Assessing the safety of bedaquiline: insight from adverse event reporting system analysis.

Background: The development and marketing of Bedaquiline (BDQ) represent significant advancements in treating tuberculosis, particularly multidrug-resistant forms. However, comprehensive research into BDQ's real-world safety remains limited. Purpose: We obtained BDQ related adverse event (AE) information from the US Food and Drug Administration's Adverse Event Reporting System (FAERS) to assess its safety and inform drug usage. Methods: The AE data for BDQ from 2012 Q4 to 2023 Q3 was collected and standardized. Disproportionality analysis, including Reporting Odds Ratio (ROR), Proportional Reporting Ratio (PRR), Multi-item Gamma Poisson Shrinker (MGPS), and Bayesian Confidence Propagation Neural Network (BCPNN) was used to quantify signals of BDQ-related AEs. Logistic regression was used to analyze the individual data of hepatotoxicity and drug-induced liver injury, and multiple linear regression models were established. Additionally, network pharmacology was employed to identify the potential biological mechanisms of BDQ-induced liver injury. Results: We identified 2017 case reports directly related to BDQ. Our analysis identified 341 Preferred Terms (PTs) characterizing these AEs across 27 System Organ Classes (SOC). An important discovery was the identification of AEs associated with ear and labyrinth disorders, which had not been documented in the drug's official leaflet before. Subgroup analysis revealed a negative correlation between BDQ-related liver injury and females (OR: 0.4, 95%CI: 0.3-0.6). In addition, via network pharmacology approach, a total of 76 potential targets for BDQ related liver injury were predicted, and 11 core target genes were selected based on the characterization of protein-protein interactions. The pathway linked to BDQ-induced liver injury was identified, and it was determined that the PI3K-Akt signaling pathway contained the highest number of associated genes. Conclusion: The analysis of the FAERS database revealed adverse events linked to BDQ, prompting the use of a network pharmacology approach to study the potential molecular mechanism of BDQ-induced liver injury. These findings emphasized the significance of drug safety and offered understanding into the mechanisms behind BDQ-induced liver injury. BDQ demonstrated distinct advantages, including reduced incidence of certain adverse events compared to traditional treatments such as injectable agents and second-line drugs. However, it is important to acknowledge the limitations of this analysis, including potential underreporting and confounding factors. This study provides valuable insights into the safety of BDQ and its role in the management of MDR-TB, emphasizing the need for continued surveillance and monitoring to ensure its safe and effective use.

Cryomicroneedle delivery of nanogold-engineered Rhodospirillum rubrum for photochemical transformation and tumor optical biotherapy.

Tumor metabolite regulation is intricately linked to cancer progression. Because lactate is a characteristic metabolite of the tumor microenvironment (TME), it supports tumor progression and drives immunosuppression. In this study, we presented a strategy for antitumor therapy by developing a nanogold-engineered Rhodospirillum rubrum (R.r-Au) that consumed lactate and produced hydrogen for optical biotherapy. We leveraged a cryogenic micromolding approach to construct a transdermal therapeutic cryomicroneedles (CryoMNs) patch integrated with R.r-Au to efficiently deliver living bacterial drugs. Our long-term storage studies revealed that the viability of R.r-Au in CryoMNs remained above 90%. We found that the CryoMNs patch was mechanically strong and could be inserted into mouse skin. In addition, it rapidly dissolved after administering bacterial drugs and did not produce by-products. Under laser irradiation, R.r-Au effectively enhanced electron transfer through Au NPs actuation into the photosynthetic system of R. rubrum and enlarged lactate consumption and hydrogen production, thus leading to an improved tumor immune activation. Our study demonstrated the potential of CryoMNs-R.r-Au patch as a minimally invasive in situ delivery approach for living bacterial drugs. This research opens up new avenues for nanoengineering bacteria to transform tumor metabolites into effective substances for tumor optical biotherapy.

Transcriptomic Investigation of the Virus Spectrum Carried by Midges in Border Areas of Yunnan Province.

