A patient-based murine model recapitulates human STAT3 gain-of-function syndrome.

STAT3 gain-of-function (GOF) variants results in a heterogeneous clinical syndrome characterized by early onset immunodeficiency, multi-organ autoimmunity, and lymphoproliferation. While 191 documented cases with STAT3 GOF variants have been reported, the impact of individual variants on immune regulation and the broad clinical spectrum remains unclear. We developed a Stat3p.L387R mouse model, mirroring a variant identified in a family exhibiting common STAT3 GOF symptoms, and rare phenotypes including pulmonary hypertension and retinal vasculitis. In vitro experiments revealed increased STAT3 phosphorylation, nuclear migration, and DNA binding of the variant. Our Stat3p.L387R model displayed similar traits from previous Stat3GOF strains, such as splenomegaly and lymphadenopathy. Notably, Stat3p.L387R/+ mice exhibited heightened embryonic lethality compared to prior Stat3GOF/+ models and ocular abnormalities were observed. This research underscores the variant-specific pathology in Stat3p.L387R/+ mice, highlighting the ability to recapitulate human STAT3 GOF syndrome in patient-specific transgenic murine models. Additionally, such models could facilitate tailored treatment development.

Overlaid Lateral Flow Immunoassay for the Simultaneous Detection of Two Variant-Specific SARS-CoV-2 Neutralizing Antibodies.

COVID-19 vaccines have been provided to the general public to build immunity since the 2019 coronavirus pandemic. Once vaccinated, SARS-CoV-2 neutralizing antibodies (NAbs-COVID-19) are needed for excellent protection against COVID-19. However, monitoring NAbs-COVID-19 is complicated and requires hospital visits. Moreover, the resulting NAbs-COVID-19 are effective against different strains of COVID-19 depending on the type of vaccine received. Here, an overlaid lateral flow immunoassay (O-LFIA) was developed for the simultaneous detection of two NAbs-COVID-19 against different virus strains, Delta and Omicron. The O-LFIA was visualized with two T-lines with a single device using competition between the free antigen and the antigen-binding antibody. Angiotensin-converting enzyme 2 (ACE2) immobilized on the T-line binds to the antigen remaining after antibody binding. Under the optimum conditions, the proposed device exhibited 50% inhibition concentrations (IC50 values) of 45.1 and 53.6 ng/mL for the Delta and Omicron variants, respectively. Additionally, the proposed platform was applied to real-world samples of animal and human serum, and the developed immunoassay provided results that were in good agreement with those obtained with the standard method. In conclusion, this developed O-LFIA can be used as an alternative method to detect NAbs-COVID-19 and can be enabled for future advancements toward commercialization.

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.

Unraveling the Immunogenetics of STAT Proteins: Clinical Perspectives on Gain-of-Function and Loss-of-Function Variants.

Signal Transducer and Activator of Transcription (STAT) proteins play pivotal roles in immune regulation. The dysregulation of these proteins, attributed to both gain-of-function (GOF) and loss-of-function (LOF) variants, has emerged as a substantial and intricate area of research. This comprehensive review delves into the intricate details of the diverse clinical spectrum associated with STAT variants and the immunological findings linked to these genetic alterations. Although this review does not encompass the treatment of each individual disease, we discuss investigative approaches ranging from immunophenotyping assessment to evaluation of STAT protein activity. These investigations play a crucial role in identifying affected patients and understanding the complexities of STAT.

Novel xeno-free and serum-free culturing condition to improve piggyBac transposon-based CD19 chimeric antigen receptor T-cell production and characteristics.

