Ingrid Cornax

Virulent Newcastle disease virus elicits a strong innate immune response in chickens

Newcastle disease virus (NDV) is an avian paramyxovirus that causes significant economic losses to the poultry industry worldwide. There is limited knowledge about the avian immune response to infection with virulent NDVs, and how this response may contribute to disease. In this study, pathogenesis and the transcriptional host response of chickens to a virulent NDV strain that rapidly causes 100% mortality was characterized. Using microarrays, a strong transcriptional host response was observed in spleens at early times after infection with the induction of groups of genes involved in innate antiviral and pro-inflammatory responses. There were multiple genes induced at 48 h post-infection including: type I and II interferons (IFNs), several cytokines and chemokines, IFN effectors and inducible nitric oxide synthase (iNOS). The increased transcription of nitric oxide synthase was confirmed by immunohistochemistry for iNOS in spleens and measured levels of nitric oxide in serum. In vitro experiments showed strong induction of the key host response genes, alpha IFN, beta interferon, and interleukin 1β and interleukin 6, in splenic leukocytes at 6 h post-infection in comparison to a non-virulent NDV. The robust host response to virulent NDV, in conjunction with severe pathological damage observed, is somewhat surprising considering that all NDV encode a gene, V, which functions as a suppressor of class I IFNs. Taken together, these results suggest that the host response itself may contribute to the pathogenesis of this highly virulent strain in chickens.

Characterization of Live LaSota Vaccine Strain–Induced Protection in Chickens upon Early Challenge with a Virulent Newcastle Disease Virus of Heterologous Genotype

SUMMARY. Newcastle disease (ND) is a major threat to the international poultry industry, causing bird mortality, reduction in growth and egg production, and trade restrictions. The primary strategy available to the poultry industry to control virulent Newcastle disease virus (NDV), the causative agent of ND, is vaccination. LaSota and other commonly used live-virus NDV vaccine strains were developed in the 1950s and 1960s and show a great degree of genetic divergence from currently circulating NDV strains. In order to characterize protective immunity induced by LaSota against a heterologous NDV strain, we vaccinated groups of specific-pathogen-free (SPF) chickens with LaSota (virus titers ranging from 102 to 108 egg infective dose 50 [EID50] in 10-fold increments) and challenged the birds 14 days later with ZJ1 strain, an NDV strain that was isolated in the year 2000 from geese in China. We monitored multiple parameters of immunity, including serum antibody titers, antigen-specific lymphocyte proliferation, and splenic cytokine expression and determined that SPF birds vaccinated with an adequate titer of LaSota strain live vaccine are fully protected from morbidity and mortality due to challenge with ZJ1 strain NDV, and we concluded that in the absence of interfering maternal antibody, protection due to vaccination increases with vaccine titer until a threshold titer is reached, beyond which, little or no further benefit can be elucidated.

In vivo transcriptional cytokine responses and association with clinical and pathological outcomes in chickens infected with different Newcastle disease virus isolates using formalin-fixed paraffin-embedded samples

Little is known about the host response of chickens infected with Newcastle disease virus (NDV) and the relationship between the innate immune response and the severity of clinical disease. Measurement of cytokine responses during infection in vivo can help to elucidate the mechanisms of virus pathogenesis. The transcriptional response of several cytokines from paraffin-embedded, formalin-fixed spleen of chicken naturally infected by NDV velogenic viscerotropic viruses was compared to the responses of atypical velogenic, velogenic neurotropic, and mesogenic strains during the first five days after infection. The RNA expression for IFN-γ and IL-6 was enhanced at day two in the highly virulent velogenic viscerotropic viruses (California and rZJ1 strains) and corresponded with the presence of the virus in tissues. However, in one atypical velogenic viscerotropic virus (Australia strain), two velogenic neurotropic viruses (Turkey ND and Texas GB) and, a mesogenic virus (Anhinga strain) the cytokine responses to infection were delayed or reduced. Increased levels of IFN-β RNA expression were only detected in the velogenic viscerotropic virus infected chickens (California and rZJ1 strains) at 3 days post-infection and one mesogenic strain (Anhinga) early in infection. The RNA expression levels of IL-2 did not increase upon infection with any of the viruses. A pronounced increase of RNA expression levels of IL-6 and IFN-γ was detected simultaneously with infiltration of macrophages and/or lymphoid necrosis in the histopathological analysis of the spleen and cecal tonsils. The differences in the RNA expression levels may help explain possible underlying mechanisms of clinical disease and/or immune responses in birds infected with strains of APMV-1 that cause distinct pathologic changes.

