrots, Europe 
Emerging Infectious Diseases  2011;17(5):950-952.
doi:10.3201/eid1705.101618
PMCID: PMC3338161  PMID: 21529428
malaria; parasites; bird; cytochrome b; Haemoproteus; Plasmodium; Leucocytozoon; host specificity; Besnoitia; Passeriformes; Psittaciformes; letter
12.  Virulence Determinants of Avian H5N1 Influenza A Virus in Mammalian and Avian Hosts: Role of the C-Terminal ESEV Motif in the Viral NS1 Protein ▿  
Journal of Virology  2010;84(20):10708-10718.
We assessed the prediction that access of the viral NS1 protein to cellular PDZ domain protein networks enhances the virulence of highly pathogenic avian influenza A viruses. The NS1 proteins of most avian influenza viruses bear the C-terminal ligand sequence Glu-Ser-Glu-Val (ESEV) for PDZ domains present in multiple host proteins, whereas no such motif is found in the NS1 homologues of seasonal human virus strains. Previous analysis showed that a C-terminal ESEV motif increases viral virulence when introduced into the NS1 protein of mouse-adapted H1N1 influenza virus. To examine the role of the PDZ domain ligand motif in avian influenza virus virulence, we generated three recombinants, derived from the prototypic H5N1 influenza A/Vietnam/1203/04 virus, expressing NS1 proteins that either have the C-terminal ESEV motif or the human influenza virus RSKV consensus or bear a natural truncation of this motif, respectively. Cell biological analyses showed strong control of NS1 nuclear migration in infected mammalian and avian cells, with only minor differences between the three variants. The ESEV sequence attenuated viral replication on cultured human, murine, and duck cells but not on chicken fibroblasts. However, all three viruses caused highly lethal infections in mice and chickens, with little difference in viral titers in organs, mean lethal dose, or intravenous pathogenicity index. These findings demonstrate that a PDZ domain ligand sequence in NS1 contributes little to the virulence of H5N1 viruses in these hosts, and they indicate that this motif modulates viral replication in a strain- and host-dependent manner.
doi:10.1128/JVI.00610-10
PMCID: PMC2950580  PMID: 20686040
13.  Does a Feline Leukemia Virus Infection Pave the Way for Bartonella henselae Infection in Cats? ▿  
Journal of Clinical Microbiology  2010;48(9):3295-3300.
Domestic cats serve as the reservoir hosts of Bartonella henselae and may develop mild clinical symptoms or none after experimental infection. In humans, B. henselae infection can result in self-limiting cat scratch disease. However, immunocompromised patients may suffer from more-severe courses of infection or may even develop the potentially lethal disease bacillary angiomatosis. It was reasoned that cats with immunocompromising viral infections may react similarly to B. henselae infection. The aim of our study was to investigate the influence of the most important viruses known to cause immunosuppression in cats—Feline leukemia virus (FeLV), Feline immunodeficiency virus (FIV), and Feline panleukopenia virus (FPV)—on natural B. henselae infection in cats. Accordingly, 142 cats from animal shelters were necropsied and tested for B. henselae and concurrent infections with FeLV, FIV, or FPV by PCR and immunohistochemistry. A significant association was found between B. henselae and FeLV infections (P = 0.00028), but not between B. henselae and FIV (P = 1.0) or FPV (P = 0.756) infection, age (P = 0.392), or gender (P = 0.126). The results suggest that susceptibility to B. henselae infection is higher in cats with concurrent FeLV infections, regardless of whether the infection is latent or progressive. Histopathology and immunohistochemistry for B. henselae failed to identify lesions that could be attributed specifically to B. henselae infection. We conclude that the course of natural B. henselae infection in cats does not seem to be influenced by immunosuppressive viral infections in general but that latent FeLV infection may predispose cats to B. henselae infection or persistence.
doi:10.1128/JCM.00750-10
PMCID: PMC2937684  PMID: 20610682
14.  Metastatic canine mammary carcinomas can be identified by a gene expression profile that partly overlaps with human breast cancer profiles 
BMC Cancer  2010;10:618.
