Cytokine Secretion Levels Research Articles

Loop33 × 123 CAR-T targeting CD33 and CD123 against immune escape in acute myeloid leukemia

BackgroundImmunotherapy, such as chimeric antigen receptor T (CAR-T) cells targeting CD33 or CD123, has been well developed over the past decade for the treatment of acute myeloid leukemia (AML). However, the inability to sustain tumor-free survival and the possibility of relapse due to antigen loss have raised concerns. A dual targeting of CD33 and CD123 is needed for better outcomes.MethodsBased on our previously constructed CD33 and CD123 monovalent CAR-T, Loop33 × 123 and Loop123 × 33 CAR-T were constructed with molecular cloning techniques. All CAR-T cells were generated by lentivirus transduction of T cells from healthy donors. Phenotype detection was evaluated on day 7 concerning activation, exhaustion, and subtype proportions. Coculture killing assays were conducted using various AML cell lines and primary AML cells. Degranulation and cytokine secretion levels were detected by flow cytometry. Cell-derived xenograft models were established using wild-type Molm 13 cell lines, or a mixture of Molm 13-KO33 and Molm 13-KO123 cells as an ideal model of immune escape. By monitoring body weight and survival of tumor-bearing mice, Loop33 × 123 and Loop123 × 33 CAR-T cells were further assessed for their efficacy in vivo.ResultsIn vitro study, our results demonstrated that Loop33 × 123 CAR-T cells could efficiently eliminate AML cell lines and primary AML cells with elevated degranulation and cytokine secretion levels. Compared with our previously constructed monovalent CD33 or CD123 CAR-T cells, Loop33 × 123 CAR-T cells showed superior advantages in an immune escape model. In vivo studies further confirmed that Loop33 × 123 CAR-T cells could effectively prolong the survival of mice without significant toxicity. However, Loop123 × 33 CAR-T cells failed to show the same effects. Furthermore, Loop33 × 123 CAR-T cells efficiently circumvented potential immune escape, a challenge where monovalent CAR-T cells failed.ConclusionsLoop33 × 123 CAR-T targeting CD33 and CD123 could efficiently eliminate AML cells and prolong survival of tumor-bearing mice, while addressing the issue of immune escape.

In Silico Studies: Stigmastan-3,6-dione in the Ethyl Acetate Fraction of Momordica charantia L Fruit has immunostimulant and anti-inflammatory activity

Momordica charantia L fruit provides an immunomodulatory effect by stimulating certain components of the body's immune system. Bioactive phytochemicals from M. charantia L function as anti-inflammatory agents by reducing levels of pro-inflammatory cytokine secretion including IL-1, IL-6, IL-8, TNF-α, NF-kB. This research looks at the insilico mechanism of action of the active ingredient of the EtOAc fraction of M. charantia L fruit as an immunostimulant and anti-inflammatory. The materials and methods used were an Intel Core i7 10 Th Gen laptop, Chem Office Professional 17.1, Chem 2D, Chem 3D software, PDB code (6W9K) and ligand (TUA), Stigmastan-3,6 dione (C29H48O2) from the EtOAc fraction of fruit M. charantia L was docked using MVD software, RMSD and RMSF values ​​were viewed using Yasara Software, pkCSM online tool to predict compound toxicity, toxicity prediction (LD50) was used by Protox online tool. The results of molecular docking of standard compounds, namely Methylprednisolone and Prednisolone and Stigmastan-3,6-dione, have Rerank Score values of -120.62, -121.47, -79.50, respectively. The lower the Rerank score value, the lower the binding energy between the protein and the ligand, causing the protein and ligand bonds to be more stable and it is predicted that the activity of the compound will be greater. The movement of RMSD values ​​between 0.6-1.9 Å for the 3 (three) compounds, is still within stable limits and does not undergo conformation. The RMSF value of the 3 (three) compounds has the same amino acid residue pattern. The insilico toxicity prediction for the 3 (three) compounds is still within safe limits. The EtOAc fraction of M. charantia L fruit with the active compound Stigmastan-3,6-Dione in its mechanism of action in silico shows activity as an anti-inflammatory and immunostimulant that works on the NF-kB pathway.

