Enhancing the efficacy of vaccinia-based oncolytic virotherapy by inhibiting CXCR2-mediated MDSC trafficking.

Oncolytic virotherapy is an innovative approach for cancer treatment. However, recruitment of myeloid-derived suppressor cells (MDSCs) into the tumor microenvironment (TME) after oncolysis-mediated local inflammation leads to tumor resistance to the therapy. Using the murine malignant mesothelioma model, we demonstrated that the in situ vaccinia virotherapy recruited primarily polymorphonuclear MDSCs (PMN-MDSCs) into the TME, where they exhibited strong suppression of cytotoxic T lymphocytes in a reactive oxygen species-dependent way. Single-cell RNA sequencing analysis confirmed the suppressive profile of PMN-MDSCs at the transcriptomic level and identified CXCR2 as a therapeutic target expressed on PMN-MDSCs. Abrogating PMN-MDSC trafficking by CXCR2-specific small molecule inhibitor during the vaccinia virotherapy exhibited enhanced antitumor efficacy in 3 syngeneic cancer models, through increasing CD8+/MDSC ratios in the TME, activating cytotoxic T lymphocytes, and skewing suppressive TME into an antitumor environment. Our results warrant clinical development of CXCR2 inhibitor in combination with oncolytic virotherapy.

RNA Interference against ATP as a Gene Therapy Approach for Prostate Cancer.

Chemotherapeutic agents targeting energy metabolism have not achieved satisfactory results in different types of tumors. Herein, we developed an RNA interference (RNAi) method against adenosine triphosphate (ATP) by constructing an interfering plasmid-expressing ATP-binding RNA aptamer, which notably inhibited the growth of prostate cancer cells through diminishing the availability of cytoplasmic ATP and impairing the homeostasis of energy metabolism, and both glycolysis and oxidative phosphorylation were suppressed after RNAi treatment. Further identifying the mechanism underlying the effects of ATP aptamer, we surprisingly found that it markedly reduced the activity of membrane ionic channels and membrane potential which led to the dysfunction of mitochondria, such as the decrease of mitochondrial number, reduction in the respiration rate, and decline of mitochondrial membrane potential and ATP production. Meanwhile, the shortage of ATP impeded the formation of lamellipodia that are essential for the movement of cells, consequently resulting in a significant reduction of cell migration. Both the downregulation of the phosphorylation of AMP-activated protein kinase (AMPK) and endoplasmic reticulum kinase (ERK) and diminishing of lamellipodium formation led to cell apoptosis as well as the inhibition of angiogenesis and invasion. In conclusion, as the first RNAi modality targeting the blocking of ATP consumption, the present method can disturb the respiratory chain and ATP pool, which provides a novel regime for tumor therapies..

SARS-CoV-2 hijacks neutralizing dimeric IgA for nasal infection and injury in Syrian hamsters1.

Prevention of robust severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in nasal turbinate (NT) requires in vivo evaluation of IgA neutralizing antibodies. Here, we report the efficacy of receptor binding domain (RBD)-specific monomeric B8-mIgA1 and B8-mIgA2, and dimeric B8-dIgA1, B8-dIgA2 and TH335-dIgA1 against intranasal SARS-CoV-2 challenge in Syrian hamsters. These antibodies exhibited comparable neutralization potency against authentic virus by competing with human angiotensin converting enzyme-2 (ACE2) receptor for RBD binding. While reducing viral loads in lungs significantly, prophylactic intranasal B8-dIgA unexpectedly led to high amount of infectious viruses and extended damage in NT compared to controls. Mechanistically, B8-dIgA failed to inhibit SARS-CoV-2 cell-to-cell transmission, but was hijacked by the virus through dendritic cell-mediated trans-infection of NT epithelia leading to robust nasal infection. Cryo-EM further revealed B8 as a class II antibody binding trimeric RBDs in 3-up or 2-up/1-down conformation. Neutralizing dIgA, therefore, may engage an unexpected mode of SARS-CoV-2 nasal infection and injury.

