Insights into estrogen impact in oral health & microbiome in COVID-19.

BACKGROUND: COVID-19 emerged in late 2019 and has occasioned more than 765 millions cumulative cases and 6.9 millions of deaths globally. Notably, around 70% of patients with severe COVID-19 are men. Therefore, it is to be presumed that women have a hormonal protector factor in inflammation and ACE2 expression. On the other hand, oral health status, and local microbiome can be key factors to respiratory viral infections control. Nevertheless, it has been poorly investigated. In our study 20 premenopausal, 18 postmenopausal and 22 men with COVID-19 were included. Oral health status, viral load, lingual ACE2 expression, as well as microbiome, estrogens and cytokines in saliva were analyzed. RESULTS: Our results showed a lower expression of ACE2 in tongue cells of postmenopausal compared with premenopausal (p = 0.05), and a strong negative correlation between saliva estrogen and viral load (r = -0.76; p = 0.001). Respect to IFN-γ (p = 0.05), IL-1ß, TNF-α, IL-18, and IL-23 levels were increased in postmenopausal. Oral microbiome signature of premenopausal was characterized by Prevotella melaninogenica (Log2 = 26.68; p = 1.34e-10), Haemophilus (Log2 = 23.99; p = 2.96e-9), and Alloprevotella (Log2 = 7.92; p = 0.0001). On the other hand, Leptotrichia (Log2 = -18.74; p = 0.001), Tanerella (Log2 = -17.08; p = 0.004), and Clostridiales (Log2 = -2.88; p = 0.04) represented the poor oral health group compared with the adequate group which was enriched with the commensal microorganism Neisseria perflava (Log2 = 26.70; p = 1.74e-7). Furthermore, the high viral load group was characterized by Prevotella nanceiensis (Log2 = 19.60; p = 6.06e-8), Prevotella melaninogenica (Log2 = 21.45; p = 9.59e-6), Alloprevotella (Log2 = 23.50; p = 2.70e-7) and bacteria from the red complex Porphyromonas endodentalis (Log2 = 21.97; p = 1.38e-7). CONCLUSIONS: Postmenopausal and men have a poor oral health status which could be related to a detrimental progression of COVID-19 also linked to a lower expression of ACE2, lower saliva estrogen levels and oral dysbiosis. Nevertheless, functional studies are required for a deeper knowledge.

Aberrant B-cell activation and B-cell subpopulations in rheumatoid arthritis: analysis by clinical activity, autoantibody seropositivity, and treatment.

Few studies analyze the role of B-cell subpopulations in rheumatoid arthritis (RA) pathophysiology. Therefore, this study aimed to analyze the differences in B-cell subpopulations and B-cell activation according to disease activity, RA subtype, and absence of disease-modifying antirheumatic drugs (DMARDs) therapy. These subgroups were compared with control subjects (CS). One hundred and thirty-nine subjects were included, of which 114 were RA patients, and 25 were controls. Patients were divided into 99 with seropositive RA, 6 with seronegative RA, and 9 without DMARDs. The patients with seropositive RA were subclassified based on the DAS28 index. A seven-color multicolor flow cytometry panel was used to identify B-cell immunophenotypes and cell activation markers. There were no changes in total B-cell frequencies between RA patients and controls. However, a lower frequency of memory B cells and pre-plasmablasts was observed in seropositive RA compared to controls (P < 0.0001; P = 0.0043, respectively). In contrast, a higher frequency of mature B cells was observed in RA than in controls (P = 0.0002). Among patients with RA, those with moderate activity had a higher percentage of B cells (P = 0.0021). The CD69+ marker was increased (P < 0.0001) in RA compared to controls, while the CD40+ frequency was decreased in patients (P < 0.0001). Transitional, naïve, and double-negative B-cell subpopulations were higher in seronegative RA than in seropositive (P < 0.01). In conclusion, in seropositive and seronegative RA patients, there are alterations in B-cell activation and B-cell subpopulations, independently of clinical activity and DMARDs therapy.

Splicing Characterization and Isoform Switch Events in Human Keratinocytes Carrying Oncogenes from High-Risk HPV-16 and Low-Risk HPV-84.

