A prospective humoral immune monitoring study of kidney transplant recipients receiving three doses of SARS-CoV-2 mRNA vaccine.

Kidney transplant recipients (KTRs), like other solid organ transplant recipients display a suboptimal response to mRNA vaccines, with only about half achieving seroconversion after two doses. However, the effectiveness of a booster dose, particularly in generating neutralizing antibodies (NAbs), remains poorly understood, as most studies have mainly focused on non-neutralizing antibodies. Here, we have longitudinally assessed the humoral response to the SARS-CoV-2 mRNA vaccine in 40 KTRs over a year, examining changes in both anti-spike IgG and NAbs following a booster dose administered about 5 months post-second dose. We found a significant humoral response increase 5 months post-booster, a stark contrast to the attenuated response observed after the second dose. Of note, nearly a quarter of participants did not achieve protective plasma levels even after the booster dose. We also found that the higher estimated glomerular filtration rate (eGFR) correlated with a more robust humoral response postvaccination. Altogether, these findings underscore the effectiveness of the booster dose in enhancing durable humoral immunity in KTRs, as evidenced by the protective level of NAbs found in 65% of the patients 5 months post- booster, especially those with higher eGFR rates.

SIRT1 is an actionable target to restore p53 function in HPV-associated cancer therapy.

BACKGROUND: Our aim was to evaluate the efficacy and anti-cancer action of a precision medicine approach involving a novel SIRT1-dependent pathway that, when disrupted, leads to the restoration of a functional p53 in human papillomavirus (HPV)-transformed cells. METHODS: The anticancer potential of inhibiting SIRT1 was evaluated by examining the effects of the specific SIRT1 inhibitor EX527 (also known as Selisistat) or genetic silencing, either individually or in conjunction with standard chemotherapeutic agents, on a range of HPV+ cancer cells and a preclinical mouse model of HPV16-induced cancer. RESULTS: We show that SIRT1 inhibition restores a transcriptionally active K382-acetylated p53 in HPV+ but not HPV- cell lines, which in turn promotes G0/G1 cell cycle arrest and inhibits clonogenicity specifically in HPV+ cells. Additionally, EX527 treatment increases the sensitivity of HPV+ cells to sublethal doses of standard genotoxic agents. The enhanced sensitivity to cisplatin as well as p53 restoration were also observed in an in vivo tumorigenicity assay using syngeneic C3.43 cells harbouring an integrated HPV16 genome, injected subcutaneously into C57BL/6J mice. CONCLUSIONS: Our findings uncover an essential role of SIRT1 in HPV-driven oncogenesis, which may have direct translational implications for the treatment of this type of cancer.

The RIG-I agonist M8 triggers cell death and natural killer cell activation in human papillomavirus-associated cancer and potentiates cisplatin cytotoxicity.

Although the activation of innate immunity to treat a wide variety of cancers is gaining increasing attention, it has been poorly investigated in human papillomavirus (HPV)-associated malignancies. Because these tumors harbor a severely impaired cGAS-STING axis, but they still retain a largely functional RIG-I pathway, another critical mediator of adaptive and innate immune responses, we asked whether RIG-I activation by the 5'ppp-RNA RIG-I agonist M8 would represent a therapeutically viable option to treat HPV+ cancers. Here, we show that M8 transfection of two cervical carcinoma-derived cell lines, CaSki and HeLa, both expressing a functional RIG-I, triggers intrinsic apoptotic cell death, which is significantly reduced in RIG-I KO cells. We also demonstrate that M8 stimulation potentiates cisplatin-mediated cell killing of HPV+ cells in a RIG-I dependent manner. This combination treatment is equally effective in reducing tumor growth in a syngeneic pre-clinical mouse model of HPV16-driven cancer, where enhanced expression of lymphocyte-recruiting chemokines and cytokines correlated with an increased number of activated natural killer (NK) cells in the tumor microenvironment. Consistent with a role of RIG-I signaling in immunogenic cell killing, stimulation of NK cells with conditioned medium from M8-transfected CaSki boosted NK cell proliferation, activation, and migration in a RIG-I-dependent tumor cell-intrinsic manner. Given the highly conserved molecular mechanisms of carcinogenesis and genomic features of HPV-driven cancers and the remarkably improved prognosis for HPV+ oropharyngeal cancer, targeting RIG-I may represent an effective immunotherapeutic strategy in this setting, favoring the development of de-escalating strategies.

