A (gamma-Aminobutyric acid) modulator, lorazepam, on behavioral and neural responses to coercive power. Here, we applied a virtual obedience to authority paradigm alongside lorazepam administration (versus placebo), and during functional magnetic resonance imaging scanning. Our results show that lorazepam administration exerted differential effects on the reaction times (RTs) when initiating harming versus helping behaviors, with longer harming RTs compared to helping RTs, despite comparable subjective ratings regarding perceived coercion. Coercive harming significantly increased activity in the amygdala, hippocampus, orbitofrontal cortex, and dorsolateral prefrontal cortex (dlPFC). Lorazepam administration decreased amygdala and hippocampus activity, but increased dlPFC and right temporoparietal junction activations. The lower activity in the hippocampus predicted higher ratings for perceived coercion. Furthermore, lorazepam significantly decreased the functional connectivity of the hippocampus with the dlPFC during coercive harming. In conclusion, we provide evidence –by incorporating multimodal indices, including neuroimaging, neuropharmacological interventions, and behavioral assessments– to posit that the GABAA agonist, lorazepam, might aid as a possible intervention in service of coping strategies against coercion.Previous research on coercion has neglected the fact that agents under authoritative pressure may also suffer from coercive power, which can trigger anxiety-like emotional negativity on its victims. Furthermore, high levels of neuroticism and/or anxiety have been found to be associated with the compliance of various forms of social pressure. In this study, we investigate the effects of the anxiolytic GABA Throughout history, it has been well documented that people are enabled to perform the most vilest and unscrupulous actions against other human beings when under the pressure of an authority figure . FurtherA) receptor antagonist bicuculline methiodide (BMI)  ; and witde (BMI) . The anxde (BMI) , as it eBefore going further, it is important to note that “coercion” itself cannot be studied directly –as if in a real-life situation– in an experimental setting due to ethical concerns. Strictly speaking, the term “coercion” encompasses actions that make use of force or violence, either directly or indirectly (through the use of threats), in order to induce an individual into doing something to which they have not given their full or partial consent. Consequently, in the present study, we use the notion of “coercion” to describe an experimental condition in which agents execute orders which lead them to inflict harm to another individual. In a recent study, we demonstrated that the effect of social coercion on the experience of agency and responsibility was modulated by individual variability in response to coercive power . In the To understand a potential intervention that may lead people to counteract the effects of coercive violence, we investigate the behavioral and neural effects of the anxiolytic drug lorazepam on the virtual obedience paradigm. Nevertheless, and contrary to the research mentioned above, the present study makes use of functional magnetic resonance imaging (fMRI). This is, firstly, due to the amygdala being a key brain structure responsible for salience detection in the environment, including threats ; thus, wWithin each participant, the mean RTs were firstly calculated and represented the reaction speed of the six different conditions  . Due to F2,152 = 3.64, P = 0.03, η2 = 0.05) as well as a main effect of scenario . Follow-up analyses indicated that the harming and helping LOG-RTs differed significantly after lorazepam administration , whereas they were comparable after placebo . The administration effect (lorazepam vs. placebo) had opposite directions depending on the factor of scenario, where lorazepam administration prolonged harming RTs, while shortening helping RTs, and did not have any effect on neutral RTs . As a consequence of that, the harming and helping LOG-RTs differed significantly after lorazepam administration , similar results were obtained with the neutral and helping LOG-RTs  (A 2 (administration: placebo vs. lorazepam) x 3  repeated ANOVA on LOG-RTs revealed an interaction between administration and scenario ( 0.4779) .F1,76 = 9.4, P = 0.003, η2 = 0.11) in addition to a main effect of scenario  that showed shorter RTs in helping behaviors . Follow-up analyses indicated that the administration effect (lorazepam vs. placebo) had opposite directions depending on the factor of scenario . Here, the descriptive statistics for the post hoc tests were conducted and supplemented for the sake of revealing how the interactions functioned. Harming and helping LOG-RTs differed significantly after lorazepam administration , whereas they were comparable after placebo . The descriptive statistics for the post hoc tests were conducted and supplemented for the sake of revealing how the interaction functioned. Complementary non-parametric analyses using raw RTs were reported in the To isolate the sedation effect (slowing) of lorazepam, the differential LOG-RTs for helping/harming actions were derived from subtracting participant’s LOG-RTs in the neutral condition from LOG-RTs in the helping or harming condition. The differential LOG-RTs subject to a 2 (administration: lorazepam vs. placebo) × 2 (scenario: harming vs. helping) repeated ANOVA further confirmed the unique interaction between administration and scenario , indicating that, under coercion, participants were less willing  to do harming (5.15 ± 0.13) than to helping (4.53 ± 0.14). Neither the administration  nor its interaction with scenario  reached significance.The subjective ratings of coercion, derived from the violation of free will, were subject to a 2 (administration: lorazepam vs. placebo) × 2 (scenario: harming vs. helping) repeated ANOVA. There was a main effect of scenario , whereas 72% of participants exhibited this pattern in the lorazepam condition. Regarding to the subjective ratings, while 70% of participants showed patterns of moral valence  in the placebo condition, 77% of participants showed this pattern in the lorazepam condition.F1,76 = 5.15, P = 0.026, η2 = 0.06), hippocampus , and dlPFC . The rTPJ had a marginal effect with a medium effect size . The follow-up analyses indicated that the administration effect in the amygdala, hippocampus, dlPFC, and rTPJ had opposite directions depending on the factor of scenarios (coercive harming vs. helping). In the amygdala, harming induced a stronger neuro-hemodynamic responserelative to helping under the placebo condition , whereas no such difference was found in the lorazepam condition . In the hippocampus, harming induced a stronger neuro-hemodynamic response relative to helping under the placebo condition , whereas no such difference was found in the lorazepam condition . In the dlPFC, helping induced a stronger neuro-hemodynamic response relative to harming under the placebo condition , whereas no such difference was found in the lorazepam condition . In the rTPJ, helping induced a stronger neuro-hemodynamic response relative to harming under the placebo condition , whereas no such difference was found in the lorazepam condition .Regarding the ROI results , signifiP < 0.05 .To examine the association between the subjective experience of coercion and the observed neural responses, we did a whole-brain correlation analysis where subjective ratings were computed as a continuous variable with FWE rate at P < 0.05 . After lLorazepam triggered distinct patterns in the functional couplings . After lWith the present fMRI study, we aimed to elucidate the effects of the drug lorazepam on the sense of agency under coercion, as well as its neural underpinnings, by means of a virtual obedience paradigm. We found that RTs to initiate moral behaviors show an interaction effect with drug administration and scenario.It is important to note that RTs may be considered an objective proxy measure for sedation, with the latter being a possible side effect of the GABA receptor agonist lorazepam , and aloWhen it comes to the sense of agency, previous literature uncovered explicit and implicit approaches to measure such a subjective phenomenon. The implicit index, as measured by the intentional binding effect, was based on the subjective perception of time compression between an action and its effects ; whereasTo explain the behavior of the agents under coercion, previous studies have set the players distributed in a rather simple model , 2018: . We thusAlongside the above findings, we also observed that coercive harming versus helping recruited the activity in the amygdala and insula. This finding is in line with previous reports which posit that directly harming others generates an aversive emotional “gut” reaction . ConflicHere, it is likely to make a case for lorazepam as a freer of cognitive power –letting participants to reason more clearly. Lorazepam has been suggested to increase ruthlessness, as it was observed that its administration drove participants to endorse harmful behaviors due to the drug’s ability to reduce threat intensity during moral dilemmas . FurtherNotably, after lorazepam administration, the weaker activity in the hippocampus during coercive harming predicted higher subjective ratings of coercion see . Based oper se. In real-life situations, individuals who obey coercive orders to harm exhibit differences in emotional responses to their immoral actions. Using the Nuremberg trials as a historical example, some war criminals being processed took their own lives out of guilt-like anxiety even before the trials began, whereas others attended the whole prosecution with seeming indifference (per se), future studies adopting a better control condition are warranted, such as a control condition with instructions simply asking participants to press the button in order to view the following (harming) pictures, without specifically priming them to simulate the action. Second, post-session questionnaires to assess the perceived interpersonal behavior of the experimenter might be helpful to evaluate the effects of response expectancy to the intervention in drug-placebo studies  affects that were caused by the aversive (or favorable) responses toward the perception of the following harming/helping pictures. However, these were similar to the affective responses incurred by coercion in a natural setting. The negative affect toward coercion was related to both the coercive power as well as the harming action fference , which sfference . To diss studies . Third, In conclusion, the present findings –which incorporate multimodal indices, including functional neuroimaging, neuropharmacological intervention, and behavioral assessments– provide evidence to corroborate the notion that agents under coercive pressure may suffer from anxiety-like emotional negativity, and that the GABA receptor agonist lorazepam might help in unveiling the power of authority and assist in the emergence of prosocial behavior. Noteworthy, in real life scenarios, coercive orders to harm third parties could as well come with negative consequences for the self in case of disobedience. While we found the freeing effect of lorazepam on the sense of agency, lorazepam might also promote harming behaviors in moral-personal dilemmas out of selfish motivation . Therefof (ranging from 0.2 to 0.22) for the primary fMRI outcomes found in previous fMRI literature using similar stimuli  on one day, and a single dose of placebo  on another day. A crossover study is a longitudinal study in which subjects receive a sequence of different treatments. Both lorazepam and placebo were administered orally. The experimental sequence of lorazepam and placebo administration was counter-balanced between participants through a Latin square design, which randomizes through having equal number of AB (lorazepam-placebo) and BA (placebo-lorazepam) sequences. Thus, half of the participants went first through the lorazepam session, and half of them went first through the placebo session. To coincide with the pharmacokinetics of lorazepam , fMRI scIn this study, we designed a virtual obedience paradigm inspired by prior studies on obedience to authority , in whicForty-five validated animations from previous fMRI studies were presented ,b. Each Participants underwent two sessions of fMRI scanning (placebo and lorazepam) in different days. Stimuli were presented with the E-prime software  and an MRI compatible goggle  in a three-level within-subject design of moral scenarios .The scanning followed a block design (22.9 ± 0.6 s ON/13.2 ± 4.4 s OFF) and had two runs. Each run consisted of six ON blocks  intermixed with six OFF blocks. Each ON block consisted of five trials, and five inter-stimulus intervals  with a fixation cross presented against a gray background. While the ISI  was set as 2,200-ms, the duration of each fMRI regressor was modeled with each participant’s actual RTs. Because the RT varies across trials and participants, the modeled duration self-served as jittering in nature, leaving the average length of each ON block 2294 ± 598 ms (mean ± SD). The sequence of the scenarios  was pseudo-randomized within each run. The order of runs was counterbalanced across participants .Scanning was performed on a 3T Siemens Magnetom Trio-Tim magnet. For functional changes, changes in blood oxygenation level-dependent (BOLD) T2* weighted MR signal were collected along the AC–PC plane using a gradient echo-planar imaging (EPI) sequence . High-resolution structural T1-weighted images were acquired using a 3D magnetization-prepared rapid gradient echo sequence  .Functional images were processed with SPM12  in MATLAB 9.0 . Structural T1 images were coregistered to the mean functional images, and a skull-stripped image was created from the segmented gray matter, white matter, and Cerebrospinal Fluid (CSF) images. These segmented images were combined to create a subject-specific brain template. EPI images were realigned and filtered (128-s cutoff), then coregistered to these brain templates, normalized to Montreal Neurologic Institute (MNI) space, and smoothed . The hemodynamic response function was time-locked to stimulus onset .P < 0.05 was determined with a threshold at uncorrected P < 0.001, cut-off, t = 3.118, and cluster extent of at least 10 contiguous voxels, using a Monte Carlo simulation built in 3dClustSim: https://afni.nimh.nih.gov/pub/dist/doc/program_help/3dClustSim.html.Data were input into a general linear model, with movement parameters as nuisance regressors. There was no significant difference in movement parameters between lorazepam and placebo. None of participants had movements greater than 2 mm of translation or 0.03 degrees of rotation . A two-sa priori coordinates. Data extraction for the ROI analyses was performed using the MarsBaR toolbox1 implemented in SPM12.To elucidate the lorazepam effect, regions of interest (ROIs) analyses in limbic areas were conducted for bilateral amygdala and hippocampus . Beyond P < 0.05 .Based on our whole-brain results and prior studies , the psyThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of National Yang-Ming University (YM104041E). The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any identifiable images or data included in this article.YC and CC conceived and conceptualized the study. Y-CC and CC collected and analyzed the data. YC, RM, and CC conducted the necessary literature reviews and drafted the first manuscript. Y-TF provided critical feedback and helped shape the manuscript. All authors contributed toward the revision and writing the final draft.


