Immunosuppressive therapy and humoral response to third mRNA COVID-19 vaccination with a six-month interval in rheumatic disease patients.

OBJECTIVES: To evaluate the long-term impact of immunosuppressive therapeutic agents on antibody response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) mRNA vaccination in patients with autoimmune rheumatic diseases (AIRD) in order to propose a strategy for annual vaccination. METHODS: This prospective multicentre cohort study evaluated the humoral response to second and third BNT162b2 and/or mRNA-1273 vaccines in 382 Japanese AIRD patients classified into 12 different medication groups and in 326 healthy controls (HCs). The third vaccination was administered six months after the second vaccination. Antibody titres were measured using the Elecsys Anti-SARS-CoV-2 S assay. RESULTS: The seroconversion rate and antibody titres were lower in AIRD patients than in HCs 3-6 weeks after the second vaccination and 3-6 weeks after the third vaccination. Seroconversion rates were <90% after the third vaccination in patients receiving mycophenolate mofetil and rituximab. Antibody levels after the third vaccination were significantly lower in the groups prescribed TNF inhibitor with or without methotrexate, abatacept and rituximab or cyclophosphamide than those of HCs in a multivariate analysis adjusting for age, sex, and glucocorticoid dosage. The third vaccination induced an adequate humoral response in patients treated with sulfasalazine, bucillamine, methotrexate monotherapy, iguratimod, interleukin-6 inhibitors or calcineurin inhibitors including tacrolimus. CONCLUSIONS: Repeated vaccinations in many immunosuppressed patients produced antibody responses similar to those observed in HCs. In contrast, annual vaccination in patients receiving TNF inhibitors, abatacept, mycophenolate mofetil and rituximab may require caution.

An integrated strategy combining network toxicology and feature-based molecular networking for exploring hepatotoxic constituents and mechanism of Epimedii Folium-induced hepatotoxicity in vitro.

Epimedii Folium (EF), a commonly used herbal medicine to treat osteoporosis, has caused serious concern due to potential hepatotoxicity. Until now, its intrinsic hepatotoxic mechanism and hepatotoxic ingredients remain unclear. Here, a novel high-throughput approach was designed to investigate the intrinsic hepatotoxic of EF. High-content screen imaging (HCS) and biochemical tests were first performed to obtain the cytotoxicity parameter matrix of 17 batch EF samples. EF-treated alpha mouse liver 12 (AML12) cells showed increased reactive oxygen species (ROS), reduced glutathione (GSH) and mitochondrial membrane potential (MMP), and apoptosis and cholestasis were further observed. Network toxicology predicted that EF-triggered hepatotoxiciy was involved in transcription factor (TF) activity. The FXR expression, screened by a TF PCR array, exhibited down-regulation following EF extract administration. Moreover, EF inhibited bile acid (BA) metabolism pathway in an FXR-dependent manner. Pearson correlation between the cytotoxicity parameter matrix and quantification feature table obtained from UHPLC-QTOF data of EF suggested 7 prenylated flavonoids possessed potent hepatotoxicities and their cytotoxicity order was further summarized. The transcriptional repression effects of them on FXR were also verified. Collectively, our findings indicate that FXR is probably responsible for EF-induced hepatotoxicity and prenylated flavonoids may be a major class of hepatotoxic constituents in EF.

Revealing active components and action mechanism of Fritillariae Bulbus against non-small cell lung cancer through spectrum-effect relationship and proteomics.

