A One Base Pair Deletion in the Canine ATP13A2 Gene Causes Exon Skipping and Late-Onset Neuronal Ceroid Lipofuscinosis in the Tibetan Terrier

Neuronal ceroid lipofuscinosis (NCL) is a progressive neurodegenerative disease characterized by brain and retinal atrophy and the intracellular accumulation of autofluorescent lysosomal storage bodies resembling lipofuscin in neurons and other cells. Tibetan terriers show a late-onset lethal form of NCL manifesting first visible signs at 5–7 years of age. Genome-wide association analyses for 12 Tibetan-terrier-NCL-cases and 7 Tibetan-terrier controls using the 127K canine Affymetrix SNP chip and mixed model analysis mapped NCL to dog chromosome (CFA) 2 at 83.71–84.72 Mb. Multipoint linkage and association analyses in 376 Tibetan terriers confirmed this genomic region on CFA2. A mutation analysis for 14 positional candidate genes in two NCL-cases and one control revealed a strongly associated single nucleotide polymorphism (SNP) in the MAPK PM20/PM21 gene and a perfectly with NCL associated single base pair deletion (c.1620delG) within exon 16 of the ATP13A2 gene. The c.1620delG mutation in ATP13A2 causes skipping of exon 16 presumably due to a broken exonic splicing enhancer motif. As a result of this mutation, ATP13A2 lacks 69 amino acids. All known 24 NCL cases were homozygous for this deletion and all obligate 35 NCL-carriers were heterozygous. In a sample of 144 dogs from eleven other breeds, the c.1620delG mutation could not be found. Knowledge of the causative mutation for late-onset NCL in Tibetan terrier allows genetic testing of these dogs to avoid matings of carrier animals. ATP13A2 mutations have been described in familial Parkinson syndrome (PARK9). Tibetan terriers with these mutations provide a valuable model for a PARK9-linked disease and possibly for manganese toxicity in synucleinopathies

Morbillivirus Glycoprotein Expression Induces ER Stress, Alters Ca2+ Homeostasis and Results in the Release of Vasostatin

Although the pathology of Morbillivirus in the central nervous system (CNS) is well described, the molecular basis of neurodegenerative events still remains poorly understood. As a model to explore Morbillivirus-mediated CNS dysfunctions, we used canine distemper virus (CDV) that we inoculated into two different cell systems: a monkey cell line (Vero) and rat primary hippocampal neurons. Importantly, the recombinant CDV used in these studies not only efficiently infects both cell types but recapitulates the uncommon, non-cytolytic cell-to-cell spread mediated by virulent CDVs in brain of dogs. Here, we demonstrated that both CDV surface glycoproteins (F and H) markedly accumulated in the endoplasmic reticulum (ER). This accumulation triggered an ER stress, characterized by increased expression of the ER resident chaperon calnexin and the proapoptotic transcription factor CHOP/GADD 153. The expression of calreticulin (CRT), another ER resident chaperon critically involved in the response to misfolded proteins and in Ca2+ homeostasis, was also upregulated. Transient expression of recombinant CDV F and H surface glycoproteins in Vero cells and primary hippocampal neurons further confirmed a correlation between their accumulation in the ER, CRT upregulation, ER stress and disruption of ER Ca2+ homeostasis. Furthermore, CDV infection induced CRT fragmentation with re-localisation of a CRT amino-terminal fragment, also known as vasostatin, on the surface of infected and neighbouring non-infected cells. Altogether, these results suggest that ER stress, CRT fragmentation and re-localization on the cell surface may contribute to cytotoxic effects and ensuing cell dysfunctions triggered by Morbillivirus, a mechanism that might potentially be relevant for other neurotropic viruses

Differential Contributions of the Complement Anaphylotoxin Receptors C5aR1 and C5aR2 to the Early Innate Immune Response against Staphylococcus aureus Infection

The complement anaphylatoxin C5a contributes to host defense against Staphylococcus aureus. In this study, we investigated the functional role of the two known C5a receptors, C5aR1 and C5aR2, in the host response to S. aureus. We found that C5aR1−/− mice exhibited greater susceptibility to S. aureus bloodstream infection than wild type and C5aR2−/− mice, as demonstrated by the significantly higher bacterial loads in the kidneys and heart at 24 h of infection, and by the higher levels of inflammatory IL-6 in serum. Histological and immunohistochemistry investigation of infected kidneys at 24 h after bacterial inoculation revealed a discrete infiltration of neutrophils in wild type mice but already well-developed abscesses consisting of bacterial clusters surrounded by a large number of neutrophils in both C5aR1−/− and C5aR2−/− mice. Furthermore, blood neutrophils from C5aR1−/− mice were less efficient than those from wild type or C5aR2−/− mice at killing S. aureus. The requirement of C5aR1 for efficient killing of S. aureus was also demonstrated in human blood after disrupting C5a-C5aR1 signaling using specific inhibitors. These results demonstrated a role for C5aR1 in S. aureus clearance as well as a role for both C5aR1 and C5aR2 in the orchestration of the inflammatory response during infection