Background
Similar to human breast cancer mammary tumors of the female dog are commonly associated with a fatal outcome due to the development of distant metastases. However, the molecular defects leading to metastasis are largely unknown and the value of canine mammary carcinoma as a model for human breast cancer is unclear. In this study, we analyzed the gene expression signatures associated with mammary tumor metastasis and asked for parallels with the human equivalent.
Methods
Messenger RNA expression profiles of twenty-seven lymph node metastasis positive or negative canine mammary carcinomas were established by microarray analysis. Differentially expressed genes were functionally characterized and associated with molecular pathways. The findings were also correlated with published data on human breast cancer.
Results
Metastatic canine mammary carcinomas had 1,011 significantly differentially expressed genes when compared to non-metastatic carcinomas. Metastatic carcinomas had a significant up-regulation of genes associated with cell cycle regulation, matrix modulation, protein folding and proteasomal degradation whereas cell differentiation genes, growth factor pathway genes and regulators of actin organization were significantly down-regulated. Interestingly, 265 of the 1,011 differentially expressed canine genes are also related to human breast cancer and, vice versa, parts of a human prognostic gene signature were identified in the expression profiles of the metastatic canine tumors.
Conclusions
Metastatic canine mammary carcinomas can be discriminated from non-metastatic carcinomas by their gene expression profiles. More than one third of the differentially expressed genes are also described of relevance for human breast cancer. Many of the differentially expressed genes are linked to functions and pathways which appear to be relevant for the induction and maintenance of metastatic progression and may represent new therapeutic targets. Furthermore, dogs are in some aspects suitable as a translational model for human breast tumors in order to identify prognostic molecular signatures and potential therapeutic targets.
doi:10.1186/1471-2407-10-618
PMCID: PMC2994823  PMID: 21062462
15.  Genetic and diet effects on Ppar-α and Ppar-γ signaling pathways in the Berlin Fat Mouse Inbred line with genetic predisposition for obesity 
Background
The Berlin Fat Mouse Inbred (BFMI) line is a new mouse model for obesity, which was long-term selected for high fatness. Peroxisome proliferator-activated receptors (PPARs) are involved in the control of energy homeostasis, nutrient metabolism and cell proliferation. Here, we studied the expression patterns of the different Ppar genes and the genes in the PPAR pathway in the BFMI line in comparison to physiological changes.
Results
At the age of 10 weeks, the BFMI mice exhibited marked obesity with enlarged adipocytes and high serum triglycerides concentrations in comparison to the often used mouse line C57BL/6 (B6). Between these two lines, gene expression analyses revealed differentially expressed genes belonging to the PPAR pathway, in particular genes of the lipogenesis and the fatty acid transport.
Conclusion
Surprisingly, the Ppar-α gene expression was up-regulated in liver and Ppar-γ gene expression was down-regulated in the white adipose tissue, indicating the activation of a mechanism that counteracts the rise of obesity.
doi:10.1186/1476-511X-9-99
PMCID: PMC2944240  PMID: 20831792
16.  HEPACAM1 and 2 are differentially regulated in canine mammary adenomas and carcinomas and its lymph node metastases 
Background
Cell adhesion is an important regulator of cell growth and motility. Recently the hepatocyte cell adhesion molecules 1 and 2 (HEPACAM1 and 2), members of the immunoglobulin family of adhesion genes, have been identified. HEPACAM1 is involved in negative cell cycle regulation via p53, p21 and p27 signalling but also mediates increased human breast cancer cell spread. The role and expression pattern of HEPACAM2 has not been analyzed so far. In the present study we quantified gene expression levels of HEPACAM1 and 2 to evaluate their possible role during the carcinogenesis of canine mammary tumours.
Results
Adenomas displayed increased HEPACAM1 and 2 mRNA expression levels and decreased HEPACAM1 protein expression levels when compared to normal gland, carcinomas and lymph node metastases. In contrast, metastatic carcinomas, intravascular tumour cells and lymph node metastases had HEPACAM 1 protein and mRNA expression levels similar to normal gland but decreased HEPACAM2 mRNA expression when compared to normal gland of the same dog.
