Construction of a recombinant avipoxvirus expressing the env gene of Zika virus as a novel putative preventive vaccine.

BACKGROUND: Zika virus (ZIKV) has been declared a public health emergency that requires development of an effective vaccine, as it might represent an international threat. METHODS: Here, two novel DNA-based (pVAXzenv) and fowlpox-based (FPzenv) recombinant putative vaccine candidates were constructed that contained the cPrME genes of ZIKV. The env gene inserted into the fowlpox vector was verified for correct transgene expression by Western blotting and by immunofluorescence in different cell lines. The production of virus-like particles as a result of env gene expression was also demonstrated by electron microscopy. BALB/c mice were immunosuppressed with dexamethasone and immunized following a prime-boost strategy in a heterologous protocol where pVAXzenv was followed by FPzenv, to evaluate the immunogenicity of the Env protein. The mice underwent a challenge with an epidemic ZIKV after the last boost. RESULTS: These data show that the ZIKV Env protein was correctly expressed in both normal human lung fibroblasts (MRC-5 cells) and green monkey kidney (Vero) cells infected with FPzenv, and that the transgene expression lasted for more than 2 weeks. After mucosal administration of FPzenv, the immunized mice showed specific and significantly higher humoral responses compared to the control mice. However, virus neutralizing antibodies were not detected using plaque reduction assays. CONCLUSIONS: Although BALB/c mice appear to be an adequate model for ZIKV infection, as it mimics the natural mild infection in human beings, inadequate immune suppression seemed to occur by dexamethasone and different immune suppression strategies should be applied before challenge to reveal any protection of the mice.

Immunotherapy of HPV-associated cancer: DNA/plant-derived vaccines and new orthotopic mouse models.

Under the optimistic assumption of high-prophylactic HPV vaccine coverage, a significant reduction of cancer incidence can only be expected after decades. Thus, immune therapeutic strategies are needed for persistently infected individuals who do not benefit from the prophylactic vaccines. However, the therapeutic strategies inducing immunity to the E6 and/or E7 oncoprotein of HPV16 are more effective for curing HPV-expressing tumours in animal models than for treating human cancers. New strategies/technologies have been developed to improve these therapeutic vaccines. Our studies focussed on preparing therapeutic vaccines with low-cost technologies by DNA preparation fused to either plant-virus or plant-toxin genes, such as saporin, and by plant-produced antigens. In particular, plant-derived antigens possess an intrinsic adjuvant activity that makes these preparations especially attractive for future development. Additionally, discrepancy in vaccine effectiveness between animals and humans may be due to non-orthotopic localization of animal models. Orthotopic transplantation leads to tumours giving a more accurate representation of the parent tumour. Since HPV can cause cancer in two main localizations, anogenital and oropharynx area, we developed two orthotopic tumour mouse models in these two sites. Both models are bioluminescent in order to follow up the tumour growth by imaging and are induced by cell injection without the need to intervene surgically. These models were utilized for immunotherapies with genetic or plant-derived therapeutic vaccines. In particular, the head/neck orthotopic model appears to be very promising for studies combining chemo-radio-immune therapy that seems to be very effective in patients.

A prime/boost strategy using DNA/fowlpox recombinants expressing the genetically attenuated E6 protein as a putative vaccine against HPV-16-associated cancers.

BACKGROUND: Considering the high number of new cases of cervical cancer each year that are caused by human papilloma viruses (HPVs), the development of an effective vaccine for prevention and therapy of HPV-associated cancers, and in particular against the high-risk HPV-16 genotype, remains a priority. Vaccines expressing the E6 and E7 proteins that are detectable in all HPV-positive pre-cancerous and cancer cells might support the treatment of HPV-related lesions and clear already established tumors. METHODS: In this study, DNA and fowlpox virus recombinants expressing the E6F47R mutant of the HPV-16 E6 oncoprotein were generated, and their correct expression verified by RT-PCR, Western blotting and immunofluorescence. Immunization protocols were tested in a preventive or therapeutic pre-clinical mouse model of HPV-16 tumorigenicity using heterologous (DNA/FP) or homologous (DNA/DNA and FP/FP) prime/boost regimens. The immune responses and therapeutic efficacy were evaluated by ELISA, ELISPOT assays, and challenge with TC-1* cells. RESULTS: In the preventive protocol, while an anti-E6-specific humoral response was just detectable, a specific CD8(+) cytotoxic T-cell response was elicited in immunized mice. After the challenge, there was a delay in cancer appearance and a significant reduction of tumor volume in the two groups of E6-immunized mice, thus confirming the pivotal role of the CD8(+) T-cell response in the control of tumor growth in the absence of E6-specific antibodies. In the therapeutic protocol, in-vivo experiments resulted in a higher number of tumor-free mice after the homologous DNA/DNA or heterologous DNA/FP immunization. CONCLUSIONS: These data establish a preliminary indication for the prevention and treatment of HPV-related tumors by the use of DNA and avipox constructs as safe and effective immunogens following a prime/boost strategy. The combined use of recombinants expressing both E6 and E7 proteins might improve the antitumor efficacy, and should represent an important approach to control HPV-associated cancers.