Infection of epithelial cells with high-risk HPV (HR-HPV) types, followed by expression of virus oncogenic proteins (E5, E6, and E7), leads to genomic imbalance, suppression of tumor inhibitors, and induction of oncogenes. Low-risk HPV (LR-HPV) may slow the rate at which cervical cancer spreads to an invasive stage since co-infection with LR-HPV is linked to a decreased risk of future invasive cancer than infection with HR-HPV alone. We then propose that cancer-progressing changes may be distinguished through identifying the functional differences between LR-HPV and HR-HPV. Lentiviral strategies were followed to establish HaCaT cells with constitutive expression of HPV oncogenes. RNAseq experiments were designed to analyze the transcriptome modulations caused by each of the E5, E6, and E7 oncogenes of HPV-16 and HPV-84 in HaCaT cells. We identified enhanced RNA degradation, spliceosome, and RNA polymerase pathways related to mRNA processing. ATTS (alternative transcription termination site) was discovered to be more prevalent in cells with HPV-16E5 than HPV-84E5. In HPV-16E6-infected cells, ATTS gain was significantly higher than ATTS loss. Cells with HPV-16E7 had more isoforms with intron retention (IR) than those with HPV-84E7. We identified switches in ADAM10, CLSPN, and RNPS1 that led to greater expression of the coding isoforms in HR-HPV. The results of this work highlight differences between LR-HPV and HR-HPV in mRNA processing. Moreover, crucial cervical cancer-related switch events were detected.

ICOS Gene Polymorphisms (IVS1 + 173 T/C and c. 1624 C/T) in Primary Sjögren's Syndrome Patients: Analysis of ICOS Expression.

Background: Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease, which affects exocrine glands. T cell activation is a trigger mechanism in the immune response. Hyperreactivity of T cells and antibody production are features in pSS. ICOS can be critical in the pathogenesis of pSS. Methods: A total of 134 pSS patients and 134 control subjects (CS) were included. Genotyping was performed by PCR-RFLP. ICOS mRNA expression was quantified by real-time PCR, and CD4+ ICOS+ T cells were determined by flow cytometry. Results: The ICOS IVS1 + 173 T>C polymorphisms were not associated with susceptibility to pSS (p = 0.393, CI = 0.503−1.311). However, the c.1624 C>T polymorphism was associated with a reduction in the risk of development of pSS (p = 0.015, CI = 0.294−0.884). An increase in ICOS mRNA expression in patients was observed (3.7-fold). Furthermore, pSS patients showed an increase in membranal-ICOS expression (mICOS). High expression of mICOS (MFI) was associated with lymphocytic infiltration. Conclusions: The IVS1 + 173 polymorphism is not a genetic marker for the development of pSS, while c.1624 T allele was associated with a low risk. However, elevated mICOS expression in pSS patients with high lymphocytic infiltration was found. ICOS may have an important role in the immunopathogenesis of pSS and should be analyzed in T cell subsets in pSS patients as a possible disease marker.

Performance evaluation of a lateral flow assay for nasopharyngeal antigen detection for SARS-CoV-2 diagnosis.

BACKGROUND: SARS-CoV-2 has become a global pandemic due to its capacity for rapid transmission. In this context, an early and rapid diagnosis of infected patients that do not require expensive equipment or highly trained personnel is crucial in order to reduce the contagious rate. The aim of this study was to evaluate a chromatographic immunoassay's performance for the rapid diagnosis of SARS-CoV-antigen. METHODS: A cross-sectional study included 369 adults from Western México with diagnosis or suspicion of SARS-CoV-2 infection. Two samples were collected; a naso-oropharyngeal was used for a molecular determination of SARS-CoV-2 RNA. The molecular analysis was carried out using DeCoV19 Kit Triplex (Genes2life S.A.P.I.) based on the CDC diagnostic panel for N1, N2, and N3 regions. The second sample was retrieved from a nasopharyngeal rub and used for the rapid diagnosis of SARS-CoV-2 antigen employing the commercial STANDARD™ Q COVID-19 Ag Test (SD BIOSENSOR). RESULTS: Overall, in 28.2% of the patients was detected the SARS-CoV-2 RNA, and 21.4% were positive for antigen detection. The rapid antigen test showed a sensitivity and specificity of 75.9% and 100%, respectively, with a positive predictive and negative values of 100% and 91%. Symptoms as anosmia presented a high OR for the positive diagnosis for both test, reverse transcription-polymerase chain reaction (RT-PCR), and the rapid antigen test of 8.86 (CI = 4.91-16) and 6.09 (CI = 3.42-10.85), respectively. CONCLUSION: SD BIOSENSOR is a useful assay, but some caveats must be considered before the general implementation.

Metabolic syndrome in rheumatoid arthritis patients: Relationship among its clinical components.