Conclusions
HEPACAM1 and 2 seem to be important for cell-cell adhesion of normal and neoplastic canine mammary cells. The loss of HEPACAM1 protein expression in adenomas but not in carcinomas questions its role as a tumour suppressor at late stages of malignant transformation and indicates that it might rather be involved in physiologic mammary cell adhesion and canine mammary tumour metastasis. Furthermore, it can be speculated, whether HEPACAM2 plays a different role in malignancy and metastasis of canine mammary tumours since its transcriptional levels are different in carcinomas and their lymph node metastases when compared to HEPACAM1.
doi:10.1186/1746-6148-6-15
PMCID: PMC2842258  PMID: 20226097
17.  Sarcocystis Species Lethal for Domestic Pigeons 
Emerging Infectious Diseases  2010;16(3):497-499.
A large number of Sarcocystis spp. infect birds as intermediate hosts, but pigeons are rarely affected. We identified a novel Sarcocystis sp. that causes lethal neurologic disease in domestic pigeons in Germany. Experimental infections indicated transmission by northern goshawks, and sequence analyses indicated transnational distribution. Worldwide spread is possible.
doi:10.3201/eid1603.090860
PMCID: PMC3322016  PMID: 20202429
Apicomplexa; protozoa; polyuria; depression; torticollis; encephalitis; ribosomal; RNA; 18S; 28S; parasites; dispatch
18.  Host Genetic Background Strongly Influences the Response to Influenza A Virus Infections 
PLoS ONE  2009;4(3):e4857.
The genetic make-up of the host has a major influence on its response to combat pathogens. For influenza A virus, several single gene mutations have been described which contribute to survival, the immune response and clearance of the pathogen by the host organism. Here, we have studied the influence of the genetic background to influenza A H1N1 (PR8) and H7N7 (SC35M) viruses. The seven inbred laboratory strains of mice analyzed exhibited different weight loss kinetics and survival rates after infection with PR8. Two strains in particular, DBA/2J and A/J, showed very high susceptibility to viral infections compared to all other strains. The LD50 to the influenza virus PR8 in DBA/2J mice was more than 1000-fold lower than in C57BL/6J mice. High susceptibility in DBA/2J mice was also observed after infection with influenza strain SC35M. In addition, infected DBA/2J mice showed a higher viral load in their lungs, elevated expression of cytokines and chemokines, and a more severe and extended lung pathology compared to infected C57BL/6J mice. These findings indicate a major contribution of the genetic background of the host to influenza A virus infections. The overall response in highly susceptible DBA/2J mice resembled the pathology described for infections with the highly virulent influenza H1N1-1918 and newly emerged H5N1 viruses.
doi:10.1371/journal.pone.0004857
PMCID: PMC2654507  PMID: 19293935
19.  Phenotypic alterations in type II alveolar epithelial cells in CD4+ T cell mediated lung inflammation 
Respiratory Research  2007;8(1):47.
Background
Although the contribution of alveolar type II epithelial cell (AEC II) activities in various aspects of respiratory immune regulation has become increasingly appreciated, our understanding of the contribution of AEC II transcriptosome in immunopathologic lung injury remains poorly understood. We have previously established a mouse model for chronic T cell-mediated pulmonary inflammation in which influenza hemagglutinin (HA) is expressed as a transgene in AEC II, in mice expressing a transgenic T cell receptor specific for a class II-restricted epitope of HA. Pulmonary inflammation in these mice occurs as a result of CD4+ T cell recognition of alveolar antigen. This model was utilized to assess the profile of inflammatory mediators expressed by alveolar epithelial target cells triggered by antigen-specific recognition in CD4+ T cell-mediated lung inflammation.
Methods
We established a method that allows the flow cytometric negative selection and isolation of primary AEC II of high viability and purity. Genome wide transcriptional profiling was performed on mRNA isolated from AEC II isolated from healthy mice and from mice with acute and chronic CD4+ T cell-mediated pulmonary inflammation.
Results
T cell-mediated inflammation was associated with expression of a broad array of cytokine and chemokine genes by AEC II cell, indicating a potential contribution of epithelial-derived chemoattractants to the inflammatory cell parenchymal infiltration. Morphologically, there was an increase in the size of activated epithelial cells, and on the molecular level, comparative transcriptome analyses of AEC II from inflamed versus normal lungs provide a detailed characterization of the specific inflammatory genes expressed in AEC II induced in the context of CD4+ T cell-mediated pneumonitis.