Women with symptomatic cervical ectopy and HPV infection had increased diversity (p<0.001), and their vaginal microbiota was enriched in bacterial vaginosis-associated anaerobes  and depleted in Lactobacillus spp. Furthermore, the vaginal microbiota of women with symptomatic cervical ectopy and HPV infection correlated with vaginal inflammation  and increased mucosal homeostatic response . Taken together, our results suggest that HPV infection and dysbiotic vaginal communities could favor a vaginal microenvironment that might delay the recovery of the cervical epithelium in women with symptomatic cervical ectopy and favor STDs acquisition.Cervical ectopy is a benign condition of the lower genital tract that is frequently detected in women of reproductive age. Although cervical ectopy is regarded as a physiological condition, some women experience symptoms such as leucorrhoea, persistent bleeding and recurrent vaginal infections that require medical intervention. Cervical ectopy has not been linked to cervical cancer, but it is thought to facilitate the acquisition of sexually transmitted diseases (STDs), like Human Papillomavirus (HPV) infection, as it provides a favorable microenvironment for virus infection and dissemination. We and others have described the presence of oncogenic HPV types in women with symptomatic cervical ectopy. The relevance of this finding and the impact of symptomatic cervical ectopy on the cervicovaginal microenvironment  are currently unknown. To shed some light into the interplay between HPV, the vaginal microbiota and mucosal immune and inflammatory responses in the context of this condition, we enrolled 156 women with symptomatic cervical ectopy and determined the presence of HPV using a type-specific multiplex genotyping assay. Overall, HPV was detected in 54.48% women, oncogenic HPV types were found in more than 90% of HPV-positive cases. The most prevalent HPV types were HPV16 (29.4%), HPV31 (21.17%) and HPV18 (15.29%). Next, we evaluated the vaginal microbial composition and diversity by 16S rDNA sequencing, and quantified levels of cytokines and chemokines by flow cytometry using bead-based multiplex assays in a sub-cohort of 63 women. IL-21 and CXCL9 were significantly upregulated in HPV-positive women ( Chlamydia trachomatis , which colonize the cervicovaginal mucus , trauma , and the pH of the cervicovaginal mucus. Indeed, squamous metaplastic activity is stimulated by the acidic pH of the vagina , which ial mucus  and consal mucus , among oal mucus  and depllus spp. . Moreovelus spp. . Very liA general feature of women with symptomatic cervical ectopy is the abnormal discharge of cervicovaginal mucus, which is produced by the everted glandular cells. Cervicovaginal mucus contributes to mucosal immune responses, because it harbors both cytokines and antibodies which are mediators of innate and adaptive immunity. Genital inflammation, often determined by levels of pro-inflammatory cytokines in cervicovaginal mucus, has been extensively studied in women with HPV-associated squamous lesions and tumors , but lesThus, in the present work we determined the prevalence of HPV genotypes in glandular tissue of women with symptomatic cervical ectopy. Mucosal immune responses were evaluated in cervicovaginal mucus of a selected group of HPV-positive and HPV-negative women, in order to determine their genital inflammation profile (cytokines and chemokines). Next, we also characterized the vaginal microbiota by 16S rRNA sequencing. Finally, associations between HPV infection, vaginal microbial communities and genital inflammation were explored. Our study aims at improving our understanding of the complex interactions of distinct elements within the cervicovaginal environment of women suffering from a common, but scarcely explored condition of the female genital tract.Female participants were selected from women attending the Colposcopy Clinic of the “Cruz-Talonia” Foundation, Mexico City, Mexico, a non-profit institution dedicated to preventing cervical cancer, between 2017 and 2018. Women who complained of abnormal vaginal discharge, intermenstrual and/or post-coital bleeding were examined and underwent colposcopy. Women with symptomatic cervical ectopy, defined as irregular patches of columnar epithelium with papillae and clefts, without ectocervical changes after acetic acid application, were invited to participate. Eligible women: older than 20 years of age, sexually active, not pregnant, without a previous history of HPV-associated pathologies, and HIV seronegative, were invited to participate. All participants agreed to participate in this study, signed an informed consent form and answered a validated questionnaire on sociodemographic characteristics, sexual behavior, and medical history. This study was approved by the Local Ethics Committee of the Colposcopy Clinic of the “Cruz-Talonia” Foundation (Reference Number: 17/02/2017/LRZ/CE05). All women consented to provide a tissue biopsy and a cervicovaginal mucus sample, and to give any remaining unwanted samples to research.All samples were obtained from participants at the seventh day of their menstrual cycle. Colposcopy-directed biopsies were obtained by trained specialists (colposcopists) experienced in the management of lower genital tract pathologies. Biopsies were transported on ice and processed immediately. Tissue biopsies were divided into fragments. One fragment was used for histopathology analysis to corroborate colposcopy-directed diagnosis, tissue biopsies were fixed in formalin and embedded in paraffin, 4 µm sections were subsequently deparaffinized, rehydrated and stained with hematoxylin-eosin to be analyzed by experienced pathologists. The second fragment was used for DNA extraction. Cervicovaginal mucus was collected by washing the cervix with 1mL of sterile phosphate-buffered saline. Mucus samples were transported on ice and stored at -20°C until used.DNA was isolated from cervical eversion biopsies using the QIAamp DNA kit , according to the manufacturer´s instructions. The presence and genotyping of HPV was performed using the type-specific multiplex genotyping (E7-MPG) assay, which combines multiplex polymerase chain reaction and bead-based Luminex technology , at the International Agency for Research on Cancer (IARC), Lyon, France, as described elsewhere . The assA total of 63 samples were available for 16S sequencing. DNA was extracted from cervicovaginal mucus using the PureLink™ Microbiome DNA Purification Kit . Libraries were prepared as described in the MiSeq 16S rRNA gene Amplicon Sequencing protocol  with modifications. Primers for the V4 region were 515F (5´- GTGCCAGCMGCCGCGGTAA-3´and 806R (5´- GGACTACHVGGGTWTCTAAT-3´) . Brieflyvia q2‐phylogeny) after aligning all ASVs with mafft  . First, th mafft . Artifacth mafft . Before th mafft . Linear th mafft . Spearmag for 15 min at 4°C, supernatants were recovered and the total protein content was calculated using the DC Protein Assay . Samples were analyzed in a FACS ARIA III flow cytometer . Cytokines and chemokines concentrations were calculated using the LEGENDplex™ Data Analysis Software v 7.0 . Cytokine and chemokine expression levels were normalized to total protein concentration. The lower limit of detection (LLOD) value, provided by the manufacturer, was assigned to those samples with cytokine or chemokine concentrations equal or below the reported limit. To control for inter-individual variation due to differences in the amount of protein contained in the samples, the final concentration of analytes was reported as pg/mg of total protein.The following cytokines and chemokines were evaluated using bead-based multiplex immune-assays by flow cytometry according to the protocols of the manufacturer: Interleukin-1β (IL-1β), and Interleukin-12p70 (IL-12p70) (BD Cytometric Bead Array (CBA) Human Inflammatory Cytokines Kit. BD Biosciences, CA, USA); Interleukin-2 (IL-2), Interleukin-4 (IL-4), Interleukin-5 (IL-5), Interleukin-6 (IL-6), Interleukin-9 (IL-9), Interleukin-10 (IL-10), Interleukin-13 (IL-13), Interleukin-17A (IL-17A), Interleukin-17F (IL-17F), Interleukin-21 (IL-21), Interleukin-22 (IL-22), Interferon-γ (IFN-γ), and Tumor Necrosis Factor- α (TNF-α) (LEGENDplex™ Human Th Cytokine Panel (13-plex), BioLegend, CA, USA). MCP-1 (CCL2), RANTES (CCL5), IP-10 (CXCL10), Eotaxin (CCL11), TARC (CCL17), MIP-1α (CCL3), MIP-1β (CCL4), MIG (CXCL9), MIP-3α (CCL20), ENA-78 (CXCL5), GROα (CXCL1), I-TAC (CXCL11) and IL-8 (CXCL8) . Briefly, cervicovaginal mucus samples were thawed and centrifuged at 3000 x p values were adjusted for multiple comparisons using Dunn’s multiple comparisons test. When using this test, adjusted-p values were reported. Two-tailed p values  lesser than 0.05 were considered statistically significant.Data was analyzed using the GraphPad Prism 7 software . Differences in demographic and clinical factors between groups were analyzed using the Mann-Whitney test for continuous variables and Chi-square test for categorical values. Comparisons between two groups were performed using the Wilcoxon Rank Sum Test. Comparisons between three or more groups were performed using Kruskal-Wallis, p=0.0459, ®, MSD), again no difference was observed between HPV-neg (8/17) and HPV-pos (9/17) women. It is worth mentioning that 3 of the HPV-vaccinated women were positive for HPV-16, whereas 6 HPV-vaccinated women were infected with HPV types not included in the vaccine. Taken the latter in consideration, and the prevalence of HR-HPV types in our cohort, these women might be at increased risk for HPV-associated pathologies.A total of 156 samples from women with symptomatic cervical ectopy were available for HPV testing. A total of 19 HPV types were detected: HPV6, 11, 16, 18, 26, 31, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82. The prevalence and distribution of HPV genotypes is shown in Subsequent analyses were performed in a