RESUMO
Oncolytic viruses and morbilliviruses in particular, represent an interesting therapeutic approach for tumors with a poor prognosis and frequent resistance to conventional therapies. Canine histiocytic sarcomas (HS) exemplify such a neoplasm in need for new curative approaches. Previous investigations demonstrated a limited success of an acute intratumoral application of canine distemper virus (CDV) on xenotransplanted canine histiocytic sarcoma cells (DH82 cells), while persistently CDV-infected DH82 cell transplants exhibited a complete spontaneous regression. Therefore, the present study focuses on an intratumoral application of persistently CDV vaccine strain Onderstepoort-infected DH82 (DH82 Ond p.i.) cells into non-infected subcutaneous DH82 cell transplants in a murine model. DH82 cell transplants that received 10 applications, two days apart, showed a transient growth retardation as well as larger areas of intratumoral necrosis, lower mitotic rates, and a decreased intratumoral vascularization compared to controls. Viral mRNA was detected in all neoplasms following application of DH82 Ond p.i. cells until 66 days after the last injection. Furthermore, infectious virus was present until 62 days after the last injection. Although complete regression was not achieved, the present application regimen provides promising results as a basis for further treatments, particularly with genetically modified viruses, to enhance the observed effects.
Assuntos
Vírus da Cinomose Canina , Sarcoma Histiocítico , Terapia Viral Oncolítica , Animais , Vírus da Cinomose Canina/patogenicidade , Vírus da Cinomose Canina/genética , Cães , Sarcoma Histiocítico/virologia , Camundongos , Terapia Viral Oncolítica/métodos , Linhagem Celular Tumoral , Cinomose/virologia , Vírus Oncolíticos/genética , Vírus Oncolíticos/fisiologiaRESUMO
Canine histiocytic sarcoma (HS) represents a neoplasia with poor prognosis. Due to the high metastatic rate of HS, there is urgency to improve treatment options and to prevent tumor metastases. Canine distemper virus (CDV) is a single-stranded negative-sense RNA (ssRNA (-)) virus with potentially oncolytic properties. Moreover, vasostatin and granulocyte-macrophage colony-stimulating factor (GM-CSF) are attractive molecules in cancer therapy research because of their anti-angiogenetic properties and potential modulation of the tumor microenvironment. In the present study, an in vitro characterization of two genetically engineered viruses based on the CDV strain Onderstepoort (CDV-Ond), CDV-Ondneon-vasostatin and CDV-Ondneon-GM-CSF was performed. Canine histiocytic sarcoma cells (DH82 cells) were persistently infected with CDV-Ond, CDV-Ondneon, CDV-Ondneon-vasostatin and CDV-Ondneon-GM-CSF and characterized on a molecular and protein level regarding their vasostatin and GM-CSF production. Interestingly, DH82 cells persistently infected with CDV-Ondneon-vasostatin showed a significantly increased number of vasostatin mRNA transcripts. Similarly, DH82 cells persistently infected with CDV-Ondneon-GM-CSF displayed an increased number of GM-CSF mRNA transcripts mirrored on the protein level as confirmed by immunofluorescence and Western blot. In summary, modified CDV-Ond strains expressed GM-CSF and vasostatin, rendering them promising candidates for the improvement of oncolytic virotherapies, which should be further detailed in future in vivo studies.