Generation of ex vivo autologous hematopoietic stem cell-derived T lymphocytes for cancer immunotherapy

CD19CAR-T cell therapy demonstrated promising outcomes in relapsed/refractory B-cell malignancies. Nonetheless, the limited T-cell function and ineffective T-cell apheresis for therapeutic purposes are still concern in heavily pretreated patients. We investigated the feasibility of generating hematopoietic stem cell-derived T lymphocytes (HSC-T) for cancer immunotherapy. The patients’ autologous peripheral blood HSCs were enriched for CD34+ and CD3+ cells. The CD34+ cells were then cultured following three steps of lymphoid progenitor differentiation, T-cell differentiation, and T-cell maturation processes. HSC-T cells were successfully generated with robust fold expansion of 3735 times. After lymphoid progenitor differentiation, CD5+ and CD7+ cells remarkably increased (65–84 %) while CD34+ cells consequentially declined. The mature CD3+ cells were detected up to 40 % and 90 % on days 42 and 52, respectively. The majority of HSC-T population was naïve phenotype compared to CD3-T cells (73 % vs 34 %) and CD8:CD4 ratio was 2:1. The higher level of cytokine and cytotoxic granule secretion in HSC-T was observed after activation. HSC-T cells were assessed for clinical application and found that CD19CAR-transduced HSC-T cells demonstrated higher cytokine secretion and a trend of superior cytotoxicity against CD19+ target cells compared to control CAR-T cells. A chronic antigen stimulation assay revealed similar T-cell proliferation, stemness, and exhaustion phenotypes among CAR-T cell types. In conclusions, autologous HSC-T was feasible to generate with preserved T-cell efficacy. The HSC-T cells are potentially utilized as an alternative option for cellular immunotherapy.

Biomimetic copper-containing nanogels for imaging-guided tumor chemo-chemodynamic-immunotherapy

Developing multifunctional nanoplatforms to comprehensively modulate the tumor microenvironment and enhance diagnostic and therapeutic outcomes still remains a great challenge. Here, we report the facile construction of a multivariate nanoplatform based on cancer cell membrane (CM)-encapsulated redox-responsive poly(N-vinylcaprolactam) (PVCL) nanogels (NGs) co-loaded with Cu(II) and chemotherapeutic drug toyocamycin (Toy) for magnetic resonance (MR) imaging-guided combination tumor chemodynamic therapy/chemoimmunotherapy. We show that redox-responsive PVCL NGs formed through precipitation polymerization can be aminated, conjugated with 3,4-dihydroxyhydrocinnamic acid for Cu(II) complexation, physically loaded with Toy, and finally camouflaged with CMs. The created ADCT@CM NGs with an average size of 113.0 nm are stable under physiological conditions and can efficiently release Cu(II) and Toy under tumor microenvironment with a high level of glutathione. Meanwhile, the developed NGs are able to enhance cancer cell oxidative stress and endoplasmic reticulum stress by synergizing the effects of chemodynamic therapy mediated by Cu-based Fenton-like reaction and Toy-mediated chemotherapy, thereby triggering significant immunogenic cell death (ICD). In a melanoma mouse model, the NGs show potent immune activation effects to reinforce tumor therapeutic efficacy through ICD induction and immune modulation including high levels of immune cytokine secretion, increased tumor infiltration of CD8+ cytotoxic T cells, and reduced tumor infiltration of regulatory T cells. With the CM coating and Cu(II) loading, the developed NG platform demonstrates homologous tumor targeting and T1-weighted MR imaging, hence providing a general biomimetic NG platform for ICD-facilitated tumor theranostic nanoplatform. Statement of significanceDeveloping multifunctional nanoplatforms to comprehensively modulate the tumor microenvironment (TME) and enhance theranostic outcomes remains a challenge. Here, a cancer cell membrane (CM)-camouflaged nanoplatform based on aminated poly(N-vinylcaprolactam) nanogels (NGs) co-loaded with Cu(II) and toyocamycin (Toy) was prepared for magnetic resonance (MR) imaging-guided combination tumor chemodynamic therapy/chemoimmunotherapy. The tumor targeting specificity and efficient TME-triggered release of Cu(II) and Toy could enhance tumor cell oxidative stress and endoplasmic reticulum stress by synergizing the effects of chemodynamic therapy mediated by Cu-based Fenton-like reaction and Toy-mediated chemotherapy, respectively, thereby leading to significant immunogenic cell death (ICD) and immune response. With the CM coating and Cu(II) loading, the developed NG platform also demonstrates good T1-weighted tumor MR imaging performance. Hence, this study provides a general biomimetic NG platform for ICD-facilitated tumor theranostics.

Dexmedetomidine leads to the amelioration of stress response to surgery.