Human Papillomavirus E7 Oncoprotein Subverts Host Innate Immunity via SUV39H1-Mediated Epigenetic Silencing of Immune Sensor Genes.

Subversion of innate immunity by oncoviruses, such as human papillomavirus (HPV), favors carcinogenesis because the mechanism(s) of viral immune evasion can also hamper cancer immunosurveillance. Previously, we demonstrated that high-risk (hr) HPVs trigger simultaneous epigenetic silencing of multiple effectors of innate immunity to promote viral persistence. Here, we expand on those observations and show that the HPV E7 oncoprotein upregulates the H3K9-specific methyltransferase, whose action shuts down the host innate immune response. Specifically, we demonstrate that SUV39H1 contributes to chromatin repression at the promoter regions of the viral nucleic acid sensors RIG-I and cGAS and the adaptor molecule STING in HPV-transformed cells. Inhibition of SUV39H1 leads to transcriptional activation of these genes, especially RIG-I, followed by increased beta interferon (IFN-ß) and IFN-λ1 production after poly(dA·dT) or RIG-I agonist M8 transfection. Collectively, our findings provide new evidence that the E7 oncoprotein plays a central role in dampening host innate immunity and raise the possibility that targeting the downstream effector SUV39H1 or the RIG-I pathway is a viable strategy to treat viral and neoplastic disease.IMPORTANCE High-risk HPVs are major viral human carcinogens responsible for approximately 5% of all human cancers. The growth of HPV-transformed cells depends on the ability of viral oncoproteins to manipulate a variety of cellular circuits, including those involved in innate immunity. Here, we show that one of these strategies relies on E7-mediated transcriptional activation of the chromatin repressor SUV39H1, which then promotes epigenetic silencing of RIG-I, cGAS, and STING genes, thereby shutting down interferon secretion in HPV-transformed cells. Pharmacological or genetic inhibition of SUV39H1 restored the innate response in HPV-transformed cells, mostly through activation of RIG-I signaling. We also show that IFN production upon transfection of poly(dA·dT) or the RIG-I agonist M8 predominantly occurs through RIG-I signaling. Altogether, the reversible nature of the modifications associated with E7-mediated SUV39H1 upregulation provides a rationale for the design of novel anticancer and antiviral therapies targeting these molecules.

HPV18 Persistence Impairs Basal and DNA Ligand-Mediated IFN-ß and IFN-λ1 Production through Transcriptional Repression of Multiple Downstream Effectors of Pattern Recognition Receptor Signaling.

Although it is clear that high-risk human papillomaviruses (HPVs) can selectively infect keratinocytes and persist in the host, it still remains to be unequivocally determined whether they can escape antiviral innate immunity by interfering with pattern recognition receptor (PRR) signaling. In this study, we have assessed the innate immune response in monolayer and organotypic raft cultures of NIKS cells harboring multiple copies of episomal HPV18 (NIKSmcHPV18), which fully recapitulates the persistent state of infection. We show for the first time, to our knowledge, that NIKSmcHPV18, as well as HeLa cells (a cervical carcinoma-derived cell line harboring integrated HPV18 DNA), display marked downregulation of several PRRs, as well as other PRR downstream effectors, such as the adaptor protein stimulator of IFN genes and the transcription factors IRF1 and 7. Importantly, we provide evidence that downregulation of stimulator of IFN genes, cyclic GMP-AMP synthase, and retinoic acid-inducible gene I mRNA levels occurs at the transcriptional level through a novel epigenetic silencing mechanism, as documented by the accumulation of repressive heterochromatin markers seen at the promoter region of these genes. Furthermore, stimulation of NIKSmcHPV18 cells with salmon sperm DNA or poly(deoxyadenylic-deoxythymidylic) acid, two potent inducers of PRR signaling, only partially restored PRR protein expression. Accordingly, the production of IFN-ß and IFN-λ1 was significantly reduced in comparison with the parental NIKS cells, indicating that HPV18 exerts its immunosuppressive activity through downregulation of PRR signaling. Altogether, our findings indicate that high-risk human papillomaviruses have evolved broad-spectrum mechanisms that allow simultaneous depletion of multiple effectors of the innate immunity network, thereby creating an unreactive cellular milieu suitable for viral persistence.