BACKGROUND: Metabolic syndrome (MetS) prevalence in rheumatoid arthritis (RA) patients is known to vary considerably across the world. This study aimed to determine the prevalence of MetS in RA patients from western Mexico and to analyze the interrelation of the MetS components with the clinical variables of RA. METHODS: This case-control study included 216 RA patients and 260 control subjects (CS). MetS prevalence was determined according to the NCEP/ATP III and the Latin American Consensus of the Latin American Diabetes Association (ALAD) criteria. RESULTS: MetS was observed in 30.6% RA patients and 33.3% of controls (p > 0.05) according to NCEP/ATP III and 28.7% in RA patients and 31.1% for controls using ALAD criteria. Total cholesterol, LDL-C, and Castelli's I-II indexes were lower in RA (p < 0.001) than in CS. The RA patients with MetS had more swollen joints than those without MetS (p = 0.018). In RA patients with MetS, DAS-28 score correlated with smoking index (rho = 0.4601, p = 0.0004) and VLDL-C (rho = 0.3108, p = 0.0056); similarly, rheumatoid factor (RF) correlated with age (rho = 0.2031, p = 0.0027), smoking index (rho = 0.3404, p < 0.0001), triglycerides (rho = 0.1958, p = 0.0039), and VLDL-C (rho = 0.1761, p = 0.0162). CONCLUSIONS: The MetS prevalence in RA patients from western Mexico is not higher than controls; however, in RA patients with MetS, some inflammatory markers are associated with MetS components; thus, the control of MetS in RA could be beneficial to regulate disease activity.

Expression patterns of CD28 and CTLA-4 in early, chronic, and untreated rheumatoid arthritis.

BACKGROUND: T-cell activation pathways have been proposed as trigger mechanisms in the pathogenesis of rheumatoid arthritis (RA). CD28 and CTLA-4 play major roles in regulating the stimulatory and inhibitory co-signals in T cells. OBJECTIVE: To analyze the association between soluble and surface expression of CD28 and CTLA-4 with the clinical parameters of RA patients. METHODS: A total of 35 RA patients classified as early RA (n = 14), chronic RA (n = 14), and untreated RA (n = 7), as well as 7 age- and sex-matched control subjects (CS) were included. Surface expression of CD28 and CTLA-4 on T cells was evaluated by flow cytometry. Soluble levels of CD28 (sCD28), CTLA-4 (sCTLA-4), and anti-CCP antibodies were measured by ELISA. RESULTS: A significant lower percentage of CD8 + T cells positive to CD28 (CS = 64.9% vs RA = 42.7%, P = .04), and diminished surface expression of CD28 (CS: MFI = 122.9 vs RA: MFI = 33.1, P = .006), were found in chronic RA patients compared to CS. Higher sCD28 were observed in early RA patients compared with chronic RA patients (P < .05). sCTLA-4 was found increased in untreated RA patients compared to early RA patients (P < .05). sCD28 concentration correlated with anti-CCP levels (rho = -0.12; P = .032). The soluble and surface expressions of CTLA-4 were not associated with RA clinical parameters. CONCLUSIONS: In RA, the percentage of CD8 + CD28+ T cells decreases and expresses fewer membrane CD28 than CS. sCD28 levels are lower in chronic RA and are associated negatively with anti-CCP levels. sCTLA 4 levels are lower in early RA patients than in untreated RA patients.

Interaction between 17ß-estradiol, prolactin and human papillomavirus induce E6/E7 transcript and modulate the expression and localization of hormonal receptors.

BACKGROUND: Cervical cancer (CC) is the second most common cancer in less developed countries and the second leading cause of death by cancer in women worldwide. The 99% of CC patients are infected with the Human Papilloma Virus (HPV), being HPV16 and HPV18 infection the most frequent. Even though HPV is considered to be a necessary factor for the development of CC, it is not enough, as it requires the participation of other factors such as the hormonal ones. Several studies have demonstrated the requirement of estrogen and its receptors (ERα, ERß, and GPER) in the precursor lesions progress towards CC. Also, prolactin (PRL) and its receptor (PRLR) have been associated with CC. The molecular mechanisms underlying the cooperation of these hormones with the viral oncoproteins are not well elucidated. For this reason, this study focused on analyzing the contribution of 17ß-estradiol (E2), PRL, and HPV on the expression and localization of hormone receptors, as well as to evaluate whether these hormones may promote greater expression of HPV oncogenes and contribute to tumor progression. METHODS: qPCR was used to evaluate the effect of E2 and PRL on the expression of E6 and E7 oncoproteins in HeLa and SiHa cervical cancer cells lines. HaCaT cells were transduced with the viral oncogenes E6 and E7 from HPV 16 and 18. ERα, ERß, GPER, and PRLR expression and localization were evaluated by qPCR, Western blot and immunofluorescence. RESULTS: E2 and PRL induce E6/E7 oncogenes expression in HeLa and SiHa cells. E6 and E7 oncogenes of HPV16/18 significantly increased the protein expression of ERα, GPER, and PRLR. ERß was positively regulated only by E6 oncogenes of HPV16/18. Besides, some of these oncogenes modify the location of PRLR toward cytoplasm, and ERα, ERß, and GPER mainly to the nucleus. CONCLUSION: Our studies suggest that the mutual regulation between E2, PRL, and HPV oncogenes could cooperate with the carcinogenesis process in CC.