BACKGROUND AIMS: Chimeric antigen receptor (CAR) T cell is a novel therapy for relapse and refractory hematologic malignancy. Characteristics of CAR T cells are associated with clinical efficacy and toxicity. The type of serum supplements used during cultivation affects the immunophenotype and function of viral-based CAR T cells. This study explores the effect of serum supplements on nonviral piggyBac transposon CAR T-cell production. METHODS: PiggyBac CD19 CAR T cells were expanded in cultured conditions containing fetal bovine serum, human AB serum or xeno-free serum replacement. We evaluated the effect of different serum supplements on cell expansion, transduction efficiency, immunophenotypes and antitumor activity. RESULTS: Xeno-free serum replacement exhibited comparable CAR surface expression, cell expansion and short-term antitumor activity compared with conventional serum supplements. However, CAR T cells cultivated with xeno-free serum replacement exhibited an increased naïve/stem cell memory population and better T-cell expansion after long-term co-culture as well as during the tumor rechallenge assay. CONCLUSIONS: Our study supports the usage of xeno-free serum replacement as an alternative source of serum supplements for piggyBac-based CAR T-cell expansion.

Self-enhancement lateral flow immunoassay for COVID-19 diagnosis.

Equipment-free colorimetric-based lateral flow immunoassay (LFIA) is the most convenient and popular tool for various applications, including diagnostic tools requiring high sensitivity for the detection of pathogens. Thus, improvements and developments of LFIA are constantly being reported. Herein, we enriched the sensitivity of LFIA using the gold enhancement principle, emphasizing needlessly complicated apparatus, only one step for the strip test operation, and typical time incubation (15 min) process. Self-enhanced LFIA was then executed for subsequent flows by overlapping the additionally enhanced pad composed of gold ions and reducing agent on the conjugate pad and the sample pad. Self-enhanced LFIA was performed to detect SARS-CoV-2 antigens in saliva. The obtained result depicted that the achieved sensitivity was up to tenfold compared with that of conventional LFIA by visual measurements. The detection limits of self-enhanced LFIA detecting nucleocapsid protein antigens in the saliva sample was 0.50 and 0.10 ng/mL employed by naked eye detection and calibration curve-based calculation, respectively. When the proposed device was applied to 207 human saliva samples, the diagnostic performance presented a 96.10 % sensitivity and 99.23 % specificity. This self-enhanced LFIA could be implemented in large-scale production and demonstrates higher sensitivity with effortless use, which meets the requirements for point-of-care testing and on-field mass screening.

The Regulatory Roles of Ezh2 in Response to Lipopolysaccharide (LPS) in Macrophages and Mice with Conditional Ezh2 Deletion with LysM-Cre System.

The responses of macrophages to lipopolysaccharide (LPS) might determine the direction of clinical manifestations of sepsis, which is the immune response against severe infection. Meanwhile, the enhancer of zeste homologue 2 (Ezh2), a histone lysine methyltransferase of epigenetic regulation, might interfere with LPS response. Transcriptomic analysis on LPS-activated wild-type macrophages demonstrated an alteration of several epigenetic enzymes. Although the Ezh2-silencing macrophages (RAW264.7), using small interfering RNA (siRNA), indicated a non-different response to the control cells after a single LPS stimulation, the Ezh2-reducing cells demonstrated a less severe LPS tolerance, after two LPS stimulations, as determined by the higher supernatant TNF-α. With a single LPS stimulation, Ezh2 null (Ezh2flox/flox; LysM-Crecre/-) macrophages demonstrated lower supernatant TNF-α than Ezh2 control (Ezh2fl/fl; LysM-Cre-/-), perhaps due to an upregulation of Socs3, which is a suppressor of cytokine signaling 3, due to the loss of the Ezh2 gene. In LPS tolerance, Ezh2 null macrophages indicated higher supernatant TNF-α and IL-6 than the control, supporting an impact of the loss of the Ezh2 inhibitory gene. In parallel, Ezh2 null mice demonstrated lower serum TNF-α and IL-6 than the control mice after an LPS injection, indicating a less severe LPS-induced hyper-inflammation in Ezh2 null mice. On the other hand, there were similar serum cytokines after LPS tolerance and the non-reduction of serum cytokines after the second dose of LPS, indicating less severe LPS tolerance in Ezh2 null mice compared with control mice. In conclusion, an absence of Ezh2 in macrophages resulted in less severe LPS-induced inflammation, as indicated by low serum cytokines, with less severe LPS tolerance, as demonstrated by higher cytokine production, partly through the upregulated Socs3.