To determine the impact of intravenous Dexmedetomidine (Dex) administered perioperatively on stress response markers in patients undergoing ENT surgeries. This randomized interventional study was conducted at POFs Hospital and NUMS affiliated WMC, Wah Cantt, in collaboration with IIMC Rawalpindi, Pakistan, from October 2021 to April 2022. One hundred patients aged between 15-60 years, after satisfying the inclusion standards were randomly divided into two groups C and D. Group-C (n=50) received normal saline in addition to the standard anesthesia protocol. The intervention Group-D (n=50) was administered 1µg/kg dexmedetomidine hydrochloride intravenously over 10 minutes just before the induction followed 0.5μg/kg/hr as maintenance dose till the end of surgery. Serum inflammatory biomarkers (interleukins-6, TNF-α and cortisol) were measured in blood samples in both groups, taken at 0 (T0), 30 minutes(T1), and two hours (T2) time intervals and analyzed by using Enzyme-linked immunosorbent assay (ELISA). Data was statistically analyzed using SPSS 23. The patients receiving Dex showed marked decrease in serum levels of cortisol, TNF- α and interleukins-6, from 139.73 to10.18, 99.51 to 0.96 and 85.09 to 0.96 respectively. Comparison between C and D groups revealed significant differences (p≤0.05) in these serum biomarkers. In the present study, intravenous Dex suppressed the intraoperative rise in levels of cytokine secretion and enhanced smooth recovery with no incidence of nausea and vomiting. These effects could be attributed to the anti-inflammatory effects of dexmedetomidine.

CpG Oligodeoxynucleotide-Coated Chitosan Nanoparticles Enhance Macrophage Proinflammatory Phenotype In Vitro.

This study aimed to investigate the effects of CpG Oligodeoxynucleotide (CpG ODNs)-Coated Chitosan Nanoparticles (CNP) on the phenotype of murine macrophages and their pro-inflammatory cytokine profile in vitro. CNP-CpG ODNs loaded with FITC-scrambled siRNA were prepared using the ionotropic gelation method. Peritoneal macrophages were isolated and exposed to CNP-CpG ODNs. Treated macrophages were assessed for uptake capacity. Flow cytometry was used to evaluate the expression levels of MHC-II, CD40, and CD86 costimulatory molecules in treated macrophages. Furthermore, the secretion levels of proinflammatory cytokines (TNF-α and IL-6) and the release of nitric oxide (NO) were measured in the culture supernatant of treated macrophages using sandwich ELISA and the Griess reaction, respectively. These in vitro studies showed that CNP-CpG ODNs had no cytotoxic effect on macrophages and were efficiently taken up by them. Additionally, CNP-CpG ODNs significantly increased the production of TNF-α, IL-6, and NO in the culture supernatant compared to CNP alone. Moreover, CNP-CpG ODNs enhanced the expression of MHC-II, CD40, and CD86 costimulatory molecules on macrophages. These findings indicate that incorporating CpG ODNs into CNPs promotes macrophage maturation and a proinflammatory phenotype. Therefore, CNP-CpG ODNs may serve as an effective system for targeted gene delivery to macrophages, enhancing immune responses.

Effects of simulated space conditions on CD4+ T cells: a multi modal analysis.

The immune system is an intricate network of cellular components that safeguards against pathogens and aberrant cells, with CD4+ T cells playing a central role in this process. Human space travel presents unique health challenges, such as heavy ion ionizing radiation, microgravity, and psychological stress, which can collectively impede immune function. The aim of this research was to examine the consequences of simulated space stressors on CD4+ T cell activation, cytokine production, and gene expression. CD4+ T cells were obtained from healthy individuals and subjected to Fe ion particle radiation, Photon irradiation, simulated microgravity, and hydrocortisone, either individually or in different combinations. Cytokine levels for Th1 and Th2 cells were determined using multiplex Luminex assays, and RNA sequencing was used to investigate gene expression patterns and identify essential genes and pathways impacted by these stressors. Simulated microgravity exposure resulted in an apparent Th1 to Th2 shift, evidenced on the level of cytokine secretion as well as altered gene expression. RNA sequencing analysis showed that several gene pathways were altered, particularly in response to Fe ions irradiation and simulated microgravity exposures. Individually, each space stressor caused differential gene expression, while the combination of stressors revealed complex interactions. The research findings underscore the substantial influence of the space exposome on immune function, particularly in the regulation of T cell responses. Future work should focus expanding the limited knowledge in this field. Comprehending these modifications will be essential for devising effective strategies to safeguard the health of astronauts during extended space missions. The effects of simulated space stressors on CD4+ T cell function are substantial, implying that space travel poses a potential threat to immune health. Additional research is necessary to investigate the intricate relationship between space stressors and to develop effective countermeasures to mitigate these consequences.

Polysaccharide of Atractylodes macrocephala Koidz alleviates NAFLD-induced hepatic inflammation in mice by modulating the TLR4/MyD88/NF-κB pathway

Non-alcoholic fatty liver disease (NAFLD) not only could cause abnormal lipid metabolism in the liver, but also could cause liver inflammation. Previous studies have shown that Polysaccharide of Atractylodes macrocephala Koidz (PAMK) could alleviate animal liver inflammatory damage and alleviate NAFLD in mice caused by high-fat diet(HFD), but regulation of liver inflammation caused by NAFLD has rarely been reported. In this study, an animal model of non-alcoholic fatty liver inflammation in the liver of mice was established to explore the protective effect of PAMK on the liver of mice. The results showed that PAMK could alleviate the abnormal increase of body weight and liver weight of mice caused by HFD, alleviate the abnormal liver structure of mice, reduce the level of oxidative stress and cytokine secretion in the liver of mice, and downregulate the mRNA expression of TLR4, MyD88, NF-κB and protein expression of P-IκB, P-NF-κB-P65, TLR4, MyD88, NF-κB in the liver. These results indicate that PAMK could alleviate hepatocyte fatty degeneration and damage, oxidative stress and inflammatory response of the liver caused by NAFLD in mice.

