Probes for INS

Crimean-Congo hemorrhagic fever virus (CCHFV) can cause severe hepatic injury in humans. However, the mechanism(s) causing this damage are poorly characterized. CCHFV produces an acute disease, including liver damage, in mice lacking type I interferon signaling (IFN-I) either due to STAT-1 gene deletion or disruption of the IFN-I receptor 1 gene. Here, we explored CCHFV-induced liver pathogenesis in mice using an antibody to disrupt IFN-I signaling. When IFN-I blockade was induced within 24 h post-exposure to CCHFV, mice developed severe disease with greater than 95% mortality by six days post-exposure. In addition, we observed increased proinflammatory cytokines, chemoattractants, and liver enzymes in these mice. Extensive liver damage was evident by 4 days post-exposure and was characterized by hepatocyte necrosis and loss of CLEC4F-positive Kupffer cells. Similar experiments in CCHFV-exposed NOD-SCID-γ (NSG), Rag2-deficient, and perforin-deficient mice also demonstrated liver injury, suggesting cytotoxic immune cells are dispensable for hepatic damage. Some apoptotic liver cells contained viral RNA while other apoptotic liver cells were negative, suggesting that cell death occurred by both intrinsic and extrinsic mechanisms. Protein and transcriptional analysis of livers revealed that activation of TNF superfamily members occurred by day four post-exposure, implicating these molecules as factors in liver cell death. These data provide insights into CCHFV-induced hepatic injury and demonstrate the utility of antibody-mediated IFN-I blockade in the study of CCHFV pathogenesis in mice.IMPORTANCE CCHFV is an important human pathogen that is both endemic and emerging throughout Asia, Africa and Europe. A common feature of acute disease is liver injury ranging from mild to fulminant hepatic failure. The processes through which CCHFV induces severe liver injury are unclear, mostly due to the limitations of existing small animal systems. The only small animal model in which CCHFV consistently produces severe liver damage are mice lacking IFN-I signaling. In this study, we used antibody-mediated blockade of IFN-I signaling in mice to study CCHFV liver pathogenesis in various transgenic mouse systems. We found liver injury did not depend on cytotoxic immune cells and observed extensive activation of death receptor signaling pathways in the liver during acute disease. Furthermore, acute CCHFV infection resulted in a near complete loss of Kupffer cells. Our model system provides insight into both the molecular and cellular features of CCHFV hepatic injury.

Background and aims

The causative role of high risk human papillomavirus (HR-HPV) in breast cancer development is controversial, though a number of reports have identified HR-HPV DNA in breast cancer specimens. Nevertheless, most studies to date have focused primarily on viral DNA rather than the viral transcription. The aim of this study was to investigate the presence of HR-HPV in breast cancer tissues at HPV DNA level and HPV oncogenes mRNA level by in situ hybridization (ISH).

Methods

One hundred and forty six (146) cases of breast invasive ductal carcinoma(IDC) and 83 cases of benign breast lesions were included in the study. Type specific oligonucleotide probes were used for the DNA detection of HPV 16,18 and 58 by ISH. HR-HPV oncogenes mRNA was assayed by novel RNAscope HR-HPV HR7 assay ISH. p16 protein expression was evaluated by immunohistochemistry (IHC).

Results

HR-HPV 16,18 and 58 DNA were detected in 52 out of 146 (35.6%) IDC and in 3 out of 83 (3.6%) benign breast lesions by ISH. The HR-HPV mRNAs was detected only in a few specimens with strong HPV DNA positivity(4/25) in a few scattered cancer cells with very weak punctate nuclear and/or cytoplasmic staining. p16 over-expression did not correlate with the HPV DNA positive breast cancer samples(17/52 HPVDNA+ vs 28/94 HPV DNA-, p = 0.731).

Conclusions

HR-HPVs certainly exist in breast cancer tissue with less active transcription, which implies that the causal role of HPV in breast cancer development need further study.

The performance characteristics of neuroendocrine-specific and squamous-specific immunohistochemical markers in head and neck squamous cell carcinomas (SCC), in particular in oropharyngeal tumors in this era of human papillomavirus (HPV)-induced cases, are not well-established. The differential diagnosis for poorly differentiated SCCs, for nonkeratinizing oropharyngeal SCCs, and for other specific SCC variants such as basaloid SCC and undifferentiated (or lymphoepithelial-like) carcinomas includes neuroendocrine carcinomas. Given that neuroendocrine carcinomas of the head and neck are aggressive regardless of HPV status, separating them from SCC is critically important. In this study, we examined the neuroendocrine markers CD56, synaptophysin, and chromogranin-A along with the squamous markers p40 and cytokeratin 5/6 in a large tissue microarray cohort of oral, oropharyngeal, laryngeal, and hypopharyngeal SCCs with known HPV results by RNA in situ hybridization for the oropharyngeal tumors. Results were stratified by site and specific SCC variant. The neuroendocrine stains were rarely expressed in SCC (<1% overall) with CD56 the least, and chromogranin-A the most, specific markers. Further, p40 and cytokeratin 5/6 were very consistently expressed in all head and neck SCC (>98% overall), including very strong, consistent staining in oropharyngeal HPV-related nonkeratinizing SCC. Undifferentiated (or lymphoepithelial-like) carcinomas of the oropharynx are more frequently p40 or cytokeratin 5/6 negative or show only weak or focal expression. In summary, markers of neuroendocrine and squamous differentiation show very high specificity and sensitivity, respectively, across the different types of head and neck SCC.

