HIV rapidly targets a diverse pool of CD4+ T cells to establish productive and latent infections.

Upon infection, HIV disseminates throughout the human body within 1-2 weeks. However, its early cellular targets remain poorly characterized. We used a single-cell approach to retrieve the phenotype and TCR sequence of infected cells in blood and lymphoid tissue from individuals at the earliest stages of HIV infection. HIV initially targeted a few proliferating memory CD4+ T cells displaying high surface expression of CCR5. The phenotype of productively infected cells differed by Fiebig stage and between blood and lymph nodes. The TCR repertoire of productively infected cells was heavily biased, with preferential infection of previously expanded and disseminated clones, but composed almost exclusively of unique clonotypes, indicating that they were the product of independent infection events. Latent genetically intact proviruses were already archived early in infection. Hence, productive infection is initially established in a pool of phenotypically and clonotypically distinct T cells, and latently infected cells are generated simultaneously.

IFN-γ ELISpot-enabled machine learning for culprit drug identification in nonimmediate drug hypersensitivity.

BACKGROUND: Diagnosing drug-induced allergy, especially nonimmediate phenotypes, is challenging. Incorrect classifications have unwanted consequences. OBJECTIVE: We sought to evaluate the diagnostic utility of IFN-γ ELISpot and clinical parameters in predicting drug-induced nonimmediate hypersensitivity using machine learning. METHODS: The study recruited 393 patients. A positive patch test or drug provocation test (DPT) was used to define positive drug hypersensitivity. Various clinical factors were considered in developing random forest (RF) and logistic regression (LR) models. Performances were compared against the IFN-γ ELISpot-only model. RESULTS: Among the 102 patients who had 164 DPTs, most patients had severe cutaneous adverse reactions (35/102, 34.3%) and maculopapular exanthems (33/102, 32.4%). Common suspected drugs were antituberculosis drugs (46/164, 28.1%) and ß-lactams (42/164, 25.6%). Mean (SD) age of patients with DPT was 52.7 (20.8) years. IFN-γ ELISpot, fixed drug eruption, Naranjo categories, and nonsteroidal anti-inflammatory drugs were the most important features in all developed models. The RF and LR models had higher discriminating abilities. An IFN-γ ELISpot cutoff value of 16.0 spot-forming cells/106 PBMCs achieved 94.8% specificity and 57.1% sensitivity. Depending on clinical needs, optimal cutoff values for RF and LR models can be chosen to achieve either high specificity (0.41 for 96.1% specificity and 0.52 for 97.4% specificity, respectively) or high sensitivity (0.26 for 78.6% sensitivity and 0.37 for 71.4% sensitivity, respectively). CONCLUSIONS: IFN-γ ELISpot assay was valuable in identifying culprit drugs, whether used individually or incorporated in a prediction model. Performances of RF and LR models were comparable. Additional test datasets with DPT would be helpful to validate the model further.

A germline STAT6 gain-of-function variant is associated with early-onset allergies.

BACKGROUND: The signal transducer and activator of transcription 6 (STAT6) signaling pathway plays a central role in allergic inflammation. To date, however, there have been no descriptions of STAT6 gain-of-function variants leading to allergies in humans. OBJECTIVE: We report a STAT6 gain-of-function variant associated with early-onset multiorgan allergies in a family with 3 affected members. METHODS: Exome sequencing and immunophenotyping of T-helper cell subsets were conducted. The function of the STAT6 protein was analyzed by Western blot, immunofluorescence, electrophoretic mobility shift assays, and luciferase assays. Gastric organoids obtained from the index patient were used to study downstream effector cytokines. RESULTS: We identified a heterozygous missense variant (c.1129G>A;p.Glu377Lys) in the DNA binding domain of STAT6 that was de novo in the index patient's father and was inherited by 2 of his 3 children. Severe atopic dermatitis and food allergy were key presentations. Clinical heterogeneity was observed among the affected individuals. Higher levels of peripheral blood TH2 lymphocytes were detected. The mutant STAT6 displayed a strong preference for nuclear localization, increased DNA binding affinity, and spontaneous transcriptional activity. Moreover, gastric organoids showed constitutive activation of STAT6 downstream signaling molecules. CONCLUSIONS: A germline STAT6 gain-of-function variant results in spontaneous activation of the STAT6 signaling pathway and is associated with an early-onset and severe allergic phenotype in humans. These observations enhance our knowledge of the molecular mechanisms underlying allergic diseases and will potentially contribute to novel therapeutic interventions.