Transforming Growth Factor ß Signaling Promotes HIV-1 Infection in Activated and Resting Memory CD4+ T Cells.

Understanding the facilitator of HIV-1 infection and subsequent latency establishment may aid the discovery of potential therapeutic targets. Here, we report the elevation of plasma transforming growth factor ß (TGF-ß) during acute HIV-1 infection among men who have sex with men (MSM). Using a serum-free in vitro system, we further delineated the role of TGF-ß signaling in mediating HIV-1 infection of activated and resting memory CD4+ T cells. TGF-ß could upregulate both the frequency and expression of the HIV-1 coreceptor CCR5, thereby augmenting CCR5-tropic viral infection of resting and activated memory CD4+ T cells via Smad3 activation. The production of live HIV-1JR-FL upon infection and reactivation was increased in TGF-ß-treated resting memory CD4+ T cells without increasing CD4 expression or inducing T cell activation. The expression of CCR7, a central memory T cell marker that serves as a chemokine receptor to facilitate T cell trafficking into lymphoid organs, was also elevated on TGF-ß-treated resting and activated memory CD4+ T cells. Moreover, the expression of CXCR3, a chemokine receptor recently reported to facilitate CCR5-tropic HIV-1 infection, was increased on resting and activated memory CD4+ T cells upon TGF-ß treatment. These findings were coherent with the observation that ex vivo CCR5 and CXCR3 expression on total resting and resting memory CD4+ T cells in combination antiretroviral therapy (cART)-naive and cART-treated patients were higher than in healthy individuals. Overall, the study demonstrated that TGF-ß upregulation induced by acute HIV-1 infection might promote latency reservoir establishment by increasing infected resting memory CD4+ T cells and lymphoid organ homing of infected central memory CD4+ T cells. Therefore, TGF-ß blockade may serve as a potential supplementary regimen for HIV-1 functional cure by reducing viral latency. IMPORTANCE Incomplete eradication of HIV-1 latency reservoirs remains the major hurdle in achieving a complete HIV/AIDS cure. Dissecting the facilitator of latency reservoir establishment may aid the discovery of druggable targets for HIV-1 cure. This study showed that the T cell immunomodulatory cytokine TGF-ß was upregulated during the acute phase of infection. Using an in vitro serum-free system, we specifically delineated that TGF-ß promoted HIV-1 infection of both resting and activated memory CD4+ T cells via the induction of host CCR5 coreceptor. Moreover, TGF-ß-upregulated CCR7 or CXCR3 might promote HIV-1 latent infection by facilitating lymphoid homing or IP-10-mediated viral entry and DNA integration, respectively. Infected resting and central memory CD4+ T cells are important latency reservoirs. Increased infection of these cells mediated by TGF-ß will promote latency reservoir establishment during early infection. This study, therefore, highlighted the potential use of TGF-ß blockade as a supplementary regimen with cART in acute patients to reduce viral latency.

Isoformic PD-1-mediated immunosuppression underlies resistance to PD-1 blockade in hepatocellular carcinoma patients.

