Probes for INS

Human Papilloma Virus (HPV) can play a causative role in the development of sinonasal tract malignancies. In fact, HPV may be the most significant causative agent implicated in sinonasal tumorigenesis and is implicated in as many as 21% of sinonasal carcinomas. To date, there are no definitive, reliable and cost-effective, diagnostic tests approved by the FDA for the unequivocal determination of HPV status in head and neck cancers. We followed an exhaustive algorithm to correctly test HPV infection, including a sequential approach with p16INK4a IHC, viral DNA genotyping and in situ hybridization for E6/E7 mRNA. Here, we report a case of sinonasal carcinoma with discordant results using HPV test assays. The tumor we describe showed an irregular immunoreactivity for p16INK4a, and it tested positive for HPV DNA; nevertheless, it was negative for HR-HPV mRNA. We discuss the possible meaning of this discrepancy. It would be advisable to test HPV transcriptional status of sinonasal carcinoma on a diagnostic routine basis, not only by p16INK4a IHC assay, but also by HPV DNA genotyping and HR-HPV mRNA assessment.

LN follicles constitute major reservoir sites for HIV/SIV persistence. Cure strategies could benefit from the characterization of CD8+ T cells able to access and eliminate HIV-infected cells from these areas. In this study, we provide a comprehensive analysis of the phenotype, frequency, localization, and functionality of follicular CD8+ T cells (fCD8+) in SIV-infected nonhuman primates. Although disorganization of follicles was a major factor, significant accumulation of fCD8+ cells during chronic SIV infection was also observed in intact follicles, but only in pathogenic SIV infection. In line with this, tissue inflammatory mediators were strongly associated with the accumulation of fCD8+ cells, pointing to tissue inflammation as a major factor in this process. These fCD8+ cells have cytolytic potential and can be redirected to target and kill HIV-infected cells using bispecific antibodies. Altogether, our data support the use of SIV infection to better understand the dynamics of fCD8+ cells and to develop bispecific antibodies as a strategy for virus eradication.

Sexual transmission of filoviruses was first reported in 1968 after an outbreak of Marburg virus (MARV) disease and recently caused flare-ups of Ebola virus disease in the 2013–2016 outbreak. How filoviruses establish testicular persistence and are shed in semen remain unknown. We discovered that persistent MARV infection of seminiferous tubules, an immune-privileged site that harbors sperm production, is a relatively common event in crab-eating macaques that survived infection after antiviral treatment. Persistence triggers severe testicular damage, including spermatogenic cell depletion and inflammatory cell invasion. MARV mainly persists in Sertoli cells, leading to breakdown of the blood-testis barrier formed by inter-Sertoli cell tight junctions. This disruption is accompanied by local infiltration of immunosuppressive CD4+Foxp3+ regulatory T cells. Our study elucidates cellular events associated with testicular persistence that may promote sexual transmission of filoviruses and suggests that targeting immunosuppression may be warranted to clear filovirus persistence in damaged immune-privileged sites.

Abstract

Background: Identifying biomarkers for cells harboring replication-competent HIV is a major research priority. Recently, there have been mixed reports addressing the possibility that CD32-expressing T-cells are enriched for HIV. There is growing evidence that CD32 expression increases with cellular activation that may be related to, but not necessarily specific for, infection with HIV.  However, the relationship of CD32 expression to HIV-infection in subtypes of tissue-resident leukocytes is unclear. 

 Methods: First, we used duplex chromogenic in situ hybridization to identify cells actively transcribing RNA for both CD32 and HIV on human gut tissues. Then we performed multiplexed immunofluorescence and in situ hybridization (mIFISH) on sections from the same tissues to determine the phenotype of individual cells co-expressing HIV-RNA and CD32-RNA. 

 Results: HIV-RNA+ cells were more abundant in tissues from viremic individuals compared to those on suppressive anti-retroviral therapy (ART). However, staining by both methods indicated that a higher proportion of HIV-RNA+ cells co-expressed CD32-RNA in ART-suppressed compared to viremic individuals. The majority of HIV-RNA+ cells were CD3+.

Conclusions: Our data suggest that the transcription of CD32-RNA is correlated with HIV transcriptional activity in CD3+ cells found within human gut tissue. Whether or not up-regulation of CD32-RNA is a direct result of HIV reactivation or more global T cell activation remains unclear.