Conclusion
An important contribution of AEC II gene expression to the orchestration and regulation of interstitial pneumonitis is suggested by the panoply of inflammatory genes expressed by this cell population, and this may provide insight into the molecular pathogenesis of pulmonary inflammatory states. CD4+ T cell recognition of antigen presented by AEC II cells appears to be a potent trigger for activation of the alveolar cell inflammatory transcriptosome.
doi:10.1186/1465-9921-8-47
PMCID: PMC1939847  PMID: 17610738
20.  Leptospirosis in Urban Wild Boars, Berlin, Germany 
Emerging Infectious Diseases  2007;13(5):739-742.
We found antibodies to leptospires in 25 (18%) of 141 wild boars from Berlin (95% confidence interval 12–25). Seropositivity was associated with chronic interstitial nephritis (odds ratio 10.5; p = 0.01), and leptospires were detected in kidney tissues. Wild boars represent a potential source for human leptospirosis in urban environments.
doi:10.3201/eid1305.061302
PMCID: PMC2738438  PMID: 17553254
Leptospirosis; Sus scrofa; urban; wildlife; zoonoses; dispatch
21.  E6/E7 Expression of Human Papillomavirus Type 20 (HPV-20) and HPV-27 Influences Proliferation and Differentiation of the Skin in UV-Irradiated SKH-hr1 Transgenic Mice▿  
Journal of Virology  2006;80(22):11153-11164.
The functional role of UV irradiation, in combination with the E6 and E7 proteins of the cutaneous human papillomavirus (HPV) types in the malignant conversion of benign papillomatous lesions, has not been elucidated. Transgenic SKH-hr1 hairless mice expressing HPV-20 and HPV-27 E6 and E7 proteins in the suprabasal compartment were generated and exposed to chronic UV irradiation. Histological and immunohistochemical examination of skin samples revealed enhanced proliferation of the epidermal layers and papilloma formation in both transgenic strains in comparison to what was observed with nontransgenic mice. Squamous cell carcinoma developed in the HPV-20 E6/E7 transgenic line as well as in the HPV-27 E6/E7 transgenic line. Several weeks after cessation of UV-B exposure, enhanced proliferation, as measured by BrdU incorporation, was maintained only in HPV-20 transgenic skin. Keratin 6 expression was increased in the transgenic mice throughout all cell layers. Expression of the differentiation markers involucrin and loricrin was reduced and disturbed. p63α expression was differentially regulated with high levels of cytoplasmic expression in clusters of cells in the granular layer of the skin in the transgenic lines 20 weeks after cessation of UV-B exposure, in contrast to uninterrupted staining in the nontransgenic lines. p53 was expressed in clusters of cells in nontransgenic and HPV-27 transgenic mice, in contrast to an even distribution in a higher number of cells in HPV-20 transgenic animals.
doi:10.1128/JVI.00954-06
PMCID: PMC1642157  PMID: 16971438
22.  Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 Regulates Tumor Necrosis Factor mRNA Stability and Translation Mainly by Altering Tristetraprolin Expression, Stability, and Binding to Adenine/Uridine-Rich Element 
Molecular and Cellular Biology  2006;26(6):2399-2407.
The mitogen-activated protein kinase (MAPK) p38/MAPK-activated protein kinase 2 (MK2) signaling pathway plays an important role in the posttranscriptional regulation of tumor necrosis factor (TNF), which is dependent on the adenine/uridine-rich element (ARE) in the 3′ untranslated region of TNF mRNA. After lipopolysaccharide (LPS) stimulation, MK2-deficient macrophages show a 90% reduction in TNF production compared to the wild type. Tristetraprolin (TTP), a protein induced by LPS, binds ARE and destabilizes TNF mRNA. Accordingly, macrophages lacking TTP produce large amounts of TNF. Here, we generated MK2/TTP double knockout mice and show that, after LPS stimulation, bone marrow-derived macrophages produce TNF mRNA and protein levels comparable to those of TTP knockout cells, indicating that in the regulation of TNF biosynthesis TTP is genetically downstream of MK2. In addition, we show that MK2 is essential for the stabilization of TTP mRNA, and phosphorylation by MK2 leads to increased TTP protein stability but reduced ARE affinity. These data suggest that MK2 inhibits the mRNA destabilizing activity of TTP and, in parallel, codegradation of TTP together, with the target mRNA resulting in increased cellular levels of TTP.
