ELISA Analysis Research Articles

Helicobacter pylori Inhibition, Gastritis Attenuation, and Gut Microbiota Protection in C57BL/6 Mice by Ligilactobacillus salivarius NCUH062003

Helicobacter pylori (H. pylori), one of the most prevalent pathogenic bacteria worldwide, is the leading cause of gastritis, gastric intestinal metaplasia, and gastric cancer. Antibiotics, the conventional treatment for eliminating H. pylori, often lead to severe bacterial resistance, gut dysbiosis, and hepatic insufficiency and fail to address the inflammatory response or gastric mucosal damage caused by H. pylori infection. In this study, based on 10-week animal experiments, two models of L. salivarius NCUH062003 for the prophylaxis and therapy of H. pylori infection in C57BL/6 mice were established; a comprehensive comparative analysis was performed to investigate the anti-H. pylori effect of probiotics, the reduction in inflammation, and repair of gastric mucosal damage. ELISA, immunohistochemistry, and pathology analyses showed that NCUH062003 decreased the expression of pro-inflammatory cytokine interleukins (IL-1β, IL-6) and myeloperoxidase (MPO) and reduced neutrophil infiltration in the gastric mucosa lamina propria. Immunofluorescence and biochemical analysis showed that NCUH062003 resisted gastric epithelial cell apoptosis, increased the level of superoxide dismutase (SOD) in gastric mucosa, and promoted the expression of tight junction protein ZO1 and Occludin. In addition, through high-throughput sequencing, in the probiotic therapy and prophylactic mode, the diversity and composition of the gut microbiota of HP-infected mice were clarified, the potential functions of the gut microbiota were analyzed, the levels of short-chain fatty acids (SCFAs) were measured, and the effects of L. salivarius NCUH062003 on the gut microbiota and its metabolites in HP-infected mice treated with amoxicillin/metronidazole were revealed. This study provides functional strain resources for the development and application of microbial agents seeking to antagonize H. pylori beyond antibiotics.

A multi-omics approach identifies the key role of disorders of sphingolipid metabolism in Ang II-induced hypertensive cardiomyopathy myocardial remodeling

Hypertension-induced myocardial remodelling encompasses both structural and functional changes in cardiac muscle tissue, such as myocardial hypertrophy, fibrosis, and inflammation. These alterations not only impair the systolic and diastolic functions of the heart but also elevate the risk of cardiovascular events and heart failure. One of the primary contributors to hypertensive cardiomyopathy (HTN-CM) is the over-activation of the renin-angiotensin-aldosterone system (RAAS), which subsequently induces myocardial remodeling. Although conventional therapeutic strategies aim to suppress RAAS and slow the progression of heart failure, the primary challenge in treating HTN-CM remains the lack of sensitive and specific biomarkers for early detection of myocardial remodelling. Combined multi-omics analyses, complemented by experimental validation, offer a systematic understanding of the landscape of gene/protein/metabolite expression in HTN-CM, revealing the underlying mechanisms of angiotensin II (Ang II)-induced myocardial remodeling in HTN-CM. Transcriptomic analysis revealed that differentially expressed genes (DEGs) are implicated in sphingolipid metabolic processes and are associated with collagen synthesis and inflammatory responses, collectively contributing to myocardial remodeling in HTN-CM. Proteomic analysis demonstrated that differentially expressed proteins (DEPs) are also involved in inflammatory and fibrotic processes, with associations to sphingolipid signaling pathways, particularly manifested through elevated expression of IL6, COL4A1, FGG, FGB, CREBBP and SPHK2 proteins. Metabolomic profiling further elucidated the increased expression of bioactive sphingolipid metabolites S1P and Sa1P in the myocardium of HTN-CM. Integrative multi-omics analysis revealed that HTN-CM is primarily influenced by the sphingolipid signaling pathway, with additional associations to the HIF-1α and FoxO signaling pathways. Correlation analysis has highlighted strong associations between sphingolipids and genes/proteins related to fibrosis and inflammation, as well as their connection to the HIF-1α and FoxO signalling pathways. Furthermore, certain key indicators were validated through ELISA and Western blot analyses in both plasma and myocardial tissue. In conclusion, the findings of this study suggest that excessive Ang II may induce abnormalities in sphingolipid metabolism, resulting in increased levels of S1P in both circulating and myocardial tissues. This elevation in S1P is implicated in myocardial inflammatory and fibrotic alterations, highlighting its pivotal role in myocardial remodeling. The specific mechanism underlying the sphingolipid signaling pathway in myocardial remodeling may involve downstream biological processes, including oxidative stress and excessive mitochondrial autophagy, mediated by HIF-1α and FoxO.

