Specific reference interval for high-sensitivity cardiac troponin I among healthy children in Wuhan, China.

Background: Troponin (Tn) is of an important biomarker for the diagnosis and prognosis of myocardial injury and for evaluating the severity of cardiac involvement due to other systemic diseases in children. Unfortunately, high-sensitivity cardiac troponin I (hs-cTnI) specific reference intervals (RIs) are extremely limited. This study aimed to establish a preliminary pediatric hs-cTnI RI for newborns, children, and adolescents in Wuhan, China. Methods: A total of 1,355 healthy participants (1 day to 19 years) were recruited from a cross-sectional study implemented in Wuhan Children's Hospital from September 2022 to August 2023. Serum hs-cTnI levels were detected via the Mindray automated chemiluminescence immunoassay analyzer (CL-6000i). Specific serum hs-cTnI RIs were established according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. The RIs were defined by the nonparametric median (P50), and 2.5th, 97.5th [P50 (P2.5-P97.5)] intervals. Results: Of the 1,355 pediatric participants, serum hs-cTnI concentrations of 1,332 children were measured. The serum overall P50 and 95% interval range (P2.5-P97.5) of serum hs-cTnI was 0.41 (0.00, 44.31) ng/L. This was higher in males of 0.47 (0.00, 44.90) ng/L than in females of 0.36 (0.00, 44.17) ng/L (P<0.01). Age- and sex-specific differences in hs-cTnI levels were observed. The levels were highly variable in children under 1 year of age (especially in newborns), deriving a P50 (P2.5-P97.5) of 22.06 (1.04, 154.22) ng/L, and gradually narrowed and decreased with increasing age, with a markedly lower established P50 (P2.5-P97.5) of 0.36 (0.00, 2.16) ng/L. However, the levels began to increase slightly at the age of 9-12 years and reached a small peak at the age range of 15 to 18 years in males with 0.71 (0.03, 3.29) ng/L, while females were less affected by puberty. Sex- and age-specific RIs for hs-cTnI were established: 5 age-specific RIs in males, 1 day-1 month: 30.16 (8.67, 171.81) ng/L; >1-12 months: 13.20 (0.63, 61.91) ng/L; >1-15 years: 0.36 (0.00, 1.86) ng/L; >15-18 years: 0.71 (0.03, 3.29) ng/L; >18-19 years: 0.52 (0.00, 1.92) ng/L; and 4 age-specific RIs in females, 1 day-1 month: 43.93 (18.82, 146.38) ng/L; >1-12 months: 5.22 (0.92, 42.54) ng/L; >1-6 years: 0.54 (0.00, 2.74) ng/L; >6-19 years: 0.23 (0.00, 1.56) ng/L. Conclusions: This study preliminarily established age- and sex-specific serum hs-cTnI RIs using the Mindray CL-6000i system in healthy children in Wuhan, China.

Epidemiological and clinical profile of pediatric hepatitis B virus infections in Wuhan: a retrospective cohort study.

BACKGROUND: Hepatitis B virus (HBV) remains a substantial public health safety concern drawing considerable attention in China and globally. The detection of HBV serological markers can enable the assessment of HBV infection and replication status in vivo and evaluate the body's protection against HBV. Therefore, this study aims to identify the epidemiological and clinical characteristics of HBV infection in children to prevent and control HBV infection in Wuhan areas. METHODS: We conducted an extensive retrospective cohort analysis of 115,029 individuals aged 0-18 years who underwent HBV serological markers detection for HBV infection in hospital between 2018 and 2021 using Electrochemiluminescence immunoassay. We generated descriptive statistics and analysed HBV infection's epidemiological and clinical characteristics between different sex and age groups. RESULTS: The overall positive detection rates of HBsAg, HBsAb, HBeAg, HBeAb, and HBcAb in all participants were 0.13%, 79.09%, 0.17%, 2.81%, and 5.82%, respectively. The positive rate of HBeAb and HBcAb in males was significantly lower than that in females (2.64% vs. 3.13%, 5.56% vs. 6.29%) (P < 0.05). Twenty-two distinct HBV serological expression patterns were revealed. Among them, 8 common expression patterns accounted for 99.63%, while the remaining 14 uncommon expression patterns were primarily observed in neonatal patients with HBV infection. There are no significant differences in serological patterns based on sex (P < 0.05). The overall HBV infection detection rate was 5.82% [range 5.68-5.95] and showed a declining yearly trend. The rate in females was higher than that in males 6.29% [6.05, 6.35] vs. 5.56% [5.39, 5.59]. The overall HBV diagnostic rate over 4 years was 0.20% [0.17, 0.22], and the rate declined yearly. The prevalence of acute infection was higher than that of other infection types before 2019, but the incidence of unclassified infection showed a significant upward trend after 2019. CONCLUSIONS: While the overall HBV infection detection rate in children has decreased year by year, the infection rate remains high in children under one year and between 4 and 18 years. This continued prevalence warrants heightened attention and vigilance.