doi:10.1128/MCB.26.6.2399-2407.2006
PMCID: PMC1430282  PMID: 16508014
23.  Deletion of the Ferric Uptake Regulator Fur Impairs the In Vitro Growth and Virulence of Actinobacillus pleuropneumoniae  
Infection and Immunity  2005;73(6):3740-3744.
In order to investigate the role of the ferric uptake regulator Fur in the porcine lung pathogen Actinobacillus pleuropneumoniae, we constructed an isogenic in-frame deletion mutant, A. pleuropneumoniae Δfur. This mutant showed constitutive expression of transferrin-binding proteins, growth deficiencies in vitro, and reduced virulence in an aerosol infection model.
doi:10.1128/IAI.73.6.3740-3744.2005
PMCID: PMC1111875  PMID: 15908404
24.  T Cell–specific Inactivation of the Interleukin 10 Gene in Mice Results in Enhanced T Cell Responses but Normal Innate Responses to Lipopolysaccharide or Skin Irritation 
The Journal of Experimental Medicine  2004;200(10):1289-1297.
Interleukin (IL)-10 is a regulator of inflammatory responses and is secreted by a variety of different cell types including T cells. T regulatory cells have been shown to suppress immune responses by IL-10–dependent, but also IL-10–independent, mechanisms. Herein, we address the role of T cell–derived IL-10 in mice with an inactivation of the IL-10 gene restricted to T cells generated by Cre/loxP-mediated targeting of the IL-10 gene. Splenocytes from this T cell–specific mutant secrete increased amounts of proinflammatory cytokines after activation in vitro compared with show enhanced contact hypersensitivity reactions, and succumb to severe immunopathology upon infection with Toxoplasma gondii. Despite intact IL-10 genes in other cell types, the dysregulation of T cell responses observed in the T cell–specific IL-10 mutant closely resembles the phenotype in complete IL-10 deficiency. However, in contrast to complete IL-10 deficiency, sensitivity to endotoxic shock and irritant responses of the skin are not enhanced in the T cell–specific IL-10 mutant. Our data highlight the importance of T cell–derived IL-10 in the regulation of T cell responses and demonstrate that endotoxic shock and the irritant response of the skin are controlled by IL-10 from other cell types.
doi:10.1084/jem.20041789
PMCID: PMC2211912  PMID: 15534372
cytokines; gene targeting; inflammation; inflammatory bowel disease; allergic contact dermatitis
25.  Identification of cardiac malformations in mice lacking Ptdsr using a novel high-throughput magnetic resonance imaging technique 
Background
Congenital heart defects are the leading non-infectious cause of death in children. Genetic studies in the mouse have been crucial to uncover new genes and signaling pathways associated with heart development and congenital heart disease. The identification of murine models of congenital cardiac malformations in high-throughput mutagenesis screens and in gene-targeted models is hindered by the opacity of the mouse embryo.
Results
We developed and optimized a novel method for high-throughput multi-embryo magnetic resonance imaging (MRI). Using this approach we identified cardiac malformations in phosphatidylserine receptor (Ptdsr) deficient embryos. These included ventricular septal defects, double-outlet right ventricle, and hypoplasia of the pulmonary artery and thymus. These results indicate that Ptdsr plays a key role in cardiac development.
Conclusions
Our novel multi-embryo MRI technique enables high-throughput identification of murine models for human congenital cardiopulmonary malformations at high spatial resolution. The technique can be easily adapted for mouse mutagenesis screens and, thus provides an important new tool for identifying new mouse models for human congenital heart diseases.
doi:10.1186/1471-213X-4-16
PMCID: PMC545075  PMID: 15615595

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