OBJECTIVE: Immune checkpoint blockade (ICB) has improved cancer treatment, yet why most hepatocellular carcinoma (HCC) patients are resistant to PD-1 ICB remains elusive. Here, we elucidated the role of a programmed cell death protein 1 (PD-1) isoform, Δ42PD-1, in HCC progression and resistance to nivolumab ICB. DESIGN: We investigated 74 HCC patients in three cohorts, including 41 untreated, 28 treated with nivolumab and 5 treated with pembrolizumab. Peripheral blood mononuclear cells from blood samples and tumour infiltrating lymphocytes from tumour tissues were isolated for immunophenotyping. The functional significance of Δ42PD-1 was explored by single-cell RNA sequencing analysis and validated by functional and mechanistic studies. The immunotherapeutic efficacy of Δ42PD-1 monoclonal antibody was determined in HCC humanised mouse models. RESULTS: We found distinct T cell subsets, which did not express PD-1 but expressed its isoform Δ42PD-1, accounting for up to 71% of cytotoxic T lymphocytes in untreated HCC patients. Δ42PD-1+ T cells were tumour-infiltrating and correlated positively with HCC severity. Moreover, they were more exhausted than PD-1+ T cells by single T cell and functional analysis. HCC patients treated with anti-PD-1 ICB showed effective PD-1 blockade but increased frequencies of Δ42PD-1+ T cells over time especially in patients with progressive disease. Tumour-infiltrated Δ42PD-1+ T cells likely sustained HCC through toll-like receptors-4-signalling for tumourigenesis. Anti-Δ42PD-1 antibody, but not nivolumab, inhibited tumour growth in three murine HCC models. CONCLUSION: Our findings not only revealed a mechanism underlying resistance to PD-1 ICB but also identified anti-Δ42PD-1 antibody for HCC immunotherapy.

Human neutralizing antibodies for SARS-CoV-2 prevention and immunotherapy.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). SARS-CoV-2 has been spreading worldwide since December 2019, resulting in the ongoing COVID-19 pandemic with 237 million infections and 4.8 million deaths by 11 October 2021. While there are great efforts of global vaccination, ending this pandemic has been challenged by issues of exceptionally high viral transmissibility, re-infection, vaccine-breakthrough infection, and immune escape variants of concern. Besides the record-breaking speed of vaccine research and development, antiviral drugs including SARS-CoV-2-specific human neutralizing antibodies (HuNAbs) have been actively explored for passive immunization. In support of HuNAb-based immunotherapy, passive immunization using convalescent patients' plasma has generated promising evidence on clinical benefits for both mild and severe COVID-19 patients. Since the source of convalescent plasma is limited, the discovery of broadly reactive HuNAbs may have significant impacts on the fight against the COVID-19 pandemic. In this review, therefore, we discuss the current technologies of gene cloning, modes of action, in vitro and in vivo potency and breadth, and clinical development for potent SARS-CoV-2-specific HuNAbs.

A Dual pH/O2 Sensing Film Based on Functionalized Electrospun Nanofibers for Real-Time Monitoring of Cellular Metabolism.

Real-time monitoring of dissolved oxygen (DO) and pH is of great significance for understanding cellular metabolism. Herein, a dual optical pH/O2 sensing membrane was prepared by the electrospinning method. Cellulose acetate (CA) and poly(ε-caprolactone) (PCL) nanofiber membrane blended with platinum (II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorophenyl)-porphyrin (PtTFPP) was used as the DO sensing matrix, upon which electrospun nanofiber membrane of chitosan (CS) coupled with fluorescein 5-isothiocyanate (FITC) was used as the pH sensing matrix. The electrospun sensing film prepared from biocompatible biomaterials presented good response to a wide range of DO concentrations and physiological pH. We used it to monitor the exracellular acidification and oxygen consumption levels of cells and bacteria. This sensing film can provide a luminescence signal change as the DO and pH change in the growth microenvironment. Due to its advantages of good biocompatibility and high stability, we believe that the dual functional film has a high value in the field of biotechnology research.

[Preparation and intestinal absorption of Panax notoginseng saponins chitosan nanoparticles].

In this experiment, Panax notoginseng saponins chitosan nanoparticles(PNS-NPs) were prepared by self-assembly and their appearance, particle size, encapsulation efficiency, drug loading, polydispersity index(PDI), Zeta potential, and microstructure were characterized. The prepared PNS-NPs were intact in structure, with an average particle size of(209±0.258) nm, encapsulation efficiency of 42.34%±0.28%, a drug loading of 37.63%±0.85%, and a Zeta potential of(39.8±3.122) mV. The intestinal absorption of PNS-NPs in rats was further studied. The established HPLC method of PNS was employed to investigate the effects of pH, perfusion rate, and different drugs(PNS raw materials, Xuesaitong Capsules, and PNS-NPs). The absorption rate constant(K_a) and apparent permeability coefficient(P_(app)) in the duodenum, jejunum, ileum, and colon were calculated and analyzed. As illustrated by the results, the intestinal absorption of PNS-NPs was increased in the perfusion solution at pH 6.8(P<0.05), and perfusion rate had no significant effect on the K_a and P_(app) of PNS-NPs. The intestinal absorption of PNS-NPs was significantly different from that of PNS raw materials and Xuesaitong Capsules(P<0.05), and the intestinal absorption of PNS-NPs was significantly improved.