Yunnan province in China shares its borders with three neighboring countries: Myanmar, Vietnam, and Laos. The region is characterized by a diverse climate and is known to be a suitable habitat for various arthropods, including midges which are notorious for transmitting diseases which pose significant health burdens affecting both human and animal health. A total of 431,100 midges were collected from 15 different locations in the border region of Yunnan province from 2015 to 2020. These midges were divided into 37 groups according to the collection year and sampling site. These 37 groups of midges were then homogenized to extract nucleic acid. Metatranscriptomics were used to analyze their viromes. Based on the obtained cytochrome C oxidase I gene (COI) sequences, three genera were identified, including one species of Forcipomyia, one species of Dasyhelea, and twenty-five species of Culicoides. We identified a total of 3199 viruses in five orders and 12 families, including 1305 single-stranded positive-stranded RNA viruses (+ssRNA) in two orders and seven families, 175 single-stranded negative-stranded RNA viruses (-ssRNA) in two orders and one family, and 1719 double-stranded RNA viruses in five families. Six arboviruses of economic importance were identified, namely Banna virus (BAV), Japanese encephalitis virus (JEV), Akabane virus (AKV), Bluetongue virus (BTV), Tibetan circovirus (TIBOV), and Epizootic hemorrhagic disease virus (EHDV), all of which are capable, to varying extents, of causing disease in humans and/or animals. The survey sites in this study basically covered the current distribution area of midges in Yunnan province, which helps to predict the geographic expansion of midge species. The complexity and diversity of the viral spectrum carried by midges identified in the study calls for more in-depth research, which can be utilized to monitor arthropod vectors and to predict the emergence and spread of zoonoses and animal epidemics, which is of great significance for the control of vector-borne diseases.

Nitrification inhibitor chlorate and nitrogen substrates differentially affect comammox Nitrospira in a grassland soil.

Introduction: Through the combined use of two nitrification inhibitors, Dicyandiamide (DCD) and chlorate with nitrogen amendment, this study aimed to investigate the contribution of comammox Nitrospira clade B, ammonia oxidizing bacteria (AOB) and archaea (AOA) to nitrification in a high fertility grassland soil, in a 90-day incubation study. Methods: The soil was treated with nitrogen (N) at three levels: 0 mg-N kg-1 soil, 50 mg-N kg-1 soil, and 700 mg-N kg-1 soil, with or without the two nitrification inhibitors. The abundance of comammox Nitrospira, AOA, AOB, and nitrite oxidising bacteria (NOB) was measured using qPCR. The comammox Nitrospira community structure was assessed using Illumina sequencing. Results and Discussion: The results showed that the application of chlorate inhibited the oxidation of both NH4+ and NO2- in all three nitrogen treatments. The application of chlorate significantly reduced the abundance of comammox Nitrospira amoA and nxrB genes across the 90-day experimental period. Chlorate also had a significant effect on the beta diversity (Bray-Curtis dissimilarity) of the comammox Nitrospira clade B community. Whilst AOB grew in response to the N substrate additions and were inhibited by both inhibitors, AOA showed litle or no response to either the N substrate or inhibitor treatments. In contrast, comammox Nitrospira clade B were inhibited by the high ammonium concentrations released from the urine substrates. These results demonstrate the differential and niche responses of the three ammonia oxidising communities to N substrate additions and nitrification inhibitor treatments. Further research is needed to investigate the specificity of the two inhibitors on the different ammonia oxidising communities.