Mucosal Alpha-Papillomavirus (HPV89) in a rare skin lesion.

BACKGROUND: Apocrine acrosyringeal keratosis is a rare skin lesion showing a unique benign keratotic lesion associated with syringocystoadenoma papilliferum. It is characterized by an exophytic proliferation of the epidermis with two distinct keratinocytic structures: a) columns of hyperkeratotic mass surrounded by acanthotic epidermis and b) papillated and/or cystic invaginations typical of syringocystoadenoma papilliferum. No causative agents were reported. FINDINGS: The present report describes a typical case of apocrine acrosyringeal keratosis localized in the right retro-auricular area of 57-year-old man in which the presence of HPV was evaluated. PCR analysis and direct sequencing revealed the presence of HPV 89. The presence of this low risk mucosal HPV in a skin localization was never reported as well as in association with this rare tumor. Furthermore rolling circle amplification, RT-PCR and in situ hybridization confirmed the presence of a transcriptionally active HPV 89. CONCLUSIONS: Taken together our results suggest that HPV89 plays a role in apocrine acrosyringeal keratosis with syringocystoadenoma papilliferum, in consideration of the documented biological activity of the virus. The association of low risk mucosal HPV infection with this skin lesion opens new perspectives in its clinical management. Further studies on samples from other patients are needed to confirm this association.

In vivo antitumor effect of an intracellular single-chain antibody fragment against the E7 oncoprotein of human papillomavirus 16.

Human papillomavirus (HPV)-associated tumors still represent an urgent problem of public health in spite of the efficacy of the prophylactic HPV vaccines. Specific antibodies in single-chain format expressed as intracellular antibodies (intrabodies) are valid tools to counteract the activity of target proteins. We previously showed that the M2SD intrabody, specific for the E7 oncoprotein of HPV16 and expressed in the endoplasmic reticulum of the HPV16-positive SiHa cells, was able to inhibit cell proliferation. Here, we showed by confocal microscopy that M2SD and E7 colocalize in the endoplasmic reticulum of SiHa cells, suggesting that the E7 delocalization mediated by M2SD could account for the anti-proliferative activity of the intrabody. We then tested the M2SD antitumor activity in two mouse models for HPV tumors based respectively on TC-1 and C3 cells. The M2SD intrabody was delivered by retroviral vector to tumor cells before cell injection into C57BL/6 mice. In both models, a marked delay of tumor onset with respect to the controls was observed in all the mice injected with the M2SD-expressing tumor cells and, importantly, a significant percentage of mice remained tumor-free permanently. This is the first in vivo demonstration of the antitumor activity of an intrabody directed towards an HPV oncoprotein. We consider that these results could contribute to the development of new therapeutic molecules based on antibodies in single-chain format, to be employed against the HPV-associated lesions even in combination with other drugs.

Evaluation of the quality of foods for special diets produced in a school catering facility within a HACCP-based approach: a case study.

A study was carried out to verify the appropriateness of the Hazard Analysis and Critical Control Point (HACCP) plan adopted in a school catering facility. To that end, the microbiological quality of foods, the correct implementation of special diets (lactose- and gluten-free) and the nutritional value of foods were assessed. Thirty-six samples of lactose-free and 87 samples of gluten-free special diet food preparations were subjected to microbiological, chemical, and nutritional analyses. The data collected demonstrate the effectiveness of the HACCP plan in reducing the occurrence of microbial and chemical (lactose and gluten) cross-contamination. The data obtained from the nutritional analyses showed that the dietary intake provided by the meals under study was satisfactory.

The landscape of circulating tumor HPV DNA and TTMV-HPVDNA for surveillance of HPV-oropharyngeal carcinoma: systematic review and meta-analysis.