sub-cohort of 63 women including 35 HPV-pos and 28 HPV-neg women. This sub-cohort was selected based on sample availability  and MIG (56.89 pg/mg in HPV-pos vs 15.34 pg/mg in HPV-neg women) were significantly higher in HPV-pos women , which is suggestive of an anti-viral cellular immune response.Next, we explored the impact of HPV infection on mucosal inflammation as it is well documented that persistent HPV infection is associated with increased levels of local and systemic inflammatory cytokines . Sixteenp<0.001, p=0.019 for weighted UniFrac and p=0.013 for Bray-Curtis dissimilarity, p=0.014 and Bray-Curtis p=0.003). Multiple HPV infection had no impact on the vaginal microbiota (weighted UniFrac PERMANOVA p=0.149 and Bray-Curtis p=0.122). Results seem to indicate that differences in vaginal microbial composition between HPV-negative and HPV-positive women might be driven by single HPV infection, rather than multiple HPV infection. Therefore, next we restricted our analysis to the 3 predominant HR-HPV types found in our study. The prevalence of HPV16, HPV18 and HPV31 single infection was 8/35 (22.9%), 5/35 (14.3%) and 7/35 (20%), respectively.Our results suggest that women with symptomatic cervical ectopy suffer from a state of generalized mucosal inflammation which is not entirely explained by the presence of HPV. Strong evidence indicates that inflammatory responses in the female genital tract are modulated by the local microbiota . Thus, wp=0.059; Shannon, adjusted p=0.030) and HPV31-positive  compared to HPV-neg women    (p=0.114 and Bray-Curtis PERMANOVA p=0.061)   between Lactobacillus spp., bacterial-vaginosis associated anaerobes and vaginal richness and diversity, as would be expected (p<0.001) were found between alpha diversity and most anaerobes. No associations were found between inflammation-associated cytokines and chemokines and the vaginal microbiota.Finally, we explored associations between genital inflammation and vaginal microbiota  in the context of HPV infection. We restricted our analysis to the cytokines and chemokines who were determined in 85% or more samples  (p=0.0004) and IL-22  as shown in p<0.001) were found between Lactobacillus spp. and alpha diversity, and anaerobes like Atopobium, Clostridium, Megasphaera, Mycoplasma, Prevotella, and Sneathia. In contrast, no correlations were observed between richness, diversity and inflammation-associated cytokines and chemokines in HPV-neg women (p<0.001) were found between alpha diversity indices and most anaerobes in both HPV-pos and HPV-neg women, however fewer correlations were found compared to HPV-pos women.When stratifying for HPV infection, distinct correlations were found. In HPV-pos women, we found positive correlations between alpha diversity and IL-1β  persist.The presence of cervical ectopy has been associated with an increased risk of sexually transmitted infections . Here, wIn women with cervical ectopy, endocervical cells protruding onto the ectocervix create an additional mucosal barrier with a natural vulnerability to physical trauma that may lead to increased genital inflammation in sexually active women . AccordiHPV is one of the most common pathogens affecting the cervical canal. Cellular immune responses are decisive for the resolution of HPV infections . Here, wLactobacillus species and a low microbial diversity. We found that the vaginal microbiota of HPV-pos women was highly diverse and differed in community composition (beta diversity) with a decrease in Lactobacillus dominance compared to HPV-neg women. We also found a strong correlation between alpha diversity metrics and IL-1β and IL-22, both of which are produced at sites of inflammation. These results are in line with a previous study showing correlations between highly diverse bacterial communities and genital pro-inflammatory cytokines  is a critical factor for the natural replacement of ectopic tissue with metaplastic squamous epithelium. Thus, the loss or decrease in the relative abundance of Lactobacilli may delay the recovery of the external stratified epithelium in women with cervical ectopy, possibly increasing their risk of acquiring STDs, including HPV and HIV.The link between vaginal dysbiosis and HPV infection, persistence, premalignant lesions , and ultysbiosis . In our currence  and cervcurrence . Furthercurrence , and inccurrence . In contOur results should be interpreted with caution due to the limitations in statistical power inherent to small cohort sizes like ours. We also acknowledge that we did not adjust for confounding variables; when comparing clinical and demographic variables between these two groups, we found that the number of sexual partners was significantly higher in HPV-positive women as previously reported. All other variables were not significant, with the exception of age. There was a tendency for HPV-negative women to be older than HPV-positive women.We are aware that our study has important limitations; first, the inclusion of a control group of women without cervical ectopy was not possible, so we cannot compare our results with those of age-matched women without cervical ectopy. However, previous reports show that cytokines, chemokines and vaginal microbial community profiles are different in women with cervical ectopy compared with women with a normal ectocervix . Taking Lactobacillus dominance decreased in HPV-pos women, and bacterial vaginosis-associated anaerobes were increased. Correlations between inflammation-associated cytokines IL-1β and IL-22 and the vaginal microbiota were exclusively found in HPV-pos women. Taken together, our observations suggest that HR-HPV infection and vaginal dysbiosis coexist in women with symptomatic cervical ectopy. These women might be more susceptible to chronic inflammation and epithelial damage, and could benefit from a closer follow-up to improve their sexual and reproductive health. Our study provides a deeper understanding of the cervicovaginal microenvironment of women with symptomatic cervical ectopy, a benign gynecological condition.In conclusion, we showed the presence of oncogenic HPV types in a high proportion of women with symptomatic cervical ectopy. Notably, HPV was associated with increased levels of IL-21 and CXCL9 suggesting an active cellular immune response. We also found the co-expression of inflammation markers in these women irrespective of HPV status. We identified that changes in vaginal microbiota composition were associated with the presence of HPV. The raw 16S sequences have been deposited at the National Center for Biotechnology Information (NCBI)-Sequence Read Archive (SRA) under project PRJNA763215.The studies involving human participants were reviewed and approved by Local Ethics Committee of the Colposcopy Clinic of the “Cruz-Talonia” Foundation (Reference Number: 17/02/2017/LRZ/CE05). All women gave written informed consent to participate in this study.LR-Z conceived the project, designed experiments, coordinated study operation, supervised laboratory staff, analyzed and discussed data, and wrote the manuscript. SP-C conducted 16S rRNA gene sequencing analysis, designed figures, analyzed and discussed data, and wrote the manuscript. ML-F performed flow cytometry analysis, sequencing reactions, analyzed data, designed figures and wrote the manuscript. FC generated clinical data base, processed tissue samples, extracted and analyzed DNA, and designed tables. TG conducted the E7-MPG assay to determine HPV genotypes. OC and FC-T coordinated study operation ant the Colposcopy Clinic of the “Cruz-Talonia” Foundation, and obtained tissue samples. MC-T extracted DNA from cervicovaginal mucus, constructed bacterial libraries, and performed 16S rRNA sequencing. CA-G analyzed cases and contributed to diagnosis confirmation. IM-H performed flow cytometry for chemokine determination. JM and VC-M performed flow cytometry analysis of cytokines. CA participated in the selection of patients, collection of patient data and processing of patient samples. MT coordinated study operation at the International Agency for Research in Cancer, analyzed and discussed data. All authors contributed to the article and approved the submitted version.This work was supported by a grant from PAPIIT, UNAM (grant number IN204419). LR-Z received a fellowship from Programa de Apoyos para la Superación del Personal Académico de la UNAM (PASPA. Number 523.01/2299DFA/2017). Grants from CONACyT  were awarded to FC. SP-C was supported by funds from the Mexican Government .Where authors are identified as personnel of the International Agency for Research on Cancer/World Health Organization, the authors alone are responsible for the views expressed in this article and they do not necessarily represent the decisions, policy or views of the International Agency for Research on Cancer/World Health Organization.