Human Cytomegalovirus Tegument Protein pp65 (pUL83) Dampens Type I Interferon Production by Inactivating the DNA Sensor cGAS without Affecting STING.

The innate immune response plays a pivotal role during human cytomegalovirus (HCMV) primary infection. Indeed, HCMV infection of primary fibroblasts rapidly triggers strong induction of type I interferons (IFN-I), accompanied by proinflammatory cytokine release. Here, we show that primary human foreskin fibroblasts (HFFs) infected with a mutant HCMV TB40/E strain unable to express UL83-encoded pp65 (v65Stop) produce significantly higher IFN-ß levels than HFFs infected with the wild-type TB40/E strain or the pp65 revertant (v65Rev), suggesting that the tegument protein pp65 may dampen IFN-ß production. To clarify the mechanisms through which pp65 inhibits IFN-ß production, we analyzed the activation of the cGAS/STING/IRF3 axis in HFFs infected with either the wild type, the revertant v65Rev, or the pp65-deficient mutant v65Stop. We found that pp65 selectively binds to cGAS and prevents its interaction with STING, thus inactivating the signaling pathway through the cGAS/STING/IRF3 axis. Consistently, addition of exogenous cGAMP to v65Rev-infected cells triggered the production of IFN-ß levels similar to those observed with v65Stop-infected cells, confirming that pp65 inactivation of IFN-ß production occurs at the cGAS level. Notably, within the first 24 h of HCMV infection, STING undergoes proteasome degradation independently of the presence or absence of pp65. Collectively, our data provide mechanistic insights into the interplay between HCMV pp65 and cGAS, leading to subsequent immune evasion by this prominent DNA virus.IMPORTANCE Primary human foreskin fibroblasts (HFFs) produce type I IFN (IFN-I) when infected with HCMV. However, we observed significantly higher IFN-ß levels when HFFs were infected with HCMV that was unable to express UL83-encoded pp65 (v65Stop), suggesting that pp65 (pUL83) may constitute a viral evasion factor. This study demonstrates that the HCMV tegument protein pp65 inhibits IFN-ß production by binding and inactivating cGAS early during infection. In addition, this inhibitory activity specifically targets cGAS, since it can be bypassed via the addition of exogenous cGAMP, even in the presence of pp65. Notably, STING proteasome-mediated degradation was observed in both the presence and absence of pp65. Collectively, our data underscore the important role of the tegument protein pp65 as a critical molecular hub in HCMV's evasion strategy against the innate immune response.

Primary trichodysplasia spinulosa polyomavirus infection in a kidney transplant child displaying virus-infected decoy cells in the urine.

We report a case of primary trichodysplasia spinulosa (TS) infection in a kidney transplant child and describe for the first time the presence of degenerated TS-associated polyomavirus (TSPyV)-infected cells in a TS patient's urine that are morphologically different from BK or JC polyomavirus-infected decoy cells.

The Nuclear DNA Sensor IFI16 Acts as a Restriction Factor for Human Papillomavirus Replication through Epigenetic Modifications of the Viral Promoters.