Downregulation of LAMB3 Altered the Carcinogenic Properties of Human Papillomavirus 16-Positive Cervical Cancer Cells.

Nearly all cervical cancer cases are caused by infection with high-risk human papillomavirus (HR-HPV) types. The mechanism of cervical cell transformation is related to the powerful action of viral oncoproteins and cellular gene alterations. Transcriptomic data from cervical cancer and normal cervical cells were utilized to identify upregulated genes and their associated pathways. The laminin subunit beta-3 (LAMB3) mRNAwas overexpressed in cervical cancer and was chosen for functional analysis. The LAMB3 was predominantly expressed in the extracellular region and the plasma membrane, which play a role in protein binding and cell adhesion molecule binding, leading to cell migration and tissue development. LAMB3 was found to be implicated in the pathway in cancer and the PI3K-AKT signaling pathway. LAMB3 knockdown decreased cell migration, invasion, anchorage-dependent and anchorage-independent cell growth and increased the number of apoptotic cells. These effects were linked to a decrease in protein levels involved in the PI3K-AKT signaling pathway and an increase in p53 protein. This study demonstrated that LAMB3 could promote cervical cancer cell migration, invasion and survival.

Beta-lactam hypersensitivity diagnosis in ambulatory and hospitalized settings require different approaches.

BACKGROUND: Data on beta-lactam hypersensitivity (BLH) are mainly focused on immediate or mild nonimmediate reactions in the ambulatory setting, but limited in patients with concurrent illness and moderate-to-severe nonimmediate reactions in the hospitalized setting. OBJECTIVE: To investigate the entire spectrum of BLH in Thai tertiary hospital. METHODS: Clinical characteristics of 357 patients with suspected BLH were evaluated in a 7-year period. Culprit drug identification was performed in 335 patients by combined skin testing, in vitro testing, or drug provocation tests. RESULTS: The predominant BLH presentations were non-immunoglobulin (Ig)E-mediated reactions with severe cutaneous adverse reactions of 18.9%, and BLH status was definitively confirmed in 18.1%. The most common verified culprits were cephalosporins (34.8%), particularly in hypersensitivity type IV reactions. Natural penicillins were the main implicated drugs in 48.5% of ambulatory patients. In contrast, cephalosporins and carbapenems were the main implicated drugs in hospitalized patients. Non-IgE-mediated anaphylaxis and serum sickness-like reaction remained diagnostically challenged. New generations of beta-lactams, hospitalized patients, recent allergic history, and underlying malignancies or autoimmune diseases were associated with increased BLH risk. CONCLUSION: At present, cephalosporins are the leading causes of BLH, particularly in non-IgE-mediated reactions. More research on the verification of non-IgE hypersensitivity reactions from new generations of beta-lactams should be better emphasized. CLINICAL TRIAL REGISTRATION: The registry was approved by the Ethics and Research Committee of the Faculty of Medicine, Chulalongkorn University, and listed on ClinicalTrials.gov (Identifier: NCT01667055; https://www. CLINICALTRIALS: gov/ct2/show/NCT01667055).

Exosomal microRNAs from PBMCs stimulated with culprit drugs enhanced keratinocyte cell death in Stevens-Johnson syndrome/toxic epidermal necrolysis.