Holstein × Montbéliarde-sired F1 generation crossbred female calves have an increased cellular immune response potential compared with purebred Holsteins

It is well known that crossbreds show many advantages over purebreds in improving calf health traits, but the immunological factors responsible for this heterosis remain largely unclear. The objective of this study was to compare the cellular immune responses and antibodies of Holstein (HO) and Montbéliarde-sired × Holstein (MH) F1 generation female calves, and investigate the effects of crossbreeding on the immunity. Fifty three-month-old healthy female calves (25 HO, 25 MH) were selected meticulously in a farm with the same criteria. Subsequently, complete blood count, flow cytometric analysis of T cell subsets and intracellular IFN-γ production, as well as indirect ELISA analysis of antibodies were performed in order to determine the immune profiles of the two groups of calves. We found that MH calves had higher percentage and number of CD4+ and CD8+ T cells than HO calves in the peripheral blood (p < 0.05), with higher MFI of CD44 on CD4+ and CD8+ T cells (p < 0.05). When stimulated by PMA and Ionomycin, the CD4+ and CD8+ T cells from MH calves secreted more IFN-γ than that of HO calves (p < 0.01). These results suggested that some immunological traits have been improved in MH calves, which may be an important cause of heterosis in crossbred animals.

USP33 promotes caerulein-induced apoptotic, oxidative and inflammatory injuries in acute pancreatitis by deubiquitinating TRAF3.

Background: Tumor necrosis factor receptor associated factor 3 (TRAF3) and deubiquitinating enzyme ubiquitin-specific protease 33 (USP33) have been identified to play important roles in inflammatory diseases, including acute pancreatitis (AP). Here, we aimed to explore whether USP33 affected AP progression by affecting TRAF3 expression through deubiquitination.Methods: Caerulein-treated HPDE6-C7 cells were used to mimic AP conditions in vitro. Levels of mRNAs and proteins were examined by qRT-PCR and Western blot. Cell proliferation and apoptosis were evaluated using CCK-8 assay, EdU assay and flow cytometry. Cell oxidative stress was assessed by detecting the production of superoxide dismutase and malonaldehyde. ELISA analysis detected IL-6 and TNF-α levels. Macrophage M1 polarization was evaluated by flow cytometry. Cellular ubiquitination analyzed the ubiquitination effect on TRAF3. Protein interaction between USP33 and TRAF3 was identified by immunofluorescence staining.Results: Caerulein dose-dependently induced apoptosis, oxidative stress, and inflammatory response in HPDE6-C7 cells and promoted macrophage M1 polarization to enhance inflammation (P < 0.05). TRAF3 was highly expressed in AP patients (3.5±1.10 vs. 1.0 ±0.74, P < 0.05) and caerulein-induced HPDE6-C7 cells (3.3 ±0.34 vs. 1.0 ±0.10, P < 0.05). Knockdown of TRAF3 protected HPDE6-C7 cells from caerulein-induced apoptotic, oxidative and inflammatory injuries. Mechanistically, USP33 interacted with TRAF3 and induced TRAF3 deubiquitination to up-regulate its expression (P < 0.05). Further analyses showed that USP33 knockdown reversed caerulein-induced apoptosis, oxidative stress and inflammation in HPDE6-C7 cells by TRAF3 (P < 0.05). Moreover, USP33-TRAF3 activated the NF-κB pathway (P < 0.05).Conclusion: USP33 promoted caerulein-induced apoptosis, oxidative stress and inflammation in pancreatic ductal cells by deubiquitinating TRAF3, indicating a novel insight into the pathogenesis of AP.

