Probes for INS

Studies utilizing highly pathogenic simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus (SHIV) have largely focused on the immunopathology of the central nervous system (CNS) during end-stage neuro AIDS and SIV encephalitis. However, this may not model pathophysiology in earlier stages of infection. In this non-accelerated SHIV model, plasma SHIV RNA levels and peripheral blood and colonic CD4 T+ cell counts mirrored early HIV infection in humans. At 12 weeks post infection, cerebrospinal fluid (CSF) detection of SHIV RNA and elevations in IP-10 and MCP-1 reflected a discrete neurovirologic process. Immunohistochemical staining revealed a diffuse, low-level CD3+, CD4- cellular infiltrate in the brain parenchyma, without a concomitant increase in CD68/CD163+ monocytes, macrophages and activated microglial cells. Rare SHIV-infected cells in the brain parenchyma and meninges were identified by RNAscope^®in situhybridization. In the meninges, there was also a trend toward increased CD4+ infiltration in SHIV-infected animals, but no differences in CD68/CD163+ cells between SHIV-infected and uninfected control animals. These data suggest that in a model that closely recapitulates human disease, CNS inflammation and SHIV in CSF may be predominantly mediated by T-cell mediated processes during early infection in both brain parenchyma and meninges. Because SHIV expresses an HIV rather than SIV envelope, this model could inform studies to understand potential HIV cure strategies targeting the HIV envelope.IMPORTANCE Animal models of the neurologic effects of HIV are needed because brain pathology is difficult to assess in humans. Many current models focus on the effects of late stage disease utilizing simian immunodeficiency virus (SIV). In the era of antiretroviral therapy, manifestations of late stage HIV are less common. Furthermore, new interventions such as monoclonal antibodies and therapeutic vaccinations target HIV envelope. We therefore describe a new model of central nervous system involvement in rhesus macaques infected with simian-human immunodeficiency virus (SHIV) expressing HIV envelope in earlier, less aggressive stages of disease. Here, we demonstrate that SHIV mimics the early clinical course in humans, and that early neurologic inflammation is characterized by predominantly T cell mediated inflammation, accompanied by SHIV infection in the brain and meninges. This model can be utilized to assess the effect of novel therapies targeted to HIV envelope on reducing brain inflammation before end stage disease.

OBJECTIVE:

The programed death-1 (PD1)/programed death-ligand 1 (PD-L1) pathway plays a critical role in balancing immunity and host immunopathology. During chronic HIV/SIV infection, there is persistent immune activation accompanied by accumulation of virus-specific cells with terminally differentiated phenotypes and expression of regulatory receptors such as PD1. These observations led us to hypothesize that the PD1/PD-L1 pathway contributes to the functional dysregulation and ineffective viral control, and its blockade may be a potential immunotherapeutic target.

METHODS:

Lymph node biopsies from HIV-infected patients (n = 23) were studied for expression of PD1 and PD-L1. In addition, we assessed the safety and biological activity of a human anti-PD-L1 antibody (Avelumab) in chronically SIV-infected rhesus macaques.

RESULTS:

PD-L1 expression was observed in cells with myloid/macrophage morphology in HIV-infected lymph nodes. Administration of anti-PD-L1 was well tolerated, and no changes in body weights, hematologic, or chemistry parameters were observed during the study. Blockade of PD-L1 led to a trend of transient viral control after discontinuation of treatment.

CONCLUSION:

Administration of anti-PD-L1 in chronic SIV-infected rhesus macaques was well tolerated. Overall, these data warrant further investigation to assess the efficacy of anti-PD-L1 treatment on viral control in chronic SIV infection as a prelude to such therapy in humans.

Nonhuman primate models are needed for evaluations of proposed strategies targeting residual virus that persists in HIV-1-infected individuals receiving suppressive combination antiretroviral therapy (cART). However, relevant nonhuman primate (NHP) models of cART-mediated suppression have proven challenging to develop. We used a novel three-class, six-drug cART regimen to achieve durable 4.0- to 5.5-log reductions in plasma viremia levels and declines in cell-associated viral RNA and DNA in blood and tissues of simian immunodeficiency virus SIVmac239-infected Indian-origin rhesus macaques, then evaluated the impact of treatment with the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA; Vorinostat) on the residual virus pool. Ex vivo SAHA treatment of CD4(+) T cells obtained from cART-suppressed animals increased histone acetylation and viral RNA levels in culture supernatants. cART-suppressed animals each received 84 total doses of oral SAHA. We observed SAHA dose-dependent increases in acetylated histones with evidence for sustained modulation as well as refractoriness following prolonged administration. In vivo virologic activity was demonstrated based on the ratio of viral RNA to viral DNA in peripheral blood mononuclear cells, a presumptive measure of viral transcription, which significantly increased in SAHA-treated animals. However, residual virus was readily detected at the end of treatment, suggesting that SAHA alone may be insufficient for viral eradication in the setting of suppressive cART. The effects observed were similar to emerging data for repeat-dose SAHA treatment of HIV-infected individuals on cART, demonstrating the feasibility, utility, and relevance of NHP models of cART-mediated suppression for in vivo assessments of AIDS virus functional cure/eradication approaches.