BACKGROUND: Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) and drug reactions with eosinophilia and systemic symptoms (DRESS) are both severe cutaneous adverse reactions. Keratinocyte death is much more prominent in SJS/TEN compared to DRESS. OBJECTIVE: This study aimed to investigate the role of exosomal miRNAs on keratinocyte death in SJS/TEN. METHODS: Peripheral blood mononuclear cells (PBMCs) from SJS/TEN and DRESS patients were stimulated with the culprit drugs. The exosomes released in cell supernatants were co-incubated with HaCaT cells to study the cytotoxic effects on keratinocytes. Exosomal miRNA sequencing analysis was performed to compare the expression patterns between SJS/TEN and DRESS subjects. HaCaT cells were then transfected with miRNA mimics and inhibitors to explore the functions of miRNAs on keratinocyte cell death. RESULTS: Cytotoxic effects of PBMC-derived exosomes on keratinocytes were demonstrated in SJS/TEN and could be neutralized with exosome inhibitors. Cytotoxic effects of PBMC-derived exosomes from SJS/TEN subjects were higher after incubating PBMCs with the culprit drugs than those incubating with irrelevant drugs and unstimulated controls. The sequencing data revealed differential expressions of 61 exosomal miRNAs between SJS/TEN and DRESS. Exosomal miR-4488 was upregulated while miR-486-5p, miR-96-5p and miR-132-3p were downregulated in SJS/TEN compared to DRESS as determined by quantitative real-time PCR. The increased percentage of apoptotic cells upon transfection of HaCat cells was 36.3% and 34.9% with miR-4488 mimic and miR-96-5p inhibitor, respectively. CONCLUSION: This study illustrated the regulatory functions of exosomal miRNAs in controlling keratinocyte death in SJS/TEN. Exosome inhibitors might have a therapeutic role in SJS/TEN.

Paradoxically Greater Persistence of HIV RNA-Positive Cells in Lymphoid Tissue When ART Is Initiated in the Earliest Stage of Infection.

Starting antiretroviral therapy (ART) in Fiebig 1 acute HIV infection limits the size of viral reservoirs in lymphoid tissues, but does not impact time to virus rebound during a treatment interruption. To better understand why the reduced reservoir size did not increase the time to rebound we measured the frequency and location of HIV RNA+ cells in lymph nodes from participants in the RV254 acute infection cohort. HIV RNA+ cells were detected more frequently and in greater numbers when ART was initiated in Fiebig 1 compared to later Fiebig stages and were localized to the T-cell zone compared to the B-cell follicle with treatment in later Fiebig stages. Variability of virus production in people treated during acute infection suggests that the balance between virus-producing cells and the immune response to clear infected cells rapidly evolves during the earliest stages of infection. Clinical Trials Registration: NCT02919306.

Transcriptomic analysis of peripheral blood mononuclear cells in head and neck squamous cell carcinoma patients.

OBJECTIVES: To investigate the gene expression profile of peripheral blood mononuclear cells (PBMCs) from head and neck squamous cell carcinoma (HNSCC), including oral cancer (OC) and oropharyngeal cancer (OPC) patients, and compare them with healthy controls (HC). MATERIALS AND METHODS: Transcriptomic analysis of PBMCs was performed by RNA-sequencing. The upregulated candidate genes were selected for validation by quantitative real-time polymerase chain reaction (qPCR). In addition, related plasma protein levels were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Three significantly upregulated genes, including high mobility group nucleosomal binding domain 2 (HMGN2), folate receptor gamma (FOLR3), and amphiregulin (AREG), were selected. In the first cohort, the results showed that only HMGN2 expression was significantly increased in OC patients. In the larger sample size, the overall results demonstrated that HMGN2 expression had a tendency to increase in both OC and OPC patients compared with HC. Interestingly, the plasma HMGN2 (HMG-17) protein level exhibited the same trend as that observed at the transcriptional level. CONCLUSION: HMGN2 expression and plasma HMG-17 (HMGN2 protein) were increased in both cancer patients compared with HC. This gene may be important for further functional studies in the PBMCs of HNSCC patients.

Correlations between serum interleukin 6, serum soluble interleukin 6 receptor, and disease activity in systemic juvenile idiopathic arthritis patients treated with or without tocilizumab.

