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Neutrophil extracellular traps detected by immunohistochemistry in selected animal diseases
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ESVP and ECVP Proceedings 2016
J. Comp. Path. 2017, Vol. 156, 54e141
Oral presentations
General Pathology RNA INTERFERENCE SCREEN REVEALS DIFFERENTIAL EFFECTS OF HOST CELL FACTORS ON TRANSDUCTION BY ADENO-ASSOCIATED VIRUS 2 (AAV2) VECTORS F.D. Franzoso *, A. Yakimovich y, K. Tobler *, A. Man *, R. Vogel *, U. Greber y, M. Ackermann * and C. Fraefel* *Institute of Virology, VetSuisse Faculty and yInstitute of Molecular Life Sciences, University of Z€urich, Z€urich, Switzerland Introduction: AAV2 is a small, non-enveloped parvovirus, with a 5 kb long, single-stranded linear DNA genome. In co-infection with a helper virus (typically a herpesvirus or an adenovirus), AAV2 can establish a productive infection. AAV2 has attracted considerable interest as a platform for the design of vectors for gene therapy, particularly in treating genetic disorders. Their efficient transduction in vivo is limited to postmitotic cells. We performed siRNA screening to assess the effect of 65 host cell proteins (identified previously as components of AAV2 replication compartments) on the transduction efficiency by single-stranded (rAAVGFP) and self-complementary (scAAVGFP) AAV2 vectors. Materials and Methods: Viral transduction (GFP fluorescence) was recorded in HeLa cells by high-throughput microscopy using an ImageXpress High Content Screening System (Molecular Devices) and further analyzed with CellProfiler (BROAD Institute, Cambridge, USA) and KNIME (KNIME.COM, Zurich Switzerland) software. The post-transcriptional silencing of the target genes was confirmed and the data on gene expression were validated by RT-qPCR and western blotting. Results: Knockdown of several cellular genes, such as Mre11, Nbs1 and Rad50, enhanced the transcription from both single-stranded and scAAV2 vectors, while knockdown of other genes, such as those encoding replication proteins (RPA1 and RPA2) and DNA mismatch repair proteins (MSH2, MSH3 and MSH6), differentially affected gene expression from the two different vectors. Conclusions: We identified a number of cellular proteins that either positively or negatively affect transduction efficiency by AAV2 vectors. These findings may have further implications for studies on AAV2 biology and for improving AAV2 vector-mediated gene therapy.
NEUTROPHIL EXTRACELLULAR TRAPS DETECTED BY IMMUNOHISTOCHEMISTRY IN SELECTED ANIMAL DISEASES M. Fragoso, S. Binder, K. Dietert and A.D. Gruber Institute for Veterinary Pathology, Freie Universit€at Berlin, Germany Introduction: Neutrophils effectively contribute to innate defence against bacterial infections through phagocytosis and degranulation (e.g. release of bactericidal peptides). Recently, a third and completely novel class of extracellular defence mechanism has been discovered in man, termed neutrophil extracellular traps (NETs). The release of NETs, a process called NETosis, includes a unique programmed cell death pathway that results in the ejection of nuclear DNA and citrullinated histones, decorated with antimicrobial peptides including neutrophil elastase (NE) and myeloperoxidase (MO). NETosis has not yet been characterized in domestic animals. Materials and Methods: Four groups of diseases were tested: bacterial (n 5 62), fungal (n 5 9) and parasitic (n 5 2) infections as well as coagulopathies (n 5 20) from 71 dogs and 22 horses. The archival formalin-fixed and paraffin wax-embedded tissues were labelled immunohistochemically with antibodies against citrullinated histone H3, NE and MO. Nuclear DNA was visualized using the DAPI stain. Results: NETs were identified by co-localized signals of NE, MO, H3 and DAPI only in acute or subacute bacterial or fungal infections, primarily adjacent to the pathogens, necrotic debris or Splendoree Hoeppli material. NETosis was also visualized in thrombi in splenic infarctions and/or hematomas. Conclusions: The methods used yielded signals consistent with the previous detection of NETosis in man, both in terms of antibody reactivity and location of the signals. Not surprisingly, NETosis seems to also occur in dogs and horses and likely other animals. This is only the start of more comprehensive and systematic studies of this newly discovered phenomenon and its relevance for veterinary pathology.