BACKGROUND: Human papilloma virus (HPV) related cancers of the oropharynx are rapidly increasing in incidence and may soon represent the majority of all head and neck cancers. Improved monitoring and surveillance methods are thus an urgent need in public health. MAIN TEXT: The goal is to highlight the current potential and limitations of liquid biopsy through a meta analytic study on ctHPVDNA and TTMV-HPVDNA. It was performed a Literature search on articles published until December 2023 using three different databases: MEDLINE, Embase, and Cochrane Library. Studies that evaluated post-treatment ctHPVDNA and TTMV-HPVDNA in patients with HPV + OPSCC, studies reporting complete data on the diagnostic accuracy in recurrence, or in which the number of true positives, false positives, true negatives, and false negatives was extractable, and methods of detection of viral DNA clearly defined. The meta-analysis was conducted following the Meta-analysis Of Observational Studies in Epidemiology (MOOSE) reporting guidelines. The aim of this meta-analysis was to evaluate the sensitivity, specificity, and accuracy of ctHPVDNA and TTMV by ddPCR to define its efficacy in clinical setting for the follow up of HPV-OPSCC. CONCLUSION: The 12 studies included in the meta-analysis provided a total of 1311 patients for the analysis (398 valuated with ctHPVDNA and 913 with TTMV-HPVDNA). Pooled sensitivity and specificity were 86% (95% CI: 78%-91%) and 96% (95% CI: 91%-99%), respectively; negative and positive likelihood ratios were 0.072 (95% CI: 0.057-0.093) and 24.7 (95% CI: 6.5-93.2), respectively; pooled DOR was 371.66 (95% CI: 179.1-918). The area under the curve (AUC) was 0.81 (95% CI, 0.67-0.91). Liquid biopsy for the identification of cell free DNA might identify earlier recurrence in HPV + OPSCC patients. At the present time, liquid biopsy protocol needs to be standardized and liquid biopsy cannot yet be used in clinical setting. In the future, a multidimensional integrated approach which links multiple clinical, radiological, and laboratory data will contribute to obtain the best follow-up strategies for the follow-up of HPV-OPSCC.

A first assessment of the safe brain initiative care bundle for addressing postoperative delirium in the postanesthesia care unit.

BACKGROUND: Postoperative delirium (POD) following surgery is a prevalent and distressing condition associated with adverse patient outcomes and an increased healthcare burden. OBJECTIVES: To assess the effectiveness of the Safe Brain Initiative care bundle (SBI-CB) in reducing POD in the postanesthesia care unit (PACU). DESIGN: A multicenter, quality-improvement initiative with retrospective analysis of collected data. SETTING: The study was conducted in the operating rooms and postanesthesia care units (PACUs) of four hospitals across Denmark and Turkey. PATIENTS: The convenience sample of patients were aged ≥18 years, scheduled for surgery, and could communicate verbally. Age, sex, preoperative delirium, and the American Society for Anesthesiology physical status classification were used in statistical methods to control for potential confounding influences. INTERVENTION: The SBI-CB, 18 delirium-reducing recommendations aligned with international guidelines. The intervention included patient education, staff training, coordination meetings across centers, and a dashboard for the monitoring of outcomes in the PACU. MAIN OUTCOME MEASURES: The primary outcome was the POD trend in the PACU during implementation months, assessed through Nu-DESC screening at up to three time points in the PACU. We also examined the length of hospital stay. RESULTS: Data were collected from 18,697 adult patients across four hospitals. Initial POD incidence in the PACU after the first three months was 16.36% across all sites (n = 1021). POD in the PACU was observed across all age groups, with peak incidence in younger (18-35 years) and older (>75 years) patients. General anesthesia and longer surgical duration (>1 h) were identified as significant risk factors for POD in the PACU. Matched patients who experienced POD in the PACU had longer stays in hospital, with a mean increase from 35 to 69 h (p < 0.001). Implementation of the SBI-CB was associated with a decreased risk of POD in the PACU for each month of SBI-CB implementation (adjusted odds ratio 0.96, 95% confidence interval: [0.94, 0.97], p < 0.001). CONCLUSIONS: The presented pragmatic implementation of a multidisciplinary care bundle, encompassing pre-, intra-, and postoperative measures alongside outcome monitoring, has the potential to significantly reduce the incidence of POD in the PACU. Improved patient outcomes may be achieved for general surgical departments with patient cohorts not typically considered at risk for developing POD. TRIAL REGISTRATION: Clinicaltrials.gov, identifier NCT05765162.