Preparation of cardamonin and IR780 Co-loaded on Lycium barbarum polysaccharide nanoparticles and anti-tumor efficacy evaluation

To improve the therapeutic efficacy of cardamonin (CRD) in the treatment of breast cancer, Lycium barbarum polysaccharide (LBP) was selected as a nanocarrier for CRD and IR780 to prepare CRD-IR780@LBP nanoparticles (NPs) via antisolvent precipitation. CRD-IR780@LBP NPs demonstrated superior characteristics, such as sustainable drug release, high photothermal conversion, and good antitumor efficacy and secretion levels of cytokines (TNF-α, IL-6 and IL-2). The IC50 value of CRD-IR780@LBP NPs was 2.31 μg/mL, which was significantly lower than that of the pure CRD group (P < 0.0001). The tumor inhibition rate of CRD-IR780@LBP NPs was 80.03 %, which was a 1.58-fold increase compared to that of PTX injection. Furthermore, the ELISA results suggested that LBP can exert therapeutic effects by upregulating the expression levels of TNF-α, IL-6 and IL-2 (P < 0.01) without causing any apparent adverse effects on major tissues and organs. In conclusion, the CRD-IR780@LBP NPs prepared in this study can enhance the therapeutic efficacy of CRD against breast cancer via multiple synergistic anti-tumor mechanisms.

Recombinant Hemagglutinin Protein from H9N2 Avian Influenza Virus Exerts Good Immune Effects in Mice.

The H9N2 subtype of avian influenza virus (AIV) causes enormous economic losses and poses a significant threat to public health; the development of vaccines against avian influenza is ongoing. To study the immunogenicity of hemagglutinin (HA) protein, we constructed a recombinant pET-32a-HA plasmid, induced HA protein expression with isopropyl β-D-1-thiogalactopyranoside (IPTG), verified it by SDS-PAGE and Western blotting, and determined the sensitivity of the recombinant protein to acid and heat. Subsequently, mice were immunized with the purified HA protein, and the immunization effect was evaluated according to the hemagglutination inhibition (HI) titer, serum IgG antibody titer, and cytokine secretion level of the mice. The results showed that the molecular weight of the HA protein was approximately 84 kDa, and the protein existed in both soluble and insoluble forms; in addition, the HA protein exhibited good acid and thermal stability, the HI antibody titer reached 6 log2-8 log2, and the IgG-binding antibody titer was 1:1,000,000. Moreover, the levels of IL-2, IL-4, and IL-5 in the immunized mouse spleen cells were significantly increased compared with those in the control group. However, the levels of IL-1β, IL-6, IL-13, IFN-γ, IL-18, TNF-α, and GM-CSF were decreased in the immunized group. The recombinant HA protein utilized in this study exhibited good stability and exerted beneficial immune effects, providing a theoretical basis for further research on influenza vaccines.

Bushen Huoxue decotion-containing serum prevents chondrocyte pyroptosis in a m6A-dependent manner in facet joint osteoarthritis

BackgroundFacet joint osteoarthritis (FJOA) is a common lumbar osteoarthritis characterized by degeneration of small joint cartilage. Bushen Huoxue decotion (BSHXD) has good therapeutic effects on OA. Our work aimed to further probe the pharmacological effects of BSHXD-containing serum (BSHXD-CS) on FJOA and define underlying the mechanisms invovled. MethodsTo establish a FJOA cell model, primary rat chondrocytes were treated with LPS. The mRNA and protein expressions were assessed using qRT-PCR and western blot, respectively. The secretion levels of pro-inflammatory cytokines were measured by ELISA. Cell viability was determined by CCK8 assay. The global m6A level was detected by the kit, and NLRP3 mRNA m6A level was determined by Me-RIP assay. The molecular interactions were analyzed by RIP and RNA pull-down assays. ResultsBSHXD-CS treatment relieved LPS-induced cell injury, inflammation, NLRP3 inflammasome and pyroptosis in chondrocytes (all p < 0.05). LPS-induced NLRP3 upregulation in chondrocytes was related to its high m6A modification level (p < 0.05). It was also observed that BSHXD-CS reduced LPS-induced m6A modification in chondrocytes via repressing STAT3 (all p < 0.05), suggesting BSHXD-CS could repress NLRP3 expression via m6A-dependent manner. Moreover, DAA, a m6A specific inhibitor, was proved to strengthen the protectively roles of BSHXD-CS on LPS-challenged pytoptosis (all p < 0.05). ConclusionBSHXD-CS inhibited NLRP3 inflammasome activation and pyroptosis in chondrocytes to repress OA progression by reducing RNA m6A modification.