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To date, incidence of HPV-related oral and oropharyngeal carcinoma is increased;Spread of HPV infection among youth is due to sexual precocity and promiscuity;Two HPV strains are primarily related to cancer onset: 16 and 18;There are different HPV vaccines but knowledge and adherence are still quite low;Not only young women must be aware on risk factors and vaccine.Human papillomavirus is a sexually transmitted virus that is responsible not only for uterine cervical cancer, but also for the oral and oropharyngeal cancers. In this latter case, the virus indiscriminately affects both sexes at any age. Vaccination plays a key role in preventing infection and its possible consequences. Therefore, this study aimed to assess the degree of awareness of papillomavirus infection and its possible prevention in the Sicilian population. A prospective observational study was carried out on the Sicilian population through a self-administered questionnaire, consisting of 10 multiple-choice questions. The survey lasted from June 2021 to December 2021. A total of 844 respondents were included: 60.90% were female and 53.55% were aged between 21 and 30 years. Most of the surveyed population knew about the virus and the existence of the vaccine, however, many of them did not know about virus’s effects on the oral and oropharyngeal sites (49.17%), or about the indication for vaccination for males (39.69%). Oral and oropharyngeal papillomavirus-related cancers are an increasingly frequent finding, especially in young adults. Therefore, it is necessary and quite mandatory to educate the population about the risks that certain voluptuous habits may cause, with the help of general practitioners, schools, social media, and social networks. Human papillomavirus (HPV) is a sexually transmitted virus and is the leading cause of uterine cervical cancer . In receTherefore, considering the widespread infection and the poor promotion and adherence to vaccination, this study aimed, through a questionnaire, to evaluate the level of knowledge of HPV infection, its implications, and its possible prevention in the Sicilian population.A prospective observational study was carried out on the Sicilian population through a self-administered questionnaire. The survey lasted from June 2021 to December 2021. The study was approved by Ethics Committee Palermo 1 of University Hospital Paolo Giaccone of Palermo (protocol code 04/2021—28 April 2021). Inclusion criteria were: (1) males and females, (2) 18–80 years old, (3) with consent to participation, and (4) having Italian proficiency. Exclusion criteria were: (1) under 18 years old; (2) not-resident in Sicily; and (3) participants who answered only the first 3 questions. Those who partially answered the questionnaire were also included in the study.https://it.surveymonkey.com/r/5W33F5H (accessed on 1 June 2021) shared through the most famous social networks  and messaging application (Whatsapp Messenger). The survey was shared among the author’s social media contacts and with the help of Sicilian social media influencers in order to spread the questionnaire to as many and as heterogeneous people as possible. Participation in the survey was voluntary and completely anonymous. To avoid bias derived from multiple answers from the same participant, the survey site prevented the possibility of answering the questionnaire several times from the same device . The order of the questions was always the same and it was not possible to skip some of them: failure to answer a question prevented the continuation of the survey.Our questionnaire consists of 10 multiple-choice questions, written in Italian to avoid the risk of language bias. The questionnaire was designed by the authors based on current literature and similar valid questionnaires ,18,19,20Are you vaccinated against papillomavirus? investigated the adherence and/or willingness to receive the vaccination against the Papillomavirus.The first 3 questions were personal and aimed to categorize the sample on the basis of: #1 gender, #2 age, and #3 own province. Items #4, #5, #6, and #7 investigated the level of knowledge of papillomavirus in terms of risk of developing uterine and oral/oropharyngeal cancer, both in the male and female population. Finally, questions #8, #9, and #10 focused on the papillomavirus vaccine. In particular, item #10 p-value < 0.05 was considered significant.The answers were collected on Excel spreadsheets, allowing the analysis of the results presented as numbers and percentages. The collected data were also represented in graphs for immediate and easy access to the results. Moreover, performing binary logistic regression, odd ratios were used to calculate the likelihood of knowledge and awareness about HPV infections and its vaccine in relation to sex . A According to data collected in 2020 by the National Institute of Statistics (ISTAT), Sicily has 4,833,705 residents. About 60% of the Sicilian population lives in Palermo, Catania, or Messina. Females represent 51.5% of population and the average age is 44.2 years. In regard to educational level, 5.7% have only a secondary education, 31.8% have a high school diploma, and 12.8% have a university degree .A total of 909 people participated in the survey. Based on the exclusion criteria, 65 individuals were excluded. So, a total of 844 individuals were included, and of these, 811 (96.09%) completed the questionnaire. Furthermore, 60.90% of respondents were female and 53.55% were aged between 21 and 30 years. Among female respondents, 66.37% belonged to the 21–30 age group. All the provinces of Sicily were represented in a rather homogeneous way by the sample examined: Agrigento (11.37%), Caltanissetta (10.78%), Catania (10.90%), Enna (9.95%), Messina (9.84%), Palermo (11.85%), Ragusa (10.19%), Syracuse (12.80%), and Trapani (12.32%).The survey found that most of the surveyed population knew about papillomavirus infection (96.33%): in particular, they were female (92.50%), aged between 21 and 30 years (54.61%) , and fro194 respondents replied that HPV can only affect females (item #7); 123 were young (between the age of 21 and 40 years old) and 179 were female. However, most of the examined population (634/844) answered this question correctly . FurtherMost (52.70%) of the respondents who replied that they did not want to vaccinate were male with two age peaks: 21–30 years (26.35%) and 51–60 years (26.35%) . Overallp-value 0.1020) and #7 . Results are statistically significant in the case of question #5 , #6  and #8   .Human papillomaviruses can be subdivided into low-risk (LR-HPV) and high-risk (HR-HPV) types. The former is associated with benign lesions, such as papilloma and warts, the latter causes malignant lesions, first of all cervical cancer . PapilloTo date, HPV is known in the general population for its role in the onset of cervical cancer , as alsoDo you know that it can cause cancer of the oral cavity? in which there was a homogeneous distribution of the population between affirmative (49.29%) and negative (49.17%) answers. This result is consistent with the study by Barnard et al. that reported a total of 57.5% correct answers [Interesting is the result obtained for question #6  answers . Additio answers .Analyzing the results, females were, overall, more informed about HPV carcinoma both at the uterine (62.78%) and oral (64.42%) sites, in accordance with the literature . In 2019Today, the detection of this virus in the oral cavity and oropharynx is very frequent, with a prevalence ranging between 40 and 90% according to studies ,28. In pTo date, there are several types of vaccines: (1) two-valent vaccine, targeting HPV types 16 and 18; (2) four-valent vaccine, targeting HPV types 6, 11, 16, and 18; and (3) nine-valent vaccine, targeting also HPV types 31, 33, 45, 52, and 58. If administered prior to HPV exposure, the two-valent vaccine reduces onset of cervical precancerous lesions and oral infection by HPV types 16 and 18 in about 90% of cases. In the same way, quadrivalent vaccine prevents HPV infections and cervical and oral precancerous lesions caused by HPV 6, 11, 16, or 18 in around 90% of cases. Nine-valent vaccine is more efficient and is shown to significantly reduce the risk of developing cervical cancer (risk reduced to about 5% if administered before HPV exposure) and other HPV-related premalignant and malignant lesions, such as penile, anal, vulvar, vaginal, and oropharyngeal ones (risk reduced by about 80%) ,33,34. IThe most alarming data, however, is that 19.79% of respondents said they did not want to get vaccinated and 52.70% were male, confirming the common opinion that HPV is responsible only for cervical cancer which, therefore, does not affect men. In fact, 22.98% of respondents replied that HPV affects females at item #7. Data are consistent with current literature .These answers, therefore, highlight the lack of knowledge of a growing widespread and dangerous infection among young people and, above all, do not consider the epidemiological data now established: HPV-related OPC affects men more often ,36. AnotTherefore, from the analysis of the results obtained, this study shows incomplete knowledge of HPV: on the one hand, in fact, most of the assessed population proved to know the virus, the HPV-related cervical cancer, and the existence of a vaccine; on the other hand, however, a significant portion of the respondents ignored the effects of the virus at the oral and oropharyngeal sites and the indication for vaccination also for males.However, the study has some limitations: (1) although the internet and social networks are now widely used, there is a poorer and illiterate part of the population that the study could not include and assess; (2) all provinces of Sicily were sufficiently represented in the sample examined, even though the percentage of responses was not correlated with the actual population of each province; (3) in item #6, the expression “oral cavity” also included oropharyngeal structures to facilitate the understanding of the question to the general population; and (4) results are based on self-reported data because we cannot prove in any way the truthfulness of the answers. However, to reduce or avoid the risk of social desirability bias in answering, the survey was anonymous.Oral and oropharyngeal HPV-related cancers are a growing frequent finding, especially in young adults. A prevention campaign, both primary and secondary, is therefore essential and necessary, addressed not only to women in certain age groups, but also to the entire young-adult population. Our study revealed an incomplete and “misleading” knowledge about papillomavirus, HPV infection, and HPV vaccination, which implies the need to educate the population about the risks that certain voluptuous habits can provoke, with the help of general practitioners, schools, social media, and social networks. In conclusion, the main message to take home is: better safe than sorry!