BACKGROUND: Fritillariae Bulbus (FB) is widely used as a traditional medicine for the treatment of lung meridian diseases. It has been proved that FB has good anti-non-small cell lung cancer (NSCLC) activity. However, the active components and potential mechanism are still not clear. PURPOSE: To reveal the bioactive components of FB against NSCLC and potential mechanism through spectrum-effect relationship and proteomics. METHOD: First, the FB extract was chemically profiled by UHPLC-QTOF-MS and the inhibitory effect of FB extract on A549 cell viability was evaluated by Cell Counting Kit-8 assay. Second, orthogonal-partial least squares-regression analysis was applied to screen potential active compounds through correlating the chemical profile with corresponding inhibitory effect. Third, the anti-NSCLC activities of potential active components were further investigated in terms of cell proliferation, cell cycle and cell apoptosis in vitro and tumor growth in vivo. Finally, proteomics was utilized to reveal the underlying anti-NSCLC mechanism. RESULTS: Six potential active components including verticine, verticinone, zhebeirine, ebeiedinone, yibeissine and peimisine were screened out by spectrum-effect relationship. Among them, zhebeirine showed higher inhibitory effect on A549 cell viability with IC50 value of 36.93 µM and dosage-dependent inhibition of A549 xenograft tumor growth in nude mice. Proteomics and western blotting assays indicated that zhebeirine could arrest cell cycle by down-regulating the expressions of CDK1, CDK2, Cyclin A2, Cyclin B2 and inhibiting the phosphorylation of p53. Moreover, the proteins participating in p53 signaling pathway including PCNA, 14-3-3σ, CHEK1 were significantly decreased, which suggested that zhebeirine affected cell cycle progression through p53 signaling pathway. CONCLUSION: This study not only provides scientific evidence to support the clinical application of FB against NSCLC, but also demonstrates that zhebeirine is a promising anti-NSCLC lead compound deserving further studies.

Antibody response to SARS-CoV-2 mRNA vaccines in patients with rheumatic diseases in Japan: Interim analysis of a multicentre cohort study.

OBJECTIVES: To evaluate the impact of medication on antibody response to severe acute respiratory syndrome coronavirus-2 mRNA vaccines in Japanese patients with rheumatic diseases. METHODS: This prospective multicentre cohort study evaluated the humoral response in 12 different medication groups. Antibody levels before the first vaccination and 3-6 weeks after the second vaccination were measured using the Elecsys Anti-SARS-CoV-2 S assay. Statistical analysis included comparing antibody titres among the different medication groups using the Kruskal-Wallis test followed by the Bonferroni-Dunn test and multiple linear regression analysis. RESULTS: 295 patients were analysed. The seroconversion rate was 92.2% and the median antibody titre was 255 U/ml (interquartile range, 34.1-685) after the second mRNA vaccination. Antibody levels were significantly lower in the groups treated with Tumour necrosis factor inhibitor with methotrexate, abatacept, mycophenolate mofetil (MMF), MMF or mizoribine combined with calcineurin inhibitor, and rituximab or cyclophosphamide compared with those treated with sulfasalazine and/or bucillamine or calcineurin inhibitor (p < 0.01). The correlation between antibody titre and treatment was significant after adjusting for age, gender, and glucocorticoid dose (p < 0.01). CONCLUSIONS: Additional early vaccination is required in patients treated with Tumour necrosis factor inhibitor and methotrexate, abatacept, MMF, MMF or mizoribine combined with calcineurin inhibitor and rituximab or cyclophosphamide.

Baohuoside I inhibits FXR signaling pathway to interfere with bile acid homeostasis via targeting ER α degradation.

Epimedii folium (EF) is an effective herbal medicine in osteoporosis treatment, but the clinical utilization of EF has been limited due to potential hepatotoxicity. The previous studies identified that baohuoside I (BI), the main active component of EF, was relevant to EF-induced liver injury. However, the mechanisms of BI causing direct injury to hepatocytes remain unclear. Here, we reveal that BI inhibits FXR-mediated signaling pathway via targeting estrogen receptor α (ER α), leading to the accumulation of bile acids (BAs). Targeted bile acid analyses show BI alters the BA composition and distribution, resulting in impaired BA homeostasis. Mechanistically, BI induces FXR-dependent hepatotoxicity at transcriptional level. Additionally, ER α is predicted to bind to the FXR promoter region based on transcription factor binding sites databases and we further demonstrate that ER α positively regulates FXR promoter activity and affects the expression of target genes involved in BA metabolism. Importantly, we discover that ER α and its mediated FXR transcription regulation might be involved in BI-induced liver injury via ligand-dependent ER α degradation. Collectively, our findings indicate that FXR is a newly discovered target gene of ER α mediated BI-induced liver injury, and suggest BI may be responsible for EF-induced liver injury.

CEACAM 1, 3, 5 and 6 -positive classical monocytes correlate with interstitial lung disease in early systemic sclerosis.

