Early corticosteroid treatment enhances recovery from SARS-CoV-2 induced loss of smell in hamster.

Among the numerous long COVID symptoms, olfactory dysfunction persists in ∼10 % of patients suffering from SARS-CoV-2 induced anosmia. Among the few potential therapies, corticoid treatment has been used for its anti-inflammatory effect with mixed success in patients. In this study, we explored its impact using hamster as an animal model. SARS-CoV-2 infected hamsters lose their smell abilities and this loss is correlated with damage of the olfactory epithelium and persistent presence of innate immunity cells. We started a dexamethasone treatment 2 days post infection, when olfaction was already impacted, until 11 days post infection when it started to recover. We observed an improvement of olfactory capacities in the animals treated with corticoid compared to those treated with vehicle. This recovery was not related to differences in the remaining damage to the olfactory epithelium, which was similar in both groups. This improvement was however correlated with a reduced inflammation in the olfactory epithelium with a local increase of the mature olfactory neuron population. Surprisingly, at 11 days post infection, we observed an increased and disorganized presence of immature olfactory neurons, especially in persistent inflammatory zones of the epithelium. This unusual population of immature olfactory neurons coincided with a strong increase of olfactory epithelium proliferation in both groups. Our results indicate that persistent inflammation of the olfactory epithelium following SARS-CoV-2 infection may alter the extent and speed of regeneration of the olfactory neuron population, and that corticoid treatment is effective to limit inflammation and improve olfaction recovery following SARS-CoV-2 infection.

Neutrophils play a major role in the destruction of the olfactory epithelium during SARS-CoV-2 infection in hamsters.

The loss of smell (anosmia) related to SARS-CoV-2 infection is one of the most common symptoms of COVID-19. Olfaction starts in the olfactory epithelium mainly composed of olfactory sensory neurons surrounded by supporting cells called sustentacular cells. It is now clear that the loss of smell is related to the massive infection by SARS-CoV-2 of the sustentacular cells in the olfactory epithelium leading to its desquamation. However, the molecular mechanism behind the destabilization of the olfactory epithelium is less clear. Using golden Syrian hamsters infected with an early circulating SARS-CoV-2 strain harboring the D614G mutation in the spike protein; we show here that rather than being related to a first wave of apoptosis as proposed in previous studies, the innate immune cells play a major role in the destruction of the olfactory epithelium. We observed that while apoptosis remains at a low level in the damaged area of the infected epithelium, the latter is invaded by Iba1+ cells, neutrophils and macrophages. By depleting the neutrophil population or blocking the activity of neutrophil elastase-like proteinases, we could reduce the damage induced by the SARS-CoV-2 infection. Surprisingly, the impairment of neutrophil activity led to a decrease in SARS-CoV-2 infection levels in the olfactory epithelium. Our results indicate a counterproductive role of neutrophils leading to the release of infected cells in the lumen of the nasal cavity and thereby enhanced spreading of the virus in the early phase of the SARS-CoV-2 infection.

HPV RNA CISH score identifies two prognostic groups in a p16 positive oropharyngeal squamous cell carcinoma population.

HPV-related and HPV-unrelated oropharyngeal squamous cell carcinomas are two distinct entities according to the Union for International Cancer Control, with a better prognosis conferred to HPV-related oropharyngeal squamous cell carcinomas. However, variable clinical outcomes are observed among patients with p16 positive oropharyngeal squamous cell carcinoma, which is a surrogate marker of HPV infection. We aimed to investigate the prognostic value of RNA CISH against E6 and E7 transcripts (HPV RNA CISH) to predict such variability. We retrospectively included 50 histologically confirmed p16 positive oropharyngeal squamous cell carcinomas (p16 positive immunostaining was defined by a strong staining in 70% or more of tumor cells). HPV RNA CISH staining was assessed semi-quantitatively to define two scores: RNA CISH "low" and RNA CISH "high". Negative HPV RNA CISH cases were scored as RNA CISH "low". This series contained 29 RNA CISH low cases (58%) and 21 RNA CISH high cases (42%). Clinical and pathologic baseline characteristics were similar between the two groups. RNA CISH high staining was associated with a better overall survival in both univariate and multivariate analyses (p = 0.033 and p = 0.042, respectively). Other recorded parameters had no prognostic value. In conclusion, HPV RNA CISH might be an independent prognostic marker in p16 positive oropharyngeal squamous cell carcinomas and might help guide therapeutics.

EZH2 inhibition reduces cartilage loss and functional impairment related to osteoarthritis.

Histone methyltransferase EZH2 is upregulated during osteoarthritis (OA), which is the most widespread rheumatic disease worldwide, and a leading cause of disability. This study aimed to assess the impact of EZH2 inhibition on cartilage degradation, inflammation and functional disability. In vitro, gain and loss of EZH2 function were performed in human articular OA chondrocytes stimulated with IL-1ß. In vivo, the effects of EZH2 inhibition were investigated on medial meniscectomy (MMX) OA mouse model. The tissue alterations were assayed by histology and the functional disabilities of the mice by actimetry and running wheel. In vitro, EZH2 overexpression exacerbated the action of IL-1ß in chondrocytes increasing the expression of genes involved in inflammation, pain (NO, PGE2, IL6, NGF) and catabolism (MMPs), whereas EZH2 inhibition by a pharmacological inhibitor, EPZ-6438, reduced IL-1ß effects. Ex vivo, EZH2 inhibition decreased IL-1ß-induced degradation of cartilage. In vivo, intra-articular injections of the EZH2 inhibitor reduced cartilage degradation and improved motor functions of OA mice. This study demonstrates that the pharmacological inhibition of the histone methyl-transferase EZH2 slows the progression of osteoarthritis and improves motor functions in an experimental OA model, suggesting that EZH2 could be an effective target for the treatment of OA by reducing catabolism, inflammation and pain.