Less Severe Sepsis in Cecal Ligation and Puncture Models with and without Lipopolysaccharide in Mice with Conditional Ezh2-Deleted Macrophages (LysM-Cre System).

Despite a previous report on less inflammatory responses in mice with an absence of the enhancer of zeste homologue 2 (Ezh2), a histone lysine methyltransferase of epigenetic regulation, using a lipopolysaccharide (LPS) injection model, proteomic analysis and cecal ligation and puncture (CLP), a sepsis model that more resembles human conditions was devised. As such, analysis of cellular and secreted protein (proteome and secretome) after a single LPS activation and LPS tolerance in macrophages from Ezh2 null (Ezh2flox/flox; LysM-Crecre/-) mice (Ezh2 null) and the littermate control mice (Ezh2fl/fl; LysM-Cre-/-) (Ezh2 control) compared with the unstimulated cells from each group indicated fewer activities in Ezh2 null macrophages, especially by the volcano plot analysis. Indeed, supernatant IL-1ß and expression of genes in pro-inflammatory M1 macrophage polarization (IL-1ß and iNOS), TNF-α, and NF-κB (a transcription factor) were lower in Ezh2 null macrophages compared with the control. In LPS tolerance, downregulated NF-κB compared with the control was also demonstrated in Ezh2 null cells. In CLP sepsis mice, those with CLP alone and CLP at 2 days after twice receiving LPS injection, representing sepsis and sepsis after endotoxemia, respectively, symptoms were less severe in Ezh2 null mice, as indicated by survival analysis and other biomarkers. However, the Ezh2 inhibitor improved survival only in CLP, but not LPS with CLP. In conclusion, an absence of Ezh2 in macrophages resulted in less severe sepsis, and the use of an Ezh2 inhibitor might be beneficial in sepsis.

Immunosuppressive Polymeric Nanoparticles Targeting Dendritic Cells Alleviate Lupus Disease in Fcgr2b-/- Mice by Mediating Antigen-Specific Immune Tolerance.

Dendritic cells (DCs) are the most potent antigen-presenting cells that have multifaceted functions in the control of immune activation and tolerance. Hyperresponsiveness and altered tolerogenicity of DCs contribute to the development and pathogenesis of system lupus erythematosus (SLE); therefore, DC-targeted therapies aimed at inducing specific immune tolerance have become of great importance for the treatment of SLE. This study developed a new nanoparticle (NP) containing a biodegradable PDMAEMA-PLGA copolymer for target-oriented delivery to DCs in situ. PDMAEMA-PLGA NPs provided sustained drug release and exhibited immunosuppressive activity in FLT3L and GM-CSF-derived bone marrow in conventional DCs (BM-cDCs). PDMAEMA-PLGA NPs improved dexamethasone capability to convert wild-type and Fcgr2b-/- BM-cDCs from an immunogenic to tolerogenic state, and BM-cDCs treated with dexamethasone-incorporated PDMAEMA-PLGA NPs (Dex-NPs) efficiently mediated regulatory T cell (Treg) expansion in vitro. Dex-NP therapy potentially alleviated lupus disease in Fcgr2b-/- mice by mediating Foxp3+ Treg expansion in an antigen-specific manner. Our findings substantiate the superior efficacy of DC-targeted therapy using the PDMAEMA-PLGA NP delivery system and provide further support for clinical development as a potential therapy for SLE. Furthermore, PDMAEMA-PLGA NP may be a versatile platform for DC-targeted therapy to induce antigen-specific immune tolerance to unwanted immune responses that occur in autoimmune disease, allergy, and transplant rejection.

Improving cytokine-induced killer cell expansion using a gas-permeable culture method for clinical-scale production.