Serum level of S100A8/A9 as a biomarker for establishing the diagnosis and severity of community-acquired pneumonia in children.

Background: S100A8/A9, which is a member of S100 proteins, may be involved in the pathophysiology of Community-acquired pneumonia (CAP) that seriously threatens children's health. However, circulating markers to assess the severity of pneumonia in children are yet to be explored. Therefore, we aimed to investigate the diagnostic performance of serum S100A8/A9 level in determining the severity of CAP in children. Methods: In this prospective and observational study, we recruited 195 in-hospital children diagnosed with CAP. In comparison, 63 healthy children (HC) and 58 children with non-infectious pneumonia (pneumonitis) were included as control groups. Demographic and clinical data were collected. Serum S100A8/A9 levels, serum pro-calcitonin concentrations, and blood leucocyte counts were quantified. Results: The serum S100A8/A9 levels in patients with CAP was 1.59 ± 1.32 ng/mL, which was approximately five and two times higher than those in healthy controls and those in children with pneumonitis, respectively. Serum S100A8/A9 was elevated parallelly with the clinical pulmonary infection score. The sensitivity, specificity, and Youden's index of S100A8/A9 ≥1.25 ng/mL for predicting the severity of CAP in children was optimal. The area under the receiver operating characteristic curve of S100A8/A9 was the highest among the indices used to evaluate severity. Conclusions: S100A8/A9 may serve as a biomarker for predicting the severity of the condition in children with CAP and establishing treatment grading.

Application value of antibody titres and RNA detection in the early prediction of Mycoplasma pneumoniae pneumonia in children: a retrospective study.

BACKGROUND: Children with Mycoplasma pneumoniae pneumonia (MPP) are prone to a missed diagnosis at the early stages of the disease, which greatly affects the prognosis of children. In this study, the application value of Mycoplasma pneumoniae (MP) antibody titres and RNA detection for diagnosing MP infection in children with community-acquired pneumonia (CAP) was evaluated. The present study aimed to seek appropriate detection methods and strategies for early rapid diagnosis in children with MPP. METHODS: A retrospective study was conducted on 563 paediatric patients aged 1 month to 15 years with CAP who were admitted to Wuhan Children's Hospital, Tongji Medical College, Huazhong University of Science and Technology between July 2021 and February 2022. In all patients, throat swabs were collected for MP-RNA detection (simultaneous amplification and testing, SAT), and paired serum samples were collected for MP total antibody detection (particle agglutination, PA). RESULTS: The classification as MPP or non-MPP was based on clinical diagnosis, serum MP antibody titre, and clinical or laboratory evidence of infection by other pathogen(s). Among the 563 patients with pneumonia, 187 patients were in the MPP group, and 376 patients were in the non-MPP group. The Kappa values between the particle agglutination test at different titres (1:80, 1:160) and MP-RNA detection were 0.612 and 0.660 (P<0.01), and the consistency of the three methods was acceptable. When the single screening method was used, MP-RNA had the highest sensitivity (93.05%), while PA (1:160) had the highest specificity (100%). PA (1:80), with an area under the curve (AUC) of 0.822, was better than PA (1:160), with an AUC of 0.783, and there was a significant difference. When the combined screening methods were used, the AUC of MP-RNA parallel PA (1:160) was significantly higher than that of titres (1:80) (z=-4.906, P < 0.01). Except for MP-80, the efficacy of the other three test methods in females was slightly better than that in males. Among the differences in age distribution, PA (1:80) was slightly less effective in the 13-72 months age group than at other ages, and MP-RNA parallel PA (1:160) was slightly better than the younger age group (≤ 36 m). In the older age group (> 36 m), PA (1:160) was just the opposite, while MP-RNA was slightly better than other age groups in the 13-72 months age group. CONCLUSIONS: For the diagnosis of MPP in children at the early of the disease, the antibody titre (1:160) parallel MP-RNA should be given preference, and then the disease should be further classified according to the antibody titre level and the age of the child. The combined application of the two detection methods could complement each other and strengthen the advantages, providing reliable laboratory evidence for the clinical diagnosis and timely treatment of MPP. When using the PA method alone to provide a reference standard to clarify MP infection, the differential diagnosis ability of 1:80 for MPP is better than 1:160, especially for children younger than 36 months.

High-Risk HPV16 E6 Activates the cGMP/PKG Pathway Through Glycosyltransferase ST6GAL1 in Cervical Cancer Cells.