The second TransatlanticEarly Career Investigator (ECI) G Protein-CoupledReceptor (GPCR) Symposium was an online scientific meeting gearedat young GPCR investigators, with the primary goal of expanding opportunitiesfor sharing research and networking among trainees in North Americaand Europe. Here, we discuss the format of our meeting, its impact,and the challenges and opportunities facing meetings like it in thefuture. Following the success of the first editionof this meeting,2 our goals as the newco-organizers for the second edition were (1) to maintain or expandthe reach to a wide audience; (2) to improve on the format of themeeting; and (3) to expand opportunities for ECIs to present theirwork. Here we discuss the general structure of our meeting, its impact,and our views on the challenges and opportunities for similar meetings.With in-person scientific meetingsbeing canceled due to the COVID-19 global pandemic, virtual conferencesserved as a crucial means for presenting research and networking.This is particularly important for early career investigators (ECIs),for whom building their scientific networks is necessary for careeradvancement. In 2021, with support from The European Research Networkon Signal Transduction (ERNEST),https://www.wonder.me/)where attendees could meet outside of the sessions to discuss collaborations,job openings, or anything else they were interested in. Our scientificprogram featured two keynote speakers, Miriam Stoeber (Universityof Geneva) and David Olson , bothof whom are young rising stars in studying GPCRs in neuroscience,as well as five oral presentation sessions  and twoposter sessions for ECIs to present their research. Notably, by having15 oral presentation slots, this was a 50% increase over 2021.The first edition of the meetingwas a 10-hour one-day symposium. However, since time zones representedin a virtual meeting spanning North America and Europe range fromPacific Daylight Time (UTC–07:00) to Eastern European DaylightTime (UTC+03:00), the one-day meeting format posed challenges withscheduling, particularly if we wanted to expand it. To overcome this,we structured the second meeting as two 6-hour half-days, each witha career development element, talks, poster presentations, and networkingopportunities. In doing so we achieved our goal of an expanded agendawithout exacerbating the logistical challenges with timing. For careerdevelopment/networking, our meeting included two informal“breakfast/lunch/dinner sessions” withfaculty members—one with established “senior”faculty members and one with “junior” faculty membersor those imminently launching their independent groups. We also includeda career panel featuring six researchers who could speak to GPCR researchopportunities in academia, industry, and biotech. Regarding the latter,we chose to focus our panelists on the discussion of research careersin different settings rather than alternative careers, given timeconstraints. Lastly, we provided an open networking space via Wonder, able tohandle the anticipated number of participants, easy to access andnavigate between sessions, easy to use without further preparation,and ideally without the need to create an account.There isa common misconception that organizing conferences online comes withlittle or no cost due to the lack of physical event space, meals,etc. While they are cheaper than their in-person counterparts, platformfees, website costs, technical support staff, and other expenses alladd up. It is important for sponsors and funders to know that forthese meetings to be successful and run smoothly a financial investmentis necessary.There is something to be said aboutconversations that develop organically at an in-person meeting, whichhas been challenging to replicate in online settings. Given that attendeesdo not see one another at a virtual meeting, there is often a senseof reduced interactivity, as was also mentioned by participants inthe exit survey. Further, in our experience, networking sessions areunfortunately often perceived as “skippable” duringa meeting, especially if the timing is inconvenient or if insufficienttime is allocated to breaks. Improvements to the attendee experience,by enhancing their sense of connection as well as giving prioritytime to networking opportunities, will be critical for online meetings,especially those focused around ECIs. While improvements in technology,such as use of environmental exploration platforms ,have helped make communication easy, there remains more to be doneto ensure that conversations occur more naturally and result in lasting interpersonal connections and that attendeesare able to interact more similarly to how they would in-person meetings.Challenges that in-person conferences do not contend with are timezones and people’s limited tolerance for screen time. Unfortunately,these must be carefully considered when hundreds convene online acrosstime zones spanning as much as a 10-hour difference in the most extremecase. This means that it is difficult to actively include an audiencefrom all parts of the world, and even with careful considerationssome attendees may find themselves attending sessions at somewhatinconvenient times. Furthermore, the time that can be dedicated tosessions during a virtual symposium is limited also by the fact thatmost people have a limited capacity for engaging with a computer screen.To make it easier for people to stay focused in front of their screen,it is helpful to have interactive sessions and breaks between talk-focusedscientific sessions.Virtualmeetings have a very low barrier for entry and almost no limit tothe number of participants. While scalability is also a strength ofonline meetings, this results in the registration of many people whowill not actually participate in all sessions or who may choose toattend only a subset of sessions. According to the exit survey, only8% of respondents attended all sessions. The most common reasons fornot attending a session cited by survey respondents were a prior commitment(54%), inconvenient timing (46%), or needed a break (31%). Importantly,only 14% indicated a lack of scientific interest was their reasonfor not attending a session. In this way, an online meeting is a double-edgedsword: it is easy to squeeze into your normal work schedule withouttraveling or having to remove yourself from ongoing work in the lab;however, this also means that people often do not dedicate their fullattention to a virtual meeting as they would to an in-person meeting.It is worth considering why we value a meeting we travel to more highlythan one we can access from our computer, and whether this can beadjusted if online meetings remain ubiquitous in our lives.either an in-person meeting or an onlinemeeting, they will choose the in-person meeting more often. However,we emphasize that the community must not a priori regard an onlinemeeting as “lesser” than an in-person meeting. We speculatethat one of the causes for the reduced participation from many wealthyNorth American and European countries is the resumption of in-personmeetings and people not seeing meetings like this one as a prioritywhen they have access to in-person meetings. Yet, for many the constraintsof traveling to an in-person meeting, whether it be cost, illness,childcare, or family obligations, make online meetings an importantdoor to networking and scientific discourse for these individuals.We encourage the reader to choose an online meeting to prioritizesimilarly as an in-person meeting and to take full advantage of allaspects of the meeting, including any networking sessions. This willensure that meetings like this one remain high-quality scientificforums where those who may not have the privilege to attend an in-personmeeting can also maximally benefit.From our own personal experiences, as well as anecdotesfrom others, it seems to be the case that if someone has a choiceto attend Having discussed some ofthe challenges meetings such as this one face, we want to end by discussingsome strengths of online meetings, and their position in the future.Indeed, 97% of survey respondents think that virtual meetings suchas this one will have value even after in-person meetings are back.Virtual meetings create an equality that in-person meetings fail toachieve, especially if the online meeting is free of charge. Thisis especially helpful for ECIs and researchers from laboratories withtighter budgets who cannot afford the cost of attending an in-personinternational meeting. Further, online meetings do not suffer fromborders: there are no visa/immigration issues. Finally, virtualmeetings are a more environmentally conscious solution to connectscientists from around world.Overall,we believe that the second TransatlanticECI GPCR Symposium was an extremely successful follow-up to the firstedition of this meeting, with 99% of survey respondents indicatingthat they would like to see the symposium happen again. Still, givenour experience and the feedback we received informally and throughour exit survey, we believe this meeting can still be improved upon.The extension from a 1-day to a 2-day meeting made it possible toexpand both the scientific and career development agendas while alsohaving shorter days and more breaks. We are excited to share thatthe Transatlantic ECI GPCR Symposium is scheduled for a third editionin summer 2023 and will be co-chaired by Franziska M. Heydenreich(MRC Laboratory of Molecular Biology), Gabriele Kockelkoren (Universityof Copenhagen), Aditya Kumar (University of Michigan), and SreeparnaMajumdar (University of North Carolina Chapel Hill), and we look forwardto seeing how the meeting evolves under their direction. Our experienceorganizing this meeting showed us the value of bringing together adiverse set of ECIs who may not otherwise have the same opportunitiesto network and present their research, and we hope that it and meetingslike it will continue to be supported by their respective researchcommunities and fill a niche distinct from the many traditional in-personmeetings.


Apis dorsata (Giant Asian Honeybee) venom is the commonest insect allergy in Sri Lanka and South East Asia. However, laboratory diagnosis is difficult as the pure venom and diagnostic reagents are not commercially available.Allergy to A. mellifera venom and the passive basophil activation test in the diagnosis of A. dorsata venom anaphylaxis.This study assessed the use of four recombinant allergens of A. mellifera, rApi m 1, 2, 5 and 10 were assessed and compared with serum IgE to the crude venom of A. mellifera or V. vulgaris by Phadia ImmunoCAP, in patients who developed anaphylaxis to A. dorsata stings. Basophil activation in response to venom of A. dorsata or V. affinis was assessed using a passive basophil activation test. Association of the severity of the reaction with basophil activation was compared.Serum IgE levels to four recombinant allergens of A. mellifera (Western honeybee) and a higher positivity rate of 90% (27/30). Whereas, IgE reactivity to rApi m 2 or 5 had significant correlation (p = 0.02 and p = 0.005 respectively) with V. vulgaris crude venom. All 30 (100%) were positive to A. dorsata venom in passive BAT; 70% (21/30) had over 80% activation, 96.7% (29/30) had over 60% activation and 100% had over 50% activation. Percentage activation of basophils in patients who had mild or moderate reactions (n = 20) was significantly low (p = 0.02) from that of patients who had severe reactions (n = 10).rApi m 1 and 10 combinedly had significant correlation  with the crude venom of A. dorsata allergy. This combination had the lowest cross-reactivity rate with Vespula vulgaris. The passive BAT is highly sensitive in A. dorsata allergy. The basophil reactivity was significantly higher in severe anaphylaxis compared to mild/moderate anaphylaxis. This finding should be further explored in further studies.rApi m 1 and 10 when combined was sensitive for the diagnosis of  Apis dorsata (Giant Asian Honeybee) is restrained to a small geographical area (South East Asian region) compared to the Western honeybee, A. mellifera [A. dorsata venom is the commonest venom allergy in Sri Lanka