Extended SARS-CoV-2 RBD booster vaccination induces humoral and cellular immune tolerance in mice.

The repetitive applications of vaccine boosters have been brought up in face of continuous emergence of SARS-CoV-2 variants with neutralization escape mutations, but their protective efficacy and potential adverse effects remain largely unknown. Here, we compared the humoral and cellular immune responses of an extended course of recombinant receptor binding domain (RBD) vaccine boosters with those from conventional immunization strategy in a Balb/c mice model. Multiple vaccine boosters after the conventional vaccination course significantly decreased RBD-specific antibody titers and serum neutralizing efficacy against the Delta and Omicron variants, and profoundly impaired CD4+ and CD8+T cell activation and increased PD-1 and LAG-3 expressions in these T cells. Mechanistically, we confirmed that extended vaccination with RBD boosters overturned the protective immune memories by promoting adaptive immune tolerance. Our findings demonstrate potential risks with the continuous use of SARS-CoV-2 vaccine boosters, providing immediate implications for the global COVID-19 vaccination enhancement strategies.

Identification of cross-reactive CD8+ T cell receptors with high functional avidity to a SARS-CoV-2 immunodominant epitope and its natural mutant variants.

Despite the growing knowledge of T cell responses in COVID-19 patients, there is a lack of detailed characterizations for T cell-antigen interactions and T cell functions. Here, with a predicted peptide library from SARS-CoV-2 S and N proteins, we found that specific CD8+ T cell responses were identified in over 75% of COVID-19 convalescent patients (15/20) and an epitope from the N protein, N361-369 (KTFPPTEPK), was the most dominant epitope from our selected peptide library. Importantly, we discovered 2 N361-369-specific T cell receptors (TCRs) with high functional avidity that were independent of the CD8 co-receptor. These TCRs exhibited complementary cross-reactivity to several presently reported N361-369 mutant variants, as to the wild-type epitope. Further, the natural functions of these TCRs in the cytotoxic immunity against SARS-CoV-2 were determined with dendritic cells (DCs) and the lung organoid model. We found that the N361-369 epitope could be normally processed and endogenously presented by these different types of antigen presenting cells, to elicit successful activation and effective cytotoxicity of CD8+ T cells ex vivo. Our study evidenced potential mechanisms of cellular immunity to SARS-CoV-2, and illuminated potential ways of viral clearance in COVID-19 patients. These results indicate that utilizing CD8-independent TCRs against SARS-CoV-2-associated antigens may provide functional superiority that is beneficial for the adoptive cell immunotherapies based on natural or genetically engineered T cells. Additionally, this information is highly relevant for the development of the next-generation vaccines with protections against continuously emerged SARS-CoV-2 mutant strains.

A Highly Conserved Peptide Vaccine Candidate Activates Both Humoral and Cellular Immunity Against SARS-CoV-2 Variant Strains.

Facing the imminent need for vaccine candidates with cross-protection against globally circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutants, we present a conserved antigenic peptide RBD9.1 with both T-cell and B-cell epitopes. RBD9.1 can be recognized by coronavirus disease 2019 (COVID-19) convalescent serum, particularly for those with high neutralizing potency. Immunization with RBD9.1 can successfully induce the production of the receptor-binding domain (RBD)-specific antibodies in Balb/c mice. Importantly, the immunized sera exhibit sustained neutralizing efficacy against multiple dominant SARS-CoV-2 variant strains, including B.1.617.2 that carries a point mutation (SL452R) within the sequence of RBD9.1. Specifically, SY451 and SY454 are identified as the key amino acids for the binding of the induced RBD-specific antibodies to RBD9.1. Furthermore, we have confirmed that the RBD9.1 antigenic peptide can induce a S448-456 (NYNYLYRLF)-specific CD8+ T-cell response. Both RBD9.1-specific B cells and the S448-456-specific T cells can still be activated more than 3 months post the last immunization. This study provides a potential vaccine candidate that can generate long-term protective efficacy over SARS-CoV-2 variants, with the unique functional mechanism of activating both humoral and cellular immunity.

Potent SARS-CoV-2 neutralizing antibodies with protective efficacy against newly emerged mutational variants.