INTRODUCTION: Interleukin (IL)-6 is a proinflammatory cytokine involved in systemic juvenile idiopathic arthritis (SJIA). Since these patients are often treated with tocilizumab (TCZ), anti-IL-6 receptor (IL-6R) antibody, we investigated correlations between serum IL-6 and soluble IL-6R-levels and disease activity in SJIA patients treated with or without TCZ. MATERIAL AND METHODS: 164 serum samples were taken from 42 SJIA patients treated with or without TCZ (69 and 95 samples, respectively). Patients were assigned to three groups according to disease status: 1) systemic (patients with systemic features and/or arthritis), 2) arthritis (patients with arthritis but no systemic features), and 3) inactive (clinically inactive disease). Disease activity was assessed using the Juvenile Arthritis Disease Activity Score-27 (JADAS-27) at the time of blood collection. RESULTS: IL-6 levels were highest in SJIA patients with predominant systemic features, while serum sIL-6R levels were highest in patients with persistent arthritis. Serum IL-6 correlated with JADAS-27 in patients treated with and without TCZ (r = 0.38 and r = 0.65, respectively), whereas serum sIL-6R levels correlated with JADAS-27 in patients treated without (r = 0.30) but not with (r = -0.14) TCZ. The sIL-6R/IL-6 ratio negatively correlated with JADAS-27 in patients treated with and without TCZ (r = -0.49 and r = -0.56, respectively). CONCLUSIONS: Serum IL-6 levels correlated more strongly with disease activity parameters than did sIL-6R levels and could be useful for monitoring disease activity in SJIA patients. The sIL-6R/IL-6 ratio might be a promising disease activity marker in both SJIA patients treated with and without TCZ.

Preservation of Peripheral T Follicular Helper Cell Function in HIV Controllers.

The maturation process of high-affinity antibodies is a result of intricate interactions between B cells and follicular helper T (Tfh) cells occurring in lymphoid germinal centers. HIV infection induces significant chronic immune activation, phenotypic skewing, and inflammation driven by years of continuous viral replication. High levels of viremia as well as immune activation and dysfunction have been demonstrated to have a perturbing impact on the B cell memory compartment and contribute to B cell exhaustion. Counterintuitively, the factors associated with perturbation of the B cell compartment seem to be favorable for the generation of highly affinity-matured Env-specific antibodies in a minority of HIV-infected individuals. Thus, the impact of HIV antigenemia on B cells and Tfh cell interactions warrants further exploration. We therefore studied immunophenotypes of HIV-specific B cells in individuals with differing levels of viral control using HIV Env gp120 probes and characterized the functionality of matched T cells in peripheral blood. While CXCR5+ CD4+ T cells were significantly diminished in HIV progressors, we found that a small subset of gp120-specific interleukin-21 (IL-21)-secreting CXCR5+ CD4+ T cells were significantly associated with gp120-specific B cell frequencies. In contrast, neither bulk CXCR5+ CD4+ T cells nor other HIV antigen specificities were associated with gp120-specific B cell levels. HIV-specific B cells derived from elite controllers displayed greater amounts of gp120-specific B cells in the resting memory subset, whereas HIV-specific B cells in progressors accumulated in tissue-like and activated memory subsets. Furthermore, CXCR5+ CD4+ T cells from elite controllers showed a stronger ex vivo capacity to induce B cell maturation and immunoglobulin class switching than cells from HIV progressors.IMPORTANCE Dissecting the factors that are involved in B cell maturation and antibody development is important for HIV vaccine design. In this study, we found that HIV Env-specific CXCR5+ CD4+ T cells that secrete interleukin-21 are strongly associated with B cell memory phenotypes and function. Moreover, we found that the immune responses of HIV controllers showed intrinsically better helper activity than those of HIV progressors.

Slow desensitization of imatinib-induced nonimmediate reactions and dynamic changes of drug-specific CD4+CD25+CD134+ lymphocytes.

BACKGROUND: Imatinib is a tyrosine kinase inhibitor indicated for the treatment of gastrointestinal stromal tumors (GISTs) and certain neoplastic diseases; however, nonimmediate adverse reactions are common. OBJECTIVE: To describe the process of imatinib slow desensitization in patients who experienced nonimmediate reactions to imatinib and the dynamic change in drug-specific CD4+CD25+CD134+ T-lymphocyte percentages. METHODS: Five patients diagnosed as having GISTs and with a recent history of imatinib-induced nonimmediate reactions (maculopapular exanthema with eosinophilia, exfoliative dermatitis, palmar-plantar erythrodysesthesia, and drug rash with eosinophilia and systemic symptoms) were desensitized using a slow desensitization protocol. The reintroduced imatinib dosage was stepped up every week starting from 10 mg/d and increasing to 25, 50, 75, 100, 150, 200, and 300 mg/d until the target dose of 400 mg/d was achieved. Prednisolone of up to 30 mg/d was allowed if allergic reactions recurred. The percentages of CD4+CD25+CD134+ T cells present after incubating peripheral blood mononuclear cells with imatinib, at baseline and after successful desensitization, were analyzed using flow cytometric analysis. RESULTS: By using a slow desensitization technique, all patients were able to receive 400 mg/d of imatinib, and prednisolone was gradually tapered off. The percentages of imatinib-induced CD4+CD25+CD134+ T cells decreased from a mean (SD) of 11.3% (6.5%) and 13.4% (7.3%) at baseline to 3.2% (0.7%) and 3.0% (1.1%) after successful desensitization, when stimulating peripheral blood mononuclear cells with 1 and 2 µM of imatinib, respectively. CONCLUSION: Slow desensitization is a helpful procedure in treating patients with imatinib-induced nonimmediate reactions other than simple maculopapular exanthema. The reduced percentages of imatinib-induced CD4+CD25+CD134+ T cells in these patients may be associated with immune tolerance.