UNLABELLED: The human interferon-inducible IFI16 protein, an innate immune sensor of intracellular DNA, was recently demonstrated to act as a restriction factor for human cytomegalovirus (HCMV) and herpes simplex virus 1 (HSV-1) infection by inhibiting both viral-DNA replication and transcription. Through the use of two distinct cellular models, this study provides strong evidence in support of the notion that IFI16 can also restrict human papillomavirus 18 (HPV18) replication. In the first model, an immortalized keratinocyte cell line (NIKS) was used, in which the IFI16 protein was knocked down through the use of small interfering RNA (siRNA) technology and overexpressed following transduction with the adenovirus IFI16 (AdVIFI16) vector. The second model consisted of U2OS cells transfected by electroporation with HPV18 minicircles. In differentiated IFI16-silenced NIKS-HPV18 cells, viral-load values were significantly increased compared with differentiated control cells. Consistent with this, IFI16 overexpression severely impaired HPV18 replication in both NIKS and U2OS cells, thus confirming its antiviral restriction activity. In addition to the inhibition of viral replication, IFI16 was also able to reduce viral transcription, as demonstrated by viral-gene expression analysis in U2OS cells carrying episomal HPV18 minicircles and HeLa cells. We also provide evidence that IFI16 promotes the addition of heterochromatin marks and the reduction of euchromatin marks on viral chromatin at both early and late promoters, thus reducing both viral replication and transcription. Altogether, these results argue that IFI16 restricts chromatinized HPV DNA through epigenetic modifications and plays a broad surveillance role against viral DNA in the nucleus that is not restricted to herpesviruses. IMPORTANCE: Intrinsic immunity is mediated by cellular restriction factors that are constitutively expressed and active even before a pathogen enters the cell. The host nuclear factor IFI16 acts as a sensor of foreign DNA and an antiviral restriction factor, as recently demonstrated by our group for human cytomegalovirus (HCMV) and herpes simplex virus 1 (HSV-1). Here, we provide the first evidence that IFI16 inhibits HPV18 replication by repressing viral-gene expression and replication. This antiviral restriction activity was observed in immortalized keratinocytes transfected with the religated genomes and in U2OS cells transfected with HPV18 minicircles, suggesting that it is not cell type specific. We also show that IFI16 promotes the assembly of heterochromatin on HPV DNA. These changes in viral chromatin structure lead to the generation of a repressive state at both early and late HPV18 promoters, thus implicating the protein in the epigenetic regulation of HPV gene expression and replication.

Human Beta-papillomavirus infection and keratinocyte carcinomas.

Although the role of oncogenic human Alpha-papillomaviruses (HPVs) in the development of mucosal carcinomas at different body sites (eg cervix, anus, oropharynx) is fully recognized, a role for HPV in keratinocyte carcinomas (KCs; basal and squamous cell carcinomas) of the skin is not yet clear. KCs are the most common cancers in Caucasians, with the major risk factor being ultraviolet (UV) light exposure. A possible role for Beta-HPV types (BetaPV) in the development of KC was suggested several decades ago, supported by a number of epidemiological studies. Our current review summarizes the recent molecular and histopathological evidence in support of a causal association between BetaPV and the development of KC, and outlines the suspected synergistic effect of viral gene expression with UV radiation and immune suppression. Further insights into the molecular pathways and protein interactions used by BetaPV and the host cell is likely to extend our understanding of the role of BetaPV in KC.

Expression of betapapillomavirus oncogenes increases the number of keratinocytes with stem cell-like properties.