Alterations in glycosylation regulate fundamental molecular and cellular processes of cancer, serving as important biomarkers and therapeutic targets. However, the potential association and regulatory mechanisms of E6 oncoprotein on glycosylation of cervical cancer cells are still unclear. Here, we evaluated the glycomic changes via using Lectin microarray and determined the corresponding enzymes associated with endogenous high-risk HPV16 E6 expression in cervical cancer cells. α-2,6 sialic acids and the corresponding glycosyltransferase ST6GAL1 were significantly increased in E6 stable-expressing HPV- cervical cancer C33A cells. Clinical validation further showed that the expression of ST6GAL1 was significantly increased in patients infected with high-risk HPV subtypes and showed a positive association with E6 in cervical scraping samples. Interfering ST6GAL1 expression markedly blocked the oncogenic effects of E6 on colony formulation, proliferation, and metastasis. Importantly, ST6GAL1 overexpression enhanced tumorigenic activities of both E6-positive and E6-negative cells. Mechanistical investigations revealed that E6 depended on activating YAP1 to stimulate ST6GAL1 expression, as verteporfin (inhibitor of YAP1) significantly suppressed the E6-induced ST6GAL1 upregulation. E6/ST6GAL1 triggered the activation of downstream cGMP/PKG signaling pathway and ODQ (inhibitor of GMP production) simultaneously suppressed the oncogenic activities of both E6 and ST6GAL1 in cervical cancer cells. Taken together, these findings indicate that ST6GAL1 is an important mediator for oncogenic E6 protein to activate the downstream cGMP/PKG signaling pathway, which represents a novel molecular mechanism and potential therapeutic targets for cervical cancer.

Helicobacter urease suppresses cytotoxic CD8+ T-cell responses through activating Myh9-dependent induction of PD-L1.

As a key virulence factor for persistent colonization, urease B subunit (UreB) is considered to be an ideal vaccine antigen against Helicobacter pylori infection. However, the role and molecular mechanisms of UreB involved in immune microenvironment dysregulation still remain largely unknown. In the present study, we evaluated the effects of UreB on macrophage activation and found that UreB induced PD-L1 accumulation on bone marrow-derived macrophages (BMDMs). Co-culture assays further revealed that UreB-induced PD-L1 expression on BMDMs significantly decreased the proliferation and secretion of cytolytic molecules (granzyme B and perforin) of splenic CD8+ T cells isolated from inactivated H. pylori-immunized mice. More importantly, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and co-immunoprecipitation techniques, it has been confirmed that myosin heavy chain 9 (Myh9) is a direct membrane receptor for UreB and is required for PD-L1 up-regulation on BMDMs. Molecular studies further demonstrated that the interaction between UreB and Myh9 decreased GCN2 autophosphorylation and enhanced the intracellular pool of amino acids, leading to the up-regulation of S6K phosphorylation, a commonly used marker for monitoring activation of mTORC1 signaling activity. Furthermore, blocking mTORC1 activation with its inhibitor Temsirolimus reversed the UreB-induced PD-L1 up-regulation and the subsequent inhibitory effects of BMDMs on activation of cytotoxic CD8+ T-cell responses. Overall, our data unveil a novel immunosuppressive mechanism of UreB during H. pylori infection, which may provide valuable clues for the optimization of H. pylori vaccine.

The novel ZEB1-upregulated protein PRTG induced by Helicobacter pylori infection promotes gastric carcinogenesis through the cGMP/PKG signaling pathway.

Helicobacter pylori (H. pylori) is listed as a class I carcinogen in human gastric cancer; however, the underlying mechanisms are poorly understood. In this study, we identified Protogenin (PRTG) was upregulated in both gastric cancer tissues and H. pylori-infected tissues by analyzing dysregulated genes in TCGA and GEO databases. Importantly, upregulated PRTG predicted poor prognosis of gastric cancer patients and integrative analysis revealed that PRTG served as an oncogenic protein in gastric cancer and was required for H. pylori-mediated tumorigenic activities in in vitro cellular and in vivo tumor-bearing mouse models. Mechanistically, H. pylori infection enhanced PRTG expression by promoting transcriptional factor ZEB1 stabilization and recruitment to the PRTG promoter, and which then activated the sub-following cGMP/PKG signaling pathway in bioinformatic and cellular studies. Cellular studies further confirmed that PRTG depended on activating cGMP/PKG axis to promote proliferation, metastasis, and chemoresistance of gastric cancer cells. The PKG inhibitor KT5823 played synergistic anti-tumor effects with cisplatin and paclitaxel to gastric cancer cells in in vitro cellular and in vivo tumor-bearing mouse models. Taken together, our findings suggested that H. pylori infection depends on ZEB1 to induce PRTG upregulation, and which leading to the development and progression of gastric cancer through activating cGMP/PKG signaling pathway. Blocking PRTG/cGMP/PKG axis, therefore, presents a promising novel therapeutic strategy for gastric cancer.