HPV infected cervical cells secrete mediators that are gradually changed and have influence on infiltrating M2 phenotypic monocytes in cervical lesions. However, profiles of circulating immune cells in women with cervical lesions and M2 phenotypic monocyte activity in HPV infected cervical lesions are limited. This study aimed to investigate circulating monocyte populations correlated with M2 phenotype density and its activity in HPV infected cervical lesions. HPV DNA was investigated in cervical tissues using PCR. High risk HPV E6/E7 mRNA was detected using in situ hybridization. CD163 immunohistochemical staining was performed for M2 macrophage. CD163 and Arg1 mRNA expression were detected using real-time PCR. Circulating monocyte subpopulations were analyzed using flow cytometry. CD163 and Arg1 mRNA expression were increased according to cervical lesion severity and corresponding with density of M2 macrophage in HSIL and SCC in stroma and peri-tumoral areas. Additionally, the relationship between M2 macrophage infiltration and high risk HPV E6/E7 mRNA expression was found and corresponded with cervical lesion severity. Circulating CD14+CD16+ and CD14+CD163+ monocytes were elevated in No-SIL and cervical lesions. Interestingly, CD14+CD64+ monocyte was greatly elevated in HSIL and SCC, whereas intracellular IL-10+monocytes were not significantly different between cervical lesions. The correlation between increasing ratio of circulating CD64+/CD163+monocyte and density of infiltrating CD163+ monocytes was associated with severity of HPV infected cervical lesions. The elevated circulating CD64+/CD163+ monocyte ratio correlates to severity of HPV infected cervical lesions and might be a prognostic marker in cervical cancer progression.

Abstract

Objectives

In the present study, we comprehensively analyzed the prevalence of transcriptionally active HPV in tissue samples of Indian patients with leukoplakia - predominantly hyperplastic lesions and HNSCC. In addition, saliva samples from patients with HNSCC were screened for HPV detection.

Study Design

p16 overexpression was analyzed by immunohistochemistry. Leukoplakia (n = 121) and HNSCC (n = 427) tissue samples and the saliva of patients with HNSCC (n = 215) were tested for HPV using nested PCR. Positive samples were sequenced for subtyping. The presence of HPV E6/E7 mRNA was confirmed by RNA in-situ hybridization.

Results

p16 expression and HPV DNA were not detected in any of the leukoplakia specimens. Of the 427 HNSCC tumors, 9 showed p16 overexpression and 7/427 cases were positive for HPV16 DNA, either in saliva and/or tissue. E6/E7 mRNA positivity was observed in eight HNSCC samples, primarily from patients with no habit of tobacco consumption. The prevalence of high-risk HPV was restricted to oropharynx and larynx with very little concordance between p16 overexpression and HPV positivity. All patients with HPV positive saliva samples had transcriptionally active HPV present in their tumors.

Conclusion

Presence of HPV-DNA does not necessarily reflect transcriptionally active virus in tumors; hence, it is important to consider this fact while categorizing HPV associated tumors.

Sonic hedgehog (SHH) is an essential morphogenetic signal dictating cell fate decisions in several developing organs in mammals. In vitrodata suggest that SHH is required to specify LHX3+/LHX4+ Rathke's pouch (RP) progenitor identity. However, in vivo studies have failed to reveal such a function, supporting instead, a critical role for SHH in promoting proliferation of these RP progenitors and for differentiation of pituitary cell types. Here, we have used a genetic approach to demonstrate that activation of the SHH pathway is necessary to induce LHX3+/LHX4+ RP identity in mouse embryos. First, we show that conditional deletion of Shh in the anterior hypothalamus results in a fully penetrant phenotype characterised by a complete arrest of RP development, with lack of Lhx3/Lhx4 expression in RP epithelium at 9.0 dpc (days post coitum) and total loss of pituitary tissue by 12.5 dpc. Conversely, over-activation of the SHH pathway by conditional deletion of Ptch1 in RP progenitors leads to severe hyperplasia and enlargement of the Sox2+ve stem cell compartment by the end of gestation.

The unique mental abilities of humans are rooted in the immensely expanded and folded neocortex, which reflects the expansion of neural progenitors, especially basal progenitors including basal radial glia (bRGs) and intermediate progenitor cells (IPCs). We found that constitutively active Sonic hedgehog (Shh) signaling expanded bRGs and IPCs and induced folding in the otherwise smooth mouse neocortex, whereas the loss of Shh signaling decreased the number of bRGs and IPCs and the size of the neocortex. SHH signaling was strongly active in the human fetal neocortex but Shh signaling was not strongly active in the mouse embryonic neocortex, and blocking SHH signaling in human cerebral organoids decreased the number of bRGs. Mechanistically, Shh signaling increased the initial generation and self-renewal of bRGs and IPC proliferation in mice and the initial generation of bRGs in human cerebral organoids. Thus, robust SHH signaling in the human fetal neocortex may contribute to bRG and IPC expansion and neocortical growth and folding.