BACKGROUND: Cytokine-induced killer (CIK) cells are a heterogeneous group of immune cells that exert potent MHC-unrestricted cytotoxicity toward various cancer cells in both solid and hematological malignancies. OBJECTIVE: The purposes of this study were to compare the expansion and characteristics of cytokine-induced killer cells between a standard culture method and a gas-permeable culture method and to develop a clinical-scale expansion protocol for cytokine-induced killer cells using a gas-permeable culture method. METHODS: We compared the absolute cell number, fold change, cell subsets, activation markers, cytokine concentrations, and cytotoxicity toward myeloid leukemia cell lines between cytokine-induced killer cells expanded using two different culture methods. Then, we determined the ability to achieve clinical-scale expansion of cytokine-induced killer cells using the gas-permeable culture method. RESULTS: Cytokine-induced killer cells in the gas-permeable culture method group exhibited significantly better expansion but maintained similar cell subsets, activation markers, and cytotoxicity to those in the standard culture method group. In addition, we successfully manufactured cytokine-induced killer cells for clinical use using the gas-permeable culture method. We also showed the clinical efficacy of allogeneic cytokine-induced killer cells produced by the gas-permeable culture method in a patient with acute myeloid leukemia that relapsed after allogeneic hematopoietic stem cell transplantation. This patient maintained ongoing disease remission for 2 years with minimal side effects after cytokine-induced killer cell infusion. CONCLUSIONS: We successfully developed a simple and effective protocol for the ex vivo expansion of cytokine-induced killer cells using the gas-permeable culture method for clinical application.

TIGIT Monoallelic Nonsense Variant in Patient with Severe COVID-19 Infection, Thailand.

A heterozygous nonsense variant in the TIGIT gene was identified in a patient in Thailand who had severe COVID-19, resulting in lower TIGIT expression in T cells. The patient's T cells produced higher levels of cytokines upon stimulation. This mutation causes less-controlled immune responses, which might contribute to COVID-19 severity.

MicroRNA-mediated metabolic reprogramming of chimeric antigen receptor T cells.

Advances made in chimeric antigen receptor (CAR) T cell therapy have revolutionized the treatment and management of certain cancers. Currently, B cell malignancies have been among the few cancers to which CAR T cells have shown persistent and resilient anti-tumor responses. A growing body of evidence suggests that the persistence of CAR T cells within patients following infusion is linked to the mitochondrial fitness of the CAR T cell, which could affect clinical outcomes. Analysis of CAR T cells from patients undergoing successful treatment has shown an increase in mitochondrial mass and fusion events, and a reduction in aerobic metabolism, highlighting the importance of mitochondria in CAR T cell function. Consequently, there has been recent interest and investment in approaches that focus on mitochondrial programming. In this regard, miRNAs are promising agents in mitochondrial reprogramming for several reasons: (1) natural and artificial miRNAs are non-immunogenic, (2) one miRNA can simultaneously modulate the expression of multiple genes within a pathway, (3) the small size of a sequence required for producing mature miRNA is ideal for use in viral vectors and (4) different precursor miRNAs (pre-miRNAs) hairpins can be incorporated into a polycistronic miRNA cluster to create a miRNA cocktail. In this perspective, we describe the latest genetic engineering strategies that can be used to achieve the optimal expression of candidate miRNAs alongside a CAR construct. In addition, we include an in silico analysis of rational candidate miRNAs that could promote the mitochondrial fitness of CAR T cells.

Host genetic factors of COVID-19 susceptibility and disease severity in a Thai population.