Diagnostic performance of culture filtered protein 10-specific perforin in pediatric patients with active tuberculosis.

BACKGROUND: Mycobacterium tuberculosis (Mtb)-specific perforin were significantly increased in patients with tuberculosis. This study aims to evaluate the diagnosis value of Mtb-specific perforin in pediatric patients with tuberculosis. METHODS: Diagnostic performance of perforin levels induced by 6-kDa early secreted antigen target (ESAT6) or culture filtered protein 10 (CFP10) were evaluated in eighty-six samples from children participants by receiver operating characteristic curve analysis. Flow cytometry was used to detect the expression of perforin and INF-γ of CD4+ , CD8+ T cells in response to CFP10 stimulation. RESULTS: After ex vivo stimulation, levels of ESAT6/CFP10-specific perforin in LTBI patients were significantly higher than active TB (ATB) patients, non-tuberculosis infection (non-TB), and health control (HC) individuals. The diagnostic efficacy of CFP10-specific perforin for TB diagnosis was significantly higher than ESAT6-specific perforin and T-SPOT assay, and when 0.74 ng/mL was taken as the cutoff value, the sensitivity, specificity, and accuracy were 97.83%, 87.5%, and 93.02%. CFP10-specific perforin in both CD4+ and CD8+ T cells were significantly higher in ATB patients compared to HCs and further increased in LTBI patients. However, INF-γ was mainly secreted by CD4+ T cells and showed no significant difference between LTBI and ATB patients. In addition, CFP10-specific perforin can effectively distinguish between ATB and LTBI with the cutoff value of 1.80 ng/mL. Sensitivity and specificity were 88.46% and 85.62%, respectively. CONCLUSIONS: CFP10-specific perforin may be used as a novel cellular immunity-based diagnostic marker of pediatric patients with tuberculosis, and with the potential for discriminating ATB from LTBI.

Efficient production of sTNFRII-gAD fusion protein in large quantity by use of the modified CHO-S cell expression system.

TNFα is one of the initial and important mediators to activate downstream signaling pathways by binding to trimerized TNFα receptors (TNFR), and thus is an ideal drug target for cancer therapy. Taking advantage of intrinsic homotimerization of the globular domain of adiponectin (gAD), we have developed a novel TNFα antagonist, the trimerized fusion protein named sTNFRII-gAD. However, our previously-used CHO expression system yielded less than 10 mg/L of sTNFRII-gAD. To produce large quantities of sTNFRII-gAD efficiently, we used a modified CHO-S cell expression system, which is based on a pMH3 vector with non-coding GC-rich DNA fragments for high-level gene expression. We obtained stable clones that produced 75 mg/L of sTNFRII-gAD in the 96-well plate, adapted the clones to 40 ml suspension serum-free batch culture, then optimized the culturing conditions to scale up the fed-batch culture in a 3 L shake-flask and finally in a 5 L AP30 bioreactor. We achieved a final yield of 52 mg/L of sTNFRII-gAD. The trimerized sTNFRII-gAD exhibited the higher affinity to TNFα with a dissociation constant (Kd) of 5.63 nM than the dimerized sTNFRII-Fc with a Kd of 13.4 nM, and further displayed the higher TNFα-neutralizing activity than sTNFRII-Fc (p<0.05) in a L929 cytotoxicity assay. Therefore, the strategy employed in this study may provide an efficient avenue for large-scale production of other recombinant proteins by use of the modified CHO-S cell expression system.

Lead induces COX-2 expression in glial cells in a NFAT-dependent, AP-1/NFκB-independent manner.

Epidemiologic studies have provided solid evidence for the neurotoxic effect of lead for decades of years. In view of the fact that children are more vulnerable to the neurotoxicity of lead, lead exposure has been an urgent public health concern. The modes of action of lead neurotoxic effects include disturbance of neurotransmitter storage and release, damage of mitochondria, as well as induction of apoptosis in neurons, cerebrovascular endothelial cells, astroglia and oligodendroglia. Our studies here, from a novel point of view, demonstrates that lead specifically caused induction of COX-2, a well known inflammatory mediator in neurons and glia cells. Furthermore, we revealed that COX-2 was induced by lead in a transcription-dependent manner, which relayed on transcription factor NFAT, rather than AP-1 and NFκB, in glial cells. Considering the important functions of COX-2 in mediation of inflammation reaction and oxidative stress, our studies here provide a mechanistic insight into the understanding of lead-associated inflammatory neurotoxicity effect via activation of pro-inflammatory NFAT3/COX-2 axis.