The replication and transcription activator of murine gammaherpesvirus 68 cooperatively enhances cytokine-activated, STAT3-mediated gene expression.

Gammaherpesviruses (γHVs) have a dynamic strategy for lifelong persistence, involving productive infection, latency, and intermittent reactivation. In latency reservoirs, such as B lymphocytes, γHVs exist as viral episomes and express few viral genes. Although the ability of γHV to reactivate from latency and re-enter the lytic phase is challenging to investigate and control, it is known that the γHV replication and transcription activator (RTA) can promote lytic reactivation. In this study, we provide first evidence that RTA of murine γΗV68 (MHV68) selectively binds and enhances the activity of tyrosine-phosphorylated host STAT3. STAT3 is a transcription factor classically activated by specific tyrosine 705 phosphorylation (pTyr705-STAT3) in response to cytokine stimulation. pTyr705-STAT3 forms a dimer that avidly binds a consensus target site in the promoters of regulated genes, and our results indicate that RTA cooperatively enhances the ability of pTyr705-STAT3 to induce expression of a STAT3-responsive reporter gene. As indicated by coimmunoprecipitation, in latently infected B cells that are stimulated to reactivate MHV68, RTA bound specifically to endogenous pTyr705-STAT3. An in vitro binding assay confirmed that RTA selectively recognizes pTyr705-STAT3 and indicated that the C-terminal transactivation domain of RTA was required for enhancing STAT3-directed gene expression. The cooperation of these transcription factors may influence both viral and host genes. During MHV68 de novo infection, pTyr705-STAT3 promoted the temporal expression of ORF59, a viral replication protein. Our results demonstrate that MHV68 RTA specifically recognizes and recruits activated pTyr705-STAT3 during the lytic phase of infection.

Tiled microarray identification of novel viral transcript structures and distinct transcriptional profiles during two modes of productive murine gammaherpesvirus 68 infection.

We applied a custom tiled microarray to examine murine gammaherpesvirus 68 (MHV68) polyadenylated transcript expression in a time course of de novo infection of fibroblast cells and following phorbol ester-mediated reactivation from a latently infected B cell line. During de novo infection, all open reading frames (ORFs) were transcribed and clustered into four major temporal groups that were overlapping yet distinct from clusters based on the phorbol ester-stimulated B cell reactivation time course. High-density transcript analysis at 2-h intervals during de novo infection mapped gene boundaries with a 20-nucleotide resolution, including a previously undefined ORF73 transcript and the MHV68 ORF63 homolog of Kaposi's sarcoma-associated herpesvirus vNLRP1. ORF6 transcript initiation was mapped by tiled array and confirmed by 5' rapid amplification of cDNA ends. The ∼1.3-kb region upstream of ORF6 was responsive to lytic infection and MHV68 RTA, identifying a novel RTA-responsive promoter. Transcription in intergenic regions consistent with the previously defined expressed genomic regions was detected during both types of productive infection. We conclude that the MHV68 transcriptome is dynamic and distinct during de novo fibroblast infection and upon phorbol ester-stimulated B cell reactivation, highlighting the need to evaluate further transcript structure and the context-dependent molecular events that govern viral gene expression during chronic infection.

The Thoracic Inlet Length as a Reference Point to Radiographically Assess Cardiac Enlargement in Dogs with Myxomatous Mitral Valve Disease.

The diagnostic value of the vertebral heart size (VHS) in dogs with mitral valve degeneration (MVD) is compromised when middle thoracic vertebral anomalies are present. The objective of this study was to assess the use of the thoracic inlet heart score (TIHS) to identify left heart enlargement (LHE) secondary to MVD. The cardiac silhouette of 50 clinically healthy dogs and 106 MVD dogs in different stages was assessed on a right lateral chest radiograph. The TIHS and VHS value were calculated for each patient and compared. The TIHS was significantly different between the control dogs and the dogs with MMVD, increasing with disease stage, control 2.91 ± 0.23, Stage B1 2.98 ± 0.36, B2 3.25 ± 0.34, and C 3.53 ± 0.36, p < 0.05. A THIS ≥3.3 showed 69% sensitivity and 81% specificity to identify LHE. The TIHS showed moderate correlation with the VHS, LA/Ao, and LVIDDN 0.59, 0.42, and 0.62, respectively. The intraobserver and interobserver agreement were almost perfect, 0.96, and substantial, 0.73. The TIHS method can be used to identify LHE secondary to MMVD on dogs' thoracic radiographs.