Circulating tumor HPV DNA in the management of HPV+ oropharyngeal cancer and its correlation with MRI.

BACKGROUND: First aim was to compare ddPCR assays of ctHPVDNA with p16 IHC and qualitative HPV PCR. Second aim was to carry out longitudinal blood sampling to test for association of ctHPVDNA with histological confirmed recurrence. Third aim was to perform a multidimensional assessment which included: (1) clinical features; (2) ctHPVDNA; (3) MRI-based tumor size measurements of primary tumor (PT) and cervical lymph node metastases (CLNM). METHODS: Plasma samples were collected before treatment and during follow-up, and ddPCR assay comprising E6 of HPV16 and HPV 33 and HPV 35 was used. RESULTS: Present study was conducted at diagnosis in 117 patients and revealed a ctHPVDNA sensitivity of 100% (95% CI 95.5-100) and a specificity of 94.4 (95% CI 81.3-99.3), positive predictive value (PPV) of 94.4 (95% CI 81.3-99.3), and negative predictive value (NPP) of 100% (95% CI 89.7-100). During follow-up ctHPVDNA had a sensitivity of 100% (95% CI 72.1-100)% and specificity of 98.4% (95% CI 91.7-100)%, PPV% of 90.9% (95% CI 62.3-98.4) and NPV% of 100% (95% CI 94.3-100) for ability to detect recurrence. Correlation between both the CLNM volume and the sum of PT and CLNM volume was observed. CONCLUSIONS: ctHPVDNA was superior to p16 in identification of HPV-OPSCC at diagnosis. Introduction of ctHPVDNA, beyond diagnostic setting, represents a great opportunity to improve follow-up protocol of OPSCC patients.

Comparative Life Cycle Assessment Between Single-Use and Reprocessed IPC Sleeves.

Purpose: Healthcare has a large environmental footprint, not least due to the wide use of single-use supplies. Reprocessing of medical devices is a well-established, regulated process, and can reduce its environmental impact. This life cycle assessment (LCA) compares the environmental footprint of a single-use and a reprocessed version of otherwise identical intermittent pneumatic compression (IPC) sleeves. Materials and Methods: The LCA was performed in accordance with the international standard ISO 14044 using the Environmental Footprint 3.0 (EF) method for the assessment. Data were obtained in cooperation with IPC sleeve manufacturers. Where no primary data were available, ecoinvent database records were used. The functional unit is five hospital treatments applying IPC. The robustness of the results was interrogated in sensitivity analyses of the energy mix, the ethylene oxide emissions during reprocessing, and the transport distances. The impact of waste reduction on hospital disposal costs was calculated. Results: The environmental footprint of reprocessed IPC sleeves was found to be reduced in all categories compared to single-use devices, leading to a weighted normalized reduction of 43% across all categories. In a breakdown of the LCA results, reprocessed IPC sleeves were found to reduce the carbon footprint by 40%, with the treatment of five patients with single-use IPC sleeves creating 7 kg CO2eq, compared to 4.2 kg CO2eq from reprocessed sleeves. Waste disposal costs were also reduced by 90%. Conclusion: Reprocessing of IPC sleeves provides an environmental and economic benefit in comparison to single-use devices.

Targeting tumor-stroma communication by blocking endothelin-1 receptors sensitizes high-grade serous ovarian cancer to PARP inhibition.

PARP inhibitors (PARPi) have changed the treatment paradigm of high-grade serous ovarian cancer (HG-SOC). However, the impact of this class of inhibitors in HG-SOC patients with a high rate of TP53 mutations is limited, highlighting the need to develop combinatorial therapeutic strategies to improve responses to PARPi. Here, we unveil how the endothelin-1/ET-1 receptor (ET-1/ET-1R) axis, which is overexpressed in human HG-SOC and associated with poor prognosis, instructs HG-SOC/tumor microenvironment (TME) communication via key pro-malignant factors and restricts the DNA damage response induced by the PARPi olaparib. Mechanistically, the ET-1 axis promotes the p53/YAP/hypoxia inducible factor-1α (HIF-1α) transcription hub connecting HG-SOC cells, endothelial cells and activated fibroblasts, hence fueling persistent DNA damage signal escape. The ET-1R antagonist macitentan, which dismantles the ET-1R-mediated p53/YAP/HIF-1α network, interferes with HG-SOC/stroma interactions that blunt PARPi efficacy. Pharmacological ET-1R inhibition by macitentan in orthotopic HG-SOC patient-derived xenografts synergizes with olaparib to suppress metastatic progression, enhancing PARPi survival benefit. These findings reveal ET-1R as a mechanistic determinant in the regulation of HG-SOC/TME crosstalk and DNA damage response, indicating the use of macitentan in combinatorial treatments with PARPi as a promising and emerging therapy.