Given the comparable strains of high-risk human papillomavirus (HPV) present in a subset of Barrett's dysplasia and esophageal adenocarcinoma as in head and neck squamous cell carcinomas and the anatomical proximity of both lesions, we hypothesized that oral sex may increase the risk of Barrett's dysplasia/esophageal adenocarcinoma. Therefore, we compared the sexual behavior of patients with Barrett's dysplasia/esophageal adenocarcinoma and controls (hospital, reflux, and Barrett's metaplasia) to explore a plausible mechanism of viral transmission to the lower esophagus. A hospital-based case-control study involving 36 Barrett's dysplasia/esophageal adenocarcinoma subjects and 55 controls with known HPV DNA status and markers of transcriptional activity i.e p16INK4A and E6/E7 mRNA of the esophageal epithelium was conducted to evaluate differences in sexual history (if any). Barrett's dysplasia/esophageal adenocarcinoma patients were more likely than controls to be positive for HPV DNA (18 of 36, 50% vs. 6/55, 11%, p for trend <0.0001), be male (P = 0.001) and in a relationship (P = 0.02). Viral genotypes identified were HPV 16 (n = 14), 18 (n = 2), 11 (n = 1) and 6 (n = 1). HPV exposure conferred a significantly higher risk for Barrett's dysplasia/esophageal adenocarcinoma as compared with hospital/reflux/Barrett's metaplasia controls (OR = 6.8, 95% CI: 2.1-23.1, adjusted P = 0.002). On univariate analysis, ≥6 lifetime oral sex partners were significantly associated with dysplastic Barrett's esophagus and adenocarcinoma (OR, 4.0; 95% CI: 1.2-13.7, P = 0.046). After adjustment for confounders, HPV exposure and men with ≥2 lifetime sexual partners were at significant risk for Barrett's dysplasia/esophageal adenocarcinoma. If these initial findings can be confirmed in larger studies, it could lead to effective prevention strategies in combating some of the exponential increase in the incidence of esophageal adenocarcinoma in the West.

Detection of human papilloma virus (HPV)-related head and neck squamous cell carcinoma (HNSCC) is important, as HPV-associated HNSCCs respond better to therapy. The RNAscope HPV-test is a novel RNA in situ hybridization (ISH) technique which strongly stains transcripts of E6 and E7 mRNA in formalin-fixed, paraffin-embedded tissue, with the potential to replace the indirect immunohistochemical (IHC) marker for p16 protein. A direct clinical comparison between p16 IHC and an automated RNA ISH using 18 probes has not been established. Samples from 27 formalin-fixed, paraffin-embedded HNSCC cases from the Emory University Hospital archives were stained using 18 individual RNA ISH probes for high-risk HPV (RNAscope 2.5 LS Probe ) on a Leica autostainer (Buffalo Grove, IL) and were compared with p16 IHC. Two pathologists reviewed and reached a consensus on all interpretations. The RNAscope technique was positive in 89% (24/27) and the p16 IHC was positive in 78% (21/27). The RNAscope was negative in 11.1% of samples (3/27) and the p16 IHC-negative in 22.2% (6/27). The RNA ISH detected 100% of the p16-positive IHC-stained slides and had a concordance of 88.9% (24/27). This easy to interpret automated staining method for 18 high-risk HPV genotypes is a feasible replacement for the indirect p16 IHC method.

Human papillomavirus (HPV)-related multiphenotypic sinonasal carcinoma is a peculiar sinonasal tract tumor that demonstrates features of both a surface-derived and salivary gland carcinoma. Implicit in its name, this tumor has a consistent association with high-risk HPV, particularly type 33. It was first described in 2013 under the designation of HPV-related carcinoma with adenoid cystic carcinoma-like features. However, since its initial description additional cases have emerged which demonstrate a wide morphologic spectrum and relatively indolent clinical behavior. Herein we report our experience with a case of HPV-related multiphenotypic sinonasal carcinoma that was initially classified as adenoid cystic carcinoma in the 1980s. The patient recurred after a 30-year disease free interval. RNA in situ hybridization confirmed the presence of high-risk HPV in both her recurrence and her initial tumor in the 1980s, which allowed for reclassification as HPV-related multiphenotypic sinonasal carcinoma. Our case adds to the literature of this relatively newly described entity and supports the indolent clinical behavior of this neoplasm but also demonstrates a potential for very late local recurrence.

The oral cavity and oropharynx have historically been viewed as a single anatomic compartment of the head and neck. The practice of combining the oral cavity and oropharynx has recently been revised, largely owing to the observation that human papillomavirus (HPV)-related carcinogenesis has a strong predilection for the oropharynx but not the oral cavity. The purpose of this study was to determine whether HPV is evenly distributed across squamous cell carcinomas of the oropharynx including those sites that do not harbor tonsillar tissues such as the soft palate. A search of the medical records of the Johns Hopkins Hospital identified 32 primary squamous cell carcinomas of the soft palate (n=31) and posterior pharyngeal wall (n=1). All were evaluated with p16 immunohistochemistry and high-risk HPV in situ hybridization (ISH) (29 by RNA ISH and 3 by DNA ISH). For comparison, we also reviewed the medical records to obtain the HPV status of patients who had undergone HPV testing of primary tonsillar carcinomas over the same time interval as part of their clinical care. High-risk HPV as detected by ISH was present in just 1 (3.1%) of the 32 oropharyngeal squamous cell carcinomas, including 1 of 2 p16-positive carcinomas. The difference in HPV detection rates between tonsillar and nontonsillar sites was significant (1/32, 3.1% vs. 917/997, 92%; P<0.0001). HPV is not frequently detected in squamous cell carcinomas arising from nontonsillar regions of the oropharynx. Indeed, squamous cell carcinomas of the soft palate more closely resemble those arising in the oral cavity than those arising in areas of the oropharynx harboring tonsillar tissue. This finding not only further sharpens our understanding of site-specific targeting by HPV, but may have practical implications regarding HPV testing and even the way the oral vault is oncologically compartmentalized to partition HPV-positive from HPV-negative cancers.

Abstract