Determination phase at transition of gonocytes to spermatogonial stem cells improves establishment efficiency of spermatogonial stem cells in domestic cats.

The development of germ cells has not been entirely documented in the cat especially the transition phase of the gonocyte to the spermatogonial stem cell (G/SSC). The aims of study were to examine testicular development and to identify the G/SSC transition in order to isolate and culture SSCs in vitro. Testes were divided into 3 groups according to donor age (I, < 4 months; II, 4-6 months; and III, > 6 months). In Exp. 1, we studied testicular development by histology, transmission electron microscopy and immunohistochemistry. In Exp. 2, we determined the expression of GFRα-1, DDX-4 and c-kit and performed flow cytometry. The SSCs isolated from groups II and III were characterized by RT-PCR and TEM (Exp. 3). Chronological changes in the G/SSC transition were demonstrated. The size, morphology and ultrastructure of SSCs were distinguishable from those of gonocytes. The results demonstrated that group II contained the highest numbers of SSCs per seminiferous cord/tubule (17.66 ± 2.20%) and GFRα-1(+) cells (14.89 ± 5.66%) compared with the other groups. The findings coincided with an increased efficiency of SSC derivation in group II compared with group III (74.33 ± 2.64% vs. 23.33 ± 2.23%). The colonies expressed mRNA for GFRA1, ZBTB16, RET and POU5F1. Our study found that the G/SSC transition occurs at 4-6 months of age. This period is useful for isolation and improves the establishment efficiency of cat SSCs in vitro.

Antibody persistence of standard versus double three-dose hepatitis B vaccine in liver transplant children: a randomized controlled trial.

Rapid hepatitis B (HB) surface antibody (anti-HBs) loss is prevalent after liver transplantation (LT). Herein, we evaluated anti-HBs persistence after HB vaccination using two regimens in LT children. We recruited 66 previously immunized LT children with anti-HBs level of < 100 mIU/mL. Participants were randomly reimmunized with standard-three-dose (SD) and double-three-dose (DD) intramuscular HB vaccination at 0, 1, and 6 months. Anti-HBs were assessed at every outpatient visit. Antibody loss defined as anti-HBs levels < 100 mIU/mL after three-dose vaccination. After three-dose vaccination, 81.8% and 78.7% of participants in the SD and DD groups, had anti-HBs levels > 100 mIU/mL, with a geometric mean titer (GMT) of 601.68 and 668.01 mIU/mL (P = 0.983). After a mean follow-up of 2.31 years, the anti-HBs GMT was 209.81 and 212.61 mIU/mL in the SD and DD groups (P = 0.969). The number of immunosuppressants used and an anti-HBs level < 1 mIU/mL at baseline were independently associated with anti-HB loss. The DD regimen strongly increased the risk of anti-HBs loss (adjusted hazard ratio, 2.97 [1.21-7.31]; P = 0.018). The SD HB reimmunization regimen effectively maintained protective anti-HBs levels in children undergoing LT, making it the preferred regimen for such children with anti-HB loss.Trial registration: TCTR20180723002.

De novo reconstruction of a functional in vivo-like equine endometrium using collagen-based tissue engineering.