It is time to improve the diagnostic workup of oropharyngeal cancer with circulating tumor HPV DNA: Systematic review and meta-analysis.

The possibility of detecting circulating tumor HPV DNA (ctHPVDNA) in plasma in patients with oropharyngeal cancer has been demonstrated in several reports. However, these data are from small cohorts and available tests for detection of ctHPVDNA are not fully validated. The aim is to evaluate sensitivity, specificity, and accuracy of ctHPVDNA by ddPCR to define its efficacy in the clinical setting for the diagnosis of HPV + OPSCC. A comprehensive search of three different databases: MEDLINE, Embase, and Cochrane Library databases. A total of 998 patients were evaluated from the 13 studies. OPSSC p16+ were 729, while controls p16- were 269. The meta-analytic study estimated the diagnostic performance of ctHPVDNA as follows: pooled sensitivity and specificity of 0.90 (95% CI: 0.82-0.94) and 0.94 (95% CI: 0.85-0.98), respectively; positive and negative likelihood ratios of 12.6 (95% CI: 4.9-32.1) and 0.05 (95% CI: 0.02-0.13), respectively. ddPCR for ctHPVDNA has good accuracy, sensitivity, and specificity for diagnosis of HPV + OPSCC. ctHPVDNA kinetic represents a great reliable opportunity to improve diagnostic and therapeutic management of cancer patients and could open new perspectives for understanding tumor biology.

hMENA isoforms regulate cancer intrinsic type I IFN signaling and extrinsic mechanisms of resistance to immune checkpoint blockade in NSCLC.

BACKGROUND: Understanding how cancer signaling pathways promote an immunosuppressive program which sustains acquired or primary resistance to immune checkpoint blockade (ICB) is a crucial step in improving immunotherapy efficacy. Among the pathways that can affect ICB response is the interferon (IFN) pathway that may be both detrimental and beneficial. The immune sensor retinoic acid-inducible gene I (RIG-I) induces IFN activation and secretion and is activated by actin cytoskeleton disturbance. The actin cytoskeleton regulatory protein hMENA, along with its isoforms, is a key signaling hub in different solid tumors, and recently its role as a regulator of transcription of genes encoding immunomodulatory secretory proteins has been proposed. When hMENA is expressed in tumor cells with low levels of the epithelial specific hMENA11a isoform, identifies non-small cell lung cancer (NSCLC) patients with poor prognosis. Aim was to identify cancer intrinsic and extrinsic pathways regulated by hMENA11a downregulation as determinants of ICB response in NSCLC. Here, we present a potential novel mechanism of ICB resistance driven by hMENA11a downregulation. METHODS: Effects of hMENA11a downregulation were tested by RNA-Seq, ATAC-Seq, flow cytometry and biochemical assays. ICB-treated patient tumor tissues were profiled by Nanostring IO 360 Panel enriched with hMENA custom probes. OAK and POPLAR datasets were used to validate our discovery cohort. RESULTS: Transcriptomic and biochemical analyses demonstrated that the depletion of hMENA11a induces IFN pathway activation, the production of different inflammatory mediators including IFNß via RIG-I, sustains the increase of tumor PD-L1 levels and activates a paracrine loop between tumor cells and a unique macrophage subset favoring an epithelial-mesenchymal transition (EMT). Notably, when we translated our results in a clinical setting of NSCLC ICB-treated patients, transcriptomic analysis revealed that low expression of hMENA11a, high expression of IFN target genes and high macrophage score identify patients resistant to ICB therapy. CONCLUSIONS: Collectively, these data establish a new function for the actin cytoskeleton regulator hMENA11a in modulating cancer cell intrinsic type I IFN signaling and extrinsic mechanisms that promote protumoral macrophages and favor EMT. These data highlight the role of actin cytoskeleton disturbance in activating immune suppressive pathways that may be involved in resistance to ICB in NSCLC.