Localization and antigenicity reduction of immunodominant conformational IgE epitopes on αs1-casein

αs1-Casein (αs1-CN) is the major allergen in cow milk; however, the understanding of its conformational epitopes remains limited due to the absence of a well-defined three-dimensional structure, which has impeded efforts to effectively reduce its antigenicity. This study employed molecular dynamics simulations (MD), ELISA, cell assays and peptidomes analysis to investigate the critical conformational epitopes of αs1-Casein. MD and immunological analyses identified a dominant conformational epitope encompassing the regions S55-E75 & Y154-T174 & F179-W199, which exhibited strong binding affinity to IgE and triggered the releasing of β-hexosaminidase, histamine and IL-6 in KU812 cells, thereby inducing allergic responses. Notably, the segments Y154-T174 and F179-W199 were particularly impactful. Furthermore, the presence of helical structures within the epitopes enhanced their binding to IgE to a certain extent. Peptidomes analysis further revealed that papain efficiently disrupted the key epitope (Y154-T174) by selectively cleaving the hotspot amino acid residues (Y154 and Y165), thereby significantly reducing the antigenicity of αs1-CN, decreasing IgE and IgG binding to 7.28 % and 10.39 %, respectively. These findings enhance the understanding of αs1-CN's antigenic epitopes and provides a theoretical and technical foundation for the targeted reduction of its antigenicity.

Synergistic Inhibition of Nasopharyngeal Carcinoma by Biyan Qingdu Granule and Micro-Radiation: An Experimental Study

Objective: To investigate the role of Biyan Qingdu Granule combined with micro-radiation in suppressing nasopharyngeal carcinoma and to explore its mechanism of action. Methods: FAT cells of nasopharyngeal carcinoma were injected into the hind limb axilla of F344 rats to establish a rat nasopharyngeal carcinoma tumor model. X-ray radiation was applied to the oral cavity of the rats, delivering a micro-dose of radiation to the distant hind limb axilla tumor. The general condition of the rats, including oral mucosal inflammation, the size of the nasopharyngeal tumor mass, pathological changes in the tumor tissue, and the levels of TNF-α, IL-1β, IL-6, IL-18, IL-12, ICAM-1, and VCAM-1 in the tumor tissue, were assessed. Results: Oral administration of Biyan Qingdu Granule improved the rats’ overall condition and reduced oral mucosal inflammation. Pathological examination revealed tumor cell degeneration and necrosis. ELISA analysis of tumor tissue showed significant reductions in TNF-α, IL-1β, IL-6, ICAM-1, and VCAM-1 levels, along with a notable increase in IL-18 levels. Conclusion: Biyan Qingdu Granule, when combined with micro-radiation, can inhibit tumor growth and reduce tumor volume. Its mechanism of action may involve enhancing immune function and suppressing inflammatory factors.

Structural insight into recognition of Clostridioides difficile toxin A by novel neutralizing nanobodies targeting QTIN-like motifs within its receptor-binding domain

Clostridioides difficile causes a large proportion of nosocomial colon infections by producing toxins TcdA and TcdB as key virulence factors. TcdA and TcdB have analogous domain structures with a receptor-binding domain containing C-terminal combined repetitive oligopeptides (CROPs), an attractive target for the development of therapeutic antibodies. Here, we identify and characterize two potent neutralizing single-domain camelid anti-CROPsA antibodies, C4.2 and H5.2, with distinct mechanisms of action. Peptide mapping, high-resolution crystal structures and site-directed mutagenesis revealed that C4.2 and H5.2 nanobodies target the same C-terminal epitope centered on a 2667QTIN2670 motif, yet utilize different paratopes. Only for C4.2 is the complex geometry compatible with multisite binding using QTIN-like repeats throughout the CROPsA domain, as supported by Western blotting, ELISA, and SEC-MALS analysis. H5.2 binding is stronger and more selective for the C-terminal epitope than C4.2, although both nanobodies are sufficient to neutralize TcdA individually. The described epitope does not overlap with previously described epitopes of anti-CROPs antibodies and provides new modalities for disease treatment and diagnostics.

Honey-fried licorice in the treatment of arrhythmia: Structure elucidation and the mechanism of antiarrhythmic activity

