Heterogeneous NLRP3 inflammasome signature in circulating myeloid cells as a biomarker of COVID-19 severity.

Dysregulated immune response is the key factor leading to unfavorable coronavirus disease 2019 (COVID-19) outcome. Depending on the pathogen-associated molecular pattern, the NLRP3 inflammasome can play a crucial role during innate immunity activation. To date, studies describing the NLRP3 response during severe acute respiratory syndrome coronavirus 2 infection in patients are lacking. We prospectively monitored caspase-1 activation levels in peripheral myeloid cells from healthy donors and patients with mild to critical COVID-19. The caspase-1 activation potential in response to NLRP3 inflammasome stimulation was opposed between nonclassical monocytes and CD66b+CD16dim granulocytes in severe and critical COVID-19 patients. Unexpectedly, the CD66b+CD16dim granulocytes had decreased nigericin-triggered caspase-1 activation potential associated with an increased percentage of NLRP3 inflammasome impaired immature neutrophils and a loss of eosinophils in the blood. In patients who recovered from COVID-19, nigericin-triggered caspase-1 activation potential in CD66b+CD16dim cells was restored and the proportion of immature neutrophils was similar to control. Here, we reveal that NLRP3 inflammasome activation potential differs among myeloid cells and could be used as a biomarker of a COVID-19 patient's evolution. This assay could be a useful tool to predict patient outcome. This trial was registered at www.clinicaltrials.gov as #NCT04385017.

Escherichia coli Rho GTPase-activating toxin CNF1 mediates NLRP3 inflammasome activation via p21-activated kinases-1/2 during bacteraemia in mice.

Inflammasomes are signalling platforms that are assembled in response to infection or sterile inflammation by cytosolic pattern recognition receptors. The consequent inflammasome-triggered caspase-1 activation is critical for the host defence against pathogens. During infection, NLRP3, which is a pattern recognition receptor that is also known as cryopyrin, triggers the assembly of the inflammasome-activating caspase-1 through the recruitment of ASC and Nek7. The activation of the NLRP3 inflammasome is tightly controlled both transcriptionally and post-translationally. Despite the importance of the NLRP3 inflammasome regulation in autoinflammatory and infectious diseases, little is known about the mechanism controlling the activation of NLRP3 and the upstream signalling that regulates the NLRP3 inflammasome assembly. We have previously shown that the Rho-GTPase-activating toxin from Escherichia coli cytotoxic necrotizing factor-1 (CNF1) activates caspase-1, but the upstream mechanism is unclear. Here, we provide evidence of the role of the NLRP3 inflammasome in sensing the activity of bacterial toxins and virulence factors that activate host Rho GTPases. We demonstrate that this activation relies on the monitoring of the toxin's activity on the Rho GTPase Rac2. We also show that the NLRP3 inflammasome is activated by a signalling cascade that involves the p21-activated kinases 1 and 2 (Pak1/2) and the Pak1-mediated phosphorylation of Thr 659 of NLRP3, which is necessary for the NLRP3-Nek7 interaction, inflammasome activation and IL-1ß cytokine maturation. Furthermore, inhibition of the Pak-NLRP3 axis decreases the bacterial clearance of CNF1-expressing UTI89 E. coli during bacteraemia in mice. Taken together, our results establish that Pak1 and Pak2 are critical regulators of the NLRP3 inflammasome and reveal the role of the Pak-NLRP3 signalling axis in vivo during bacteraemia in mice.

Problematic molecular diagnosis of HSV-1 infection due to a single nucleotide polymorphism in the US7 gene.