Accumulating mutations in the SARS-CoV-2 Spike (S) protein can increase the possibility of immune escape, challenging the present COVID-19 prophylaxis and clinical interventions. Here, 3 receptor binding domain (RBD) specific monoclonal antibodies (mAbs), 58G6, 510A5 and 13G9, with high neutralizing potency blocking authentic SARS-CoV-2 virus display remarkable efficacy against authentic B.1.351 virus. Surprisingly, structural analysis has revealed that 58G6 and 13G9 both recognize the steric region S470-495 on the RBD, overlapping the E484K mutation presented in B.1.351. Also, 58G6 directly binds to another region S450-458 in the RBD. Significantly, 58G6 and 510A5 both demonstrate prophylactic efficacy against authentic SARS-CoV-2 and B.1.351 viruses in the transgenic mice expressing human ACE2 (hACE2), protecting weight loss and reducing virus loads. Together, we have evidenced 2 potent neutralizing Abs with unique mechanism targeting authentic SARS-CoV-2 mutants, which can be promising candidates to fulfill the urgent needs for the prolonged COVID-19 pandemic.

MiRNAs and lncRNAs in NK cell biology and NK/T-cell lymphoma.

The important role of lncRNAs and miRNAs in directing immune responses has become increasingly clear. Recent evidence conforms that miRNAs and lncRNAs are involved in NK cell biology and diseases through RNA-protein, RNA-RNA, or RNA-DNA interactions. In this view, we summarize the contribution of miRNAs and lncRNAs to NK cell lineage development, activation and function, highlight the biological significance of functional miRNAs or lncRNAs in NKTL and discuss the potential of these miRNAs and lncRNAs as innovative biomarkers/targets for NKTL early diagnosis, target treatment and prognostic evaluations.

KDM6B promotes ESCC cell proliferation and metastasis by facilitating C/EBPß transcription.

BACKGROUND: As an H3K27me3 demethylase and counteracts polycomb-mediated transcription repression, KDM6B has been implicated in the development and malignant progression in various types of cancers. However, its potential roles in esophageal squamous cell carcinoma (ESCC) have not been explored. METHODS: The expression of KDM6B in human ESCC tissues and cell lines was examined using RT-qPCR, immunohistochemical staining and immunoblotting. The effects of KDM6B on the proliferation and metastasis of ESCC were examined using in vitro and in vivo functional tests. RNA-seq and ChIP-seq assay were used to demonstrate the molecular biological mechanism of KDM6B in ESCC. RESULTS: We show that the expression level of KDM6B increased significantly in patients with lymph node metastasis. Furthermore, we confirmed that KDM6B knockdown reduces proliferation and metastasis of ESCC cells, while KDM6B overexpression has the opposite effects. Mechanistically, KDM6B regulates TNFA_SIGNALING_VIA_NFκB signalling pathways, and H3K27me3 binds to the promoter region of C/EBPß, leading to the promotion of C/EBPß transcription. Besides, we show that GSK-J4, a chemical inhibitor of KDM6B, markedly inhibits proliferation and metastasis of ESCC cells. CONCLUSIONS: The present study demonstrated that KDM6B promotes ESCC progression by increasing the transcriptional activity of C/EBPß depending on its H3K27 demethylase activity.

A Rapid and Efficient Screening System for Neutralizing Antibodies and Its Application for SARS-CoV-2.

After the pandemic of COVID-19, neutralizing antibodies (NAbs) against SARS-CoV-2 have been developed for the prophylactic and therapeutic purposes. However, few methodologies are described in detail on how to rapidly and efficiently generate effective NAbs to SARS-CoV-2. Here, we integrated and optimized a strategically screening method for NAbs, which has enabled us to obtain SARS-CoV-2 receptor-binding domain (RBD) specific NAbs within 6 days, followed by additional 9 days for antibody production and function analysis. Using this method, we obtained 198 specific Abs against SARS-CoV-2 RBD from the blood samples of COVID-19 convalescent patients, and 96 of them showed neutralizing activity. At least 20% of these NAbs exhibited advanced neutralizing potency and high affinity, with the top two NAbs showing half-maximal inhibitory concentration (IC50) to block authentic SARS-CoV-2 at 9.88 and 11.13 ng/ml, respectively. Altogether, our study provides an effective methodology with high applicable value for discovering potential preventative and therapeutic NAbs for the emerging infectious diseases.

Clinical spectrum of intra-abdominal abscesses in children admitted to the pediatric emergency department.