Prolactin receptor potentiates chemotherapy through miRNAs-induced G6PD/TKT inhibition in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies, with a notoriously dismal prognosis. As a competitive inhibitor of DNA synthesis, gemcitabine is the cornerstone drug for treating PDAC at all stages. The therapeutic effect of gemcitabine, however, is often hindered by drug resistance, and the underlying mechanisms remain largely unknown. It is unclear whether their response to chemotherapeutics is regulated by endocrine regulators, despite the association between PDAC risk and endocrine deregulation. Here, we show that prolactin receptor (PRLR) synergizes with gemcitabine in both in vitro and in vivo treatment of PDAC. Interestingly, PRLR promotes the expression of miR-4763-3p and miR-3663-5p, two novel miRNAs whose functions are unknown. Furthermore, the analysis of transcriptome sequencing data of tumors from lactating mouse models enriches the PPP pathway, a multifunctional metabolic pathway. In addition to providing energy, the PPP pathway mainly provides a variety of raw materials for anabolism. We demonstrate that two key enzymes of the pentose phosphate pathway (PPP), G6PD and TKT, are directly targeted by miR-4763-3p and miR-3663-5p. Notably, miR-4763-3p and miR-3663-5p diminish the nucleotide synthesis of the PPP pathway, thereby increasing gemcitabine sensitivity. As a result, PRLR harnesses these two miRNAs to suppress PPP and nucleotide synthesis, subsequently elevating the gemcitabine sensitivity of PDAC cells. Also, PDAC tissues and tumors from LSL-KrasG12D/+, LSL-Trp53R172H/+, and PDX1-cre (KPC) mice exhibit downregulation of PRLR. Bisulfite sequencing of PDAC tissues revealed that PRLR downregulation is due to epigenetic methylation. In this study, we show for the first time that the endocrine receptor PRLR improves the effects of gemcitabine by boosting two new miRNAs that block the PPP pathway and nucleotide synthesis by inhibiting two essential enzymes concurrently. The PRLR-miRNAs-PPP axis may serve as a possible therapeutic target to supplement chemotherapy advantages in PDAC.

Higher diversity of sulfur-oxidizing bacteria based on soxB gene sequencing in surface water than in spring in Wudalianchi volcanic group, NE China.

INTRODUCTION: Sulfur-oxidizing bacteria (SOB) play a key role in the biogeochemical cycling of sulfur. OBJECTIVES: To explore SOB diversity, distribution, and physicochemical drivers in five volcanic lakes and two springs in the Wudalianchi volcanic field, China. METHODS: This study analyzed microbial communities in samples via high-throughput sequencing of the soxB gene. Physical-chemical parameters were measured, and QIIME 2 (v2019.4), R, Vsearch, MEGA7, and Mothur processed the data. Alpha diversity indices and UPGMA clustering assessed community differences, while heat maps visualized intra-sample variations. Canoco 5.0 analyzed community-environment correlations, and NMDS, Adonis, and PcoA explored sample dissimilarities and environmental factor correlations. SPSS v.18.0 tested for statistical significance. RESULTS: The diversity of SOB in surface water was higher than in springs (more than 7.27 times). We detected SOB affiliated to ß-proteobacteria (72.3 %), α-proteobacteria (22.8 %), and γ-proteobacteria (4.2 %) distributed widely in these lakes and springs. Rhodoferax and Cupriavidus were most frequent in all water samples, while Rhodoferax and Bradyrhizobium are dominant in surface waters but rare in springs. SOB genera in both habitats were positively correlated. Co-occurrence analysis identified Bradyrhizobium, Blastochloris, Methylibium, and Metyhlobacterium as potential keystone taxa. Redundancy analysis (RDA) revealed positive correlations between SOB diversity and total carbon (TC), Fe2+, and total nitrogen (TN) in all water samples. CONCLUSION: The diversity and community structure of SOB in volcanic lakes and springs in the Wudalianchi volcanic group were clarified. Moreover, the diversity and abundance of SOB decreased with the variation of water openness, from open lakes to semi-enclosed lakes and enclosed lakes.

Prognostic Significance of Preoperative and Postoperative Evaluation of Combined Tumor Markers for Patients With Colon Cancer.

BACKGROUND: The combined value of the tumor markers carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) in patients with colon cancer (CC) is unclear. This study aimed to investigate the role of composite tumor markers in the prognosis of CC. METHODS: Patients who underwent curative resection of colon adenocarcinoma were enrolled. The tumor marker status before and after the operation was used to divide the patients into groups according to the number of tumor markers with abnormal expression, and recurrence-free survival (RFS) and overall survival (OS) of different groups were compared. The impact of changes in composite tumor markers in the perioperative period on outcomes was further explored. RESULTS: Ultimately, 531 patients were enrolled in the study. As the number of preoperative and postoperative elevated tumor markers increased, both RFS and OS rates became lower (both P<0.05). Further analysis revealed that the number of elevated tumor markers after resection can significantly affect the outcomes (both P<0.05). In patients with abnormal preoperative tumor markers, normalization of markers after surgery was a protective factor for prognosis (both P<0.05), and patients with postoperative elevated levels of both tumor markers had a 5.5-fold and 6-fold increase in the risk of recurrence and death. In addition, patients with elevated markers after surgery had a high risk of recurrence within 5 years after colectomy. CONCLUSIONS: Postoperative tumor markers had a better ability to differentiate postoperative outcomes in patients with CC than preoperative tumor markers. Patients whose tumor markers normalized after surgery had a better prognosis.