NS1 and VP2 parvoviral protein expression in Purkinje cells of cats wuth feline panleukopenia virus-associated cerebellar hypoplasia

info:eu-repo/semantics/publishe

Infection of porcine enteroids and 2D differentiated intestinal epithelial cells with rotavirus A to study cell tropism and polarized immune response

AbstractIntestinal epithelial cell interactions with enteric pathogens have been incompletely elucidated owing to the lack of model systems that recapitulate the cellular diversity, architecture and functionality of the intestine. To analyze rotavirus (RV) infection and the subsequent innate immune response, we established cultures of differentiated porcine intestinal epithelial cells in three different variations: basolateral-out enteroids, apical-out enteroids and two-dimensional (2D) filter-grown intestinal epithelial cells. Application of specific antibodies for fluorescent staining indicated that enteroids and enteroid-derived cell cultures contain multiple intestinal epithelial cell types. Infection studies indicated that both apical-out enteroids and 2D intestinal epithelial cells are susceptible to porcine RV infection. However, 2D intestinal epithelial cells are more useful for a detailed characterization and comparison of apical and basolateral infection than apical-out enteroids. Virus-induced apoptosis was observed in apical-out enteroids at 24 hours post infection but not at earlier time points after infection. RV infected not only enterocytes but also goblet cells and Paneth cells in apical-out enteroids and 2D intestinal epithelial cells. Interestingly, despite the lack of significant differences in the efficiency of infection after apical and basolateral infection of 2D intestinal epithelial cells, stronger innate immune and inflammatory responses were observed after basolateral infection as compared to infection via the apical route. Therefore, apical-out enteroids and 2D intestinal epithelial cells provide useful primary cell culture models that can be extended to analyze invasion and replication strategies of agents implicated in enteric diseases or to study immune and inflammatory responses of the host induced by enteric pathogens

Infection of porcine enteroids and 2D differentiated intestinal epithelial cells with rotavirus A to study cell tropism and polarized immune response

Intestinal epithelial cell interactions with enteric pathogens have been incompletely elucidated owing to the lack of model systems that recapitulate the cellular diversity, architecture and functionality of the intestine. To analyze rotavirus (RV) infection and the subsequent innate immune response, we established cultures of differentiated porcine intestinal epithelial cells in three different variations: basolateral-out enteroids, apical-out enteroids and two-dimensional (2D) filter-grown intestinal epithelial cells. Application of specific antibodies for fluorescent staining indicated that enteroids and enteroid-derived cell cultures contain multiple intestinal epithelial cell types. Infection studies indicated that both apical-out enteroids and 2D intestinal epithelial cells are susceptible to porcine RV infection. However, 2D intestinal epithelial cells are more useful for a detailed characterization and comparison of apical and basolateral infection than apical-out enteroids. Virus-induced apoptosis was observed in apical-out enteroids at 24 h post infection but not at earlier time points after infection. RV infected not only enterocytes but also goblet cells and Paneth cells in apical-out enteroids and 2D intestinal epithelial cells. Interestingly, despite the lack of significant differences in the efficiency of infection after apical and basolateral infection of 2D intestinal epithelial cells, stronger innate immune and inflammatory responses were observed after basolateral infection as compared to infection via the apical route. Therefore, apical-out enteroids and 2D intestinal epithelial cells provide useful primary cell culture models that can be extended to analyze invasion and replication strategies of agents implicated in enteric diseases or to study immune and inflammatory responses of the host induced by enteric pathogens

Prognostic value and therapeutic potential of TREM-1 in Streptococcus pyogenes- induced sepsis.

TREM-1 (triggering receptor expressed on myeloid cells) is a surface molecule expressed on neutrophils and macrophages which has been implicated in the amplification of inflammatory responses triggered during infection. In the present study, we have investigated the clinical significance of TREM-1 in Streptococcus pyogenes-induced severe sepsis in both experimentally infected mice as well as in patients with streptococcal toxic shock. We found that S. pyogenes induced a dose-dependent upregulation of TREM-1 in in vitro cultured phagocytic cells and in the organs of S. pyogenes-infected mice. Furthermore, we reported a positive correlation between serum levels of soluble TREM-1 (sTREM-1) and disease severity in infected patients as well as in experimentally infected mice. Hence, sTREM-1 may represent a useful surrogate marker for streptococcal sepsis. We found that modulation of TREM-1 by administration of the TREM-1 decoy receptor rTREM-1/Fc substantially attenuated the synthesis of inflammatory cytokines. More importantly, treatment of S. pyogenes-infected septic mice with rTREM-1/Fc or the synthetically produced conserved extracellular domain LP17 significantly improved disease outcome. In summary, our data suggest that TREM-1 may not only represent a valuable marker for S. pyogenes infection severity but it may also be an attractive target for the treatment of streptococcal sepsis