IL-4 acts on skin-derived dendritic cells to promote the TH2 response to cutaneous sensitization and the development of allergic skin inflammation

Atopic dermatitis is characterized by scratching and a TH2-dominated local and systemic response to cutaneously encountered antigens. Dendritic cells (DCs) capture antigens in the skin and rapidly migrate to draining lymph nodes (dLNs) where they drive the differentiation of antigen-specific naive T cells. We sought to determine whether non-T-cell-derived IL-4 acts on skin-derived DCs to promote the TH2 response to cutaneously encountered antigen and allergic skin inflammation. DCs from dLNs of ovalbumin (OVA)-exposed skin were analyzed by flow cytometry and for their ability to polarize OVA-specific naive CD4+ T cells. Skin inflammation following epicutaneous sensitization of tape-stripped skin was assessed by flow cytometry of skin cells and real-time quantitative PCR of cytokines. Cytokine secretion and antibody levels were evaluated by ELISA. Scratching upregulated IL4 expression in human skin. Similarly, tape stripping caused rapid basophil-dependent upregulation of cutaneous Il4 expression in mouse skin. Invitro treatment of DCs from skin dLNs with IL-4 promoted their capacity to drive TH2 differentiation. DCs from dLNs of OVA-sensitized skin of Il4-/- mice and CD11c-CreIl4rflox/- mice, which lack IL-4Rα expression in DCs (DCΔ/Δll4ra mice), were impaired in their capacity to drive TH2 polarization compared with DCs from controls. Importantly, OVA-sensitized DCΔ/Δll4ra mice demonstrated impaired allergic skin inflammation and OVA-specific systemic TH2 response evidenced by reduced TH2 cytokine secretion by OVA-stimulated splenocytes and lower levels of OVA-specific IgE and IgG1 antibodies, compared with controls. Mechanical skin injury causes basophil-dependent upregulation of cutaneous IL-4. IL-4 acts on skin DCs that capture antigen and migrate to dLNs to promote their capacity for TH2 polarization and drive allergic skin inflammation.

Co-delivery of STING and TLR7/8 agonists in antigen-based nanocapsules to dendritic cells enhances CD8+ T cell-mediated melanoma remission

Insufficient efficacy of tumor vaccines still represents a major challenge due to poor adjuvant potency. Combining antigen and adjuvants of different classes bears the potential to induce a broad spectrum of anti-tumor immune responses. Here we demonstrate a novel nanocarrier (NC)-based vaccine combining the type I interferon-triggering STING agonist diamidobenzimidazole (diABZI) compound 3 and the well-established TLR7/8 agonist resiquimod (R848). Encapsulation of both adjuvants into polymeric nanocapsules enables the simultaneous transport of immunostimulatory molecules with tumor antigens. Thereby achieved co-delivery further improved DC stimulation and subsequent anti-tumor immune responses.Combined encapsulation of R848 and diABZI enhanced DC activation and induced stronger antigen-specific T cell responses compared to the single adjuvant NC treatment or using soluble forms of antigens and adjuvants in vitro and in vivo. This was determined by the vigorous expression of CD80, CD83, and CD86. Furthermore, the dual adjuvant therapy initiated the highest secretion levels of different pro-inflammatory cytokines and chemokines.Moreover, a substantial antigen-specific T cell proliferation led to robust tumor remission in a murine B16 melanoma model. Subcutaneous administration of R848/diABZI-loaded NCs induced enhanced infiltration of CD4+ and CD8+ T cells as well as neutrophils in tumor-draining lymph nodes (LN) and tumor tissue. Encapsulating the melanoma-specific antigenic peptide of TRP2 into the adjuvant-loaded NCs reduced the growth of B16 melanoma and prolonged the overall survival. The herein presented novel anti-tumor vaccination strategy avoids the use of structural compounds, increases the antigen load of dendritic cells, uses a fixed combination of antigen and two potent adjuvants and bears the potential to induce vigorous antigen-specific anti-cancer immunity.