Aim of the studyTo evaluate the therapeutic mechanism of Honey-fried licorice on arrhythmia, to explore the distribution of main components of Honey-fried licorice in vivo before and after processing, and to elucidate the active ingredient of Honey-fried licorice on arrhythmia. Materials and methodsUPLC-Q-TOF/MS were used to analyze the common and different components of raw and honey-fried licorice before and after processing. Yin deficiency syndrome was established by continuous irritability and water platform sleep deprivation, and then ventricular arrhythmia model was established by injection of calcium chloride into the tail vein. Applying the electrocardiograph changes in heart rate in rats. Subsequently, ELISA and histopathological examinations were conducted to assess the therapeutic effects of honey-fried licorice on arrhythmia. Metabonomics analysis was employed to predict key regulatory pathways involved in the treatment response. Finally, RT-PCR and enzyme activity assays were utilized to verify the expression and function of key genes and proteins, providing insights into the underlying mechanisms. ResultsThe heart rate of rats increased after injection of Cacl2 solution into the tail vein. Honey-fried licorice has a certain improvement effect on heart injury and tachycardia, and its mechanism may be through the obvious correction effect on SOD, MDA, LDH, Na+-K+-ATPase, CaM and CAMK2 in the arrhythmia model. Under pathological conditions, Metabonomics revealed that the heart was highly exposed to glycyrrhetic acid 3-O-glucuronide, isoformononetin, araboglycyrrhizin, 18β-glycyrrhetinic acid, liquiritigenin, licoflavonol and isoliquiritigenin are known to have anti-arrhythmic effects through immune regulation and oxidation. Notably, both PCR and ELISA analyses indicated that honey-fried licorice may effectively treat arrhythmia, potentially through the modulation of the arachidonic acid pathway. ConclusionThese results suggested that honey-fried licorice could protect against arrhythmia and alleviate oxidative stress and tissue damage caused by arrhythmia. Through correlation analysis and metabolomics, it was found that glycyrrhetic acid 3-O-glucuronide, isoformononetin, araboglycyrrhizin, 18β-glycyrrhetinic acid, liquiritigenin, licoflavonol and isoliquiritigenin can be used as the active ingredient of honey-fried licorice in the treatment of arrhythmia. Moreover, our results suggested that the therapeutic effect of honey-fried licorice on arrhythmia may be linked to the regulation of the arachidonic acid pathway. This study elucidates the mechanisms by which honey-fried licorice treats arrhythmia from a metabolic perspective, highlighting its role in "tonifying the spleen and stomach, supplementing qi, and replenishing the pulse." These findings provide a foundation for the further application of honey-fried licorice and the development of related products.

Efficacy and safety of novel multiple-chain DAP-CAR-T cells targeting mesothelin in ovarian cancer and mesothelioma: a single-arm, open-label and first-in-human study

BackgroundDespite remarkable achievements in applying chimeric antigen receptor (CAR)-T cells to treat hematological malignancies, they remain much less effective against solid tumors, facing several challenges affecting their clinical use. We previously showed that multichain DNAX-activating protein (DAP) CAR structures could enhance the safety and efficacy of CAR-T cells when used against solid tumors. In particular, mesothelin (MSLN)-targeted CAR-T cell therapy has therapeutic potential in MSLN-positive solid tumors, including ovarian cancer and mesothelioma.MethodsIn vitro cell killing assays and xenograft model were utilized to determine the anti-tumor efficacy of MSLN targeting DAP-CAR-T cells and other CAR-T cells. ELISA and flow cytometry analysis were used to assess the cytokine secretion capacity and proliferation ability. Eight patients with MSLN expression were enrolled to evaluate the safety and efficacy of MSLN-DAP CAR-T cell therapy. Single-cell sequencing was performed to explore the dynamics of immune cells in patients during treatment and to identify the transcriptomic signatures associated with efficacy and toxicity.ResultsWe found that multichain DAP-CAR formed by combining a natural killer cell immunoglobulin-like receptor truncator and DAP12 exhibited better cytotoxicity and tumor-killing capacity than other natural killer cell-activated receptors associated with DAP12, DAP10, or CD3Z. The safety and efficacy of MSLN-DAP CAR-T cell therapy in patients with ovarian cancer and mesothelioma were evaluated in a single-arm, open-label clinical trial (ChiCTR2100046544); two patients achieved partial response, while four patients had a stable disease status. Furthermore, single-cell sequencing analysis indicated that KT032 CAR-T cell infusion could recruit more immune cells and temporarily remodel the TME.ConclusionsOur study highlights the safety and therapeutic efficacy of multiple-chain DAP-CAR-T cell therapy targeting MSLN to treat patients with ovarian cancer and mesothelioma.Trial registrationChiCTR.org.cn, ChiCTR2100046544. May 21, 2021.