Human papillomaviruses (HPV) of genus Betapapillomavirus (betaPV) are associated with nonmelanoma skin cancer development in epidermodysplasia verruciformis (EV) and immunosuppressed patients. Epidemiological and molecular studies suggest a carcinogenic activity of betaPV during early stages of cancer development. Since viral oncoproteins delay and perturb keratinocyte differentiation, they may have the capacity to either retain or confer a "stem cell-like" state on oncogene-expressing cells. The aim of this study was to determine (i) whether betaPV alters the expression of cell surface markers, such as CD44 and epithelial cell adhesion molecule (EpCAM), that have been associated with epithelial stemness, and (ii) whether this confers functional stem cell-like properties to human cutaneous keratinocytes. Fluorescence-activated cell sorter (FACS) analysis revealed an increase in the number of cells with high CD44 and EpCAM expression in keratinocyte cultures expressing HPV type 8 (HPV8) oncogenes E2, E6, and E7. Particularly through E7 expression, a distinct increase in clonogenicity and in the formation and size of tumor spheres was observed, accompanied by reduction of the epithelial differentiation marker Calgranulin B. These stem cell-like properties could be attributed to the pool of CD44(high) EpCAM(high) cells, which was increased within the E7 cultures of HPV5, -8, and -20. Enhanced EpCAM levels were present in organotypic skin cultures of primary keratinocytes expressing E7 of the oncogenic HPV types HPV5, -8, and -16 and in clinical samples from EV patients. In conclusion, our data show that betaPV may increase the number of stem cell-like cells present during early carcinogenesis and thus enable the persistence and accumulation of DNA damage necessary to generate malignant stem cells.

Improved detection reveals active ß-papillomavirus infection in skin lesions from kidney transplant recipients.

The aim of this study was to determine whether detection of ß-HPV gene products, as defined in epidermodysplasia verruciformis skin cancer, could also be observed in lesions from kidney transplant recipients alongside the viral DNA. A total of 111 samples, corresponding to 79 skin lesions abscised from 17 kidney transplant recipients, have been analyzed. The initial PCR analysis demonstrated that ß-HPV-DNA was highly present in our tumor series (85%). Using a combination of antibodies raised against the E4 and L1 proteins of the ß-genotypes, we were able to visualize productive infection in 4 out of 19 actinic keratoses, and in the pathological borders of 1 out of 14 squamous cell carcinomas and 1 out of 31 basal cell carcinomas. Increased expression of the cellular proliferation marker minichromosome maintenance protein 7 (MCM7), that extended into the upper epithelial layers, was a common feature of all the E4-positive areas, indicating that cells were driven into the cell cycle in areas of productive viral infections. Although the present study does not directly demonstrate a causal role of these viruses, the detection of E4 and L1 positivity in actinic keratosis and the adjacent pathological epithelium of skin cancer, clearly shows that ß-HPV are actively replicating in the intraepidermal precursor lesions of kidney transplant recipients and can therefore cooperate with other carcinogenic agents, such as UVB, favoring skin cancer promotion.

α- and ß-papillomavirus infection in a young patient with an unclassified primary T-cell immunodeficiency and multiple mucosal and cutaneous lesions.

BACKGROUND: Correlating human papillomavirus (HPV) type with the clinical and histopathological features of skin lesions (from genital and nongenital sites) can present a diagnostic challenge. OBJECTIVE: In this study, HPV infection patterns were correlated with pathology and clinical presentation in lesional and nonlesional body sites from a young patient with a primary T-cell immunodeficiency. METHODS: HPV infection was evaluated at both DNA and protein levels by polymerase chain reaction and immunohistochemistry. RESULTS: The patient's genital lesions were caused exclusively by α-genotypes (high-risk type HPV-51 in the anal and low-risk type HPV-72 in the penile condylomas). The opposite was true for the skin lesions, which were infected by ß-genotypes alone (HPV-8 and HPV-24). HPV-24 was the predominant type in terms of viral load, and the only one found in productive areas of infection. The patient had already developed high-grade dysplasia in the anal condyloma-like lesions, and showed areas of early-stage dysplasia in the lesions caused by the ß-genotype HPV-24. LIMITATIONS: The basic origin of the immunodeficiency is not yet defined. CONCLUSION: These findings provide proof of principle that both α- and ß-genotypes can cause overt dysplastic lesions when immunosurveillance is lost, which is not restricted to epidermodysplasia verruciformis.

HPV8-induced STAT3 activation led keratinocyte stem cell expansion in human actinic keratoses.