Glycerides of lauric acid supplementation in the chicken diet enhances the humoral and cellular immune response to infectious bronchitis virus

Controlling pathogenic infections while reducing antibiotic usage is an important challenge during poultry production. In addition to vaccination strategies, several solutions to enhance the immune response against pathogens are evaluated. In this study, we aim to determine the effects of the glycerides of lauric acid (GLA) supplementation in chickens’ diets on humoral and cellular immune response pathogenic infections, using an in vivo model of infectious bronchitis virus (IBV). One-day-old Ross 308 broilers were vaccinated with live attenuated IBV and fed diets supplemented with or without GLA at 3 kg/ton. The levels of early (day 7) specific anti-IBV in sera were significantly increased in broilers fed GLA, compared to the control groups (P<0.05), showing a stronger primary humoral response. The secretion levels of main cytokines remained similar in spleens of all the experimental groups. However, the splenocytes from broilers fed GLA showed higher activation and effector abilities when measured by IFN-γ ELISpot in presence of N-261–280 IBV peptide or Concanavalin A (Con A), a pan T lymphocytes mitogen. In response to N-261–280 peptide, GLA group showed a 2-fold increase of spot numbers (P < 0.05) and 3-fold increase of spot surfaces (P < 0.01) compared to the control groups. Similarly, Con A stimulation showed a 2-fold increases in spot surfaces and numbers in the GLA supplemented group compared to the control group (P < 0.01). In summary, GLA supplementation in chicken feed enhances the primary humoral immune response and strengthen the T lymphocytes mediated cellular immune response. These findings demonstrate how GLA can improve chicken resilience against pathogenic challenges by enhancing their immune responses.

DUSP1 Mitigates MSU-Induced Immune Response in Gouty Arthritis Reinforcing Autophagy.

Persistent hyperuricemia can lead to the generation and deposition of monosodium urate (MSU) crystals. This can trigger gouty arthritis (GA), which in turn induces inflammation. Activation of the Nod-like receptor pyrin domain containing 3 (NLRP3) inflammasome plays a critical role in the onset and progression of GA. Autophagy may have a dual effect on GA with regard to the NLRP3 inflammasome. Therefore, the present study aimed to gain a deeper comprehension of the interaction between autophagy and NLRP3 inflammasome activation is imperative for developing more efficacious treatments for GA. Peripheral blood monocytes (PBMCs) were first isolated from GA patients and healthy controls and underwent bulk RNA sequencing analysis. Overexpression and knockdown of dual specificity phosphatase 1 (DUSP1) was performed in THP-1 monocytes to investigate its role in the immune response and mitochondrial damage. The luciferase assay and Western blot analysis were used to study the interaction between autophagy and NLRP3 inflammasome activation. Bulk RNA sequencing analysis showed significant upregulation of DUSP1 expression in PBMCs from GA patients compared to healthy controls. This result was subsequently verified by reverse transcription quantitative polymerase chain reaction (RT-qPCR). DUSP1 expression in human THP-1 monocytes was also shown to increase after MSU treatment. Downregulation of DUSP1 expression increased the secretion of inflammatory cytokines after MSU treatment, whereas the overexpression of DUSP1 decreased the secretion levels. Lipopolysaccharides (LPS) combined with adenosine-triphosphate (ATP) led to mitochondrial damage, which was rescued by overexpressing DUSP1. DUSP1 overexpression further increased the level of autophagy following MSU treatment, whereas downregulation of DUSP1 decreased autophagy. Treatment with the autophagy inhibitor 3-Methyladenine (3-MA) restored inflammatory cytokine secretion levels in the DUSP1 overexpression group. MSU caused pronounced pathological ankle swelling in vivo. However, DUSP1 overexpression significantly mitigated this phenotype, accompanied by significant downregulation of inflammatory cytokine secretion levels in the joint tissues. This study revealed a novel function and mechanism for DUSP1 in promoting autophagy to mitigate the MSU-induced immune response in GA. This finding suggests potential diagnostic biomarkers and anti-inflammatory targets for more effective GA therapy.

Differential Cytokine Responses of APOE3 and APOE4 Blood-brain Barrier Cell Types to SARS-CoV-2 Spike Proteins.

SARS-CoV-2 spike proteins have been shown to cross the blood-brain barrier (BBB) in mice and affect the integrity of human BBB cell models. However, the effects of SARS-CoV-2 spike proteins in relation to sporadic, late onset, Alzheimer's disease (AD) risk have not been extensively investigated. Here we characterized the individual and combined effects of SARS-CoV-2 spike protein subunits S1 RBD, S1 and S2 on BBB cell types (induced brain endothelial-like cells (iBECs) and astrocytes (iAstrocytes)) generated from induced pluripotent stem cells (iPSCs) harboring low (APOE3 carrier) or high (APOE4 carrier) relative Alzheimer's risk. We found that treatment with spike proteins did not alter iBEC integrity, although they induced the expression of several inflammatory cytokines. iAstrocytes exhibited a robust inflammatory response to SARS-CoV-2 spike protein treatment, with differences found in the levels of cytokine secretion between spike protein-treated APOE3 and APOE4 iAstrocytes. Finally, we tested the effects of potentially anti-inflammatory drugs during SARS-CoV-2 spike protein exposure in iAstrocytes, and discovered different responses between spike protein treated APOE4 iAstrocytes and APOE3 iAstrocytes, specifically in relation to IL-6, IL-8 and CCL2 secretion. Overall, our results indicate that APOE3 and APOE4 iAstrocytes respond differently to anti-inflammatory drug treatment during SARS-CoV-2 spike protein exposure with potential implications to therapeutic responses.