The Impact of Fermentation on Aflatoxin and Fumonisin Levels in Sorghum Kernels and Flour Used to Prepare Complementary Food in Kerio Valley, Kenya

ABSTRACTSorghum is a major ingredient used in the production of complementary foods in Kenya's drylands, particularly in areas like Kerio Valley. However, it is known to be susceptible to aflatoxin and fumonisin contamination, which have adverse effects on human health. The current study aimed to assess the levels of aflatoxin and fumonisin in sorghum kernels and flour from Kerio Valley and to investigate whether fermentation (spontaneous or innoculum facilated) could reduce the levels of toxins. The sorghum samples were obtained through a cross‐sectional survey and subjected to ELISA analysis for quantification of aflatoxin and fumonisin levels. The aflatoxin levels ranged from 0 to 119.91 ppb for sorghum kernels and from 2.70 to 89.36 ppb in the flour, while fumonisin concentrations ranged from 0 to 30.65 ppm in sorghum kernels and 0.22 to 27.27 ppm in the flour. Fermentation significantly reduced the levels of the toxins in sorghum samples (p ≤ 0.05). The type of fermentation (p = 0.001), sample fermented (kernels or flour) (p = 0.004), and duration of fermentation (p = 0.037) significantly impacted the reduction of both toxins. Therefore, there is a need to integrate the adoption of fermentation with other effective postharvest practices to mitigate mycotoxin contamination in sorghum and other cereal‐based foods. In addition to the customary health and nutrition messages, promoting proper food handling, storage, and processing can significantly contribute to improving food safety and the overall health and nutritional status of young children.

Aramchol attenuates fibrosis in mouse models of biliary fibrosis and blocks the TGFβ-induced fibroinflammatory mediators in cholangiocytes.

Fibroinflammatory cholangiopathies, such as primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC), are characterized by inflammation and biliary fibrosis, driving disease-related complications. In biliary fibrosis, cholangiocytes activated by transforming growth factor-β (TGFβ) release signals that recruit immune cells to drive inflammation and activate hepatic myofibroblasts to deposit the extracellular matrix (ECM). TGFβ regulates stearoyl-CoA desaturase (SCD), an enzyme that catalyzes the synthesis of monounsaturated fatty acids, in stimulating fibroinflammatory lipid signaling. However, the role of SCD or its inhibitor, Aramchol, has not been investigated in biliary fibrosis or TGFβ-mediated cholangiocyte activation. 10-16-week-old multi-drug resistance 2 knockout (Mdr2 -/- ) and 3,5-diethoxycarboncyl-1,4-dihydrocollidine (DDC) diet-fed mice were orally gavaged daily with Aramchol at 12.5 mg/kg/day for 4 and 3 weeks, respectively. Liver and serum were harvested for the assessment of fibrosis and inflammation. Transformed human cholangiocyte cells (H69) and mouse large biliary epithelial cells (MLEs) were used to test the effects of the SCD inhibitor, Aramchol, at varying doses on TGFβ-mediated expression of fibroinflammatory signals and were confirmed in PSC-derived cholangiocytes (PSC-Cs) using ELISA, qPCR, and Western blot analyses. Aramchol treatment of Mdr2 -/- mice with established biliary fibrosis (treatment) and DDC diet-induced (prevention) models of cholestatic injury and fibrosis demonstrated significant reductions in both measures of ECM synthesis (mRNA expression of ECM components in the liver), collagen content of the liver (picrosirius red staining and hydroxyproline content) and myofibroblast activation (αSMA staining). Il6 and Tnfa were also reduced with Aramchol in the liver. RNA-seq analysis of H69 cells showed that Aramchol co-treatment led to significant inhibition of TGFβ-induced hepatic fibrosis pathways while upregulating peroxisome proliferator-activated receptor (PPAR) signaling. SCD expression was significantly increased in TGFβ-treated H69 cells (2-fold, p<0.05). Aramchol in a dose-dependent manner significantly attenuated the increased expression of the fibrotic marker, plasminogen activator inhibitor-1 (PAI-1/SERPINE1), and hepatic stellate cell-activating genes ( VEGFA and PDGFB ) in TGFβ-activated H69 and MLEs. Aramchol also markedly reduced the expression of the inflammatory cytokine, interleukin 6 (IL6). SCD siRNA knockdown produced similar results in H69 cells. Furthermore, in PSC-Cs, the expressions of SCD, VEGFA and IL6 were significantly reduced with Aramchol. The expression of the anti-fibroinflammatory factors PPARα and -γ were modestly increased in cholangiocyte cell lines with increased expression of PPAR-responsive genes and increased nuclear binding of DNA PPAR response elements with Aramchol co-treatment compared to TGFβ only. Aramchol, an SCD inhibitor, both attenuates and prevents biliary fibrosis in mouse models of cholestatic injury and fibrosis. This effect is partially due to Aramchol inhibiting TGFβ-induced fibroinflammatory mediators in cholangiocytes by upregulating PPARα and -γ expression and activity. These findings, along with Aramchol's excellent safety profile in clinical trials, provide the rationale for assessing Aramchol in further clinical studies in patients with biliary fibrosis, particularly PSC, where a treatment is desperately needed.