The Thoracic Inlet Heart Size, a New Approach to Radiographic Cardiac Measurement.

In 1995, the Vertebral Heart Size (VHS) method for measuring the cardiac silhouette on thoracic radiographs was published, becoming a quantifiable and objective reference way of assessing the heart size. Since then, many studies have showed that VHS is influenced by breed variations, vertebral malformations, reference points selection, and short and long axes dimensions conversion into vertebral units. The Thoracic Inlet Heart Size (TIHS) normalizes heart size to body size using the thoracic inlet length. The lengths of the long and short axes of the heart of 144 clinically normal dogs were measured on right lateral thoracic radiographs. The sum of both measures was indexed to the thoracic inlet length. For comparison, dogs of the most represented breeds in our hospital were selected to measure their heart size using the TIHS protocol. The mean TIHS value for the population studied was 2.86 ± 0.27, and 90% of dogs had a TIHS value of less than 3.25. There was no difference in TIHS between male and female, and between small and large dogs (p-value < 0.01). There was no difference in the TIHS value between Yorkshire Terrier, Chihuahua, and Labrador retriever breeds, and between each of those three breeds and the general population. The TIHS is a simple, straightforward and accurate way to measure heart size.

Decreased cytotoxic T cells and TCR clonality in organ transplant recipients with squamous cell carcinoma.

T-cell landscape differences between cutaneous squamous cell carcinoma (cSCC) tumors in immune competent (SCC in IC) and immunocompromised organ transplant recipients (TSCC in OTR) are unclear. We developed an analytical method to define tumor infiltrating lymphocyte (TIL) phenotype in cSCC from immune competent and immune suppressed patients using single-cell TCR sequencing and gene expression data. TSCC exhibits reduced proportions of cytotoxic and naïve TILs and similar numbers of regulatory TILs. Fewer, more heterogeneous TCR clonotypes are observed in TIL from OTR. Most TCR sequences for top ten clonotypes correspond to known antigens, while 24% correspond to putative neoantigens. OTR show increased cSCC events over 12 months possibly due to reduced cytotoxic T-cells. Our novel method of barcoding CD8+ T-cells is the first providing gene expression and TCR sequences in cSCC. Knowledge regarding putative antigens recognized by TCRs with phenotypic function of T-cells bearing those TCRs could facilitate personalized cSCC treatments.

MAGE-A3 is a prognostic biomarker for poor clinical outcome in cutaneous squamous cell carcinoma with perineural invasion via modulation of cell proliferation.

Perineural invasion is a pathologic process of neoplastic dissemination along and invading into the nerves. Perineural invasion is associated with aggressive disease and a greater likelihood of poor outcomes. In this study, 3 of 9 patients with cutaneous squamous cell carcinoma and perineural invasion exhibited poor clinical outcomes. Tumors from these patients expressed high levels of MAGE-A3, a cancer testis antigen that may contribute to key processes of tumor development. In addition to perineural invasion, the tumors exhibited poor differentiation and deep invasion and were subsequently classified as Brigham and Women's Hospital tumor stage 3. Cyclin E, A and B mRNA levels were increased in these tumors compared with normal skin tissues (102.93±15.03 vs. 27.15±4.59, 36.83±19.41 vs. 11.59±5.83, 343.77±86.49 vs. 95.65±29.25, respectively; p<0.05). A431 cutaneous squamous cell carcinoma cells pretreated with MAGE-A3 antibody exhibited a decreased percentage S-phase cells (14.13±2.8% vs. 33.97±1.1%; p<0.05) and reduced closure in scratch assays (43.88±5.49% vs. 61.17±3.97%; p = 0.0058). In a syngeneic animal model of squamous cell carcinoma, immunoblots revealed overexpression of MAGE-A3 and cyclin E, A, and B protein in tumors at 6 weeks. However, knockout of MAGE-A3 expression caused a reduction in tumor growth (mean tumor volume 155.3 mm3 vs. 3.2 mm3) compared with parental cells. These results suggest that MAGE-A3 is a key mediator in cancer progression. Moreover, elevated collagen XI and matrix metalloproteases 3, 10, 11, and 13 mRNA levels were observed in poorly differentiated cutaneous squamous cell carcinoma with perineural invasion compared with normal skin tissue (1132.56±882.7 vs. 107.62±183.62, 1118.15±1109.49 vs. 9.5±5, 2603.87±2385.26 vs. 5.29±3, 957.95±627.14 vs. 400.42±967.66, 1149.13±832.18 vs. 19.41±35.62, respectively; p<0.05). In summary, this study highlights the potential prognostic value of MAGE-A3 in clinical outcomes of cutaneous squamous cell carcinoma patients.