Host genetic factors have been shown to play a role in SARs-CoV-2 infection in diverse populations. However, the genetic landscape differs among various ethnicities; therefore, we explored the host genetic factors associated with COVID-19 disease susceptivity and disease severity in a Thai population. We recruited and genotyped 212 unrelated COVID-19 Thai patients and 36 controls using AxiomTM Human Genotyping SARs-COV-2 array, including 847,384 single nucleotide polymorphisms related to SARs-COV-2 pathogenesis, immune response, and related comorbidity No SNPs passed the genome-wide significance threshold of p value <1 × 10-8. However, with a threshold of p value <1 × 10-5, a locus on chromosome 5q32 was found to have a suggestive association with COVID-19 disease susceptibility (p value 6.9 × 10-6; Q-Q plot λ = 0.805, odds ratio 0.02). Notably, IL17B is a gene located in this linkage disequilibrium block and is previously shown to play a part in inflammation and pneumonia. Additionally, a suggestive locus on chromosome 12q22, harboring EEA1 and LOC643339, was associated with COVID-19 disease severity (p value 1.3 × 10-6 - 4.4 × 10-6, Q-Q plot λ = 0.997, odds ratio 0.28-0.31). EEA1 is involved in viral entry into cells, while LOC643339 is a long non-coding RNA. In summary, our study suggested loci on chromosomes 5q32 and 12q22 to be linked to COVID-19 disease susceptibility and disease severity, respectively. The small sample size of this study may lessen the likelihood that the association found is real, but it could still be true. Further study with a larger cohort is required to confirm these findings.

Anti-Inflammatory Effects and Decreased Formation of Neutrophil Extracellular Traps by Enoxaparin in COVID-19 Patients.

Neutrophil Extracellular Traps (NETs) are a contributing factor of vascular thrombosis and alveolar damage in COVID-19 patients. As enoxaparin is currently used to inhibit vascular thrombosis, this study aimed to investigate whether enoxaparin also reduced inflammation and NETs in COVID-19 patients. Patients with COVID-19 infection were classified into three groups: mild, moderate, and severe (n = 10 for all groups). Plasma was collected from patients and healthy donors (n = 10). Neutrophils isolated from healthy controls were incubated with COVID-19 or healthy plasma, and with or without enoxaparin pretreatment in vitro. Neutrophils and plasma isolated from patients treated with enoxaparin were also investigated. The levels of inflammatory cytokines and NET products such as dsDNA, NE, MPO−DNA and Histone−DNA complexes in plasma and supernatants were measured using immunofluorescence staining and ELISA kits. The expression of inflammatory signaling genes by neutrophils (RELA, SYK, ERK and PKC) was measured using real-time qPCR. The levels of NET products were elevated in the plasma of COVID-19 patients, particularly in the severe group (p < 0.01). Moreover, plasma from the severe group enhanced NET formation (p < 0.01) from neutrophils in vitro. Enoxaparin pretreatment in vitro decreased plasma-induced NETs in a dose-dependent manner and down-regulated the expression of inflammatory genes (p < 0.05). Patients treated with prophylactic enoxaparin showed lower inflammatory cytokine levels and expression of inflammatory genes (p < 0.05). Increased NETs were associated with the severity of COVID-19 infection, particularly in patients with severe pneumonia, and could be used as biomarkers to assess disease severity. Enoxaparin pretreatment inhibited NETs and reduced the expression of inflammatory cytokines, and these effects mostly persisted in patients treated with prophylactic enoxaparin.

MicroRNA-21 in plasma exosome, but not from whole plasma, as a biomarker for the severe interstitial fibrosis and tubular atrophy (IF/TA) in post-renal transplantation.