Expression of WNT genes in cervical cancer-derived cells: Implication of WNT7A in cell proliferation and migration.

According to the multifactorial model of cervical cancer (CC) causation, it is now recognized that other modifications, in addition to Human papillomavirus (HPV) infection, are necessary for the development of this neoplasia. Among these, it has been proposed that a dysregulation of the WNT pathway might favor malignant progression of HPV-immortalized keratinocytes. The aim of this study was to identify components of the WNT pathway differentially expressed in CC vs. non-tumorigenic, but immortalized human keratinocytes. Interestingly, WNT7A expression was found strongly downregulated in cell lines and biopsies derived from CC. Restoration of WNT7A in CC-derived cell lines using a lentiviral gene delivery system or after adding a recombinant human protein decreases cell proliferation. Likewise, WNT7A silencing in non-tumorigenic cells markedly accelerates proliferation. Decreased WNT7A expression was due to hypermethylation at particular CpG sites. To our knowledge, this is the first study reporting reduced WNT7A levels in CC-derived cells and that ectopic WNT7A restoration negatively affects cell proliferation and migration.

An approach to the immunophenotypic features of circulating CD4⁺NKG2D⁺ T cells in invasive cervical carcinoma.

BACKGROUND: NKG2D, an activating immunoreceptor, is primarily restricted to NK cells and CD8(+) T cells. The existence of an atypical cytotoxic CD4(+)NKG2D(+) T cell population has also been found in patients with autoimmune dysfunctions. Nonetheless, contradictory evidence has categorized this population with a regulatory rather than cytotoxic role in other situations. These confounding data have led to the proposal that two distinct CD4(+)NKG2D(+) T cell subsets might exist. The immune response elicited in cervical cancer has been characterized by apparent contradictions concerning the role that T cells, in particular T-helper cells, might be playing in the control of the tumor growth. Interestingly, we recently reported a substantial increase in the frequency of CD4(+)NKG2D(+) T cells in patients with cervical intraepithelial neoplasia grade-1. However, whether this particular population is also found in patients with more advanced cervical lesions or whether they express a distinctive phenotype remains still to be clarified. In this urgent study, we focused our attention on the immunophenotypic characterization of CD4(+)NKG2D(+) T cells in patients with well-established cervical carcinoma and revealed the existence of at least two separate CD4(+)NKG2D(+) T cell subsets defined by the co-expression or absence of CD28. RESULTS: Patients with diagnosis of invasive cervical carcinoma were enrolled in the study. A group of healthy individuals was also included. Multicolor flow cytometry was used for exploration of TCR alpha/beta, CD28, CD158b, CD45RO, HLA-DR, CD161, and CD107a. A Luminex-based cytokine kit was used to quantify the levels of pro- and anti-inflammatory cytokines. We found an increased percentage of CD4(+)NKG2D(+) T cells in patients with cervical cancer when compared with controls. Accordingly with an increase of CD4(+)NKG2D(+) T cells, we found decreased CD28 expression. The activating or degranulation markers HLA-DR, CD161, and CD107a were heterogeneously expressed. The levels of IL-1beta, IL-2, TNF-alpha, and IL-10 were negatively correlated with the percentages of CD4(+)NKG2D(+) T cells in patients with cervical carcinoma. CONCLUSIONS: Taken together, our results reveal the existence of two separate CD4(+)NKG2D(+) T cell subsets defined by the co-expression or absence of CD28, the latter more likely to be present in patients with cervical cancer.

Substantial increase in the frequency of circulating CD4+NKG2D+ T cells in patients with cervical intraepithelial neoplasia grade 1.