To better understand molecular aspects of equine endometrial function, there is a need for advanced in vitro culture systems that more closely imitate the intricate 3-dimensional (3D) in vivo endometrial structure than current techniques. However, development of a 3D in vitro model of this complex tissue is challenging. This study aimed to develop an in vitro 3D endometrial tissue (3D-ET) with an epithelial cell phenotype optimized by treatment with a Rho-associated protein kinase (ROCK) inhibitor. Equine endometrial epithelial (eECs) and mesenchymal stromal (eMSCs) cells were isolated separately, and eECs cultured in various concentrations of Rock inhibitor (0, 5, 10 µmol) in epithelial medium (EC-medium) containing 10% knock-out serum replacement (KSR). The optimal concentration of Rock inhibitor for enhancing eEC proliferation and viability was 10 µM. However, 10 µM Rock inhibitor in the 10% KSR EC-medium was able to maintain mucin1 (Muc1) gene expression for only a short period. In contrast, fetal bovine serum (FBS) was able to maintain Muc1 gene expression for longer culture durations. An in vitro 3D-ET was successfully constructed using a collagen-based scaffold to support the eECs and eMSCs. The 3D-ET closely mimicked in vivo endometrium by displaying gland-like eEC-derived structures positive for the endometrial gland marker, Fork headbox A2 (FOXA2), and by mimicking the 3D morphology of the stromal compartment. In addition, the 3D-ET expressed the secretory protein MUC1 on its glandular epithelial surface and responded to LPS challenge by upregulating the expression of the interleukin-6 (IL6) and prostaglandin F synthase (PGFS) genes (P < 0.01), along with an increase in their secretory products, IL-6 (P < 0.01) and prostaglandin F2alpha (PGF2α) (P < 0.001) respectively. In the future, this culture system can be used to study both normal physiology and pathological processes of the equine endometrium.

Phase II prefusion non-stabilised Covid-19 mRNA vaccine randomised study.

ChulaCov19 mRNA vaccine demonstrated promising phase 1 results. Healthy adults aged 18-59 years were double-blind randomised 4:1 to receive two intramuscular doses of ChulaCov19 50 µg or placebo. Primary endpoints were safety and microneutralization antibody against-wild-type (Micro-VNT50) at day 50. One hundred fifty adults with median (IQR) age 37 (30-46) years were randomised. ChulaCov19 was well tolerated, and most adverse events were mild to moderate and temporary. Geometric mean titres (GMT) of neutralizing titre against wild-type for ChulaCov19 on day 50 were 1367 IU/mL. T-cell IFN-γ-ELISpot showed the highest responses at one week (Day29) after dose 2 then gradually declined. ChulaCov19 50 µg is well tolerated and elicited high neutralizing antibodies and strong T-cell responses in healthy adults.Trial registration number: ClinicalTrials.gov Identifier NCT04566276, 28/09/2020.

The potential of using enzyme-linked immunospot to diagnose cephalosporin-induced maculopapular exanthems.

There is no reliable test to diagnose cephalosporin-induced maculopapular exanthems (MPE). This study aimed to evaluate the role of enzyme-linked immunospot assay in the diagnosis of cephalosporin-induced MPE compared with skin testing. A total of 25 patients with a history of cephalosporin-induced MPE were skin tested and the frequencies of cephalosporin-specific interferon-γ-, interleukin-5-, and interleukin-10-releasing cells/10(6) peripheral blood mononuclear cells were measured after stimulating with the culprit drug, compared with 20 non-allergic controls. Values greater than means+2 standard deviations of the values in non-allergic controls were considered diagnostic. The study showed that the combination of interferon-γ and interleukin-5 enzyme-linked immunospot assays was more sensitive than skin testing to diagnose cephalosporin allergy (40% vs. 8%, p = 0.008) and sensitivity increased to 57.1% when the test was performed within 2 years of the drug reaction. Enzyme-linked immunospot assay is a promising tool for confirming the diagnosis of cephalosporin-induced MPE.

Interleukin-2 levels in exhaled breath condensates, asthma severity, and asthma control in nonallergic asthma.