Natural Bioactives: Back to the Future in the Fight against Human Papillomavirus? A Narrative Review.

Human papillomavirus (HPV) still represents an important threat to health worldwide. Better therapy in terms of further improvement of outcomes and attenuation of related side-effects is desirable. The pharmaceutical industry has always targeted natural substances-phytochemicals in particular-to identify lead compounds to be clinically validated and industrially produced as antiviral and anticancer drugs. In the field of HPV, numerous naturally occurring bioactives and dietary phytochemicals have been investigated as potentially valuable in vitro and in vivo. Interference with several pathways and improvement of the efficacy of chemotherapeutic agents have been demonstrated. Notably, some clinical trials have been conducted. Despite being endowed with general safety, these natural substances are in urgent need of further assessment to foresee their clinical exploitation. This review summarizes the basic research efforts conducted so far in the study of anti-HPV properties of bio-actives with insights into their mechanisms of action and highlights the variety of their natural origin in order to provide comprehensive mapping throughout the different sources. The clinical studies available are reported, as well, to highlight the need of uniformity and consistency of studies in the future to select those natural compounds that may be suited to clinical application.

The Effectiveness of Therapeutic Vaccines for the Treatment of Cervical Intraepithelial Neoplasia 3: A Systematic Review and Meta-Analysis.

Cervical cancer (CC) is a disease that affects many women worldwide, especially in low-income countries. The human papilloma virus (HPV) is the main causative agent of this disease, with the E6 and E7 oncoproteins being responsible for the development and maintenance of transformed status. In addition, HPV is also responsible for the appearance of cervical intraepithelial neoplasia (CIN), a pre-neoplastic condition burdened by very high costs for its screening and therapy. So far, only prophylactic vaccines have been approved by regulatory agencies as a means of CC prevention. However, these vaccines cannot treat HPV-positive women. A search was conducted in several databases (PubMed, Scopus, Web of Science, and ClinicalTrials.gov) to systematically identify clinical trials involving therapeutic vaccines against CIN 3. Histopathological regression data, immunological parameters, safety, DNA clearance, and vaccine efficacy were considered from each selected study, and from the 102 articles found, 8 were selected based on the defined inclusion criteria. Histopathological regression from CIN 3 to CIN < 1 was 22.1% (95% CI: 0.627−0.967; p-value = 0.024), showing a vaccine efficacy of 23.6% (95% CI; 0.666−0.876; p-value < 0.001). DNA clearance was assessed, and the risk of persistent HPV DNA was 23.2% (95% CI: 0.667−0.885; p-value < 0.001). Regarding immunological parameters, immune responses by specific T-HPV cells were more likely in vaccinated women (95% CI: 1.245−9.162; p-value = 0.017). In short, these studies favored the vaccine group over the placebo group. This work indicated that therapeutic vaccines are efficient in the treatment of CIN 3, even after accounting for publication bias.

Modifying Anthocyanins Biosynthesis in Tomato Hairy Roots: A Test Bed for Plant Resistance to Ionizing Radiation and Antioxidant Properties in Space.

Gene expression manipulation of specific metabolic pathways can be used to obtain bioaccumulation of valuable molecules and desired quality traits in plants. A single-gene approach to impact different traits would be greatly desirable in agrospace applications, where several aspects of plant physiology can be affected, influencing growth. In this work, MicroTom hairy root cultures expressing a MYB-like transcription factor that regulates the biosynthesis of anthocyanins in Petunia hybrida (PhAN4), were considered as a testbed for bio-fortified tomato whole plants aimed at agrospace applications. Ectopic expression of PhAN4 promoted biosynthesis of anthocyanins, allowing to profile 5 major derivatives of delphinidin and petunidin together with pelargonidin and malvidin-based anthocyanins, unusual in tomato. Consistent with PhAN4 features, transcriptomic profiling indicated upregulation of genes correlated to anthocyanin biosynthesis. Interestingly, a transcriptome reprogramming oriented to positive regulation of cell response to biotic, abiotic, and redox stimuli was evidenced. PhAN4 hairy root cultures showed the significant capability to counteract reactive oxygen species (ROS) accumulation and protein misfolding upon high-dose gamma irradiation, which is among the most potent pro-oxidant stress that can be encountered in space. These results may have significance in the engineering of whole tomato plants that can benefit space agriculture.

Deconvolution of malignant pleural effusions immune landscape unravels a novel macrophage signature associated with worse clinical outcome in lung adenocarcinoma patients.