BACKGROUND: The NKG2D receptor confers important activating signals to NK cells via ligands expressed during cellular stress and viral infection. This receptor has generated great interest because not only is it expressed on NK cells, but it is also seen in virtually all CD8+ cytotoxic T cells and is classically considered absent in CD4+ T cells. However, recent studies have identified a distinctive population of CD4+ T cells that do express NKG2D, which could represent a particular cytotoxic effector population involved in viral infections and chronic diseases. On the other hand, increased incidence of human papillomavirus-associated lesions in CD4+ T cell-immunocompromised individuals suggests that CD4+ T cells play a key role in controlling the viral infection. Therefore, this study was focused on identifying the frequency of NKG2D-expressing CD4+ T cells in patients with cervical intraepithelial neoplasia (CIN) 1. Additionally, factors influencing CD4+NKG2D+ T cell expansion were also measured. RESULTS: Close to 50% of patients with CIN 1 contained at least one of the 37 HPV types detected by our genotyping system. A tendency for increased CD4+ T cells and CD8+ T cells and decreased NK cells was found in CIN 1 patients. The percentage of circulating CD4+ T cells co-expressing the NKG2D receptor significantly increased in women with CIN 1 versus control group. Interestingly, the increase of CD4+NKG2D+ T cells was seen in patients with CIN 1, despite the overall levels of CD4+ T cells did not significantly increase. We also found a significant increase of soluble MICB in CIN 1 patients; however, no correlation with the presence of CD4+NKG2D+ T cells was seen. While TGF-beta was significantly decreased in the group of CIN 1 patients, both TNF-alpha and IL-15 showed a tendency to increase in this group. CONCLUSIONS: Taken together, our results suggest that the significant increase within the CD4+NKG2D+ T cell population in CIN 1 patients might be the result of a chronic exposure to viral and/or pro-inflammatory factors, and concomitantly might also influence the clearance of CIN 1-type lesion.

HPV-Negative and HPV-Positive Oral Cancer Cells Stimulate the Polarization of Neutrophils towards Different Functional Phenotypes In Vitro.

High-risk human papillomavirus (HPV) infection is one of the leading causes of oropharyngeal squamous cell carcinoma (OPSCC), while the correlation between HPV and oral squamous cell carcinoma (OSCC) remains controversial. The inflammatory infiltrate involved in these epithelial neoplasms differs based on their association with HPV. HPV- tumors show higher tumor-associated neutrophil (TAN) infiltration. It is believed that TANs can play a dual role in cancer by exerting either anti-tumorigenic or pro-tumorigenic effects. However, the impact of HPV status on neutrophil polarization remains unknown. Therefore, this study aimed to investigate the effect of OSCC cells, both HPV- and HPV16+, on the functional phenotype of neutrophils. Peripheral blood neutrophils were stimulated with supernatants from OSCC cell lines and non-tumorigenic HaCaT keratinocytes transduced with HPV16 E6/E7 oncogenes. Subsequently, cytokine production, cell viability, metabolism, expression of degranulation markers, and PD-L1 expression were evaluated. Our findings demonstrate that in contrast to UPCI:SCC154 (HPV+ OSCC) cells, the SCC-9 (HPV- OSCC) cell line induced a highly activated functional state in neutrophils, which is potentially associated with a pro-tumorigenic effect. The HaCaT 16-E7 supernatant only stimulated the activation of some neutrophil functions. Understanding the complex interplay between neutrophils and their microenvironment has the potential to identify TANs as viable therapeutic targets.

Beyond SARS-CoV-2: epidemiological surveillance of respiratory viruses in Jalisco, Mexico.

Introduction: Respiratory viral infections represent a significant global health burden. Historically, influenza, rhinovirus, respiratory syncytial virus, and adenovirus have been the prevalent viruses; however, the landscape shifted with the widespread emergence of SARS-CoV-2. The aim of this study is to present a comprehensive epidemiological analysis of viral respiratory infections in Jalisco, Mexico. Methods: Data encompassing individuals with flu-like symptoms from July 2021 to February 2023 was scrutinized for viral diagnosis through PCR multiplex. The effect of social mobility on the increase in respiratory viral diagnosis infection was considered to estimate its impact. Additionally, sequences of respiratory viruses stored in public databases were retrieved to ascertain the phylogenetic classification of previously reported viruses in Mexico. Results: SARS-CoV-2 was the most detected virus (n = 5,703; 92.2%), followed by influenza (n = 479; 7.78%). These viruses were also found as the most common co-infection (n = 11; 50%), and for those with influenza, a higher incidence of severe disease was reported (n = 122; 90.4%; p < 0.001). Regarding comorbidities and unhealthy habits, smoking was found to be a risk factor for influenza infection but a protective factor for SARS-CoV-2 (OR = 2.62; IC 95%: 1.66-4.13; OR = 0.65; IC 95%: 0.45-0.94), respectively. Furthermore, our findings revealed a direct correlation between mobility and the prevalence of influenza infection (0.214; p < 0.001). Discussion: The study presents evidence of respiratory virus reemergence and prevalence during the social reactivation, facilitating future preventive measures.