Nasal prevention of SARS-CoV-2 infection by intranasal influenza-based boost vaccination in mouse models.

BACKGROUND: Vaccines in emergency use are efficacious against COVID-19, yet vaccine-induced prevention against nasal SARS-CoV-2 infection remains suboptimal. METHODS: Since mucosal immunity is critical for nasal prevention, we investigated the efficacy of an intramuscular PD1-based receptor-binding domain (RBD) DNA vaccine (PD1-RBD-DNA) and intranasal live attenuated influenza-based vaccines (LAIV-CA4-RBD and LAIV-HK68-RBD) against SARS-CoV-2. FINDINGS: Substantially higher systemic and mucosal immune responses, including bronchoalveolar lavage IgA/IgG and lung polyfunctional memory CD8 T cells, were induced by the heterologous PD1-RBD-DNA/LAIV-HK68-RBD as compared with other regimens. When vaccinated animals were challenged at the memory phase, prevention of robust SARS-CoV-2 infection in nasal turbinate was achieved primarily by the heterologous regimen besides consistent protection in lungs. The regimen-induced antibodies cross-neutralized variants of concerns. Furthermore, LAIV-CA4-RBD could boost the BioNTech vaccine for improved mucosal immunity. INTERPRETATION: Our results demonstrated that intranasal influenza-based boost vaccination induces mucosal and systemic immunity for effective SARS-CoV-2 prevention in both upper and lower respiratory systems. FUNDING: This study was supported by the Research Grants Council Collaborative Research Fund, General Research Fund and Health and Medical Research Fund in Hong Kong; Outbreak Response to Novel Coronavirus (COVID-19) by the Coalition for Epidemic Preparedness Innovations; Shenzhen Science and Technology Program and matching fund from Shenzhen Immuno Cure BioTech Limited; the Health@InnoHK, Innovation and Technology Commission of Hong Kong; National Program on Key Research Project of China; donations from the Friends of Hope Education Fund; the Theme-Based Research Scheme.

Robust SARS-CoV-2 infection in nasal turbinates after treatment with systemic neutralizing antibodies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is characterized by a burst in the upper respiratory portal for high transmissibility. To determine human neutralizing antibodies (HuNAbs) for entry protection, we tested three potent HuNAbs (IC50 range, 0.0007-0.35 µg/mL) against live SARS-CoV-2 infection in the golden Syrian hamster model. These HuNAbs inhibit SARS-CoV-2 infection by competing with human angiotensin converting enzyme-2 for binding to the viral receptor binding domain (RBD). Prophylactic intraperitoneal or intranasal injection of individual HuNAb or DNA vaccination significantly reduces infection in the lungs but not in the nasal turbinates of hamsters intranasally challenged with SARS-CoV-2. Although postchallenge HuNAb therapy suppresses viral loads and lung damage, robust infection is observed in nasal turbinates treated within 1-3 days. Our findings demonstrate that systemic HuNAb suppresses SARS-CoV-2 replication and injury in lungs; however, robust viral infection in nasal turbinate may outcompete the antibody with significant implications to subprotection, reinfection, and vaccine.

Host and viral determinants for efficient SARS-CoV-2 infection of the human lung.