BACKGROUND: Non-invasive diagnosis of interstitial fibrosis and tubular atrophy (IF/TA), a major cause of chronic allograft dysfunction in post-kidney transplantation (post-KT), is needed. OBJECTIVE: Several candidates of microRNAs (miRs) in plasma exosome or whole plasma were evaluated for IF/TA biomarker. METHODS: Kidney samples from biopsy and plasma were tested for miRs expression. RESULTS: Expression of miR-21, miR-142-3p and miR-221 in renal histology with high fibrosis score (Banff classification) was higher than the samples with lesser score (n = 17/group). However, expression of these miRs from plasma exosome or from whole plasma of post-KT patients with different severity of IF/TA as determined by percentage of IF/TA including; grade I (5-25%) (n = 15), grade II (26-50%) (n = 15), grade III (≥ 50%) (n = 6) versus stable graft function (no IF/TA) (n = 15) was not different. However, high expression of miR-21 in exosome, but not from whole plasma, was demonstrated in IF/TA grade II and III compared with IF/TA grade I. In contrast, serum creatinine (Scr) and proteinuria, the current standard biomarkers, could not differentiate IF/TA grade I out of grade II/III. There was no correlation between exosome miR-21 versus the current standard renal injury biomarkers, including Scr, blood urea nitrogen and proteinuria, in IF/TA grade II or grade III. CONCLUSIONS: High miR-21 in plasma exosome, but not in whole plasma, indicated high grade IF/TA in post-KT patients. This non-invasive monitoring biomarker allows the more frequent evaluation on IF/TA than renal biopsy (a standard but more invasive procedure) resulting in the earlier management. More studies on patients are warrant.

Short-term immune response after inactivated SARS-CoV-2 (CoronaVac®, Sinovac) and ChAdOx1 nCoV-19 (Vaxzevria®, Oxford-AstraZeneca) vaccinations in health care workers.

BACKGROUND: Inactivated SARS-CoV-2 (CoronaVac®, Sinovac, or SV) and ChAdOx1 nCoV-19 (Vaxzevria®, Oxford-Astra Zeneca, or AZ) vaccines have been administered to the health care workers (HCWs). OBJECTIVE: To determine the short-term immune response after the SV and AZ vaccinations in HCWs. METHODS: In this prospective cohort study, HCWs who completed a 2-dose regimen of the SV or AZ were included. Immune response was evaluated by surrogate viral neutralization test (sVNT) and anti-SARS-CoV-2 total antibody. Blood samples were analyzed at 4 and 12 weeks after the complete vaccination. The primary outcome was the seroconversion rate at 4-weeks after complete immunization. RESULTS: Overall, 185 HCWs with a median (IQR) age of 40.5 (30.3-55.8) years (94 HCWs in the SV group and 91 in the AZ group) were included. At 4 weeks after completing the SV vaccination, 60.6% (95%CI: 50.0-70.6%) had seroconversion evaluated by sVNT (≥ 68% inhibition), comparable to the patients recovered from mild COVID-19 infection (69.0%), with a rapid reduction to 12.2% (95%CI: 6.3-20.8) at 12 weeks. In contrast, 85.7% (95%CI: 76.8-92.2%) HCWs who completed two doses of the AZ for 4 weeks had seroconversion, comparable to the COVID-19 pneumonia patients (92.5%), with a reduction to 39.2% (95%CI: 28.4-50.9%) at 12 weeks. When using the anti-SARS-CoV-2 total antibody level (≥ 132 U/ml) criteria, only 71.3% HCWs in the SV group had seroconversion, compared to 100% in the AZ group at 4 weeks. CONCLUSIONS: A rapid decline of short-term immune response in the HCWs after the SV vaccination indicates the need for a vaccine booster, particularly during the ongoing spreading of the SARS-CoV-2 variants of concern.

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.

Innate immunity in COVID-19: Drivers of pathogenesis and potential therapeutic targets.

A novel severe acute respiratory syndrome COVID-19 caused by coronavirus SARS-CoV-2 has been confirmed to infect more than 100 million people globally, with mortality reaching nearly 3 million as of March 2021. The symptoms vary widely, from the absence of any symptoms to death. The severity of COVID-19 relates to hyperinflammatory conditions with acute respiratory distress syndrome (ARDS), which leads to multiple-organ failure and death. Innate immunity plays an important role in the early response to SARS-CoV-2 infection and regulates the pathogenesis and its clinical outcomes. The most severe cases of COVID-19 present with increased innate immune cell infiltration in the lung, and elevated pro-inflammatory cytokines in the blood serum that are associated with disease severity. Here we review the innate immune response to SARS-CoV-2 infection based on the recent reports and discuss the potential roles of innate immune cells and their mediators in pathogenesis that dictate the outcome of the disease. Understanding the roles of innate immune responses at the initial stages of infection may provide early windows into treatment and clues for vaccine development.