Prevalence of symptoms, comorbidities, and reinfections in individuals infected with Wild-Type SARS-CoV-2, Delta, or Omicron variants: a comparative study in western Mexico.

Introduction: The variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been classified into variants of interest (VOIs) or concern (VOCs) to prioritize global monitoring and research on variants with potential risks to public health. The SARS-CoV-2 high-rate mutation can directly impact the clinical disease progression, epidemiological behavior, immune evasion, vaccine efficacy, and transmission rates. Therefore, epidemiological surveillance is crucial for controlling the COVID-19 pandemic. In the present study, we aimed to describe the prevalence of wild-type (WT) SARS-CoV-2 and Delta and Omicron variants in Jalisco State, Mexico, from 2021 to 2022, and evaluate the possible association of these variants with clinical manifestations of COVID-19. Methods: Four thousand and ninety-eight patients diagnosed with COVID-19 by real-time PCR (COVIFLU, Genes2Life, Mexico) from nasopharyngeal samples from January 2021 to January 2022 were included. Variant identification was performed by the RT-qPCR Master Mut Kit (Genes2Life, Mexico). A study population follow-up was performed to identify patients who had experienced reinfection after being vaccinated. Results and Discussion: Samples were grouped into variants according to the identified mutations: 46.3% were Omicron, 27.9% were Delta, and 25.8% were WT. The proportions of dry cough, fatigue, headache, muscle pain, conjunctivitis, fast breathing, diarrhea, anosmia, and dysgeusia were significantly different among the abovementioned groups (p < 0.001). Anosmia and dysgeusia were mainly found in WT-infected patients, while rhinorrhea and sore throat were more prevalent in patients infected with the Omicron variant. For the reinfection follow-up, 836 patients answered, from which 85 cases of reinfection were identified (9.6%); Omicron was the VOC that caused all reported reinfection cases. In this study, we demonstrate that the Omicron variant caused the biggest outbreak in Jalisco during the pandemic from late December 2021 to mid-February 2022 but with a less severe form than the one demonstrated by Delta and WT. The co-analysis of mutations and clinical outcomes is a public health strategy with the potential to infer mutations or variants that could increase disease severity and even be an indicator of long-term sequelae of COVID-19.

Specific T-Cell Immune Response to SARS-CoV-2 Spike Protein over Time in Naïve and SARS-CoV-2 Previously Infected Subjects Vaccinated with BTN162b2.

Due to the COVID-19 pandemic, the rapid development of vaccines against SARS-CoV-2 has been promoted. BNT162b2 is a lipid-nanoparticle mRNA vaccine with 95% efficacy and is the most administered vaccine globally. Nevertheless, little is known about the cellular immune response triggered by vaccination and the immune behavior over time. Therefore, we evaluated the T-cell immune response against the SARS-CoV-2 spike protein and neutralization antibodies (nAbs) in naïve and SARS-CoV-2 previously infected subjects vaccinated with BTN162b2. Methods: Forty-six BTN162b2 vaccinated subjects were included (twenty-six naïve and twenty SARS-CoV-2 previously infected subjects vaccinated with BTN162b2). Blood samples were obtained at basal (before vaccination), 15 days after the first dose, and 15 days after the second dose, to evaluate cellular immune response upon PBMC's stimulation and cytokine levels. The nAbs were determined one and six months after the second dose. Results: SARS-CoV-2 previously infected subjects vaccinated with BTN162b2 showed the highest proportion of nAbs compared to naïve individuals one month after the second dose. However, women were more prone to lose nAbs percentages over time significantly. Furthermore, a diminished CD154+ IFN-γ+ CD4+ T-cell response was observed after the second BTN162b2 dose in those with previous SARS-CoV-2 infection. In contrast, naïve participants showed an overall increased CD8+ IFN-γ+ TNF-α+ T-cell response to the peptide stimulus. Moreover, a significant reduction in IP-10, IFN-λI, and IL-10 cytokine levels was found in both studied groups. Additionally, the median fluorescence intensity (MFI) levels of IL-6, IFNλ-2/3, IFN-𝛽, and GM-CSF (p < 0.05) were significantly reduced over time in the naïve participants. Conclusion: We demonstrate that a previous SARS-CoV-2 infection can also impact cellular T-cell response, nAbs production, and serum cytokine concentration. Therefore, the study of T-cell immune response is essential for vaccination scheme recommendations; future vaccine boost should be carefully addressed as continued stimulation by vaccination might impact the T-cell response.

Differential modulation of natural killer cell cytotoxicity by 17ß-estradiol and prolactin through the NKG2D/NKG2DL axis in cervical cancer cells.