ESVP and ECVP Proceedings 2016
J. Comp. Path. 2017, Vol. 156, 54e141
Oral presentations
General Pathology RNA INTERFERENCE SCREEN REVEALS DIFFERENTIAL EFFECTS OF HOST CELL FACTORS ON TRANSDUCTION BY ADENO-ASSOCIATED VIRUS 2 (AAV2) VECTORS F.D. Franzoso *, A. Yakimovich y, K. Tobler *, A. Man *, R. Vogel *, U. Greber y, M. Ackermann * and C. Fraefel* *Institute of Virology, VetSuisse Faculty and yInstitute of Molecular Life Sciences, University of Z€urich, Z€urich, Switzerland Introduction: AAV2 is a small, non-enveloped parvovirus, with a 5 kb long, single-stranded linear DNA genome. In co-infection with a helper virus (typically a herpesvirus or an adenovirus), AAV2 can establish a productive infection. AAV2 has attracted considerable interest as a platform for the design of vectors for gene therapy, particularly in treating genetic disorders. Their efficient transduction in vivo is limited to postmitotic cells. We performed siRNA screening to assess the effect of 65 host cell proteins (identified previously as components of AAV2 replication compartments) on the transduction efficiency by single-stranded (rAAVGFP) and self-complementary (scAAVGFP) AAV2 vectors. Materials and Methods: Viral transduction (GFP fluorescence) was recorded in HeLa cells by high-throughput microscopy using an ImageXpress High Content Screening System (Molecular Devices) and further analyzed with CellProfiler (BROAD Institute, Cambridge, USA) and KNIME (KNIME.COM, Zurich Switzerland) software. The post-transcriptional silencing of the target genes was confirmed and the data on gene expression were validated by RT-qPCR and western blotting. Results: Knockdown of several cellular genes, such as Mre11, Nbs1 and Rad50, enhanced the transcription from both single-stranded and scAAV2 vectors, while knockdown of other genes, such as those encoding replication proteins (RPA1 and RPA2) and DNA mismatch repair proteins (MSH2, MSH3 and MSH6), differentially affected gene expression from the two different vectors. Conclusions: We identified a number of cellular proteins that either positively or negatively affect transduction efficiency by AAV2 vectors. These findings may have further implications for studies on AAV2 biology and for improving AAV2 vector-mediated gene therapy.
NEUTROPHIL EXTRACELLULAR TRAPS DETECTED BY IMMUNOHISTOCHEMISTRY IN SELECTED ANIMAL DISEASES M. Fragoso, S. Binder, K. Dietert and A.D. Gruber Institute for Veterinary Pathology, Freie Universit€at Berlin, Germany Introduction: Neutrophils effectively contribute to innate defence against bacterial infections through phagocytosis and degranulation (e.g. release of bactericidal peptides). Recently, a third and completely novel class of extracellular defence mechanism has been discovered in man, termed neutrophil extracellular traps (NETs). The release of NETs, a process called NETosis, includes a unique programmed cell death pathway that results in the ejection of nuclear DNA and citrullinated histones, decorated with antimicrobial peptides including neutrophil elastase (NE) and myeloperoxidase (MO). NETosis has not yet been characterized in domestic animals. Materials and Methods: Four groups of diseases were tested: bacterial (n 5 62), fungal (n 5 9) and parasitic (n 5 2) infections as well as coagulopathies (n 5 20) from 71 dogs and 22 horses. The archival formalin-fixed and paraffin wax-embedded tissues were labelled immunohistochemically with antibodies against citrullinated histone H3, NE and MO. Nuclear DNA was visualized using the DAPI stain. Results: NETs were identified by co-localized signals of NE, MO, H3 and DAPI only in acute or subacute bacterial or fungal infections, primarily adjacent to the pathogens, necrotic debris or Splendoree Hoeppli material. NETosis was also visualized in thrombi in splenic infarctions and/or hematomas. Conclusions: The methods used yielded signals consistent with the previous detection of NETosis in man, both in terms of antibody reactivity and location of the signals. Not surprisingly, NETosis seems to also occur in dogs and horses and likely other animals. This is only the start of more comprehensive and systematic studies of this newly discovered phenomenon and its relevance for veterinary pathology.