Newcastle disease virus fusion and haemagglutinin-neuraminidase proteins contribute to its macrophage host range

The fusion (F) and haemagglutinin-neuraminidase (HN) proteins of Newcastle disease virus (NDV) are multifunctional proteins that play critical roles during infection. Here, we assessed the ability of NDV to replicate in macrophages and investigated the contribution of the F and HN proteins to NDV infection/replication in these cells. Results of our study revealed that, while presenting similar replication kinetics in a fibroblast cell line (DF1) or in primary non-adherent splenocytes, the NDV strain CA02 replicates better in macrophages (HD11 and primary adherent splenocytes) than the NDV strain Anhinga/93. Notably, exchange of the HN or both F and HN genes of NDV Anhinga/93 by the corresponding genes from NDV CA02 markedly improved the ability of the chimeric viruses to replicate in macrophages. These results indicate that the F and HN proteins are determinants of NDV macrophage host range. This represents the first description of productive NDV infection in macrophages.

Aberrant Retinoblastoma (RB)-E2F Transcriptional Regulation Defines Molecular Phenotypes of Osteosarcoma

Background: Gene expression signatures define prognostically significant osteosarcoma phenotypes. Results: Deregulation of the RB-E2F pathway establishes more aggressive phenotype. Inhibitors of DNA and chromatin remodeling promote comparable transcriptional changes as genetic restoration of RB. Conclusion: Aberrant RB-E2F pathway alters epigenetic landscape and biological behavior of osteosarcoma. Significance: Epigenetic remodeling regulated by RB-E2F gives rise to patterns of gene expression that are associated with different biological behavior and progression of osteosarcoma. We previously identified two distinct molecular subtypes of osteosarcoma through gene expression profiling. These subtypes are associated with distinct tumor behavior and clinical outcomes. Here, we describe mechanisms that give rise to these molecular subtypes. Using bioinformatic analyses, we identified a significant association between deregulation of the retinoblastoma (RB)-E2F pathway and the molecular subtype with worse clinical outcomes. Xenotransplantation models recapitulated the corresponding behavior for each osteosarcoma subtype; thus, we used cell lines to validate the role of the RB-E2F pathway in regulating the prognostic gene signature. Ectopic RB resets the patterns of E2F regulated gene expression in cells derived from tumors with worse clinical outcomes (molecular phenotype 2) to those comparable with those observed in cells derived from tumors with less aggressive outcomes (molecular phenotype 1), providing a functional association between RB-E2F dysfunction and altered gene expression in osteosarcoma. DNA methyltransferase and histone deacetylase inhibitors similarly reset the transcriptional state of the molecular phenotype 2 cells from a state associated with RB deficiency to one seen with RB sufficiency. Our data indicate that deregulation of RB-E2F pathway alters the epigenetic landscape and biological behavior of osteosarcoma.

Eradication of Canine Diffuse Large B-Cell Lymphoma in a Murine Xenograft Model with CD47 Blockade and Anti-CD20

Targeting CD47 and CD20 to stimulate macrophage phagocytosis was effective in preclinical xenograft models of canine lymphoma in mice. This immunotherapeutic strategy has the potential to benefit companion animals and to inform future targeting studies in humans. Cancer immunotherapies hold much promise, but their potential in veterinary settings has not yet been fully appreciated. Canine lymphomas are among the most common tumors of dogs and bear remarkable similarity to human disease. In this study, we examined the combination of CD47 blockade with anti-CD20 passive immunotherapy for canine lymphoma. The CD47/SIRPα axis is an immune checkpoint that regulates macrophage activation. In humans, CD47 is expressed on cancer cells and enables evasion from phagocytosis. CD47-blocking therapies are now under investigation in clinical trials for a variety of human cancers. We found the canine CD47/SIRPα axis to be conserved biochemically and functionally. We identified high-affinity SIRPα variants that antagonize canine CD47 and stimulate phagocytosis of canine cancer cells in vitro. When tested as Fc fusion proteins, these therapeutic agents exhibited single-agent efficacy in a mouse xenograft model of canine lymphoma. As robust synergy between CD47 blockade and tumor-specific antibodies has been demonstrated for human cancer, we evaluated the combination of CD47 blockade with 1E4-cIgGB, a canine-specific antibody to CD20. 1E4-cIgGB could elicit a therapeutic response against canine lymphoma in vivo as a single agent. However, augmented responses were observed when combined with CD47-blocking therapies, resulting in synergy in vitro and in vivo and eliciting cures in 100% of mice bearing canine lymphoma. Our findings support further testing of CD47-blocking therapies alone and in combination with CD20 antibodies in the veterinary setting. Cancer Immunol Res; 4(12); 1072–87. ©2016 AACR.

Mobilization of ectopic yolk in Gallus gallus domesticus : a novel reverse lipid transport process