Covalent binding of reactive anhydride of cantharidin to biological amines.

Cantharidin is a terpenoid from coleoptera beetles. Cantharidin has been used to treat molluscum contagiosum and some types of tumors. Cantharidin is highly toxic and cantharidin poisoning and fatal cases have been reported worldwide. The mechanisms underlying cantharidin-induced toxicity remain unclear. Cantharidin contains anhydride, which may react with biological amines. This study aimed to examine the chemical reactivity of cantharidin toward nucleophiles and characterize adducts of cantharidin with biological amines in vitro and in mice. Here, two types of conjugates were formed in the incubation of cantharidin under physiologic conditions with free amino acids, a mimic peptide, or amine-containing compounds, respectively. Amide-type conjugates were produced by the binding of cantharidin anhydride with the primary amino group of biological amines. Imide-type conjugates were generated from the dehydration and cyclization of amide-type conjugates. The structure of the conjugates was characterized by using the high-resolution mass spectrometry. We introduced the 14N/15N and 79Br/81Br isotope signatures to confirm the formation of conjugates using L-(ε)15N-lysine, L-lysine-15N2, and bromine-tagged hydrazine, respectively. The structure of imide conjugate was also confirmed by NMR experiments. Furthermore, the amide and imide conjugates of cantharidin with amino acids or N-acetyl-lysine were detected in mouse liver and urine. Cantharidin was found to modify lysine residue proteins in mouse liver. Pan-P450 inhibitor 1-aminobenzotriazole significantly increased the urine cantharidin-N-acetyl-lysine conjugates whereas decreased cantharidin metabolites. In summary, cantharidin anhydride can covalently bind to biological amines nonenzymatically, which facilitates a better understanding of the role of nonenzymatic reactivity in cantharidin poisoning. Significance Statement Anhydride moiety of cantharidin can covalently bind to the primary amino group of biological amines nonenzymatically. Amide and imide conjugates were generated after the covalent binding of cantharidin anhydride with the primary amino groups of amino acids, a mimic peptide, and protein lysine residues. The structure of conjugates was confirmed by 14N/15N and 79Br/81Br isotope signatures using isotope-tagged reagents and NMR experiments. This study will facilitate the understanding of the role of nonenzymatic reactivity in cantharidin poisoning.

Glycolysis inhibition induces anti-tumor central memory CD8+T cell differentiation upon combination with microwave ablation therapy.

Minimally invasive thermal therapy is a successful alternative treatment to surgery in solid tumors with high complete ablation rates, however, tumor recurrence remains a concern. Central memory CD8+ T cells (TCM) play important roles in protection from chronic infection and cancer. Here we find, by single-cell RNA analysis of human breast cancer samples, that although the memory phenotype of peripheral CD8+ T cells increases slightly after microwave ablation (MWA), the metabolism of peripheral CD8+ T cells remains unfavorable for memory phenotype. In mouse models, glycolysis inhibition by 2-deoxy-D-glucose (2DG) in combination with MWA results in long-term anti-tumor effect via enhancing differentiation of tumor-specific CD44hiCD62L+CD8+ TCM cells. Enhancement of CD8+ TCM cell differentiation determined by Stat-1, is dependent on the tumor-draining lymph nodes (TDLN) but takes place in peripheral blood, with metabolic remodeling of CD8+ T cells lasting the entire course of the the combination therapy. Importantly, in-vitro glycolysis inhibition in peripheral CD8+ T cells of patients with breast or liver tumors having been treated with MWA thrice leads to their differentiation into CD8+ TCM cells. Our work thus offers a potential strategy to avoid tumor recurrence following MWA therapy and lays down the proof-of-principle for future clinical trials.

The application of graphic language personalized emotion in graphic design.