Assuntos
Vírus da Cinomose Canina , Sarcoma Histiocítico , Animais , Calreticulina , Linhagem Celular , Vírus da Cinomose Canina/genética , Cães , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Sarcoma Histiocítico/genética , Neônio , Fragmentos de Peptídeos , Infecção Persistente , RNA Mensageiro , Microambiente TumoralRESUMO
Similar to many other respiratory viruses, SARS-CoV-2 targets the ciliated cells of the respiratory epithelium and compromises mucociliary clearance, thereby facilitating spread to the lungs and paving the way for secondary infections. A detailed understanding of mechanism involved in ciliary loss and subsequent regeneration is crucial to assess the possible long-term consequences of COVID-19. The aim of this study was to characterize the sequence of histological and ultrastructural changes observed in the ciliated epithelium during and after SARS-CoV-2 infection in the golden Syrian hamster model. We show that acute infection induces a severe, transient loss of cilia, which is, at least in part, caused by cilia internalization. Internalized cilia colocalize with membrane invaginations, facilitating virus entry into the cell. Infection also results in a progressive decline in cells expressing the regulator of ciliogenesis FOXJ1, which persists beyond virus clearance and the termination of inflammatory changes. Ciliary loss triggers the mobilization of p73+ and CK14+ basal cells, which ceases after regeneration of the cilia. Although ciliation is restored after two weeks despite the lack of FOXJ1, an increased frequency of cilia with ultrastructural alterations indicative of secondary ciliary dyskinesia is observed. In summary, the work provides new insights into SARS-CoV-2 pathogenesis and expands our understanding of virally induced damage to defense mechanisms in the conducting airways.
Assuntos
COVID-19 , Animais , Cílios/metabolismo , Cricetinae , Epitélio , Homeostase , Mesocricetus , Mucosa Respiratória/metabolismo , SARS-CoV-2RESUMO
Sarcomas especially of histiocytic origin often possess a poor prognosis and response to conventional therapies. Interestingly, tumours undergoing mesenchymal to epithelial transition (MET) are often associated with a favourable clinical outcome. This process is characterized by an increased expression of epithelial markers leading to a decreased invasion and metastatic rate. Based on the failure of conventional therapies, viral oncolysis might represent a promising alternative with canine distemper virus (CDV) as a possible candidate. This study hypothesizes that a CDV infection of canine histiocytic sarcoma cells (DH82 cells) triggers the MET process leading to a decreased cellular motility. Immunofluorescence and immunoblotting were used to investigate the expression of epithelial and mesenchymal markers followed by scratch assay and an invasion assay as functional confirmation. Furthermore, microarray data were analysed for genes associated with the MET process, invasion and angiogenesis. CDV-infected cells exhibited an increased expression of epithelial markers such as E-cadherin and cytokeratin 8 compared to controls, indicating a MET process. This was accompanied by a reduced cell motility and invasiveness. Summarized, these results suggest that CDV infection of DH82 cells triggers the MET process by an increased expression of epithelial markers resulting in a decreased cell motility in vitro.
Assuntos
Movimento Celular , Vírus da Cinomose Canina/patogenicidade , Cinomose/complicações , Doenças do Cão/prevenção & controle , Transição Epitelial-Mesenquimal , Sarcoma Histiocítico/prevenção & controle , Neovascularização Patológica/prevenção & controle , Animais , Cinomose/virologia , Doenças do Cão/metabolismo , Doenças do Cão/virologia , Cães , Sarcoma Histiocítico/metabolismo , Sarcoma Histiocítico/veterinária , Sarcoma Histiocítico/virologia , Técnicas In Vitro , Análise em Microsséries , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neovascularização Patológica/virologiaRESUMO
Oxidative stress and inflammation are major contributors to acrolein toxicity. Peroxisome proliferator activated receptor gamma (PPARγ) has antioxidant and anti-inflammatory effects. We investigated the contribution of PPARγ ligand GW1929 to the attenuation of oxidative stress in acrolein-induced insult. Male gp91phox knock-out (KO) mice were treated with acrolein (0.5 mg·(kg body mass)-1 by intraperitoneal injection for 7 days) with or without GW1929 (GW; 0.5 mg·(kg body mass)-1·day-1, orally, for 10 days). The livers were processed for further analyses. Acrolein significantly increased 8-isoprostane and reduced PPARγ activity (P < 0.05) in the wild type (WT) and KO mice. GW1929 reduced 8-isoprostane (by 32% and 40% in WT and KO mice, respectively) and increased PPARγ activity (by 81% and 92% in WT and KO, respectively). Chemokine activity was increased (by 63%) in acrolein-treated WT mice, and was reduced by GW1929 (by 65%). KO mice exhibited higher xanthine oxidase (XO). Acrolein increased XO and COX in WT mice and XO in KO mice. GW1929 significantly reduced COX in WT and KO mice and reduced XO in KO mice. Acrolein significantly reduced the total antioxidant status in WT and KO mice (P < 0.05), which was improved by GW1929 (by 75% and 74%). The levels of NF-κB were higher in acrolein-treated WT mice. GW1929 reduced NF-κB levels (by 51%) in KO mice. Acrolein increased CD36 in KO mice (by 43%), which was blunted with GW1929. Data confirms that the generation of free radicals by acrolein is mainly through NAD(P)H, but other oxygenates play a role too. GW1929 may alleviate the toxicity of acrolein by attenuating NF-κB, COX, and CD36.