Background: Systemic sclerosis (SSc) is a multiple-organ disease characterized by vascular damage, autoimmunity, and tissue fibrosis. Organ injuries such as interstitial lung diseases (ILD), resulting from inflammatory and fibrosis processes, lead to poor prognosis. Although autoantibodies are detected in the serum of patients with SSc, the mechanisms by which immune cells are involved in tissue inflammation and fibrosis is not fully understood. Recent studies have revealed carcinoembryonic antigen related cell adhesion molecule (CEACAM)-positive monocytes are involved in murine bleomycin-induced lung fibrosis. We investigated CEACAM-positive monocytes in patients with SSc to clarify the role of monocytes in the pathogenesis of SSc. Methods: The proportion of of CEACAM-positive classical monocytes in healthy controls (HCs) and patients with rheumatoid arthritis (RA) and SSc was evaluated using flow cytometry. The correlation between the proportion of CEACAM-positive monocytes and clinical parameters was analyzed in patients with SSc. Gene expression microarrays were performed in CEACAM-positive and negative monocytes in patients with SSc. Infiltration of CEACAM-positive monocytes into scleroderma skin was evaluated by immunohistochemical staining. Results: The proportion of CEACAM-positive classical monocytes was increased in patients with early SSc within 2 years after diagnosis, which positively correlated with ESR, serum IgG, and serum KL-6 and negatively correlated with %forced vital capacity. The percentage of CEACAM-positive monocytes decreased after immunosuppressive therapy. CEACAM6-positive cells among classical monocytes were significantly increased in patients with SSc compared with HCs and patients with rheumatoid arthritis. SSc serum induced CEACAM6 expression on monocytes from HCs. Functionally, CEACAM-positive monocytes produced higher levels of TNF-α and IL-1ß compared to CEACAM-negative cells and showed activation of the NF-κB pathway. Furthermore, CEACAM6-positive monocytes infiltrated the dermis of SSc. Conclusions: CEACAM-positive monocytes showed inflammatory phenotypes and may be involved in the tissue inflammation and fibrosis in early SSc. CEACAM-positive monocytes may be one of biomarkers to detect patients with progressive ILD, requiring therapeutic intervention.

Methotrexate Enhances Apoptosis of Transmembrane TNF-Expressing Cells Treated With Anti-TNF Agents.

Background: Concomitant use of methotrexate (MTX) improves the clinical efficacy of anti-TNF agents in the treatment of rheumatoid arthritis (RA). We aimed to clarify the cytotoxic effect of MTX on transmembrane TNF (tmTNF)-expressing cells treated with anti-TNF agents. Methods: Jurkat T cells stably expressing tmTNF were used for the following experiments. Cytotoxicity induced by an anti-TNF agent (infliximab, adalimumab, or certolizumab pegol) with concomitant MTX were compared with that by MTX alone or by an anti-TNF agent alone using flow cytometry. Apoptosis-induction mediated by reverse signal through tmTNF, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP) were evaluated. Folic acid and Y-27632, a Rho kinase inhibitor, were used as inhibitors to study intracellular signaling pathway in apoptosis induced by MTX and anti-TNF agents. Results: Apoptosis of tmTNF-expressing cells was significantly increased by the concomitant administration of MTX and an anti-TNF agent, compared with MTX alone or an anti-TNF agent alone. The apoptosis induction by concomitant MTX was most pronounced in infliximab-treatment. Reverse signal transduction, but not CDC or ADCC/ADCP, was responsible for the coordinate effect of MTX and an anti-TNF agent on tmTNF-expressing cells. Folic acid inhibited MTX-mediated apoptosis, while Y-27632 suppressed JNK activation and infliximab-induced apoptosis via revere signal through tmTNF. Conclusion: The apoptotic effect was enhanced by combination of MTX and an anti-TNF agent in tmTNF-expressing cells. The intracellular pathways induced by MTX and anti-TNF agents seem to be independent. These findings might explain at least in part improved the clinical response upon co-therapy of MTX and an anti-TNF agent in RA.

Ovalbumin-modified nanoparticles increase the tumor accumulation by a tumor microenvironment-mediated "giant".