Angiogenic response in an in vitro model of dog microvascular endothelial cells stimulated with antigenic extracts from Dirofilaria immitis adult worms.

BACKGROUND: Angiogenesis can occur under pathological conditions when stimuli such as inflammation, vascular obstruction or hypoxia exist. These stimuli are present in cardiopulmonary dirofilariosis (Dirofilaria immitis). The aim of this study was to analyze the capacity of D. immitis antigens to modify the expression of angiogenic factors and trigger the formation of pseudocapillaries (tube-like structures) in an in vitro model of endothelial cells. METHODS: The expression of VEGF-A, sFlt, mEndoglin and sEndoglin in cultures of canine microvascular endothelial cells stimulated with extract of adult worms of D. immitis obtained from an untreated dog (DiSA) and from a dog treated for 15 days with doxycycline (tDiSA), was determined by using commercial kits. The capacity of pseudocapillary formation was evaluated analyzing cell connections and cell groups in Matrigel cell cultures stimulated with DiSA and tDiSA. In both cases non-stimulated cultures were used as controls. RESULTS: First, we demonstrated that worms obtained from the dog treated with doxycycline showed a significantly lower amount of Wolbachia (less than 60%) than worms removed from the untreated dog. Only DiSA was able to significantly increase the expression of the proangiogenic factor VEGF-A in the endotelial cells cultures. None of the D. immitis extracts modified the expression of sFlt. tDiSA extract was able to modify the expression of the endoglins, significantly decreasing the expression of the pro-angiogenic mEndoglin and increasing the anti-angiogenic sEndoglin. The formation of pseudocapillaries was negatively influenced by tDiSA, which reduced the organization and number of cellular connections. CONCLUSIONS: The ability of antigens from adult D. immitis worms to modify the expression of pro and anti-angiogenic factors in endotelial cell cultures was demonstrated, as well as the trend to form pseudocapillaries in vitro. The capacity of stimulation may be linked to the amount of Wolbachia present in the antigenic extracts.

Ruxolitinib inhibits cyclosporine-induced proliferation of cutaneous squamous cell carcinoma.

Organ transplant recipients (OTRs) on cyclosporine A (CSA) are prone to catastrophic cutaneous squamous cell carcinoma (SCC). Allograft-sparing, cancer-targeting systemic treatments are unavailable. We have shown increased risk for catastrophic SCC in OTRs via CSA-mediated induction of IL-22. Herein, we found that CSA drives SCC proliferation and tumor growth through IL-22 and JAK/STAT pathway induction. We in turn inhibited SCC growth with an FDA-approved JAK1/2 inhibitor, ruxolitinib. In human SCC cells, the greatest proliferative response to IL-22 and CSA treatment occurred in nonmetastasizing lines. IL-22 treatment upregulated JAK1 and STAT1/3 in A431 SCC cells. JAK/STAT pathway genes were highly expressed in tumors from a cohort of CSA-exposed OTRs and in SCC with high risk for metastasis. Compared with immunocompetent SCC, genes associated with innate immunity, response to DNA damage, and p53 regulation were differentially expressed in SCC from OTRs. In nude mice engrafted with human A431 cells, IL-22 and CSA treatment increased tumor growth and upregulated IL-22 receptor, JAK1, and STAT1/3 expression. Ruxolitinib treatment significantly reduced tumor volume and reversed the accelerated tumor growth. CSA and IL-22 exacerbate aggressive behavior in SCC. Targeting the IL-22 axis via selective JAK/STAT inhibition may reduce the progression of aggressive SCC in OTRs, without compromising immunosuppression.