Emotion Recognition is the experience of attitude in graphic language expression and composition. People use both verbal and non-verbal behaviours to communicate their emotions. Visual communication and graphic design are always evolving to meet the demands of an increasingly affluent and culturally conscious populace. When graphic designing works, designers should consider their own opinions about related works from the audience's or customer's standpoint so that the emotion between them can resonate. Hence, this study proposes a personalized emotion recognition framework based on convolutional neural networks (PERF-CNN) to create visual content for graphic design. Graphic designers prioritize the logic of showing objects in interactive designs and use visual hierarchy and page layout approaches to respond to users' demands via typography and imagery. This ensures that the user experience is maximized. This research identifies three tiers of emotional thinking: expressive signal, emotional experience, and emotional infiltration, all of which affect graphic design. This article explores the subject of graphic design language and its ways of emotional recognition, as well as the relationship between graphic images, shapes, and feelings. CNN initially extracted expressive features from the user's face images and the poster's visual information. The clustering process categorizes the poster or advertisement images into positive, negative, and neutral classes. Research and applications of graphic design language benefit from the proposed method's experimental results, demonstrating that it outperforms conventional classification approaches in the dataset. In comparison to other popular models, the experimental results demonstrate that the proposed PERF-CNN model improves each of the following: classification accuracy (97.4 %), interaction ratio (95.6 %), emotion recognition ratio (98.9 %), rate of influence of pattern and colour features (94.4 %), and prediction error rate (6.5 %).

Self-perceived upper extremity motor function predicts health-related quality of life in chronic stroke survivors.

PURPOSE: To examine whether the Upper Extremity Functional Index (UEFI) score independently contributes to the Stroke Impact Scale (SIS) score and quantified its relative contribution to SIS scores in chronic stroke survivors. MATERIALS AND METHODS: A cross-sectional study in a university-based rehabilitation centre with people with chronic stroke (N = 95) aged ≥ 50 years. The outcome measures included paretic hand grip strength, Fugl-Meyer Upper Extremity Assessment (FMA-UE), Wolf Motor Function Test (WMFT), UEFI, and SIS. RESULTS: Correlation analysis revealed that paretic hand grip strength, FMA-UE, UEFI, and WMFT scores exhibited a significant moderate positive correlation with SIS scores (r = 0.544-0.687, p < 0.001). The results of a regression model indicated that after adjustment for demographic factors and stroke-related impairments, the UEFI scores remained independently associated with SIS scores, accounting for 18.8% of the variance. The entire model explained 60.3% of the variance in SIS scores. CONCLUSIONS: Self-perceived UE motor function is a crucial component to be included in rehabilitation programmes aimed at enhancing quality of life and participation among chronic stroke survivors.

Observation-based outcome measures, e.g., Fugl­Meyer Assessment for Upper Extremity (FMA-UE), Wolf Motor Function Test (WMFT) could not predict the health-related quality of life (Stroke Impact scale (SIS)) in chronic stroke survivors in our study, which was contradictory with current studies.A self-perceived outcome measure to evaluate upper extremity function (Upper Extremity Functional Index (UEFI)) could independently predict the health-related quality of life (SIS), accounting for 18.8% of the variance.Our study demonstrated that self-perceived UE motor function would be an important component to optimize the rehabilitation programmes aimed at enhancing quality of life and social participation among chronic stroke survivors.

B Cells and IL-21-Producing Follicular Helper T Cells Cooperate to Determine the Dynamic Alterations of Premetastatic Tumor Draining Lymph Nodes of Breast Cancer.

Metastasis is the major cause of cancer-related death, and lymph node is the most common site of metastasis in breast cancer. However, the alterations that happen in tumor-draining lymph nodes (TDLNs) to form a premetastatic microenvironment are largely unknown. Here, we first report the dynamic changes in size and immune status of TDLNs before metastasis in breast cancer. With the progression of tumor, the TDLN is first enlarged and immune-activated at early stage that contains specific antitumor immunity against metastasis. The TDLN is then contracted and immunosuppressed at late stage before finally getting metastasized. Mechanistically, B and follicular helper T (Tfh) cells parallelly expand and contract to determine the size of TDLN. The activation status and specific antitumor immunity of CD8+ T cells in the TDLN are determined by interleukin-21 (IL-21) produced by Tfh cells, thus showing parallel changes. The turn from activated enlargement to suppressed contraction is due to the spontaneous contraction of germinal centers mediated by follicular regulatory T cells. On the basis of the B-Tfh-IL-21-CD8+ T cell axis, we prove that targeting the axis could activate TDLNs to resist metastasis. Together, our findings identify the dynamic alterations and regulatory mechanisms of premetastatic TDLNs of breast cancer and provide new strategies to inhibit lymph node metastasis.