Understanding the factors that contribute to efficient SARS-CoV-2 infection of human cells may provide insights on SARS-CoV-2 transmissibility and pathogenesis, and reveal targets of intervention. Here, we analyze host and viral determinants essential for efficient SARS-CoV-2 infection in both human lung epithelial cells and ex vivo human lung tissues. We identify heparan sulfate as an important attachment factor for SARS-CoV-2 infection. Next, we show that sialic acids present on ACE2 prevent efficient spike/ACE2-interaction. While SARS-CoV infection is substantially limited by the sialic acid-mediated restriction in both human lung epithelial cells and ex vivo human lung tissues, infection by SARS-CoV-2 is limited to a lesser extent. We further demonstrate that the furin-like cleavage site in SARS-CoV-2 spike is required for efficient virus replication in human lung but not intestinal tissues. These findings provide insights on the efficient SARS-CoV-2 infection of human lungs.

Improved Cell Viability and Biocompatibility of Bacterial Cellulose through in Situ Carboxymethylation.

Bacterial cellulose (BC) is a natural product with multiple properties, which has been utilized in tissue engineering. However, cell adhesion and proliferation are reported to be weaker on native BC, providing less support compared to other types of biomaterials, like collagen. To increase the biocompatibility and the medical performance of BC, in situ modification is used to add carboxymethyl group to BC. By partially changing the structure and physical properties of BC, carboxymethylation significantly increases cell affinity and viability, especially on the initial cell adhesion. Furthermore, in the in vivo implantation, the tissue reaction shows that carboxymethylation significantly increases the biocompatibility of BC, exhibiting better tissue condition and a lower inflammatory reaction which are proved through HE staining and immunohistochemistry. The data prove that in situ carboxymethylation is a simple and direct way of improving the performance of BC in medical applications.

Efficacy of four different irrigation techniques combined with 60 °C 3% sodium hypochlorite and 17% EDTA in smear layer removal.

BACKGROUND: Efforts to improve the efficacy of smear layer removal by applying irrigant activation at the final irrigation or by elevating the temperature of the irrigant have been reported. However, the combination of such activation protocols with 60 °C 3% sodium hypochlorite (NaOCl) has seldom been mentioned. The aim of this study was to compare the efficacy in smear layer removal of four different irrigation techniques combined with 60 °C 3% NaOCl and 17% EDTA. METHODS: Fifty single-rooted teeth were randomly divided into five groups (n = 10) according to the irrigant agitation protocols used during chemomechanical preparation(Dentsply Maillefer, Ballaigues, Switzerland): a side-vented needle group, a ultrasonic irrigation (UI) group, a NaviTip FX group, an EndoActivator group, and a control group (no agitation). After each instrumentation, the root canals were irrigated with 1 mL of 3% NaOCl at 60 °C for 1 minute, and after the whole instrumentation, the root canals were rinsed with 1 mL of 17% EDTA for 1 minute. Both NaOCl and EDTA were activated with one of the five irrigation protocols. The efficacy of smear layer removal was scored at the apical, middle and coronal thirds. The Data were statistically analyzed using SAS version 9.2 for Windows (rank sum test for a randomised block design and ANOVA). RESULTS: No significant differences among the NaviTip FX group, EndoActivator group and control groups, and each of these groups showed a lower score than that of UI group (P < 0.05). Within each group, all three thirds were ranked in the following order: coronal > middle > apical (P < 0.05). In the coronal third, the NaviTip FX group was better than UI group. In the middle and apical third, the differences were not significant among any of the groups. CONCLUSIONS: Even without any activation, the combination of 60 °C 3% NaOCl and 17% EDTA could remove the smear layer effectively, similar to NaviTip FX or EndoActivator, and these three protocols were more effective than UI. However, regardless of different types of irrigation technique applied, complete removal of the smear layer was not achieved, particularly in the apical third.

[Establishment of surgical blood ordering schedule and relative influential factors for tumor patients].