BACKGROUND: To analyze clinical spectrum of intra-abdominal abscesses in children and find helpful clinical parameters could aid physicians in earlier detection and differential diagnosis. METHODS: From 2004 to 2011, we retrospectively analyzed 66 pediatric patients, aged 18 years or younger with intra-abdominal abscesses. The data were obtained and studied: demographics, clinical presentations, etiologies, laboratory tests, microbiology, imaging studies, treatment modalities, complications and long-term outcomes. RESULTS: There were 66 patients (mean age, 9.27 ± 4.16 years) diagnosed as intra-abdominal abscesses. The two most common presented symptoms were fever and abdominal pain (90.9%; 78.8%, respectively). Most patients presented with leukocytosis (81.8%) and elevated C-reactive protein (CRP) levels (95.5%). In patients with abscesses in solid organs, urine white blood cell counts, nitrate and leukocyte esterase were all significant parameters (all P < 0.05), and urine pH and specific gravity were both lower than those in non-solid organs (P = 0.026; P = 0.043, respectively). Escherichia coli (E. coli) was the most common organism cultured from renal abscess. Streptococcus viridans was the most common organism cultured from liver abscess. Moreover, the two most predominant bacteria in periappendical and intraperitoneal abscesses were E. coli and Bacteroides fragilis. CONCLUSIONS: We suggest that primary physicians should keep this disease in mind when children present with predisposing risk factors, fever, abdominal pain, leukocytosis and elevated CRP level. Besides, we recommend the urinary analysis or ultrasonography (US) is valuable in patients with fever and abdominal pain.

CLDN1 induces autophagy to promote proliferation and metastasis of esophageal squamous carcinoma through AMPK/STAT1/ULK1 signaling.

Tight junction is a structural constitution in cell-cell adhesion and play an important role in the maintenance of permeability and integrity of normal epithelial cell barrier. The protein encoded by Claudin 1 (CLDN1), a member of the claudin family, is an integral membrane protein and a component of tight junction strands. CLDN1 has been proved to regulate the proliferation and metastasis of multiple tumors, but little is known about its role in esophageal squamous cell carcinoma (ESCC). Here, we found that CLDN1 was aberrantly increased in ESCC tissues and cell lines, and mainly distributed in the nucleus of tumor cells. Furthermore, we confirmed that CLDN1 promoted the proliferation and metastasis of ESCC by triggering autophagy both in vitro and in vivo. Mechanically, we validated that CLDN1-induced autophagy via increasing Unc-51 like autophagy activating kinase 1 (ULK1) expression through AMP-activated protein kinase (AMPK)/signal transducer and activator of transcription 1 (STAT1) signaling pathway in ESCC cells. Taken together, our findings demonstrated that aberrant expression and distribution of CLDN1 promoted the proliferation and metastasis of esophageal squamous carcinoma by triggering autophagy through AMPK/STAT1/ULK1 signaling pathway.

IL-6 and IL-8 secreted by tumour cells impair the function of NK cells via the STAT3 pathway in oesophageal squamous cell carcinoma.

BACKGROUND: Recurrence and metastasis are the leading causes of tumour-related death in patients with oesophageal squamous cell carcinoma (ESCC). Tumour-infiltrating natural killer cells (NK cells) display powerful cytotoxicity to tumour cells and play a pivotal role in tumour therapy. However, the phenotype and functional regulation of NK cells in oesophageal squamous cell carcinoma (ESCC) remains largely unknown. METHODS: Single cell suspensions from blood and tissue samples were isolated by physical dissociation and filtering through a 70 µm cell strainer. Flow cytometry was applied to profile the activity and function of NK cells, and an antibody chip experiment was used to identify and quantitate cytokine levels. We studied IL-6 and IL-8 function in primary oesophageal squamous carcinoma and NK cell co-cultures in vitro and by a xenograft tumour model in vivo. Western blotting was used to quantitate STAT3 (signal transducer and activator of transcription 3) and p-STAT3 levels. Finally, we performed an IHC array to analyse IL-6/IL-8 (interleukin 6/interleukin 8) expression in 103 pairs of tumours and matched adjacent tissues of patients with ESCC to elucidate the correlation between IL-6 or IL-8 and clinical characteristics. RESULTS: The percentages of NK cells in both peripheral blood and tumour tissues from patients with ESCC were significantly increased in comparison with those in the controls and correlated with the clinical characteristics. Furthermore, the decrease in activating receptors and increase in inhibitory receptors on the surface of tumour-infiltrating NK cells was confirmed by flow cytometry. The level of granzyme B, the effector molecule of tumour-infiltrating NK cells, was also decreased. Mechanistically, primary ESCC cells activated the STAT3 signalling pathway on NK cells through IL-6 and IL-8 secretion, leading to the downregulation of activating receptors (NKp30 and NKG2D) on the surface of NK cells. An ex vivo study showed that blockade of STAT3 attenuated the IL-6/IL-8-mediated impairment of NK cell function. Moreover, the expression of IL-6 or IL-8 in tumour tissues was validated by immunohistochemistry to be positively correlated with tumour progression and poor survival, respectively. CONCLUSIONS: Tumour cell-secreted IL-6 and IL-8 impair the activity and function of NK cells via STAT3 signalling and contribute to oesophageal squamous cell carcinoma malignancy.