Construction of CD138-targeted chimeric antigen receptor- modified T cells and their effect in multiple myeloma therapy

Objective: To construct a novel chimeric antigen receptor T (CAR-T) cell targeting CD138 and to investigate its cytotoxicity against myeloma cells. Methods: The hybridoma strain that can stably secrete the CD138 monoclonal antibody (mAb) was prepared and obtained through monoclonal antibody screening technology. The hybridoma strain cells were intraperitoneally injected into mice to produce ascites containing monoclonal antibodies, which were then collected and purified to obtain pure CD138 mAb. Further examinations were performed to assess the biological characteristics of CD138 mAb. The variable region sequence of this antibody was amplified through reverse transcription polymerase chain reaction and was used as the antigen recognition domain of CD138 CAR, which was subsequently expressed on the surface of T cells by lentiviral infection. Flow cytometry was employed to assess the phenotype of CD138 CAR-T cells. In vitro cytotoxicity and degranulation assays were performed to evaluate their antitumor effects. Results: ① We successfully prepared anti-human CD138 antibody hybridoma cell lines and screened a hybridoma cell strain, 5G2, which could persistently and stably secrete the anti-CD138 antibody. ② The purified CD138 (5G2) mAb can especially recognize CD138(+) cells with a binding affinity constant (K(D)) of 6.011×10(-9) mol/L and showed no significant binding activity with CD138(-) cells. ③The variable region sequence of the CD138 (5G2) antibody was obtained using molecular cloning technology, and CD138 (5G2) CAR was successfully constructed and expressed on T cells through lentivirus infection and, concurrently, demonstrated effective binding to recombinant human CD138 protein.④ The proliferation of T cells transduced with the CD138 (5G2) CAR was highly efficient. The phenotype analysis revealed that CD138 (5G2) CAR-T cells exhibited a greater tendency to differentiate into central memory T cells and memory stem T cells, with a reduced proportion of terminally differentiated effector memory subsets. ⑤CD138 (5G2) CAR-T cells demonstrated specific cytotoxicity against CD138(+) myeloma cell line H929, whereas CD138(-) cell line K562 remained unaffected. The percentage of residual H929 cells was (12.92±8.02) % after co-culturing with CD138 (5G2) CAR-T cells, while (54.25±15.79) % was left in the Vector-T group (E∶T=1∶2; P<0.001). ⑥Results of degranulation assays demonstrated a significant activation of CD138 (5G2) CAR-T cells after co-culture with the H929 cell line, whereas no significant activation was observed in Vector-T cells [ (25.78±3.35) % vs (6.13±1.30) %, P<0.001]. ⑦After co-culturing with CD138(+) cells, CD138 (5G2) CAR-T cells exhibited a significant increase in cytokine secretion compared to the Vector-T group [interleukin-2: (1 697.52±599.05) pg/ml vs (5.07±1.17) pg/ml, P<0.001; interferon-γ: (3 312.20±486.38) pg/ml vs (9.28±1.46) pg/ml, P<0.001; and tumor necrosis factor-α: (1 837.43±640.49) pg/ml vs (8.75±1.65) pg/ml, P<0.001]. However, no significant difference was observed in cytokine secretion levels between the two groups after co-culturing with CD138(-) cells. Conclusion: This study successfully prepared a novel monoclonal antibody against CD138, and CAR-T cells constructed with the antigen recognition domain derived from this 5G2 mAb demonstrated effective antitumor activity against myeloma cells. This can be used as a new option for the detection of the CD138 antigen and proposes a novel strategy for multiple myeloma immunotherapy.

Genetically Engineered CLDN18.2 CAR-T Cells Expressing Synthetic PD1/CD28 Fusion Receptors Produced Using a Lentiviral Vector.