Assuntos
Acroleína/farmacologia , Inflamação/tratamento farmacológico , PPAR gama/metabolismo , Receptores Imunológicos/deficiência , Transdução de Sinais/efeitos dos fármacos , Animais , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismoRESUMO
Theiler's murine encephalomyelitis virus (TMEV) induces an acute polioencephalomyelitis and a chronic demyelinating leukomyelitis in SJL mice. C57BL/6 (B6) mice generally do not develop TMEV-induced demyelinating disease (TMEV-IDD) due to virus elimination. However, TMEV can persist in specific immunodeficient B6 mice such as IFNß-/- mice and induce a demyelinating process. The proinflammatory cytokines IL-1ß and IL-18 are activated by the inflammasome pathway, which consists of a pattern recognition receptor molecule sensing microbial pathogens, the adaptor molecule Apoptosis-associated speck-like protein containing a CARD (ASC), and the executioner caspase-1. To analyze the contribution of the inflammasome pathway to the resistance of B6 mice to TMEV-IDD, ASC- and caspase-1-deficient mice and wild type littermates were infected with TMEV and investigated using histology, immunohistochemistry, RT-qPCR, and Western Blot. Despite the antiviral activity of the inflammasome pathway, ASC- and caspase-1-deficient mice eliminated the virus and did not develop TMEV-IDD. Moreover, a similar IFNß and cytokine gene expression was found in the brain of immunodeficient mice and their wild type littermates. Most importantly, Western Blot showed cleavage of IL-1ß and IL-18 in all investigated mice. Consequently, inflammasome-dependent activation of IL-1ß and IL-18 does not play a major role in the resistance of B6 mice to TMEV-IDD.
Assuntos
Doenças Desmielinizantes , Theilovirus , Animais , Camundongos , Caspase 1/genética , Citocinas , Doenças Desmielinizantes/patologia , Inflamassomos , Interleucina-18/genética , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Theilovirus/fisiologiaRESUMO
The immune response plays a key role in the treatment of malignant tumors. One important molecule promoting humoral and cellular immunity is granulocyte-macrophage colony-stimulating factor (GM-CSF). Numerous successful trials have led to the approval of this immune-stimulating molecule for cancer therapy. However, besides immune stimulation, GM-CSF may also accelerate tumor cell proliferation, rendering this molecule a double-edged sword in cancer treatment. Therefore, detailed knowledge about the in vitro function of GM-CSF produced by infected tumor cells is urgently needed prior to investigations in an in vivo model. The aim of the present study was to functionally characterize a persistent infection of canine histiocytic sarcoma cells (DH82 cells) with the canine distemper virus strain Onderstepoort genetically engineered to express canine GM-CSF (CDV-Ondneon-GM-CSF). The investigations aimed (1) to prove the overall functionality of the virally induced production of GM-CSF and (2) to determine the effect of GM-CSF on the proliferation and motility of canine HS cells. Infected cells consistently produced high amounts of active, pH-stable GM-CSF, as demonstrated by increased proliferation of HeLa cells. By contrast, DH82 cells lacked increased proliferation and motility. The significantly increased secretion of GM-CSF by persistently CDV-Ondneon-GM-CSF-infected DH82 cells, the pH stability of this protein, and the lack of detrimental effects on DH82 cells renders this virus strain an interesting candidate for future studies aiming to enhance the oncolytic properties of CDV for the treatment of canine histiocytic sarcomas.