Natural killer (NK) cells play a crucial role in cervical cancer (CC). As estrogens and prolactin (PRL) have been reported to be involved in CC, the present study attempted to elucidate the effects of both hormones on NK cells in CC. For this purpose, NKL cells, as well as CC-derived cell lines (HeLa, SiHa and C33A) and non-tumorigenic keratinocytes (HaCaT cells) were stimulated with 17ß-estradiol (E2; 10 nM), PRL (200 ng/ml), or both (E2 and PRL) for 48 h. The expression of hormone receptors (estrogen receptor α and ß, G protein-coupled estrogen receptor 1 and PRL receptor) and NK cell activating receptors [natural killer group 2D (NKG2D), natural cytotoxicity triggering receptor 3, natural cytotoxicity triggering receptor 2 and natural cytotoxicity triggering receptor 1] were measured using western blot analysis and flow cytometry, respectively. In the HeLa, SiHa, C33A and HaCaT cells stimulated with the hormones, the expression of NKG2D ligands [MHC class I polypeptide-related sequence A/B (MICA/B)] on the membrane and the soluble form of MICA was evaluated using flow cytometry and ELISA. Cytotoxicity assay was performed using GFP-transfected K562 cells as target cells. E2 reduced NKL cell-mediated cytotoxicity, while PRL exerted the opposite effect. NKL cells expressed different hormone receptor forms, of which PRL only induced a decrease in NKG2D expression compared to the untreated control NKL cells. PRL increased MICA/B expression in HeLa cells and E2 and PRL reversed this effect. However, in SiHa cells, the concurrent incubation with the two hormones decreased MICA/B expression. E2 and PRL, either alone or in combination, decreased soluble MICA secretion in all CC cell lines, while E2 solely increased soluble MICA secretion in SiHa cells. On the whole, the present study provides evidence that E2 and PRL mediate the mechanisms through which NK and CC cells mediate a cytotoxic response and these have an antagonistic effect on NK cell-mediated cytotoxicity.

Transcriptomic Analysis of Breast Cancer Patients Sensitive and Resistant to Chemotherapy: Looking for Overall Survival and Drug Resistance Biomarkers.

Worldwide breast cancer ranks first in mortality and incidence rates in women over 20 years old. Rather than one disease, breast cancer is a heterogeneous group of diseases that express distinct molecular profiles. Neoadjuvant chemotherapy is an important therapeutic strategy for breast cancer patients independently of their molecular subtype, with the drawback of resistance development. In addition, chemotherapy has adverse effects that combined with resistance could contribute to lower overall survival. Although great efforts have been made to find diagnostic and prognostic biomarkers for breast cancer and for response to targeted and immune therapy for this pathology, little has been explored regarding biomarkers of response to anthracyclines and taxanes based neoadjuvant chemotherapy. This work aimed to evaluate the molecular profile of patients who received neoadjuvant chemotherapy to identify differentially expressed genes (DEGs) that could be used as biomarkers of chemotherapy response and overall survival. Breast cancer patients who were candidates for neoadjuvant chemotherapy were enrolled in this study. After treatment and according to their pathological response, they were assigned as sensitive or resistant. To evaluate DEGs, Gene Ontology, Kyoto Encyclopedia Gene and Genome (KEGG), and protein-protein interactions, RNA-seq information from all patients was obtained by next-generation sequencing. A total of 1985 DEGs were found, and KEGG analysis indicated a great number of DEGs in metabolic pathways, pathways in cancer, cytokine-cytokine receptor interactions, and neuroactive ligand-receptor interactions. A selection of 73 DEGs was used further for an analysis of overall survival using the METABRIC study and the ductal carcinoma dataset of The Cancer Genome Atlas (TCGA) database. Nine DEGs correlated with overall survival, of which the subexpression of C1QTNF3, CTF1, OLFML3, PLA2R1, PODN, KRT15, HLA-A, and the overexpression of TUBB and TCP1 were found in resistant patients and related to patients with lower overall survival.

A gene expression signature in HER2+ breast cancer patients related to neoadjuvant chemotherapy resistance, overall survival, and disease-free survival.