Clinical efficacy and safety of erlotinib combined with chemotherapy in the treatment of advanced pancreatic cancer: A meta-analysis.

BACKGROUND: Advanced pancreatic cancer is resistant to chemotherapeutic drugs, resulting in limited treatment efficacy and poor prognosis. Combined administration of the chemotherapeutic gemcitabine and erlotinib is considered a potential first-line treatment for advanced pancreatic cancer. However, their comparative benefits and potential risks remain unclear. AIM: To assess the clinical efficacy and safety of erlotinib combined with other chemotherapy regimens for the treatment of advanced pancreatic cancer. METHODS: Literature on the clinical efficacy and safety of erlotinib combined with chemotherapy for advanced pancreatic cancer was retrieved through an online search. The retrieved literature was subjected to a methodological qualitative assessment and was analyzed using the RevMan 5.3 software. Ten randomized controlled trials involving 2444 patients with advanced pancreatic cancer were included in the meta-analysis. RESULTS: Compared with chemotherapeutic treatment, erlotinib combined with chemotherapy significantly prolonged the progression-free survival time of pancreatic cancer patients [hazard ratio (HR) = 0.78, 95%CI: 0.66-0.92, P = 0.003]. Meanwhile, the overall survival (HR= 0.99, 95%CI: 0.72-1.37, and P = 0.95) and disease control rate (OR = 0.93, 95%CI: 0.45-0.91, P = 0.84) were not significantly favorable. In terms of safety, the erlotinib and chemotherapy combination was associated with a significantly higher risk of diarrhea (OR = 3.59, 95%CI: 1.63-7.90, P < 0.05) and rash (OR = 3.63, 95%CI: 1.64-8.01, P < 0.05) compared with single-agent chemotherapy. Moreover, the risk of vomiting (OR = 1.27, 95%CI: 0.62-2.59, P = 0.51), regurgitation/anorexia (OR = 1.61, 95%CI: 0.25-10.31, P = 0.62), and infection (OR = 0.72, 95%CI: 0.28-1.87, P = 0.50) were not significant in either group. CONCLUSION: Compared with a single chemotherapeutic modality, erlotinib combined with gemcitabine can prolong progression-free survival in pancreatic cancer, but does not improve survival benefit or disease control rate, and can increase the risk of diarrhea and rash.

Immunogenicity and safety of an ORF7-deficient skin-attenuated and neuro-attenuated live vaccine for varicella: a randomised, double-blind, controlled, phase 2a trial.