BACKGROUND: HSV-1 infection is very common worldwide and can be associated with severe medical conditions such as encephalitis and neonatal herpes. Current diagnosis relies heavily on molecular assays targeting the viral genome. One of the pitfalls of these assays is genetic variability, as viral polymorphisms located in the target region can impair the detection of HSV-1. OBJECTIVES: The aim of this study was to determine if genetic diversity of HSV-1 could account for equivocal assay results we obtained during routine diagnosis of HSV-1 in a genital specimen with the HSV-1 HSV-2 VZV R-gene® assay. STUDY DESIGN: The presence of HSV-1 in the genital swab studied was assessed using the HSV-1 HSV-2 VZV R-gene® qPCR assay and viral culture. Genetic variability of the patient's HSV-1 isolate was determined using molecular cloning, sequencing and comparison of the sequence of the isolate with those of previously published strains. RESULTS: We identified an HSV-1 isolate carrying a novel single-nucleotide polymorphism (SNP) located in the US7 gene from a genital swab. This SNP resulted in a missense substitution in the gI protein. It was responsible for the generation of an altered amplification curve when the genital specimen was checked for HSV-1 presence with the Argene HSV-1 HSV-2 VZV R-gene® assay, preventing unequivocal determination of the HSV-1 status of the sample analyzed. CONCLUSIONS: This study raises awareness of the risk of misdiagnosis of HSV-1 infections when "single-target" molecular assays are employed.

Quantification of HPV16 E6/E7 mRNA Spliced Isoforms Viral Load as a Novel Diagnostic Tool for Improving Cervical Cancer Screening.

High-risk human papillomaviruses (HPVs) have been identified as the main contributors to cervical cancer. Despite various diagnostic tools available, including the predominant Papanicolaou test (Pap test), technical limitations affect the efficiency of cervical cancer screening. The aim of this study was to evaluate the diagnostic performance of spliced HPV16 E6/E7 mRNA viral loads (VL) for grade 2 or higher cervical intraepithelial neoplasia diagnosis. A new dedicated (quantitative reverse transcription polymerase chain reaction) qRT-PCR assay was developed, allowing selective quantification of several HPV16 E6/E7 mRNA: Full length (FL) with or without all or selected spliced forms (total E6/E7 mRNA corresponding to SP + E6^E7 mRNA (T), + spliced E6/E7 mRNA containing intact E7 ORF (SP), and E6/E7 mRNA containing disrupted E6 and E7 ORFs calculated by the following subtraction T-SP (E6^E7)). Twenty HPV16 DNA and mRNA positive uterine cervical smears representative of all cytological and histological stages of severity were tested. We have shown that all E6/E7 mRNA isoforms expression levels were significantly increased in high grade cervical lesions. Statistical analysis demonstrated that the SP-E6/E7 VL assay exhibited: (i) The best diagnostic performance for identification of both cervical intraepithelial neoplasia (CIN)2+ (90% (56⁻100) sensitivity and specificity) and CIN3+ (100% (72⁻100) sensitivity and 79% (49⁻95) specificity) lesions; (ii) a greater sensitivity compared to the Pap test for CIN2+ lesions detection (80% (44⁻97)); (iii) a predictive value of the histological grade of cervical lesions in 67% of atypical squamous cells of unknown significance (ASC-US) and 100% of low-grade (LSIL) patients. Overall, these results highlight the value of SP-E6/E7 mRNA VL as an innovative tool for improving cervical cancer screening.

Molecular characterization of Wolbachia infection in bed bugs (Cimex lectularius) collected from several localities in France.

Wolbachia symbionts are maternally inherited intracellular bacteria that have been detected in numerous insects including bed bugs. The objective of this study, the first epidemiological study in Europe, was to screen Wolbachia infection among Cimex lectularius collected in the field, using PCR targeting the surface protein gene (wsp), and to compare obtained Wolbachia strains with those reported from laboratory colonies of C. lectularius as well as other Wolbachia groups. For this purpose, 284 bed bug specimens were caught and studied from eight different regions of France including the suburbs of Paris, Bouches-du-Rhône, Lot-et-Garonne, and five localities in Alpes-Maritimes. Among the samples, 166 were adults and the remaining 118 were considered nymphs. In all, 47 out of 118 nymphs (40%) and 61 out of 166 adults (37%) were found positive on wsp screening. Among the positive cases, 10 samples were selected randomly for sequencing. The sequences had 100% homology with wsp sequences belonging to the F-supergroup strains of Wolbachia. Therefore, we confirm the similarity of Wolbachia strains detected in this epidemiological study to Wolbachia spp. reported from laboratory colonies of C. lectularius.

Characterization of a cluster of oncogenic mutations in E6 of a human papillomavirus 83 variant isolated from a high-grade squamous intraepithelial lesion.