Breast cancer ranks first in terms of mortality and incidence rates worldwide among women. The HER2+ molecular subtype is one of the most aggressive subtypes; its treatment includes neoadjuvant chemotherapy and the use of a HER2 antibody. Some patients develop resistance despite positive results obtained using this therapeutic strategy. OBJECTIVE: To identify prognostic markers for treatment and survival in HER2+ patients. METHODS: Patients treated with neoadjuvant chemotherapy were assigned to sensitive and resistant groups based on their treatment response. Differentially expressed genes (DEGs) were identified using RNA-seq analysis. KEGG pathway, gene ontology, and interactome analyses were performed for all DEGs. An enrichment analysis Gene set enrichment analysis was performed. All DEGs were analyzed for overall (OS) and disease-free survival (DFS). RESULTS: A total of 94 DEGs were related to treatment resistance. Survival analysis showed that 12 genes (ATF6B, DHRS13, DIRAS1, ERAL1, GRIN2B, L1CAM, IRX3, PRTFDC1, PBX2, S100B, SLC9A3R2, and TNXB) were good predictors of disease-free survival, and eight genes (GNG4, IL22RA2, MICA, S100B, SERPINF2, HLA-A, DIRAS1, and TNXB) were good predictors of overall survival (OS). CONCLUSION: We highlighted a molecular expression signature that can differentiate the treatment response, overall survival, and DFS of patients with HER2+ breast cancer.

Differences in B-Cell Immunophenotypes and Neutralizing Antibodies Against SARS-CoV-2 After Administration of BNT162b2 (Pfizer-BioNTech) Vaccine in Individuals with and without Prior COVID-19 - A Prospective Cohort Study.

Purpose: Understanding the humoral immune response dynamics carried out by B cells in COVID-19 vaccination is little explored; therefore, we analyze the changes induced in the different cellular subpopulations of B cells after vaccination with BNT162b2 (Pfizer-BioNTech). Methods: This prospective cohort study evaluated thirty-nine immunized health workers (22 with prior COVID-19 and 17 without prior COVID-19) and ten subjects not vaccinated against SARS-CoV-2 (control group). B cell subpopulations (transitional, mature, naïve, memory, plasmablasts, early plasmablast, and double-negative B cells) and neutralizing antibody levels were analyzed and quantified by flow cytometry and ELISA, respectively. Results: The dynamics of the B cells subpopulations after vaccination showed the following pattern: the percentage of transitional B cells was higher in the prior COVID-19 group (p < 0.05), whereas virgin B cells were more prevalent in the group without prior COVID-19 (p < 0.05), mature B cells predominated in both vaccinated groups (p < 0.01), and memory B cells, plasmablasts, early plasmablasts, and double-negative B cells were higher in the not vaccinated group (p < 0.05). Conclusion: BNT162b2 vaccine induces changes in B cell subpopulations, especially generating plasma cells and producing neutralizing antibodies against SARS-CoV-2. However, the previous infection with SARS-CoV-2 does not significantly alter the dynamics of these subpopulations but induces more rapid and optimal antibody production.

Th1/Th2 Balance in Young Subjects: Relationship with Cytokine Levels and Metabolic Profile.

PURPOSE: We aim to identify Th1 and Th2 cell clusters in young subjects, including their clinical and metabolic characteristics and the Th1/Th2 balance. PATIENTS AND METHODS: A total of 100 participants were included. The frequencies of Th1 and Th2 cells in peripheral blood were determined by flow cytometry. Serum C-reactive protein was measured using a turbidimetric assay, and insulin levels were quantified with an enzyme-linked immunosorbent assay. Circulating cytokine levels were analyzed using a multiplex system. RESULTS: A cluster analysis was performed to determine the Th1/Th2 balance in a group of young people, and 3 clusters were formed with the following characteristics: 1) subjects with a higher prevalence of hyperglycemia (38%), dyslipidemia (38-75%), and insulin resistance (50%), as well as a higher percentage of Th1 cells and Th1/Th2 ratio, including elevated IFN-É£ levels; 2) subjects with a lower prevalence of hyperglycemia (23%) and insulin resistance (15.4%), but a higher prevalence of dyslipidemia (8-85%) with a predominance of Th2 cells, and lower Th1/Th2 ratio; 3) subjects with a lower prevalence of hyperglycemia (6%), insulin resistance (41%), and dyslipidemia (10-63%), as well as a balance of Th1 and Th2 cells and lower Th1/Th2 ratio, including low IFN-É£ levels. Positive correlations between Th1 cells with IFN-γ, IL-12, and IL-1ß and between Th2 cells with IFN-γ, IL-2, and IL-4 were found (p < 0.05). A significant increase in Th1 cells was observed in the presence of hyperglycemia and high LDL-C levels, as well as increased Th2 cells in the absence of abdominal obesity and high blood pressure, including low HDL-C levels. The Th1/Th2 ratio was higher in the group with high cardiometabolic risk (p = 0.03). CONCLUSION: Th1/Th2 balance is related to metabolic abnormalities that may occur in young population, and thus the timely identification of different phenotypes may help predict an increased cardiometabolic risk.