HPV detection and genotyping of head and neck cancer biopsies by molecular testing with regard to the new oropharyngeal squamous cell carcinoma classification based on HPV status.

Recently, both the World Health Organization/International Agency for Research on Cancer (WHO/IARC) and the American Joint Committee on Cancer (AJCC) have classified oropharyngeal squamous cell carcinoma (OPSCC) on the basis of HPV status. For this purpose, the WHO/IARC recommended direct molecular HPV testing. In practice, formalin-fixed, paraffin-embedded (FFPE) biopsy specimens are frequently the only available samples. We herein compared in parallel two commercially available molecular assays that were first designed for cervical HPV detection and genotyping: Inno-Lipa HPV Genotyping Extra II (IL) and Anyplex II HPV28 (AP28). A total of 55 samples were tested. By IL assay, chosen as reference assay, 27 (49.1%) biopsies were positive for HPV16, 10 (18.2%) were positive for HPV but negative for HPV16, and 18 (32.7%) were negative for HPV. A valid result with AP28 was obtained for 51 biopsy samples (92.7%). Among 37 HPV positive samples by IL, 33 (89.2%) were positive by AP28. The agreement between both assays was good (Cohen's κ = 0.78). Among the six discrepancies between assays, always associated with low HPV16 viral load, four biopsies positive for HPV16 by IL could not be detected by AP28. Taken together, these observations demonstrate that both assays could be used in routine HPV detection and genotyping on FFPE biopsy samples of head and neck tumours.

Chromogenic In Situ Hybridization as a Tool for HPV-Related Head and Neck Cancer Diagnosis.

Human papillomavirus (HPV) infection is a major risk factor for a subtype of oropharyngeal squamous cell carcinoma (OPSCC), which tends to be associated with a better outcome than alcohol- and tobacco-related OPSCC. Chromogenic in situ hybridization (CISH) of HPV viral RNA could allow the semiquantitative evaluation of viral transcripts of the oncogenic proteins E6 and E7 and an in situ visualization with a good spatial resolution. This technique allows the diagnosis of an active infection with the visualization of HPV transcription in the tumoral HPV-infected cells. An advantage of this technique is the avoidance of contamination from nonneoplastic HPV-infected cells adjacent to the tumor. Overall, its good diagnosis performances have it considered to be the gold standard for active HPV infection identification. Since E6 and E7 viral protein interaction with cell proteins pRb and p53 is mandatory for cell transformation, HPV RNA CISH is functionally relevant and acutely reflects active oncogenic HPV infection. This technique is clinically relevant as well since "low" or "high" HPV transcription levels helped the identification of two prognosis groups among HPV-related p16-positive head and neck cancer patients. Here we present the protocol for manual HPV RNA CISH performed on formalin-fixed paraffin-embedded (FFPE) slides with a kit obtained from the manufacturer. Instead of chromogenic revelation, RNA in situ hybridization may also be performed with fluorescent revelation (RNA FISH). It may also be combined with conventional immunostaining.

Evaluation of the efficacy of the 4 tests (p16 immunochemistry, polymerase chain reaction, DNA, and RNA in situ hybridization) to evaluate a human papillomavirus infection in head and neck cancers: a cohort of 348 French squamous cell carcinomas.

It is now established that human papillomavirus (HPV) plays a role in the development of a subset of head and neck squamous cell carcinomas (SCCs), notably oropharyngeal (OP) SCCs. However, it is not clear which test one should use to detect HPV in OP and non-OP SCCs. In this study, using 348 head and neck SCCs (126 OP SCCs and 222 non-OP SCCs), we evaluated diagnostic performances of different HPV tests in OP and non-OP SCCs: polymerase chain reaction, p16 immunostaining, in situ hybridization targeting DNA (DNA-CISH) and RNA (RNA-CISH), combined p16 + DNA-CISH, and combined p16 + RNA-CISH. HPV DNA (polymerase chain reaction) was detected in 26% of all tumors (44% of OP SCCs and 17% of non-OP SCCs). For OP SCCs, RNA-CISH was the most sensitive stand-alone test (88%), but p16 + RNA-CISH was even more sensitive (95%). Specificities were the same for RNA-CISH and DNA-CISH (97%), but it was better for p16 + RNA-CISH (100%). For non-OP SCCs, all tests had sensitivities less than 50%, and RNA-CISH, DNA-CISH, and p16 + DNA-CISH had 100%, 97%, and 99% specificities, respectively. As a stand-alone test, RNA-CISH is the most performant assay to detect HPV in OP SCCs, and combined p16 + RNA-CISH test slightly improves its performances. However, RNA-CISH has the advantage of being one single test. Like p16 and DNA-CISH, RNA-CISH performances are poor in non-OP SCCs to detect HPV, and combining tests does not improve performances.