BACKGROUND: The Oka varicella vaccine strain remains neurovirulent and can establish lifelong latent infection, raising safety concerns about vaccine-related herpes zoster. In this study, we aimed to evaluate the immunogenicity and safety of a skin-attenuated and neuro-attenuated varicella vaccine candidate (v7D vaccine). METHODS: We did this randomised, double-blind, controlled, phase 2a clinical trial in Jiangsu, China. Healthy children aged 3-12 years with no history of varicella infection or vaccination were enrolled and randomly assigned (1:1:1:1) to receive a single subcutaneous injection of the v7D vaccine at 3·3 log10 plaque forming units (PFU; low-dose v7D group), 3·9 log10 PFU (medium-dose v7D group), and 4·2 log10 PFU (high-dose v7D group), or the positive control varicella vaccine (vOka vaccine group). All the participants, laboratory personnel, and investigators other than the vaccine preparation and management staff were masked to the vaccine allocation. The primary outcome was assessment of the geometric mean titres (GMTs) and seroconversion rates of anti-varicella zoster virus immunoglobulin G (IgG) induced by different dose groups of v7D vaccine at 0, 42, 60, and 90 days after vaccination in the per-protocol set for humoral immune response analysis. Safety was a secondary outcome, focusing on adverse events within 42 days post-vaccination, and serious adverse events within 6 months after vaccination. This study was registered on Chinese Clinical Trial Registry, ChiCTR2000034434. FINDINGS: On Aug 18-21, 2020, 842 eligible volunteers were enrolled and randomly assigned treatment. After three participants withdrew, 839 received a low dose (n=211), middle dose (n=210), or high dose (n=210) of v7D vaccine, or the vOka vaccine (n=208). In the per-protocol set for humoral immune response analysis, the anti-varicella zoster virus IgG antibody response was highest at day 90. At day 90, the seroconversion rates of the low-dose, medium-dose, and high-dose groups of v7D vaccine and the positive control vOka vaccine group were 100·0% (95% CI 95·8-100·0; 87 of 87 participants), 98·9% (93·8-100·0; 87 of 88 participants), 97·8% (92·4-99·7; 91 of 93 participants), and 96·4% (89·8-99·2; 80 of 83 participants), respectively; the GMTs corresponded to values of 30·8 (95% CI 26·2-36·0), 31·3 (26·7-36·6), 28·2 (23·9-33·2), and 38·5 (31·7-46·7). The v7D vaccine, at low dose and medium dose, elicited a humoral immune response similar to that of the vOka vaccine. However, the high-dose v7D vaccine induced a marginally lower GMT compared with the vOka vaccine at day 90 (p=0·027). In the per-protocol set, the three dose groups of the v7D vaccine induced a similar humoral immune response at each timepoint, with no statistically significant differences. The incidence of adverse reactions in the low-dose, medium-dose, and high-dose groups of v7D vaccine was significantly lower than that in the vOka vaccine group (17% [35 of 211 participants], 20% [41 of 210 participants], and 13% [27 of 210 participants] vs 24% [50 of 208 participants], respectively; p=0·025), especially local adverse reactions (10% [22 of 211 participants], 14% [30 of 210 participants] and 9% [18 of 210 participants] vs 18% [38 of 208 participants], respectively; p=0·016). None of the serious adverse events were vaccine related. INTERPRETATION: The three dose groups of the candidate v7D vaccine exhibit similar humoral immunogenicity to the vOka vaccine and are well tolerated. These findings encourage further investigations on two-dose vaccination schedules, efficacy, and the potential safety benefit of v7D vaccine in the future. FUNDING: The National Natural Science Foundation of China, CAMS Innovation Fund for Medical Sciences, the Fundamental Research Funds for the Central Universities, and Beijing Wantai. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

Magnetic CAR T cell purification using an anti-G4S linker antibody.

Chimeric antigen receptor (CAR) redirected T cells are successfully employed in the combat against several hematological malignancies, however, are often compromised by low transduction rates making refinement of the CAR T cell products necessary. Here, we report a broadly applicable enrichment protocol relying on marking CAR T cells with an anti-glycine4-serine (G4S) linker antibody followed by magnetic activated cell sorting (MACS). The protocol is broadly applicable since the G4S peptide is an integral part of the vast majority of CARs as it links the VH and VL recognition domains. We demonstrate the feasibility by using the canonical second generation CARs specific for CEA and Her2, respectively, obtaining highly purified CAR T cell products in a one-step procedure without impairing cell viability. The protocol is also applicable to a dual specific CAR (tandem CAR). Except for CD39, T cell activation/exhaustion markers were not upregulated after separation. Purified CAR T cells retained their functionality with respect to antigen-specific cytokine secretion, cytotoxicity, and the capacity to proliferate and eliminate cognate tumor cells upon repetitive stimulation. Collectively, the one-step protocol for purifying CAR T cells extends the toolbox for preclinical research and specifically for clinical CAR T cell manufacturing.

Oral administration of PEDV-dissolved Alg-CS gel induces high and sustained mucosal immunity in mice.

Porcine epidemic diarrhea (PED) is a serious disease in piglets that leads to high mortality. An effective measure that provides higher IgA levels in the intestine and milk is required to decrease losses. Porcine epidemic diarrhea virus (PEDV) was dissolved in calcium alginate (Alg) and combined with chitosan (CS) via electrostatic interactions between cationic chitosan and anionic alginate to create a porous gel (Alg-CS+PEDV). The gel was used to immunize mice orally or in combination with subcutaneous injections of inactivated PEDV vaccine. At 12 and 24 days after immunization, levels of IgA and IgG in Alg-CS+PEDV were higher than with normal PEDV oral administration. At 24 days after immunization, the concentration of IFN-γ in Alg-CS+PEDV was higher than with normal PEDV oral administration. Furthermore, oral administration combining subcutaneous immunization induced higher levels of IgG and IgA than oral administration alone. Our study provides a new method for the preparation and administration of oral vaccines to achieve enhanced mucosal immunity against PEDV.