OBJECTIVE: To design a blood ordering schedule and explore the influencing factors of blood utilizing and ordering for tumor surgical patients. METHODS: For a total of 58 306 tumor surgical patients, 22 643 applications of blood ordering and 7430 person-times of blood utilization from October 2002 to May 2012 were retrospectively analyzed at Cancer Hospital of Chinese Academy of Medical Sciences. Their clinical profiles and test results were analyzed. RESULTS: The operative transfusion rate was 32.81%. According to the operation position and the blood transfusion and preparation data, the surgical blood ordering schedule of tumor patients was established. Patient gender, hemoglobin, hematocrit, platelet, total protein and albumin level test results had significant effect on the transfusion of red blood cells (OR = 0.797, 9.614, 1.949, 0.437,0.444, 2.038, all P < 0.05). Patient gender, hemoglobin, hematocrit, activated partial thromboplastin time, prothrombin time, albumin and total protein level test results had significant effect on the transfusion of plasma (OR = 0.851, 1.367, 1.801, 1.652, 2.922, 2.224, 1.362, all P < 0.05) . CONCLUSION: For surgical tumor patients, blood ordering should be based upon the test results of routine blood, blood coagulation and protein level test results to ensure that blood transfusion is both rational and safe.

Short communication: broader T cell responses directed against human immunodeficiency virus type 1 in infected Chinese individuals through blood-borne transmission in comparison with mucosal transmission.

Cellular immune responses play a critical role in the control of human immunodeficiency virus type 1 (HIV-1), but less is known about the impact of transmission routes on immune defenses against HIV-1. Here, we report that subjects infected with HIV-1 through contaminated blood showed stronger HIV-specific T cell responses than those infected through mucosa, both in breadth (6.9±2.5 vs. 2.3±0.5, p=0.0293) and in magnitude [1270.0±544.9 vs. 409.5±121.3 SFU per million peripheral blood mononuclear cells (PBMCs), p=0.0223], by using a matrix of 404 overlapping peptides spanning all expressed HIV-1 proteins in an interferon (IFN)-γ enzyme-linked immunospot (ELISpot) assay. Our observation indicates that different mechanisms might be involved in the priming/generating of anti-HIV-specific T cell responses through different transmission routes.

Association between obesity and dental caries in Chinese children.

The study sought to analyze the association between dental caries and obesity in Chinese children, and to investigate the protective and risk factors of dental caries. A total of 280 children aged 7-12 years voluntarily answered the caries examination and questionnaire. Caries was measured using the International Caries Detection and Assessment System. According to the Chinese body mass index, the participants were grouped overweight or normal-weight. The logistic regression model showed no correlation between dental caries and obesity. Drinking yogurt and chewing gum are protective factors, whereas oral breathing and genetic predisposition to caries are risk factors.

Comparison of the effect of four decalcifying agents combined with 60°C 3% sodium hypochlorite on smear layer removal.

INTRODUCTION: The objective of this study was to compare the efficacy on smear layer removal of 4 decalcifying agents: 17% ethylenediaminetetraacetic acid (EDTA), 20% citric acid, BioPure MTAD, and SmearClear. METHODS: Forty-five single-rooted human teeth were used and prepared up to ProTaper F3. After each instrumentation, 1 mL of 3% NaOCl at 60°C was irrigated for 1 minute. All the samples were randomly divided into 5 groups according to the final irrigants: 17% EDTA, 20% citric acid, BioPure MTAD, SmearClear, and 3% NaOCl (control). The smear layer removal of all groups at the apical, middle, and coronal thirds was observed under the thermal field emission scanning electron microscope. RESULTS: The EDTA group was significantly more effective than the SmearClear and MTAD groups (P < .05). The efficacy of all three thirds in the EDTA, MTAD, and SmearClear groups can be arranged as coronal, middle, and apical thirds (P < .05) from excellent to poor. However, in the citric acid group, the coronal two thirds were significantly better than the apical third (P < .05), and there was no statistical significance between the coronal and middle thirds (P > .05). CONCLUSIONS: The 4 decalcifying agents could effectively, but not completely, remove the smear layer, especially in the apical third. The efficacy of 17% EDTA was better than that of MTAD and SmearClear.