[Effect of Epithelial-to-mesenchymal Transition on Biological Activity of NK Cells in Esophageal Squamous Cell Carcinoma].

OBJECTIVE: To investigate the effect of epithelial-mesenchymal transition (EMT) on biological activity of natural killer (NK) cells in esophageal squamous cell carcinoma (ESCC). METHODS: Western blot selected EMT EC9706 and non EMT KYSE150 from six esophageal cancer cell lines. NK cells were collected from 10 cases of healthy volunteers. According to different co-culture conditions, the medium was divided into 1640 normal medium stimulus-NK group (NC group), EC9706 cell supernatant stimulus-NK group (EC9706 group) and KYSE150 cell supernatant stimulus-NK group (KYSE150 group). The expression of NK cells surface/intracellular molecules was detected by flow cytometry after co-culture of NK cells with different conditioned medium for 72 h. 10 ng/mL transforming growth factor-ß (TGF-ß) treated KYSE150 cell line for 7 d to induce EMT. KYSE150 EMT and KYSE150 supernatant were collected and co-cultured with NK cells, respectively. 72 h later, flow cytometry was used to detect the expression of surface/intracellular molecules of NK cells, degranulation ability of CD107a, killing effect of target cell K562, proliferation and apoptosis of NK cells. RESULTS: Two EMT-treated and four non-EMT-treated esophageal squamous cell carcinoma lines were selected from the six strains, and one EC9706 and one KYSE150 respectively were selected for subsequent experiments. The purity of NK cells was more than 90% and Tcells <1%. After the supernatant of esophageal squamous cell carcinoma cells was stimulated, the surface activation type and inhibitory type receptors of NK cells in the three groups were stimulated. The effector molecule results showed that: compared with NC group and KYSE150 group, the expressions of activated type receptors NKP30, NKG2D and NKP44 in EC9706 group were decreased, and the release of granulozyme was decreased ( P<0.05). Expressions of inhibitory receptor NKG2A and CD158b increased ( P<0.05). NKP46, CD226, CD16 expressions and perforin release showed no statistically significant difference among the three groups. Compared with KYSE150 supernatant stimulation, the expressions of activated receptors NKP30, NKG2D and NKP44 decreased after KYSE150 EMT supernatant stimulation, and perforin release decreased. The degranulation of CD107a and the killing effect of target cell K562 were decreased, and the proliferation index of NK cell Ki67 was decreased ( P<0.05). Expressions of inhibitory receptor NKG2A and CD158b increased ( P<0.05). The expressions of NKP46, CD226 and CD16, granulozyme release and apoptosis of NK cells were not statistically significant. CONCLUSION: EMT of esophageal cancer cells can escape the immune surveillance by inhibit the activity of NK cells and reduce the release of effective molecules.

Hypertension and chronic kidney disease affect long-term outcomes in patients with stable coronary artery disease receiving percutaneous coronary intervention.

Percutaneous coronary intervention (PCI) is commonly used for patients with coronary artery disease (CAD). However, the effects of chronic kidney disease (CKD) and hypertension (HT) on long-term outcomes in patients with stable CAD receiving PCI are still unclear. A total of 1,676 patients treated with PCI were prospectively enrolled and divided into 4 groups according to the presence or absence of HT or CKD. General characteristics, clinical medications, risk factors, angiographic findings, and long-term outcomes were analyzed. Patients with CKD had the highest rate of all-cause and cardiovascular (CV) mortality (both P < 0.01). Patients with CKD alone had the lowest event-free rate of all-cause and CV deaths (both P < 0.001). Based on Cox proportional hazard model, patients with CKD alone had the highest risk of all-cause death (HR:2.86, 95% CI:1.73-4.75) and CV death (HR: 3.57,95% CI:2.01-6.33); while patients with both CKD and HT had the highest risk of repeat PCI (HR: 1.42, 95% CI:1.09-1.85).We found that in stable CAD patients after undergoing PCI, those with CKD alone had the highest long-term mortality. Comorbid CKD appears to increase risk in patient with HT, whereas comorbid HT doesn't seem to increase risk in patients with CKD.

Predictive power of a single body temperature at different cutoff values for neonates in the nursery transferring to special care nursery.