We designed a pH intelligently driven self-assembled nano-platform (GOx@ZIF-OVA). The nano-platform was composed of glucose oxidase (GOx), ovalbumin (OVA) and zeolitic imidazolate skeleton-8 (ZIF-8). The goal was to address the depth and cumulative limits of the drug at the tumor site. Firstly, OVA-modified GOx@ZIF could greatly increase tumor accumulation due to spontaneous self-assembly from 142.2 ± 9.1 to 705.5 ± 52.1 nm and the 5779.4 ± 598.3 nm giant at pH values of 7.4, 6.5, and 5.0, respectively. More importantly, the tumor-like sphere experiments demonstrated that the encapsulated GOx "vampires" can cut off the energy source of tumors and poisonous tumor cells without depth limitations. Furthermore, immunofluorescence assay and cytotoxicity assay tests in vivo proved that T cell infiltration could be significantly increased, triggering an effective anti-tumor immune response and inhibiting lung metastasis. Therefore, the experimental results demonstrated that the acid-smart-driven nano-platform has the potential to address the limitations of tumor depth and drug accumulation in solid tumors.

ROS-boosted photodynamic therapy against metastatic melanoma by inhibiting the activity of antioxidase and oxygen-producing nano-dopants.

The antioxidant effect weakens the ability of PDT to resist melanoma, and the hypoxic tumor environment further restricts the application of photosensitizers in tumors. Therefore, to enhance the ability of PDT to resist melanoma, we designed a sequential enhanced PDT theranostic platform (Au@MTM-HA). Firstly, the nanotherapeutic platform uses TiO2 as a photosensitizer, which is doped with MnO2 to form a mesoporous MTM. The MTM can continuously provide oxygen, thereby increasing the level of reactive oxygen species (ROS) and reducing the metastatic effect by alleviating tumor hypoxia. Furthermore, the released Au25Sv9 could inhibit the activity of antioxidant defense enzymes and reduce the scavenging of ROS and further enhance the PDT effect. Simultaneously, surface-modified HA could not only recognize CD44 receptor but also act as a sealing agent for carriers. Result: Au@MTM-HA could explosively produce a 3-fold higher ROS and improve the PDT effect. Therefore, this work may provide strong evidence for Au@MTM-HA as a new and promising PDT candidate for the treatment of metastatic melanoma.

Construction and evaluation of tumor nucleus-targeting nanocomposite for cancer dual-mode imaging - Guiding photodynamic therapy/photothermal therapy.

To tackle the barrier of the insufficient intra-cellular delivery of reactive oxygen species (ROS) and heat, we designed a multifunctional nanoplatform to release ROS and heat directly in the cell nucleus for enhancing combined photodynamic therapy (PDT) and photothermal therapy (PTT) of tumors. As a photothermal agent, WS2 nanoparticles were adsorbed photosensitive Au25(Captopril)18- (Au25) nanoclusters via electrostatic interaction. And Dexamethasone (Dex), a glucocorticoid with nucleus targeting capability, played a key role in the intra-nuclear process of heat and ROS. PTT can increase intra-tumoral blood flow to promote Au25 produce more ROS for PDT. Under near infrared (NIR) laser irradiation at a single 808 nm, these nucleus targeting WS2 nanoplatforms showed a significant decreased cell viability of 18.2 ±â€¯1.7% and a high DNA damage degree of 59.6 ±â€¯8.3%. Furthermore, the WS2 nanoplatform could be further used for X-ray computed tomography (CT) images. Taken together, our study provided a new prospect for effectively diagnostic and enhancing PTT/PDT efficacy.

Ultrahigh performance heat spreader based on gas-liquid exfoliation boron nitride nanosheets.

Due to their high thermal conductivity and insulation performance, boron nitride nanosheets (BNNS) have great promise to fabricate thermal management equipment for application in power electronics. The liquid-phase exfoliation route has been regarded as the most commonly used approach to produce single and few-layered BNNS for many research fields. However, this process takes a long time, and the production yield is extremely low. In this work, an efficient technique to obtain few-layered (mostly < 5 layers), high-yield (∼33%), and plane-defect-free BNNS by the combination of liquid N2 (L-N2) gasification and liquid exfoliation was developed. The as-obtained BNNS suspensions could be vacuum filtered to make a thermal conductive film named a BNNS heat spreader which possessed a superior thermal conductivity of 61.2 W m-1 K-1 at room temperature. In addition, we also proved that the thermal conductivity of the BNNS heat spreader increased with the increase of density, creating an approach for fine tuning the thermal property of this heat spreader.