Despite epidermal turnover, the skin is host to a complex array of microbes including viruses, such as the human papillomavirus (HPV), which must infect and manipulate skin keratinocyte stem cells (KSC) to survive. This crosstalk between the virome and KSC populations remains largely unknown. Here, we investigated the effect of HPV8 on KSCs using various mouse models. We observed that the HPV8 early region gene E6 specifically caused Lrig1+ hair follicle junctional zone KSC proliferation and expansion, which would facilitate viral transmission. Within Lrig1+ KSCs specifically, HPV8 E6 bound intracellular p300 to phosphorylate the STAT3 transcriptional regulatory node. This induces ΔNp63 expression, resulting in KSC expansion into the overlying epidermis. HPV8 was associated with 70% of human actinic keratoses (AK). Together these results define the "hit and run" mechanism for HPV8 in human actinic keratosis as an expansion of KSCs, which lacks melanosome protection and is thus susceptible to sun-light-induced malignant transformation.

Enhanced Spontaneous Skin Tumorigenesis and Aberrant Inflammatory Response to UVB Exposure in Immunosuppressed Human Papillomavirus Type 8‒Transgenic Mice.

Human papillomaviruses (HPVs) from the beta genus are commensal viruses of the skin usually associated with asymptomatic infection in the general population. However, in individuals with specific genetic backgrounds, such as patients with epidermodysplasia verruciformis, or those with immune defects, such as organ transplant recipients, they are functionally involved in sunlight-induced skin cancer development, mainly keratinocyte carcinoma. Despite their well-established protumorigenic role, the cooperation between ß-HPV infection, impaired host immunosurveillance, and UVB exposure has never been formally shown in animal models. In this study, by crossing skin-specific HPV8-transgenic mice with Rag2-deficient mice, we have generated a preclinical mouse model, named Rag2‒/‒:K14-HPV8. These mice display an unhealthy skin phenotype and spontaneously develop papilloma-like lesions spreading to the entire skin much more rapidly compared with Rag2+/+:K14-HPV8 mice. Exposure to low doses of UVB radiation is sufficient to trigger severe skin inflammation in Rag2‒/‒:K14-HPV8 but not in Rag2+/+:K14-HPV8 mice. Their inflamed skin very much resembled that observed in cutaneous field cancerization in organ transplant recipients, showing high levels of UVB-damaged cells, enhanced production of proinflammatory cytokines, and mast cell recruitment to the dermis. Overall, this immunocompromised HPV8-transgenic mouse model shows that the coexistence of immune defects, ß-HPV, and UVB exposure promotes skin cancer development.

Cancer-Associated Fibroblasts Exert Proangiogenic Activity in Merkel Cell Carcinoma.

The tumor microenvironment is a complex niche enveloping a tumor formed by extracellular matrix, blood vessels, immune cells, and fibroblasts constantly interacting with cancer cells. Although tumor microenvironment is increasingly recognized as a major player in cancer initiation and progression in many tumor types, its involvement in Merkel cell carcinoma (MCC) pathogenesis is currently unknown. In this study, we provide a molecular and functional characterization of cancer-associated fibroblasts (CAFs), the major tumor microenvironment component, in patient-derived xenografts of patients with MCC. We show that subcutaneous coinjection of patient-derived CAFs and human MCC MKL-1 cells into severe combined immunodeficient mice significantly promotes tumor growth and metastasis. These fast-growing xenografts are characterized by areas densely populated with human CAFs, mainly localized around blood vessels. We provide evidence that the growth-promoting activity of MCC-derived CAFs is mediated by the aminopeptidase A/angiotensin II and III/angiotensin II type 1 receptor axis, with the expression of aminopeptidase A in CAFs being a triggering event. Together, our findings point to aminopeptidase A as a potential marker for MCC prognostic stratification and as a candidate for therapeutic intervention.

Longitudinal monitoring of SARS-CoV-2 viral load in self-collected saliva from health care workers during breakthrough infections to spare working days.