We previously isolated human papillomavirus 83 (HPV83m) from a cervical smear. Sequence analysis of E6 and E7 proteins highlighted five mutations located in the second putative zinc-finger region of E6 (E6m), an important domain for protein-protein or protein-DNA interactions. Here, we show that E6m of HPV83m can trigger human primary cell proliferation and anchorage-independent growth properties, similarly to E6 of HPV16, a high-risk HPV (HR-HPV). Interestingly, we demonstrate that, in contrast to E6 of HPV16, E6m corrupts neither p53 stability nor telomerase activity, but acts as a specific modulator of the transcriptional machinery. By studying E6m reversion mutants, we confirmed the importance of the second zinc-finger domain in triggering the observed upregulation of cell growth and of the transcriptional machinery. Reversion of these mutations in E6m (to yield strain E6r) fully abolished the oncogenic potential of E6m, transforming the phenotype of E6 from a high-risk to a low-risk phenotype. Importantly, our data define the importance of a cluster of mutations in the second zinc finger of E6m in increasing the oncogenic potential of HPV83.

Can the microRNA signature distinguish between thyroid tumors of uncertain malignant potential and other well-differentiated tumors of the thyroid gland?

The term 'thyroid tumors of uncertain malignant potential' (TT-UMP) was coined by surgical pathologists to define well-differentiated tumors (WDT) showing inconclusive morphological evidence of malignancy or benignity. We have analyzed the expression of microRNA (miRNA) in a training set of 42 WDT of different histological subtypes: seven follicular tumors of UMP (FT-UMP), six WDT-UMP, seven follicular thyroid adenomas (FTA), 11 conventional papillary thyroid carcinomas (C-PTC), five follicular variants of PTC (FV-PTC), and six follicular thyroid carcinomas (FTC), which led to the identification of about 40 deregulated miRNAs. A subset of these altered miRNAs was independently validated by qRT-PCR, which included 18 supplementary TT-UMP (eight WDT-UMP and ten FT-UMP). Supervised clustering techniques were used to predict the first 42 samples. Based on the four possible outcomes (FTA, C-PTC, FV-PTC, and FTC), about 80% of FTA and C-PTC and 50% of FV-PTC and FTC samples were correctly assigned. Analysis of the independent set of 18 WDT-UMP by quantitative RT-PCR for the selection of the six most discriminating miRNAs was unable to separate FT-UMP from WDT-UMP, suggesting that the miRNA signature is insufficient in characterizing these two clinical entities. We conclude that considering FT-UMP and WDT-UMP as distinct and specific clinical entities may improve the diagnosis of WDT of the thyroid gland. In this context, a small set of miRNAs (i.e. miR-7, miR-146a, miR-146b, miR-200b, miR-221, and miR-222) appears to be useful, though not sufficient per se, in distinguishing TT-UMP from other WDT of the thyroid gland.

Thyroid tumours of uncertain malignant potential: frequency and diagnostic reproducibility.

The term thyroid tumours of uncertain malignant potential (TT-UMP) has been proposed for a subgroup of follicular-patterned thyroid tumours for which benignancy or malignancy cannot be assessed with certainty. The frequency, diagnostic reproducibility, immunohistochemistry and molecular genetic profiling of such tumours have been poorly explored. We, therefore, investigated (1) the frequency of TT-UMP diagnosed in a single institution (Nice, France: 2004-2008), (2) the observer variation among four pathologists, (3) whether immunohistochemical and molecular genetic profiling of TT-UMP provide additional information concerning such lesions. A series of 31 diagnosed TT-UMP (2.9%) out of 1,078 consecutive thyroidectomies were analysed. It comprised 15 follicular thyroid tumours of UMP (FT-UMP) and 16 well-differentiated tumours of UMP (WDT-UMP). Observer concordance was 70% for all TT-UMP. More than 50% of FT-UMP expressed galectin-3 and CK19, whereas more than 50% of WDT-UMP expressed HBME-1. Five cases of TT-UMP showed N-RAS mutations, while one showed H-RAS mutation and another PAX8/PPARgamma rearrangement. In conclusion, the frequency of TT-UMP is low in our institution. Diagnostic reproducibility is within the same range as other published data on follicular-patterned thyroid tumours. The ancillary methods have a low impact on aiding diagnosis of such lesions.