Alkaline sphingomyelinase deficiency impairs intestinal mucosal barrier integrity and reduces antioxidant capacity in dextran sulfate sodium-induced colitis.

BACKGROUND: Ulcerative colitis is a chronic inflammatory disease of the colon with an unknown etiology. Alkaline sphingomyelinase (alk-SMase) is specifically expressed by intestinal epithelial cells, and has been reported to play an anti-inflammatory role. However, the underlying mechanism is still unclear. AIM: To explore the mechanism of alk-SMase anti-inflammatory effects on intestinal barrier function and oxidative stress in dextran sulfate sodium (DSS)-induced colitis. METHODS: Mice were administered 3% DSS drinking water, and disease activity index was determined to evaluate the status of colitis. Intestinal permeability was evaluated by gavage administration of fluorescein isothiocyanate dextran, and bacterial translocation was evaluated by measuring serum lipopolysaccharide. Intestinal epithelial cell ultrastructure was observed by electron microscopy. Western blotting and quantitative real-time reverse transcription-polymerase chain reaction were used to detect the expression of intestinal barrier proteins and mRNA, respectively. Serum oxidant and antioxidant marker levels were analyzed using commercial kits to assess oxidative stress levels. RESULTS: Compared to wild-type (WT) mice, inflammation and intestinal permeability in alk-SMase knockout (KO) mice were more severe beginning 4 d after DSS induction. The mRNA and protein levels of intestinal barrier proteins, including zonula occludens-1, occludin, claudin-3, claudin-5, claudin-8, mucin 2, and secretory immunoglobulin A, were significantly reduced on 4 d after DSS treatment. Ultrastructural observations revealed progressive damage to the tight junctions of intestinal epithelial cells. Furthermore, by day 4, mitochondria appeared swollen and degenerated. Additionally, compared to WT mice, serum malondialdehyde levels in KO mice were higher, and the antioxidant capacity was significantly lower. The expression of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) in the colonic mucosal tissue of KO mice was significantly decreased after DSS treatment. mRNA levels of Nrf2-regulated downstream antioxidant enzymes were also decreased. Finally, colitis in KO mice could be effectively relieved by the injection of tertiary butylhydroquinone, which is an Nrf2 activator. CONCLUSION: Alk-SMase regulates the stability of the intestinal mucosal barrier and enhances antioxidant activity through the Nrf2 signaling pathway.

T cell cascade regulation initiates systemic antitumor immunity through living drug factory of anti-PD-1/IL-12 engineered probiotics.

Immune checkpoint blockade (ICB) has revolutionized cancer therapy but only works in a subset of patients due to the insufficient infiltration, persistent exhaustion, and inactivation of T cells within a tumor. Herein, we develop an engineered probiotic (interleukin [IL]-12 nanoparticle Escherichia coli Nissle 1917 [INP-EcN]) acting as a living drug factory to biosynthesize anti-PD-1 and release IL-12 for initiating systemic antitumor immunity through T cell cascade regulation. Mechanistically, INP-EcN not only continuously biosynthesizes anti-PD-1 for relieving immunosuppression but also effectively cascade promote T cell activation, proliferation, and infiltration via responsive release of IL-12, thus reaching a sufficient activation threshold to ICB. Tumor targeting and colonization of INP-EcNs dramatically increase local drug accumulations, significantly inhibiting tumor growth and metastasis compared to commercial inhibitors. Furthermore, immune profiling reveals that anti-PD-1/IL-12 efficiently cascade promote antitumor effects in a CD8+ T cell-dependent manner, clarifying the immune interaction of ICB and cytokine activation. Ultimately, such engineered probiotics achieve a potential paradigm shift from T cell exhaustion to activation and show considerable promise for antitumor bio-immunotherapy.