SUMMARY In many oviparous animals, bursting type atresia of ovarian follicles occurs during the reproductive cycle, resulting in the escape of yolk into the extracellular compartment. In birds, this ectopic yolk is rapidly cleared by an unknown process that involves the appearance of yolk-engorged macrophage-like cells. To study this unique type of lipid transport, we injected young male chickens intra-abdominally with egg yolk. Absorption of egg yolk from the body cavity markedly increased the triacylglyceride-rich fraction (TRL) of plasma lipoproteins and was coincident with increased levels of plasma triacylglycerides (TAGs) but not non-esterified fatty acids (NEFAs). Thus, the transport of yolk lipids from the abdominal cavity appears to occur in lipoproteins and be more similar to the transport of hepatic TAGs to the periphery via lipoproteins than to transport of adipose TAGs to the periphery via NEFAs released by the action of lipases. When macrophages were exposed to yolk in vitro, they quickly phagocytized yolk; however, it is unclear whether this level of phagocytosis contributes significantly to total yolk clearance. Instead, the chicken macrophage may function more as a facilitator of yolk clearance through the modification of yolk lipoproteins and the regulation of the local and systemic immune response to ectopic yolk. Yolk appears to be anti-inflammatory in nature. Yolk did not increase levels of the inflammatory cytokines IL-1, IL-6 and IFNγ either in vivo or in vitro; in fact, yolk dampened many inflammatory changes caused by lipopolysaccharide (LPS). Conversely, LPS-induced inflammation retarded yolk clearance from the abdominal cavity and plasma TAG levels.

Triptolide suppresses the in vitro and in vivo growth of lung cancer cells by targeting hyaluronan-CD44/RHAMM signaling

Higher levels of hyaluronan (HA) and its receptors CD44 and RHAMM have been associated with poor prognosis and metastasis in NSCLC. In the current study, our goal was to define, using cellular and orthotopic lung tumor models, the role of HA-CD44/RHAMM signaling in lung carcinogenesis and to assess the potential of triptolide to block HA-CD44/RHAMM signaling and thereby suppress the development and progression of lung cancer. Triptolide reduced the viability of five non-small cell lung cancer (NSCLC) cells, the proliferation and self-renewal of pulmospheres, and levels of HA synthase 2 (HAS2), HAS3, HA, CD44, RHAMM, EGFR, Akt and ERK, but increased the cleavage of caspase 3 and PARP. Silencing of HAS2, CD44 or RHAMM induced similar effects. Addition of excess HA to the culture media completely abrogated the effects of triptolide and siRNAs targeting HAS2, CD44, or RHAMM. In an orthotopic lung cancer model in nude rats, intranasal administration of liposomal triptolide (400 μg/kg) for 8 weeks significantly reduced lung tumor growth as determined by bioluminescence imaging, lung weight measurements and gross and histopathological analysis of tumor burden. Also, triptolide suppressed expressions of Ki-67, a marker for cell proliferation, HAS2, HAS3, HA, CD44, and RHAMM in lung tumors. Overall, our results provide a strong rationale for mitigating lung cancer by targeting the HA-CD44/RHAMM signaling axis.

Experimental evaluation of Peromyscus leucopus as a reservoir host of the Ehrlichia muris-like agent

BackgroundThe Ehrlichia muris-like agent (EMLA) is a newly recognized human pathogen in the North Central United States. Although blacklegged ticks (Ixodes scapularis) have been identified as capable vectors, wild reservoirs have not yet been established for EMLA. As key hosts for I. scapularis, white-footed mice (Peromyscus leucopus) are important reservoirs for various tick-borne pathogens, and potentially, for EMLA. The objective of this study was to evaluate reservoir competence in P. leucopus using a natural vector.ResultsMice acquired EMLA infection from feeding ticks and were able to transmit infection to naïve ticks. Transmission between simultaneously feeding tick life stages was also demonstrated. Infections in mice were acute and severe, with systemic dissemination. Limited host survival and clearance of infection among survivors resulted in a narrow interval where EMLA could be acquired by feeding ticks.ConclusionsPeromyscus leucopus is a competent reservoir of EMLA and likely to play a role in its enzootic transmission cycle. The duration and severity of EMLA infection in these hosts suggests that tick phenology is a critical factor determining the geographic distribution of EMLA in North America.

Slit-Robo GTPase-Activating Protein 2 as a metastasis suppressor in osteosarcoma

Osteosarcoma is the most common primary bone tumor, with metastatic disease responsible for most treatment failure and patient death. A forward genetic screen utilizing Sleeping Beauty mutagenesis in mice previously identified potential genetic drivers of osteosarcoma metastasis, including Slit-Robo GTPase-Activating Protein 2 (Srgap2). This study evaluates the potential role of SRGAP2 in metastases-associated properties of osteosarcoma cell lines through Srgap2 knockout via the CRISPR/Cas9 nuclease system and conditional overexpression in the murine osteosarcoma cell lines K12 and K7M2. Proliferation, migration, and anchorage independent growth were evaluated. RNA sequencing and immunohistochemistry of human osteosarcoma tissue samples were used to further evaluate the potential role of the Slit-Robo pathway in osteosarcoma. The effects of Srgap2 expression modulation in the murine OS cell lines support the hypothesis that SRGAP2 may have a role as a suppressor of metastases in osteosarcoma. Additionally, SRGAP2 and other genes in the Slit-Robo pathway have altered transcript levels in a subset of mouse and human osteosarcoma, and SRGAP2 protein expression is reduced or absent in a subset of primary tumor samples. SRGAP2 and other axon guidance proteins likely play a role in osteosarcoma metastasis, with loss of SRGAP2 potentially contributing to a more aggressive phenotype.