The aim of this study was to identify the clinical parameters indicative of serious etiology of neonatal hyperthermia and to determine the appropriate cutoff value of body temperature (BT) for predicting the need to transfer the newborn to the special care (SC) nursery.The nursery records of newborns diagnosed with hyperthermia between 2007 and 2013 were retrospectively reviewed. The clinical characteristics of newborns with hyperthermia remained in the nursery were compared with those transferred to the SC nursery. In addition, the receiver operating characteristic analysis was used to determine the appropriate cutoff BT for predicting further septic workup in the SC nursery.Among the 92 newborns with hyperthermia evaluated, 30 (32.6%) were transferred to the SC nursery and 62 (67.4%) remained in the nursery. Clinical characteristics associated with transfer to the SC nursery included the highest BT, BT at first measurement during hyperthermia, frequency of hyperthermia, duration of hyperthermia, irritable crying, decreased appetite, poor activity, vomiting with abdominal distension, tachypnea, and tachycardia (all P < .05). BT for predicting the need for transferring newborns with hyperthermia to the SC nursery had an area under the curve of 0.976 (P < .001). A BT of 38 °C was determined as the optimal cutoff value for predicting the need to monitoring for suspicious clinical symptoms (sensitivity (Sn), 93%; specificity (Sp), 87%). Furthermore, BT≥38.2 °C (Sn, 70%; Sp 100%) and BT≤37.8 °C (Sn, 100%; Sp, 61%) respectively were determined as the cutoff values for transferring newborns to the SC nursery or allowing them to remain in the regular nursery.Our results suggest a BT of 38 °C represents the optimal cutoff indicating newborns for close monitoring for suspicious clinical presentations including irritable crying, decreased appetite, poor activity, vomiting with abdominal distension, tachypnea, and tachycardia. Newborns with BT < 37.8 °C may remain in the nursery but should be transferred to the SC nursery for septic workup and empiric antibiotics if the BT is above 38.2 °C.

miR-503 suppresses the proliferation and metastasis of esophageal squamous cell carcinoma by triggering autophagy via PKA/mTOR signaling.

MicroRNA (miR)-503 is involved in the regulation of the malignant phenotype in multiple tumor types, and has been proven to be a novel diagnostic and therapeutic target; however, its function and mechanisms of action have not yet been fully elucidated in esophageal squamous cell carcinoma (ESCC). In the current study, we detected miR­503 expression by RT­qPCR and found that miR­503 expression was increased in ESCC, but negatively correlated with lymph node metastasis, TNM stage and tumor differentiation. Functionally, we confirmed that miR­503 inhibited the proliferation and metastasis of ESCC cells by triggering cellular autophagy. Mechanistically, we confirmed that miR­503 exerted its biological effects by targeting protein kinase CAMP­activated catalytic subunit alpha (PRKACA) in ESCC by dual luciferase reporter assay. Moreover, miR­503 was found to trigger autophagy in ESCC cells through the protein kinase A (PKA)/mammalian target of rapamycin (mTOR) pathway. Taken together, our results demonstrate that miR­503 suppresses the proliferation and metastasis of ESCC via the activation of autophagy, mediated by the PKA/mTOR signaling pathway.

Ultrathin ß-Ni(OH)2 nanoplates vertically grown on nickel-coated carbon nanotubes as high-performance pseudocapacitor electrode materials.

Various metal hydroxides/oxides grown on conductive substrates such as nickel foam have been reported and studied as supercapacitor electrode materials. However, the capacitances of these electrodes are extremely limited because of the low content of active materials grown on the limited surface of nickel foam. To achieve high capacitance, we use nickel-coated carbon nanotubes (Ni-CNTs) as the conductive substrate for the growth of ß-Ni(OH)2. By a facile chemical method, ultrathin ß-Ni(OH)2 nanoplates are vertically grown on the surface of Ni-CNTs. The density, thickness, and content of ß-Ni(OH)2 can be easily controlled by modulating the ratio of NiCl2·6H2O to Ni-CNTs. This hierarchical nanostructure can provide remarkable synergistic effects: facilitate electron and ion transport and accelerate the reversible redox reactions. As-prepared Ni-CNTs@ß-Ni(OH)2 composites exhibit high specific capacitances (∼1807 F g(-1) at 2 A g(-1), based on the mass of ß-Ni(OH)2; ∼1283 F g(-1) at 2 A g(-1), based on the mass of composite), good rate capabilities, and excellent cycling stabilities. This strategy has potential for large-scale production and can be applied to the preparation of other hierarchical nanostructured metal hydroxide/oxide composites.