This study aimed to develop synthetic Claudin18.2 (CLDN18.2) chimeric antigen receptor (CAR)-T (CAR-T) cells as a treatment for advanced gastric cancer using lentiviral vector genetic engineering technology that targets the CLDN18.2 antigen and simultaneously overcomes the immunosuppressive environment caused by programmed cell death protein 1 (PD-1). Synthetic CAR T cells are a promising approach in cancer immunotherapy but face many challenges in solid tumors. One of the major problems is immunosuppression caused by PD-1. CLDN18.2, a gastric-specific membrane protein, is considered a potential therapeutic target for gastric and other cancers. In our study, CLDN18.2 CAR was a second-generation CAR with inducible T-cell costimulatory (CD278), and CLDN18.2-PD1/CD28 CAR was a third-generation CAR, wherein the synthetic PD1/CD28 chimeric-switch receptor (CSR) was added to the second-generation CAR. In vitro, we detected the secretion levels of different cytokines and the killing ability of CAR-T cells. We found that the secretion of cytokines such as interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) secreted by three types of CAR-T cells was increased, and the killing ability against CLDN18.2-positive GC cells was enhanced. In vivo, we established a xenograft GC model and observed the antitumor effects and off-target toxicity of CAR-T cells. These results support that synthetic anti-CLDN18.2 CAR-T cells have antitumor effect and anti-CLDN18.2-PD1/CD28 CAR could provide a promising design strategy to improve the efficacy of CAR-T cells in advanced gastric cancer.

Evaluation of antioxidant activity and fermentation properties of potential probiotic strain Lactiplantibacillus plantarum HY7720 in plant-based materials

People on vegan diets are at risk of being deficient in varied nutrients such as vitamin B12 and certain amino acids. In this study, we investigated vitamin B12-producing lactic acid bacteria (LAB) as well as the probiotic and antioxidant properties. Lactiplantibacillus plantarum HY7720 was screened from 22 strains of LAB that were isolated from different plant foods, and its growth ability and extracellular vitamin B12-producing capacity in vitamin B12-deficient medium were investigated. To determine whether HY7720 functions as a probiotic, survival rate in the simulated gastrointestinal tract and adhesion property to human intestinal epithelial cells of HY7720 were compared with positive control, Lacticaseibacillus rhamnosus GG (LGG). Moreover, the results showed that HY7720 recovered the gene expression levels of tight junction-associated proteins (TJPs), including TJP1, TJP2, occludin (OCLN), and claudin-1 (CLDN1) and inhibited the secretion levels of pro-inflammatory cytokines, including interleukin (IL)-6 and IL-8, in tumor necrosis factor (TNF)-α-stimulated Caco-2 cells. Furthermore, we verified that HY7720 exhibit the antioxidant potential, by showing its intracellular reactive oxygen species (ROS) scavenging ability in hydrogen peroxide (H2O2)-stimulated Caco-2 cells. The ability of HY7720 to ameliorate H2O2-induced cytotoxicity in Caco-2 cells was inhibited by mitogen-activated protein kinase (MAPK) inhibitors, indicating that its antioxidant responses are related to extracellular signal-regulated kinase (ERK) and c-JUN N-terminal kinase (JNK). This study also investigated the nutritional qualities of three plant-based materials (brown rice, white rice, and soy milk) fermented using HY7720. Collectively, HY7720 could be used as a promising probiotic strain for the prevention of nutritional deficiencies among people on vegetarian diets.

Transport of Non-Steroidal Anti-Inflammatory Drugs across an Oral Mucosa Epithelium In Vitro Model.

Non-steroidal anti-inflammatory drugs (NSAIDs) are one of the most prescribed drugs to treat pain or fever. However, oral administration of NSAIDs is frequently associated with adverse effects due to their inhibitory effect on the constitutively expressed cyclooxygenase enzyme 1 (COX-1) in, for instance, the gastrointestinal tract. A systemic delivery, such as a buccal delivery, of NSAIDs would be beneficial and additionally has the advantage of a non-invasive administration route, especially favourable for children or the elderly. To investigate the transport of NSAIDs across the buccal mucosa and determine their potential for buccal therapeutic usage, celecoxib, diclofenac, ibuprofen and piroxicam were tested using an established oral mucosa Transwell® model based on human cell line TR146. Carboxyfluorescein and diazepam were applied as internal paracellular and transcellular marker molecule, respectively. Calculated permeability coefficients revealed a transport ranking of ibuprofen > piroxicam > diclofenac > celecoxib. Transporter protein inhibitor verapamil increased the permeability for ibuprofen, piroxicam and celecoxib, whereas probenecid increased the permeability for all tested NSAIDs. Furthermore, influence of local inflammation of the buccal mucosa on the transport of NSAIDs was mimicked by treating cells with a cytokine mixture of TNF-α, IL-1ß and IFN-γ followed by transport studies with ibuprofen (+ probenecid). Cellular response to pro-inflammatory stimuli was confirmed by upregulation of cytokine targets at the mRNA level, increased secreted cytokine levels and a significant decrease in the paracellular barrier. Permeability of ibuprofen was increased across cell layers treated with cytokines, while addition of probenecid increased permeability of ibuprofen in controls, but not across cell layers treated with cytokines. In summary, the suitability of the in vitro oral mucosa model to measure NSAID transport rankings was demonstrated, and the involvement of transporter proteins was confirmed; an inflammation model was established, and increased NSAID transport upon inflammation was measured.