There was evidence that interleukin (IL)-2 and IL-10 in the airways play roles in regulating the asthmatic inflammatory response. The purpose of this study was to measure the levels of these cytokines in exhaled breath condensates (EBCs) from asthmatic airways and their correlation with a clinical assessment of asthma severity. The levels of IL-2 and IL-10 in EBC, Asthma Control Test (ACT) score, and the forced expiratory volume in 1 second (FEV1) were studied in 28 steroid-free asthmatic patients and 10 healthy volunteers. The results were analyzed according to their allergic status, asthma severity, and body weight. The correlations between IL-2 and IL-10 levels, percent predicted FEV1, ACT score, and body mass index were also determined. Both IL-2 and IL-10 levels in EBC significantly increased in asthmatic patients, especially in patients with moderate-to-severe persistent asthma, compared with those in normal controls. However, the signification correlations between IL-2 levels and ACT (r = -0.684; p = 0.007), as well as with percent predicted FEV1 (r = -0.671; p = 0.009), were established only in patients with nonallergic asthma. The elevation of IL-2 levels in EBC in obese subjects was observed but was probably related to asthma severity. The levels of IL-2 and IL-10 in EBC increase in asthmatic patients but only IL-2 levels significantly correlate with the ACT score and percent predicted FEV1 in nonallergic asthma. Additional studies should be explored to confirm the reliability of ACT score as a predictor of inflammatory response in asthmatic airways. Clinical trial NCT01246414, http://www.clinicaltrials.gov.

Efficiency of CIN2+ Detection by Thyrotropin-Releasing Hormone (TRH) Site-Specific Methylation.

Cervical cancer screening typically involves a Pap smear combined with high-risk human papillomavirus (hr-HPV) detection. Women with hr-HPV positivity but normal cytology, as well as those with precancerous abnormal cytology, such as low-grade squamous intraepithelial lesions (LSIL) and high-grade SIL (HSIL), are referred for colposcopy and histology examination to identify abnormal lesions, such as cervical intraepithelial neoplasia (CIN) and cervical cancer. However, in order to enhance the accuracy of detection, bioinformatics analysis of a microarray database was performed, which identified cg01009664, a methylation marker of the thyrotropin-releasing hormone (TRH). Consequently, a real-time PCR assay was developed to distinguish CIN2+ (CIN2, CIN3, and cervical cancer) from CIN2- (CIN1 and normal cervical epithelia). The real-time PCR assay utilized specific primers targeting methylated cg01009664 sites, whereas an unmethylated reaction was used to check the DNA quality. A cut-off value for the methylated reaction of Ct < 33 was established, resulting in improved precision in identifying CIN2+. In the first cohort group, the assay demonstrated a sensitivity of 93.7% and a specificity of 98.6%. In the cytology samples identified as atypical squamous cells of undetermined significance (ASC-US) and LSIL, the sensitivity and specificity for detecting CIN2+ were 95.0% and 98.9%, respectively. However, when self-collected samples from women with confirmed histology were tested, the sensitivity for CIN2+ detection dropped to 49.15%, while maintaining a specificity of 100%. Notably, the use of clinician-collected samples increased the sensitivity of TRH methylation testing. TRH methylation analysis can effectively identify women who require referral for colposcopy examinations, aiding in the detection of CIN2+.

Effects of Chrysin on Oral Squamous Cell Carcinoma In Vitro.

OBJECTIVE: Chrysin is a hydroxylated flavonoid derived from "propolis or bee glue," a natural product. Previous research on chrysin's biological functions, including anticancer activity, had been reported. However, chrysin's effect on oral squamous cell carcinoma (OSCC) is still scarce. This article aimed to test the cytotoxicity, antiproliferative, antimigration, anti-invasion, and apoptotic effects of purified chrysin in two OSCC cell lines, HSC4 and SCC25. MATERIALS AND METHODS: The malignant phenotype was assessed using cell proliferation, wound healing, and transwell assays. Cell apoptosis was determined using flow cytometry. The positive control was OSCC cells treated with cisplatin, and the negative control was OSCC cells incubated with 0.1% dimethyl sulfoxide. RESULTS: Chrysin at concentrations of 100 and 200 µM could inhibit OSCC cell proliferation, migration, and invasion, as well as enhance cell apoptosis, particularly in the early stages of apoptosis. CONCLUSION: In OSCC cell lines, chrysin has been demonstrated to be an effective antioncogenic agent. Additional research is required to confirm the results. Chrysin should be suggested as a possible alternative therapeutic application for OSCC.