Interleukin-22 and Cyclosporine in Aggressive Cutaneous Squamous Cell Carcinoma.

Cutaneous squamous cell carcinomas (SCCs) account for up to 10,000 deaths annually in the United States. Most of the more than 700,000 SCCs diagnosed are cured by excision with clear margins; however, metastasis can occur despite seemingly adequate treatment in some cases. Immune-suppressed organ transplant recipients are 60 to 100 times more likely to develop SCC than immune-competent individuals. Transplant-associated SCCs occur more frequently and behave more aggressively, showing higher risk of recurrence and metastasis. This article identifies a potential role for interleukin-22 in driving SCC proliferation, particularly in solid organ transplant recipients taking cyclosporine.

RTA Occupancy of the Origin of Lytic Replication during Murine Gammaherpesvirus 68 Reactivation from B Cell Latency.

RTA, the viral Replication and Transcription Activator, is essential for rhadinovirus lytic gene expression upon de novo infection and reactivation from latency. Lipopolysaccharide (LPS)/toll-like receptor (TLR)4 engagement enhances rhadinovirus reactivation. We developed two new systems to examine the interaction of RTA with host NF-kappaB (NF-κB) signaling during murine gammaherpesvirus 68 (MHV68) infection: a latent B cell line (HE-RIT) inducible for RTA-Flag expression and virus reactivation; and a recombinant virus (MHV68-RTA-Bio) that enabled in vivo biotinylation of RTA in BirA transgenic mice. LPS acted as a second stimulus to drive virus reactivation from latency in the context of induced expression of RTA-Flag. ORF6, the gene encoding the single-stranded DNA binding protein, was one of many viral genes that were directly responsive to RTA induction; expression was further increased upon treatment with LPS. However, NF-κB sites in the promoter of ORF6 did not influence RTA transactivation in response to LPS in HE-RIT cells. We found no evidence for RTA occupancy of the minimal RTA-responsive region of the ORF6 promoter, yet RTA was found to complex with a portion of the right origin of lytic replication (oriLyt-R) that contains predicted RTA recognition elements. RTA occupancy of select regions of the MHV-68 genome was also evaluated in our novel in vivo RTA biotinylation system. Streptavidin isolation of RTA-Bio confirmed complex formation with oriLyt-R in LPS-treated primary splenocytes from BirA mice infected with MHV68 RTA-Bio. We demonstrate the utility of reactivation-inducible B cells coupled with in vivo RTA biotinylation for mechanistic investigations of the interplay of host signaling with RTA.

Interplay of Murine Gammaherpesvirus 68 with NF-kappaB Signaling of the Host.

Herpesviruses establish a chronic infection in the host characterized by intervals of lytic replication, quiescent latency, and reactivation from latency. Murine gammaherpesvirus 68 (MHV68) naturally infects small rodents and has genetic and biologic parallels with the human gammaherpesviruses (gHVs), Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus. The murine gammaherpesvirus model pathogen system provides a platform to apply cutting-edge approaches to dissect the interplay of gammaherpesvirus and host determinants that enable colonization of the host, and that shape the latent or lytic fate of an infected cell. This knowledge is critical for the development of novel therapeutic interventions against the oncogenic gHVs. The nuclear factor kappa B (NF-κB) signaling pathway is well-known for its role in the promotion of inflammation and many aspects of B cell biology. Here, we review key aspects of the virus lifecycle in the host, with an emphasis on the route that the virus takes to gain access to the B cell latency reservoir. We highlight how the murine gammaherpesvirus requires components of the NF-κB signaling pathway to promote replication, latency establishment, and maintenance of latency. These studies emphasize the complexity of gammaherpesvirus interactions with NF-κB signaling components that direct innate and adaptive immune responses of the host. Importantly, multiple facets of NF-κB signaling have been identified that might be targeted to reduce the burden of gammaherpesvirus-associated diseases.