BACKGROUND: Immune checkpoint inhibitors are still unable to provide clinical benefit to the large majority of non-small cell lung cancer (NSCLC) patients. A deeper characterization of the tumor immune microenvironment (TIME) is expected to shed light on the mechanisms of cancer immune evasion and resistance to immunotherapy. Here, we exploited malignant pleural effusions (MPEs) from lung adenocarcinoma (LUAD) patients as a model system to decipher TIME in metastatic NSCLC. METHODS: Mononuclear cells from MPEs (PEMC) and peripheral blood (PBMC), cell free pleural fluid and/or plasma were collected from a total of 24 LUAD patients and 12 healthy donors. Bulk-RNA sequencing was performed on total RNA extracted from PEMC and matched PBMC. The DEseq2 Bioconductor package was used to perform differential expression analysis and CIBERSORTx for the regression-based immune deconvolution of bulk gene expression data. Cytokinome analysis of cell-free pleural fluid and plasma samples was performed using a 48-Plex Assay panel. THP-1 monocytic cells were used to assess macrophage polarization. Survival analyses on NSCLC patients were performed using KM Plotter (LUAD, N=672; lung squamous cell carcinoma, N=271). RESULTS: Transcriptomic analysis of immune cells and cytokinome analysis of soluble factors in the pleural fluid depicted MPEs as a metastatic niche in which all the components required for an effective antitumor response are present, but conscripted in a wound-healing, proinflammatory and tumor-supportive mode. The bioinformatic deconvolution analysis revealed an immune landscape dominated by myeloid subsets with the prevalence of monocytes, protumoral macrophages and activated mast cells. Focusing on macrophages we identified an MPEs-distinctive signature associated with worse clinical outcome in LUAD patients. CONCLUSIONS: Our study reports for the first time a wide characterization of MPEs LUAD microenvironment, highlighting the importance of specific components of the myeloid compartment and opens new perspectives for the rational design of new therapies for metastatic NSCLC.

Papillomavirus E5: the smallest oncoprotein with many functions.

Papillomaviruses (PVs) are established agents of human and animal cancers. They infect cutaneous and mucous epithelia. High Risk (HR) Human PVs (HPVs) are consistently associated with cancer of the uterine cervix, but are also involved in the etiopathogenesis of other cancer types. The early oncoproteins of PVs: E5, E6 and E7 are known to contribute to tumour progression. While the oncogenic activities of E6 and E7 are well characterised, the role of E5 is still rather nebulous. The widespread causal association of PVs with cancer makes their study worthwhile not only in humans but also in animal model systems. The Bovine PV (BPV) system has been the most useful animal model in understanding the oncogenic potential of PVs due to the pivotal role of its E5 oncoprotein in cell transformation. This review will highlight the differences between HPV-16 E5 (16E5) and E5 from other PVs, primarily from BPV. It will discuss the targeting of E5 as a possible therapeutic agent.

PBMCs are additional sites of productive infection of bovine papillomavirus type 2.

Bovine papillomavirus type 2 (BPV-2) is an oncogenic virus infecting both epithelial and mesenchymal cells. Its life cycle, similar to other papillomaviruses (PVs), appears to be linked to epithelial differentiation. Human and bovine PVs have been known to reside in a latent, episomal form in PBMCs; therefore, it is believed that blood cells, like all mesenchymal cells, function as non-permissive carriers. Here, for the first time in veterinary and comparative medicine, the BPV-2 E5 oncoprotein and the major structural L1 capsid protein, known to be expressed only in productive infections, were shown to occur in defined subsets of PBMCs. E5 oncoprotein was detected in sorted T- and B-cells as well as in monocytes by flow cytometry and Western blot analysis. However, CD4(+) and CD8(+) lymphocytes appeared to be the main circulating targets of the virus, thus possibly representing the most important reservoir of active BPV-2 in blood. L1 protein was identified by flow cytometry in a population of blood cells recognized as lymphocytes by morphological scatter properties. Western blot analysis was performed on lysates obtained from the sorted subpopulations of PBMCs and detected L1 protein in CD4(+) and CD8(+) cells only. Thus, this study showed that CD4(+) and CD8(+) lymphocytes are permissive for BPV-2 and are new, hitherto unknown sites of productive PV infection. In light of these observations, the life cycle of PVs needs to be revisited to gain novel insights into the epidemiology of BPV infection and the pathogenesis of related diseases.