Notch signaling regulates function of human mucosal-associated invariant T (MAIT) cells.

BACKGROUND: Notch signaling plays an important role in the development of T lymphocytes and regulates their effector functions. The regulatory roles of Notch signaling on T cells have been intensely investigated, but whether it involves in effector functions of mucosal-associated invariant T (MAIT) cells has never been reported. OBJECTIVE: To elucidate the expression profiles of Notch receptors/ligands and to investigate their roles in human MAIT cell function. METHODS: Peripheral blood mononuclear cells (PBMCs) from health donors were stimulated with or without anti-CD3/ CD28-coupled beads, recombinant IL-12/IL-18 cytokines, riboflavin- or non-riboflavin-synthesizing bacterial cultured supernatant for 24 hours. The expression profiles of Notch receptors and ligands on MAIT cells were detected by flow cytometry. PBMCs were treated with a Notch signaling inhibitor, gamma secretase inhibitor (GSI), before stimulation to investigate the impact of interfering with Notch signaling on activation and function of MAIT cells. RESULTS: Resting MAIT cells predominantly expressed Notch2 receptor and the ligand, Jagged 2, on their surface. Upon stimulation, MAIT cells further upregulated Notch2 and also Notch1 with its cleaved form, indicating active Notch signaling. Cytokines and cytotoxic molecules which are secreted by activated MAIT cells, were suppressed by treatment with GSI. Moreover, both TCR-dependent MAIT cell activation by microbial-derived riboflavin intermediates and TCR-independent MAIT cell activation driven by IL-18 in synergy with IL-12, were blocked by GSI treatment. CONCLUSIONS: Notch signaling is operating in MAIT cells and is involved in their activation both in a TCR-independent and -dependent manners.

Plasma B-cell activating factor levels and polymorphisms in hepatitis B-related hepatocellular carcinoma: Clinical correlation and prognosis.

BACKGROUND: B-cell activating factor (BAFF), an important cytokine for B lymphocyte activation, has been shown to be increased in chronic hepatitis B virus (HBV) infection. OBJECTIVE: This study aimed at evaluating clinical correlation and prognostic role of plasma BAFF and related polymorphisms in patients with HBV-related hepatocellular carcinoma (HCC). METHODS: Plasma BAFF levels were measured from 100 healthy controls and 490 patients with chronic HBV infection (200 with HCC and 290 without HCC). The rs9514828 and rs12583006 polymorphisms were determined by allelic discrimination. RESULTS: The HCC group had significantly higher BAFF levels compared with the non-HCC group and healthy controls. Among the non-HCC group, the HBeAg-positive subgroup had higher BAFF levels compared with the HBeAg-negative subgroup. In the HCC group, high BAFF levels at initial presentation significantly correlated with alpha-fetoprotein levels, Child-Pugh classification, tumor size and BCLC stage. Multivariate analyses showed that elevated BAFF concentration (± 1,100 pg/ml) was a significant and independent prognostic factor of overall survival in patients with HCC (OR = 2.28, 95%CI: 1.07-4.87; P = 0.034). HCC patients with high BAFF levels (± 1,100 pg/ml) had a poorer median survival than those with low levels (P < 0.001, log-rank test). Regarding BAFF polymorphisms, the frequency of rs9514828 CT + TT genotypes was higher distributed in patients with chronic HBV infection compared with healthy controls (58.0% vs. 46.0%, P = 0.029). CONCLUSIONS: Our data demonstrate for the first time that elevated plasma BAFF levels at baseline exhibit clinical correlation in terms of disease severity and overall survival in HCC patients. Thus, plasma BAFF at initial diagnosis could serve as a prognostic marker for HBV-related HCC.