and South East Asia [A. dorsata and Vespa ssp) venom allergic patients from September 2011 to August 2013 [A. dorsata stings. An epidemiological study [A. dorsata and V. affinis) sting allergies. Of those, 92% were from Primary Care Hospitals (PCHs) and only 8% were from Tertiary Care Hospitals (TCHs). The study also identified underreporting of symptoms and adverse reactions, mainly systemic reactions, in Hymenoptera sting allergy [A. dorsata and Vespa ssp) was 4.6% (15/322) in the study from Deniyaya, Sri Lanka [A. dorsata venom allergy are reported in Sri Lanka [A. dorsata venom are common, diagnostic grade venom preparations of A. dorsata are not commercially available. Our previous studies [A. dorsata with that of A. mellifera, and the possible cross-reactivity of phospholipase A2 (PLA2) and hyaluronidase of A. dorsata and A. mellifera venom. This cross-reactivity indicated the possibility of using A. mellifera venom/components in the diagnosis of A. dorsata venom allergy until the diagnostic grade venom preparations of A. dorsata are commercially available.ellifera , 2. Alleast Asia –5. A stual study  in the N allergy . Therefori Lanka , Pakistari Lanka  and Nepa2 (rApi m 1) was the first allergen of the Western Honeybee used in CRD. However, due to the low sensitivity (ranging from 58 to 80%) [Spodoptera frugiperda (Sf9) insect cell-based expression system [Venom specific IgE (sIgE) detection using in vivo  or in vitro (i.e. Phadia ImmunoCAP) tests are widely used in the diagnosis of Hymenoptera insect venom allergies –16. Howe to 80%) –25 assocn system . These an system . Howevern system .A. dorsata venom allergy as basophils have surface markers that are upregulated following cross-linking of surface IgE [V. affinis (a hornet species which is native to Sri Lanka) [A. dorsata venom allergic patients. With adequate activation, the passive BAT could be considered as a useful diagnostic test for A. dorsata venom allergy.The conventional basophil activation test (BAT) may be useful in the diagnosis of face IgE . The patface IgE . This isface IgE , 29. We i Lanka) . HoweverA. mellifera in the diagnosis of A. dorsata venom allergy. In addition, we assessed the possibility of using membrane-bound IgE depleted healthy donor basophils. These basophils were then tested for activation following the addition of serum from patients who developed anaphylaxis to A. dorsata, a passive BAT. The analyses were prospective and blinded. If a higher percentage of activation could be determined compared to the Phadia ImmunoCAP, then the passive BAT could be useful in the diagnosis of A. dorsata venom allergy, and potentially, allergy in other instances as well.In the present study, we used four commercially available recombinant allergens of Ethics approval for this study was obtained from the Ethics Review Committee, Medical Research Institute, Colombo, Sri Lanka (No: 46/2013).A. dorsata venom anaphylaxis patients  from Deniyaya Base Hospital (n = 13) and Bandarawela District Hospital (n = 17), Sri Lanka. Serum from each patient was collected after obtained written consent from each patient. All patients were shown a panel of dead specimens of various Hymenoptera stinging insects to confirm the insect responsible for stinging. Patients who identified the responsible insect as A. dorsata and who had no previous allergies to other Hymenoptera insects were selected. Through a questionnaire, relevant clinical history  and ImmunoCAP i3 containing V. vulgaris crude venom. Phadia ImmunoCAP of rApi m 1, 2, 5 and 10 were purchased and IgE reactivity was detected using the Phadia 100 system. The cutoff was set as > 0.35 kUA/L for sIgE positivity. The patients who had positive IgE to both A. mellifera and V. vulgaris crude venom were considered as double-positives. The samples which had double-positivity were pooled into three categories according to the IgE positivity to V. vulgaris (wasp) venom; Category 1—low IgE (0.35–3.4 KUA/ L), Category 2—moderate IgE (3.5–17.4 KUA/ L) and Category 3—high IgE (17.5–50 KUA/ L). IgE quantity to A. mellifera venom and its components in two severity groups were compared.Venom specific IgE reactivity was detected using ImmunoCAP i1 containing crude venom of g for 5 min) at 4 °C. The supernatant was discarded and the pellet was resuspended in 100 µL of 0.13 M PBS. Afterward, the samples were incubated with 50 µL of serum of either A. dorsata venom allergic patients or patients having different allergies (IgE control sera) for 60 min at 25 °C.Passively immune basophils were prepared according to a method developed and used by us  and otheA. dorsata venom was used in this study. Following incubation, 1 µg/mL of A. dorsata venom was added to each tube comprising serum and was incubated for 20 min at 37 °C. Activated basophils were stained for 20 min using a 20 µL mixture of anti-IgE-PE and anti-CD63-FITC provided with the kit on an ice bath.The passive BAT was carried out using the sera from 30 patients selected. Two positive controls were used; polyclonal anti-IgE (Sigma Aldrich) at 1:100 dilution and fMLP provided with the kit (100 µL of 0.002 mM/ mL). Two sets of negative controls were used in the test, i.e., (i) donor basophil samples (without replacing IgE) were incubated with 100 µL of the wash buffer and (ii) IgE depleted donor basophils were incubated with 100 µL of pooled control sera from 7 patients who were not allergic to hymenopteran insects. Gunasekara et al. , 33–35 e2) was then calculated from the gated population. The cutoff point in passive BAT was set as mean of negative controls + 3.3 SD [A. dorsata venom in the two severity groups was compared. Three categories of double-positives (by Phadia ImmunoCAP) were tested using the passive BAT.Lymphocyte/basophil fraction was selected by gating side scatter vs forward scatter. Basophils were selected using the side scatter low-IgE high cell population. The tests were analyzed acquiring at least 200 basophils using FACSCalibur flow cytometer (BD Bioscience) . The per+ 3.3 SD , 36. In A, dorsata venom (1 µg/mL) and percentage CD63 activation were determined.Basophils from a second donor were used to determine the donor dependency of the test. Two tests were repeated using those basophils; sera of two patients (patient# 5 and 10) were incubated with the IgE depleted donor basophils. These were then incubated with A. mellifera (or venom components) with the crude venom of V. vulgaris were determined using Spearman correlation. IgE positivity to crude venom of A. dorsata in the two severity groups in the passive BAT: group 1 (mild + moderate) and group 2 (severe) was compared using Mann–Whitney U test. Values of P < 0.05 at 95% confidence level were considered significant.SPSS package  and Prism 8.0  were used for the statistical analyses. Non parametric tests were used since the data was not normally distributed. Possible correlations in IgE positivity to A. dorsata venom, 21 (70%), 25 (83%), 27 (90%) and 23 (76.6%) reacted to rApi m 1, rApi m 2, rApi m 5 and to rApi m 10 respectively, whereas 25 (83%) had positive sIgE to the crude venom of A. mellifera and 20 (66.3%) had positive sIgE to the crude venom of V. vulgaris venom  and higher positivity rate of 90% (27/ 30). IgE reactivity to rApi m 2 or 5 had significant correlations of r = 0.414; p = 0.02 and r = 0.502; p = 0.005 respectively with V. vulgaris crude venom and rApi m 1 and 10 combine had lower correlation with the wasp venom  had very low (< 10%) basophil activation. The cutoff was set as 13% activation of basophils (mean + 3.3SD of negative controls). None of the double-positives in Phadia ImmunoCAP gave positive results with V. affinis venom in passive BAT. On repeating the test with a second donor, similar results were obtained as the first donor  is shown in Fig. nor Fig. .Fig. 3UpA. dorsata venom allergy were positive to A. dorsata venom in the passive BAT; 100% had over 50% activation, 96. 7% (29/ 30) had over 60% activation and 70% (21/ 30) had over 80% activation. Percentage activation of basophils in group 2 (n = 20: patients who had severe reactions) was significantly higher  patients with anaphylaxis to A. mellifera venom in diagnosis of A. dorsata venom allergic patients. We found a high rate of positivity to a combination of rApi m 1 and 10, compared to a lower rate of IgE positivity with rApi m 1 and 10 when done individually. A study from Germany and Sweden [A. mellifera. The use of single components in the diagnosis of allergy to A. mellifera or A. dorsata showed lower positivity in previous studies as well. For example, the sensitivity was 72.2% and 61.8% for rApi m 1 or rApi m 10 respectively in A. mellifera allergy [A. dorsata in Thailand [A. dorsata venom allergy. There was a significant correlation of a combination of rApi m 1 and 10 with A. mellifera crude venom in our study, whereas there was no such correlation noted with V. vulgaris venom. rApi m 10 is clinically relevant but underrepresented in diagnostic venom extracts of A. mellifera [A. dorsata venom allergy and could distinguish A. dorsata venom allergy from V. affinis venom allergy.This study was the first that evaluated the use of a panel of recombinant allergens of d Sweden  showed a allergy  and 44% ellifera , 38. On V. vulgaris crude venom. Several previous reports from the West have also demonstrated the cross-reactivity of rApi m 2 and 5 with wasp venom. The cross-reactivity of rApi m 2 and 5 of A. mellifera and V. vulgaris may be due to the structural homology of these components [A. dorsata venom and V. affinis/ V. vulgaris venom may be the reason for all 20 patients who had positive IgE to V. vulgaris venom having IgE to rApi m 2 or 5. This indicates that the use of these two components to diagnose A. dorsata venom allergy has limited specificity and hence may impede the specific diagnosis of A. dorsata and V. affinis venom allergies.A high IgE positivity rate with rApi m 2  and rApi m 5 (dipeptidyl peptidase IV) was seen in our study; however, we found significant correlations of rApi m 2 or 5 with mponents . We beliThe reactivity of donor basophils in patients with severe anaphylaxis compared to those with mild/moderate anaphylaxis was significantly high. As far as we are aware, this finding has not been evaluated with the conventional BAT for venom allergies and should be explored in further studies. Few studies on food allergies, particularly on peanut allergy , 41 haveIn contrast to the results from Phadia ImmunoCAP, the passive BAT showed a sensitivity of 100%. Reported sensitivity and specificity, in patients who had systemic allergic reactions (Muller grade II-IV), was 60–80% , 42, in A. dorsata [V. vulgaris by Phadia ImmunoCAP. However, the passive BAT showed a low activation of basophils for hornet venom. This indicates that it is useful in differentiating single positivity from false double positivity which is an important issue in the diagnosis of insect allergy.In vitro testing by the Phadia ImmunoCAP indicated double-positivity with both bee and wasp venom in patients who reacted only to  dorsata . This ma dorsata . All ourEven though venom immunotherapy (VIT) is the only possible curative treatment for venom allergy , to dateThe conventional BAT needs to be done within 4 h of venipuncture, whereas the passive BAT is less time-limited, as the patient’s serum rather than basophils are used. The passive BAT is more suitable than the conventional method, especially in Asia, where more stings occur in rural areas.A dorsata allergy. The passive BAT is highly sensitive in A. dorsata allergy diagnosis. Therefore, the diagnosis of A. dorsata venom allergy should be done initially with a combination of recombinant rApi m 1 and 10 and in instances of inconclusive results or double-positivity, the passive BAT should be included. The basophil reactivity in severe anaphylaxis compared to mild/moderate anaphylaxis was significantly higher. This finding should be explored in further studies.A combination of rApi m 1 and 10 is sensitive in the diagnosis of