IMPORTANCE: Real-time quantitative PCR (RT-qPCR) on nasopharyngeal swabs (NPS) has been used as the standard method for detecting and monitoring SARS-CoV-2 infection during the pandemic. However, NPS collection often causes discomfort and poses a higher risk of transmission to health care workers (HCW). Furthermore, RT-qPCR only provides relative quantification and does not allow distinguishing those samples with residual, no longer active infection, whereas droplet digital PCR (ddPCR) allows for precise quantification of viral load, offering greater sensitivity and reproducibility. This study highlights the effectiveness of using self-collected saliva as a convenient and reliable sampling method. By utilizing ddPCR to measure the SARS-CoV-2 viral load in saliva samples, individuals with low or undetectable viral loads can be quickly identified. This approach is particularly advantageous for surveillance programs targeting HCW, as it enables the early identification and release of uninfected personnel, minimizing lost workdays. Additionally, analyzing viral load in saliva samples by ddPCR is valuable in determining virus shedding duration across different SARS-CoV-2 variants, informing transmission and disease control. Finally, testing saliva could overcome the detection of historic cases due to prolonged RNA swabbing past-infection and the unnecessary exclusion of those individuals from the workplace.

Apoptosis in Buruli ulcer: a clinicopathological study of 45 cases.

AIMS: To investigate the presence and pathogenetic role of apoptosis in Buruli ulcer (BU), a highly destructive skin disease caused by Mycobacterium ulcerans. METHODS AND RESULTS: Forty-five skin biopsies obtained from 30 Beninese patients affected by BU, in different clinical and therapeutic periods, were analysed for the main histopathological features (inflammatory infiltration, necrosis, sclerosis, oedema, granulomas and nerve damage). Immunofluorescent detection of antigens (anti-Bax, anti-caspases-3 and -8), together with deoxyuridine, 5'-triphosphate (dUTP) nick end labelling (TUNEL) assay, were also performed. A significant decrease in inflammatory infiltration (P = 0.0001) was detected between the beginning and end of antibiotic treatment. Neutrophils predominated in the first phase, while lymphocytes and plasma cells were increased at the end of the therapy. An inverse correlation between tissue necrosis and sclerosis was observed (P = 0.001). In 11 cases, inflammatory and regressive changes involved the nerve bundles with axonal degeneration and disruption of nerve fibres. TUNEL assay detected apoptotic bodies within nerve bundles, and these decreased from beginning to end of therapy. Bax, caspase-3 and -8 were down-regulated over the course of antibiotic therapy. CONCLUSIONS: In BU, apoptosis plays a role in promoting and sustaining the destructive changes and is implicated in the neural pathology that is associated with clinically detected anaesthesia.

#Stayathome If You Have a Cold: High SARS-CoV-2 Salivary Viral Loads in Pediatric Patients with Nasopharyngeal Symptoms.

The choice of the best SARS-CoV-2 detection approach is crucial to predict which children with SARS-CoV-2 are at high risk of spreading the virus in order to manage public health measures and policies. In this prospective observational study of 35 children admitted to the Pediatric Emergency Departments of two tertiary hospitals in Northern Italy who tested positive for SARS-CoV-2 by standard RT-PCR in nasopharyngeal swab (NPS), we evaluated their presenting symptoms according to their salivary viral load (SVL) determined by droplet digital PCR (ddPCR). Despite an overall low concordance between SARS-CoV-2 detected by salivary ddPCR and NPS RT-PCR (54.3%), when only patients with nasopharyngeal symptoms were analyzed, the sensitivity of ddPCR in saliva specimens increased to 71.4%, and over half of these patients had high SVL (>105 copies/mL), which was significantly more frequent than in children without nasopharyngeal symptoms (57.1% vs. 14.3%, OR = 8, CI 95% 1.28−50.03, p = 0.03). All asymptomatic children had low SVL values. Our findings support the hypothesis that children with nasopharyngeal symptoms are at higher risk of spreading SARS-CoV-2 due to their high SVL and, conversely, asymptomatic children are unlikely to spread the virus due to their low SVL, regardless of their NPS positivity.