Although human papillomavirus (HPV)-related oropharyngeal carcinomas (HPV-OPCs) are generally regarded as "poorly differentiated," they actually maintain a close resemblance to the lymphoepithelium of the tonsillar crypts from which they arise: they are basaloid, exhibit minimal keratinization, and are often permeated by lymphocytes. In rare cases, the presence of cilia in a primary HPV-OPC and their persistence in lymph node metastasis can confound the distinction between a benign and malignant process. Three cases of ciliated HPV-OPCs were identified from the archives of The Johns Hopkins Head and Neck Pathology consultation service. HPV status was determined using p16 immunohistochemistry and high-risk HPV in situ hybridization. All 3 patients presented with a cystic lymph node metastasis without a known primary carcinoma. One metastasis was originally diagnosed as a branchial cleft cyst only to regionally recur 7 years later. In 2 cases, a primary HPV-OPC was found in the tonsil. The carcinomas exhibited both nonkeratinizing squamous epithelium and cystic/microcystic spaces lined by ciliated columnar cells. Both the squamous and ciliated cells were HPV positive. This report draws attention to a novel variant of HPV-related head and neck cancer that exhibits ciliated columnar cells. This variant challenges prevailing notions that: (1) HPV-OPCs are uniformly poorly differentiated cancers; (2) cilia are an infallible feature of benignancy; and (3) presence of cilia is a reliable criterion for establishing branchial cleft origin when dealing with cystic lesions of the lateral neck.

The emergence of Zika virus (ZIKV) and its association with congenital malformations has prompted the rapid development of vaccines. Although efficacy with multiple viral vaccine platforms has been established in animals, no study has addressed protection during pregnancy. We tested in mice two vaccine platforms, a lipid nanoparticle-encapsulated modified mRNA vaccine encoding ZIKV prM and E genes and a live-attenuated ZIKV strain encoding an NS1 protein without glycosylation, for their ability to protect against transmission to the fetus. Vaccinated dams challenged with a heterologous ZIKV strain at embryo day 6 (E6) and evaluated at E13 showed markedly diminished levels of viral RNA in maternal, placental, and fetal tissues, which resulted in protection against placental damage and fetal demise. As modified mRNA and live-attenuated vaccine platforms can restrict in utero transmission of ZIKV in mice, their further development in humans to prevent congenital ZIKV syndrome is warranted.

While HIV kills most of the cells it infects, a small number of infected cells survive and become latent viral reservoirs, posing a significant barrier to HIV eradication. However, the mechanism by which immune cells resist HIV-induced apoptosis is still incompletely understood. Here, we demonstrate that while acute HIV infection of human microglia/macrophages results in massive apoptosis, a small population of HIV-infected cells survive infection, silence viral replication, and can reactivate viral production upon specific treatments. We also found that HIV fusion inhibitors intended for use as antiretroviral therapies extended the survival of HIV-infected macrophages. Analysis of the pro- and anti-apoptotic pathways indicated no significant changes in Bcl-2, Mcl-1, Bak, Bax or caspase activation, suggesting that HIV blocks a very early step of apoptosis. Interestingly, Bim, a highly pro-apoptotic negative regulator of Bcl-2, was upregulated and recruited into the mitochondria in latently HIV-infected macrophages both in vitro and in vivo. Together, these results demonstrate that macrophages/microglia act as HIV reservoirs and utilize a novel mechanism to prevent HIV-induced apoptosis. Furthermore, they also suggest that Bim recruitment to mitochondria could be used as a biomarker of viral reservoirs in vivo.

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.

Progress toward understanding Zika virus (ZIKV) pathogenesis is hindered by lack of immunocompetent small animal models, in part because ZIKV fails to effectively antagonize Stat2-dependent interferon (IFN) responses in mice. To address this limitation, we first passaged an African ZIKV strain (ZIKV-Dak-41525) through Rag1-/- mice to obtain a mouse-adapted virus (ZIKV-Dak-MA) that was more virulent than ZIKV-Dak-41525 in mice treated with an anti-Ifnar1 antibody. A G18R substitution in NS4B was the genetic basis for the increased replication, and resulted in decreased IFN-β production, diminished IFN-stimulated gene expression, and the greater brain infection observed with ZIKV-Dak-MA. To generate a fully immunocompetent mouse model of ZIKV infection, human STAT2 was introduced into the mouse Stat2 locus (hSTAT2 KI). Subcutaneous inoculation of pregnant hSTAT2 KI mice with ZIKV-Dak-MA resulted in spread to the placenta and fetal brain. An immunocompetent mouse model of ZIKV infection may prove valuable for evaluating countermeasures to limit disease.