We also review the evidence on the crosstalk between chronic HPV infection and PGE2 signaling, leading to immune response weakening and cervical cancer development. Finally, the last section updates the current therapeutic and preventive options targeting PGE2-derived inflammation and HPV infection in cervical cancer. These treatments include nonsteroidal anti-inflammatory drugs, prophylactic and therapeutical vaccines, immunomodulators, antivirals, and nanotechnology. Inflammatory signaling pathways are closely related to the carcinogenic nature of the virus, highlighting inflammation as a co-factor for HPV-dependent carcinogenesis. Therefore, blocking inflammatory signaling pathways, modulating immune response against HPV, and targeting the virus represent excellent options for anti-tumoral therapies in cervical cancer.Chronic infection by high-risk human papillomaviruses (HPV) and chronic inflammation are factors associated with the onset and progression of several neoplasias, including cervical cancer. Oncogenic proteins E5, E6, and E7 from HPV are the main drivers of cervical carcinogenesis. In the present article, we review the general mechanisms of HPV-driven cervical carcinogenesis, as well as the involvement of cyclooxygenase-2 (COX-2)/prostaglandin E Cancer encompasses a group of complex diseases characterized by uncontrolled growth, evasion of anti-growth signaling, resistance to cell death, and colonization of distant niches by malignant cells. Cancer develops by a progressive multistep process from normal diploid cells, which are spontaneously or carcinogen-transformed to cancer cells. These initial cancer cells without a distinctive phenotype progressively evolve towards a malignant phenotype by the driving force of DNA alterations or genetic instability . Several2 (PGE2) is a lipid mediator of inflammation, which is one of the hallmarks of cancer [2 bioavailability and signaling are highly regulated processes, and disruption of this regulatory axis is related to various human cancers, including cervical cancer [2 signaling in cancer progression. Crosstalk between HPV infection and PGE2 is also addressed in this review. Finally, therapeutic options in cervical cancer based on HPV and PGE2 signaling are presented.On the other hand, prostaglandin Ef cancer . Chronicf cancer . PGE2 bil cancer . This reCervical cancer is by far the most clinically relevant HPV-related disease. In 2020, more than 600,000 women were diagnosed with cervical cancer worldwide, and about 342,000 women died. Accordingly, cervical cancer is the fourth most prevalent cancer among women globally, ranking after breast, colorectal, and lung cancer . AlthougHPVs are DNA viruses that probably represent the most prevalent sexually transmitted infection in both women and men worldwide. Based on their DNA sequence, more than 200 types of HPVs have been described; however, only a few HPV types are associated with health problems. HPVs are considered cutaneous or mucosal types according to their ability to infect epithelial cells of the skin or the inner lining of tissues. The last group of HPVs is important for cancer biology because they infect the lining of the mouth, respiratory tract, and anogenital epithelium. At least 20 HPVs are associated with lesions of the anogenital tract, which are further categorized as low-risk HPV  and high-risk HPVs , which are associated with premalignant squamous lesions that are precursors of cancer [HPVs are small non-enveloped double-stranded circular DNA viruses, in which only one of the DNA strands is transcribed. HPV DNA comprises approximately 8 kb organized into three functional regions: the early region encoding the non-structural proteins E1, E2, E4, E5, E6, and E7 that regulate viral gene expression and some of them, such as E1 and E2, also participate in DNA replication; the late region that encodes the structural proteins L1 and L2, needed for the viral capsid, and a long control region (LCR) that contains the regulatory sequences required for replication and transcription, namely, the origin of viral replication and the early promoter [Although HPV replication requires E1 and E2 proteins, the participation of host proteins in this process are also required. HPVs infect only human epithelial cells, mainly keratinocytes. Cells located in the basal layer of the mucosa are the only cell type with the ability to divide and proliferate. This reservoir of basal cells is replenished by symmetric cell division of basal cells. However, asymmetric division of these cells generates two cells, one of them is used to renovate the basal cell population while the other one leaves the lower layer. Basal cells selected for terminal differentiation exit the cell cycle and stop DNA replication after they move out from the basal layer to the suprabasal layers. During this upward journey, cells acquire specialized properties and die when they arrive at the skin surface .The HPV life cycle is closely linked with this renewal process occurring in the stratified squamous epithelium of the cervix. Because HPV infection is sexually transmitted, the viruses present in the anogenital region of HPV-infected men  are introduced in the inner female genital tract during sexual intercourse. HPVs gain access to the deepest layer via microabrasions or via hair follicles, finally reaching the single layer of epithelial cells located in the squamocolumnar junction between the endocervix and ectocervix. There, basal cells are infected by HPVs through the interaction of L1 capsid protein with heparan sulfate-containing proteoglycans which are thought to be the main HPV receptor . In addiIn the initial phase of HPV replication, the viral genome in the form of episomes is maintained at a low copy number in infected basal cells . HPV DNAWhen HPV-infected basal cells move towards the epithelium surface, high levels of the HPV genome are synthesized, leading to the generation of progeny virions, which are sloughed from infected epithelia in the form of virion-laden squames. However, the HPV genome is often integrated into the host DNA in premalignant and malignant lesions of the cervix. HPV DNA integration represents a dead-end for the viral life cycle because infectious particles are no longer produced .How does HPV maintain active DNA replication in arrested cell-cycle cells? HPVs have evolved successful strategies to actively replicate in infected growth-arrested host cells that are efficiently evading apoptotic signals. Among them, early proteins E5, E6, and E7 combined function are critical drivers of persistent viral infection and cellular transformation .Unlike HPV E6 and E7 oncoproteins, little is known of the role of the E5 in the malignant transformation of the cervical epithelium. E5 is a multifunctional small hydrophobic polypeptide that is encoded by LR- and HR-HPVs and participates in key carcinogenesis points. Both HR-HPV E6 and E7 are small proteins that cooperate together to keep HPV DNA replication and prevent apoptosis while inducing genome instability and immortalization of HPV-infected cells [Transforming properties of HR-HPV E6 are closely linked to P53 activity, a tumor suppressor gene mutated in nearly half of human cancers . P53 supNormal cells maintain very low levels of P53 protein through proteasomal degradation mainly by the action of E3 ligase murine double minute 2 (MDM2)  Figure a. HoweveGiven the potential association of types 5 and 8 HPVs with non-melanoma skin cancer , it seemHPV E6 is also required to extend the life span indefinitely in infected keratinocytes. Immortalization properties of E6 are achieved by a range of cellular proteins. Both abrogation of the retinoblastoma protein (pRB) pathway and overexpression of telomerase reverse transcriptase (TERT) are the drivers for the immortalization of normal keratinocytes . It was This mechanism and those summarized in Thus, early proteins E5, E6, and E7 encoded by HPVs play a pivotal role in the HPV life cycle and are required for the malignant transformation of the cervical epithelium by increasing host genomic instability and by blocking pivotal cell cycle checkpoints . Other H2 exerts diverse physiologic and pathologic effects [2 evokes important actions in bone metabolism [PGE effects ,78, regu effects , renal [ effects , cardiov effects , gastroi effects ,83, resp effects , and rep effects ,86. Additabolism , hematoptabolism , and is tabolism ,90,91,922)  D2, E2, F2α, and I2 are eicosanoids that belong to the prostanoid family, which also includes thromboxanes (TXs) and prostacyclins. The basic chemical structure of prostanoids is a prostanoic acid with a cyclopentane ring and two carbon chains. The precursor molecule for these lipid mediators is arachidonic acid (AA), an essential polyunsaturated fatty acid that is stored in membrane phospholipids and released by the action of phospholipase A2 (PLA2)  [93,94,9 calcium .2, which is then reduced to the intermediate prostaglandin H2 (PGH2). COX-1 is constitutively expressed in most tissues and is responsible for the production of ‘housekeeping’ PGs that control a wide range of physiological effects, including maintaining homeostasis and gastrointestinal protection. On the other hand, COX-2 isoform expression is inducible at sites of inflammation and vascular trauma by a range of pro-inflammatory stimuli such as cytokines, growth factors, and ulcerogenic stimuli but particularly during infection and inflammation [AA is afterward bis-dioxygenated by cyclooxygenase (COX) enzymes COX-1 and COX-2  to generate hydroperoxy endoperoxide PGGammation . Howeverammation . COX-2 eammation .2 to the different types of PGs, such as PGE2, PGD2, PGI2, PGF2α, and TXA2 [2, which is the most widely produced prostanoid in the body, is synthesized by PGE2 synthases (PGES) in a broad range of cell types [2 is transported into the extracellular microenvironment by multidrug resistance-associated protein 4 [2 is metabolized mainly by nicotinamide adenine dinucleotide (NAD+)-dependent 15-hydroxyprostaglandin dehydrogenase to generate 15-keto-prostaglandin E2, which has significantly lower biological activity [Several specific isomerases and oxidoreductases convert PGHand TXA2 ,94,95. Bll types . After irotein 4 ,101. PGEactivity .2 performs its biological functions via interaction with four prostaglandin E receptor subtypes  [2 binds PTGER1, PTGER2, PTGER3, and PTGER4 with Kd values of 21, 40, 3, and 11 nM, respectively [2 also has an affinity for other prostanoid receptors, such as the PGD2 DP1 receptor, and the PGF2α FP receptor [PGEs EP1-4) ,104. Thes EP1-4) ,106,107.ectively . PGE2 alreceptor .2, PTGER1 couples with G proteins and the Gαq subunit promotes the activation of phospholipase C (PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), leading to intracellular calcium mobilization and activation of protein kinase C (PKC) (Upon being activated by PGE C (PKC) . Additio C (PKC)  [103,1042-PTGER4 are faster than PGE2-PTGER2 [Even though fewer intracellular cAMP formation has been reported in cells expressing PTGER4 than in those expressing PTGER2 , the des2-PTGER2 .PTGER2 and PTGER4 activate multiple signaling pathways; PTGER2, besides causing AMP production and protein kinase A (PKA) activation, increases the epidermal growth factor receptor (EGFR) and Src through its association with β-arrestin1, this leading to the subsequent activation of H-Ras/extracellular signal-regulated kinases (ERK), and phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) . PTGER4 PTGER3 activation induces its coupling to Gαi proteins, which inhibit AC decreasing intracellular cAMP concentration ,104,111 2/PTGERs is involved in cancer progression through multiple pathways that regulate fundamental oncogenic process as cell proliferation, metastasis, angiogenesis, immune evasion, and cell death , cytokines such as tumor necrosis factor-alpha (TNFα), diverse interleukins, and COX-2. In particular, proinflammatory cytokines’ constant release activates signaling pathways that result in the formation of ROS and RNS . Particuogenesis . In thisoduction , convergogenesis . PTGER2/ogenesis ,155,156.er cells .In cervical cancer, immune evasion is of the greatest importance for HPV to persist and cause malignant transformation. Viral oncoproteins and inflammation can both cause a local protolerogenic immune response, converging in the modulation of the activity of antigen-presenting cells (APCs), such as DCs, which can polarize the immune response. A tolerogenic immune response may help tumor cells and viral agents escape the immune system, allowing them to survive and grow . In this2 in cervical lesions, which is another interconnecting point between HPV infection and PGE2 signaling [Notably, E6 was also described to inhibit the migration of DCs by increasing PGEignaling . In addiignaling .On the other hand, infected cancer cells per se also contribute to polarizing the tumor immune milieu, as it was found that supernatants from HPV-transformed cell cultures contained IL-10 and transforming growth factor (TGFβ), both immunosuppressive cytokines, while the analysis of HPV-positive cervical cancer biopsies showed a predominant immunosuppressive expression profile .2, this prostaglandin is known to promote immune tolerance through PTGER2 and PTGER3 receptors by inducing secretion of IL-10 in DCs [2 levels and other inflammatory mediators were also associated with stimulation of COX-2 expression in monocytes, contributing to the induction and persistence of immunosuppressive myeloid-derived suppressor cells (MDSC), which highlights the central role of COX-2/PGE2 signaling in tumor progression [2, via PTGER2 and PTGER4 expressed on MDSCs, inhibits the development of CTLs, impairing T cell functions [2 is able to induce the rapid accumulation of cAMP, a process coupled with PTGER2/PTGER4 receptors [2, given the well-known immunosuppressive effects of cAMP [In a similar manner, in the case of inflammation, although counterintuitive to the proinflammatory activity of PGE0 in DCs . Of note0 in DCs ,168. Higgression . Indeed,gression . In partunctions . As menteceptors . This is of cAMP .2-PTGERs axis and cAMP/CREB signaling improve the oncogenic ability of HPV by promoting chronic inflammation in cervical cancer. In addition, the immunodepression caused by both HPV infection and inflammatory response is an additional pro-tumorigenic factor in cervical cancer progression. Thus, inflammation and immune modulation remain the main targets for therapies in cervical cancer.Considering this all together, it is clear that the COX-2-PGECOX enzymes are the primary target of non-steroidal anti-inflammatory drugs (NSAIDs), which are widely used to relieve pain, fever, and other inflammatory processes. These drugs are classified according to their selectivity into non-selective NSAIDS and COX-2-selective NSAIDs .The non-selective NSAIDs inhibit both COX-1 and COX-2 enzymes, and it was demonstrated that their long-term use may decrease the risk of colorectal, esophageal, breast, lung, prostate, liver, skin, and cervical cancers , which i2, and microvessel density in tumor biopsies [In addition to its typical therapeutical effects, some selective COX-2 inhibitors have chemopreventive and chemotherapeutic effects . The mecbiopsies . Additiobiopsies . In anotbiopsies . Moreovebiopsies ,186, incbiopsies ,188.Corticosteroids may inhibit the inflammatory process through modulating the transcription of anti-inflammatory and pro-inflammatory genes, including the down-regulation of COX-2 in monocytes and epithelial cells, so they are used to treat several inflammatory and immune diseases . Taking Epidemiological studies have shown the presence of HPV in up to 99% of cervical cancers and in a high percentage of other genital tract cancers . Indeed,Prophylactic vaccines are prepared using purified virus-like particles (VLPs) self-assembled by the major capsid protein L1, generating the recombinant proteins L1-VLPs, which are morphologically and immunologically similar to HPV virions, but that do not contain a viral genome. The LI-VLPs promote the generation of neutralizing antibodies directed against viral capsid proteins, blocking the adherence and internalization of HPV in the basal cells of the epithelium . SeveralVaccines made against HPV oncoproteins may induce an immune response able to kill cervical cancer cells, representing a good therapeutic strategy and a promising treatment alternative to clear HR-HPV infections and associated malignancies. Among the proteins encoded by HPV, E6 and E7 are the main targets for the development of potential therapeutic vaccines due to: (1) their constitutive expression in infected cells, (2) both are functionally important for the development of tumor cells, and (3) they are readily recognized by the adaptive immune system as tumor antigens ,198. OthLactobacillus casei, Listeria monocytogenes, Lactobacillus lactis, Lactobacillus plantarum, and Salmonella species.Bacterial vector vaccines utilize attenuated bacteria to transport genes of interest into the cell. The bacterial vectors include Lactobacillus casei modified HPV16 antigen E7 vaccine completed the phase I/IIa clinical trial in patients diagnosed with CIN-3 positive to HPV16; interestingly, nine of these patients experienced disease regression to CIN-2, while five patients remarkably regressed to LSIL [Listeria monocytogenes (Lm), which is internalized via phagocytosis by APCs, where they can follow two routes. In the first, most bacteria are killed, providing a source of antigens for major histocompatibility complex (MHC) class II for activation of CD4+ helper T-cell. In the second, Lm being inside of phagolysosome, secretes the listeriolysin O (LLO), a pore-forming toxin that destroys the phagosomal membrane allowing it to escape and enter the cytosol, where it can be degraded by the proteasome and loaded onto MHC class I molecules for presentation to cytotoxic T cells [https://www.clinicaltrials.gov/ct2/results?cond=cervical+cancer&term=ADXS11-001+&cntry=&state=&city=&dist; accessed on 1 August 2022). The GTL001 vaccine was designed to induce a T-cell immune response to HPV16 and HPV18 and prevent the development of high-grade lesions in infected women who still have normal cervical cytology or minor abnormalities [Bordetella pertussis [Bordetella pertussis secrets the AC-E7 protein fusion, which binds to the αMβ2 integrin receptor on the cell membrane of DCs and delivers its N-terminal catalytic AC domain into the cytoplasm, whose characteristic is used as the vector for antigen delivery into APC and subsequent antigen presentation [A recombinant  to LSIL . Another T cells . The nat T cells . In a fi T cells . Afterwa T cells . The saf T cells . After tertussis . Bordeteentation .Viral vectors are able to infect a variety of cell types and efficiently express encoded antigens, which makes them attractive candidates for the development of therapeutic HPV vaccines. Among the potential vectors evaluated in the development of therapeutic vaccines are vaccinia virus, adenovirus, adeno-associated virus and alphavirus .Vaccinia virus is used to deliver E6 and E7 antigens for therapeutic HPV vaccination. Among the therapeutic vaccinia based-vaccine developed to treat genital cancers is the tissue-antigen HPV vaccine (TA-HPV), which expresses modified forms of E6 and E7 fusion proteins from HPV16 and HPV18, whose safety, immunogenicity, and efficacy were confirmed in patients with clinical early and late-stage of cervical cancer as well as vulval intraepithelial neoplasia and vaginal intraepithelial neoplasia ,207,208.Nucleic acid vaccines are a method of immunization where DNA or mRNA sequences are delivered into the body to generate proteins, mimicking pathogens’ antigens to stimulate immune response . VGX-310Peptide-based vaccines have the potential to induce cellular immune responses mainly by the induction of cytotoxic T cells, with the capacity to recognize non-auto immunogenic cancer antigens eliminating malignant cells . The pepThese vaccines typically have increased stability during storage and transport as compared to DNA vaccines; however, they induce lower immunogenicity and poor stability in vivo, which remain their major drawbacks . AdditioImiquimod, an imidazoquinoline amine, is an immunomodulator approved for the treatment of external anogenital warts, small superficial basal cell carcinoma, and other malignancies ,238. ImiHPV DNA replication is an attractive target for HPV antivirals because its inhibition would result in fewer genomes available for viral protein synthesis and integration, a critical step in the development of HPV-dependent cancers . SeveralAmong the acyclic nucleoside phosphonates, cidofovir and adefovir are acyclic analogs of the corresponding monophosphates of deoxycytidine and deoxyadenosine. The active dephosphorylated forms of these antivirals are deoxynucleoside triphosphate analogs, potent inhibitors of viral DNA polymerases, reducing viral DNA synthesis . CidofovHPV-E1 and -E2 proteins are excellent targets for developing antivirals; on one side, HPV-E1 is an ATPase and helicase required for viral DNA replication, while on the other hand, E2 is required for transcription activation and repression. E2 recruits E1 at the beginning of replication, forming a ternary complex with viral DNA and the inhibition of this interaction blocks viral DNA replicating activity . The ind2 each 250 µm long, enhancing the antigenicity of the vaccine [Nanotechnology is the approach to manufacturing nanoparticles, nanotubes, nanosheets, and nanorods, with a size from 1 to 100 nm in at least one dimension . The dev vaccine . Additio vaccine ,256. One vaccine . Moreove vaccine .2–PTGERs inflammatory signaling pathway, together with the cAMP/CREB/CRE cascade, are closely intermingled with the carcinogenic nature of the virus, highlighting inflammation as a co-factor for HPV-dependent carcinogenesis. Furthermore, the attenuation of the immune response caused by both the virus and inflammatory mediators represents an additional pro-tumorigenic process involved with cervical cancer development. Therefore, inflammation and immune modulation remain the main targets for anti-tumoral therapies in cervical cancer. In this regard, accessible therapeutic options for cervical cancer treatment and prevention have been developed in the last years. For instance, anti-inflammatory drugs constitute an excellent therapeutic strategy in cervical cancer patients. Indeed, it was suggested that some of these drugs may be re-purposed as antineoplastic agents, such as aspirin and celecoxib. On the other hand, HPV infection is considered the leading causative agent of genital tract cancers, representing an attractive therapeutic target for preventing and treating cervical cancer. Prophylactic vaccines against HPV made with recombinant DNA technology have been very effective in preventing persistent HPV infection and lesions that lead to cervical cancer. Highly immunogenic prophylactic vaccines induce a strong humoral immune response with the production of HPV-neutralizing antibodies that inhibit viral infection. The development of therapeutic vaccines for cervical cancer is based on activating cellular immunity against cancer cells instead of neutralizing antibodies. Among the different therapeutic vaccines developed against cervical cancer, the best results were achieved with MVA-E2, VGX-3100, GX-188E, and pBI-11. Particularly, MVA-E2 and VGX-3100 have reached the phase III trial; however, they have not been licensed for use yet. Additionally, the clinical benefits of HPV-therapeutic vaccines were enhanced by combining them with each other and local treatments or immunomodulators such as Imiquimod. The potency of therapeutic vaccines has been increased in preclinical studies employing nanotechnology, such as in the case of Nanopatch, which delivers a Gardasil prophylactic vaccine intradermally using microneedles. Finally, another way to target HPV is through antivirals, resulting in fewer genomes available for viral protein synthesis and integration, a critical step in the development of HPV-dependent cancers.Standard treatments for advanced cervical cancer include surgery, radiotherapy, and chemotherapy, some of which are invasive and others have adverse side effects. In HPV-related malignancies such as cervical cancer, the COX-2–PGE