IMMU-61. THE LAST OF US: A FUNGI BASED DENDRITIC CELL VACCINE

Abstract BACKGROUND Glioblastoma (GBM) is the most prevalent primary malignant brain tumor in adults. The current standard treatment for newly diagnosed GBM involves maximal surgical resection followed by radiotherapy and temozolomide. Despite these interventions, the median overall survival for GBM patients remains approximately 15 months, highlighting the critical need for innovative therapeutic approaches. OBJECTIVE Our laboratory has previously developed a subcutaneous autologous tumor vaccine incorporating irradiated (IR) tumor cells with phagocytosis-stimulating ligands (Mannan-BAM), toll-like receptor (TLR) agonists, and an immunostimulant anti-CD40 antibody (collectively termed MBT). This combination has shown efficacy in mouse models of colon carcinoma, breast cancer, melanoma, and, more recently, gliomas. Our study aimed to create a dendritic cell (DC) vaccine using this strategy. METHODS To evaluate whether MBT could induce the maturation of DCs in vitro, we harvested BMDCs from mice and co-cultured them in the presence of MBT-tagged irradiated tumor cells. We then analyzed the maturation markers using flow cytometry and assessed cytokine production via ELISA. To test the efficacy of this approach in vivo, we utilized two different murine glioma models (SB28 and GL261). RESULTS Using flow cytometry, we observed a significant upregulation of important co-stimulatory molecules. Additionally, ELISA analysis demonstrated that MBT-treated DCs significantly increased the secretion of Type 1 cytokines while limiting the secretion of the regulatory cytokine IL-10. MBT-primed DCs combined with immune checkpoint inhibitor PD-1 increased mouse survival in a glioma model compared to WT mice. However, mice eventually succumbed to cerebral edema. Histology demonstrated significant treatment effects. CONCLUSION Our results demonstrate that MBT effectively matures DCs ex-vivo. Further research is required to immunophenotype the response to vaccination in glioma models and explore ways to overcome resistance.

FGL2172-220 peptides improve the antitumor effect of HCMV-IE1mut vaccine against glioblastoma by modulating immunosuppressive cells in the tumor microenvironment

ABSTRACT Glioblastoma multiforme (GBM) is a highly aggressive primary brain tumor characterized by poor prognosis and lack of effective treatments. In recent years, peptide vaccines that use sequences based on tumor-specific or tumor-associated antigens to activate immune responses against tumor cells have emerged as a new therapeutic strategy. In this study, we developed a novel therapeutic polypeptide vaccine targeting the tumor-associated antigen Fibrinogen-Like Protein 2 (FGL2), whose dominant epitope peptide was tandemly linked to the C-terminus of HCMV-IE1mut via a linker. We used this vaccine to compare the therapeutic efficacy of HCMV-IE1mut alone versus HCMV-IE1mut-FGL2172-220 and investigate the potential mechanism of action of HCMV-IE1mut-FGL2172-220 in glioma treatment. An in situ GBM model (GL261-IE1-luc cells) was used to determine the efficacy of the vaccine. Treatment with HCMV-IE1mut-FGL2172-220 exerted antitumor effects and extended the survival of the GL261 animal model. We observed reduced proportions of microglia, regulatory T cells (Treg), and myeloid-derived suppressor cells (MDSC) in the tumor microenvironment (TME) by immunofluorescence. Flow cytometry showed that compared to HCMV-IE1mut alone, treatment with HCMV-IE1mut-FGL2172-220 increased the proportion of CD8+ T cells and tissue-resident memory T cells (TRM). ELISA analysis showed that it improved the secretion of tumor-specific IFN-γ and TNF-α by these cells and downregulated the expression of IL-6 and IL-10. Our study demonstrates that the long-peptide FGL2172-220 improves the antitumor efficacy of HCMV-IE1mut, possibly by reshaping immune cells in the glioma microenvironment. These findings lay the groundwork for the development of therapeutic antigenic peptide vaccines to improve antitumor effects for cancer.

EGFR-TKIs induce acneiform rash and xerosis via Caspase-3/GSDME-mediated pyroptosis of keratinocytes and sebocytes

Skin toxicities are the most common adverse effects of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). While EGFR-TKIs induce pyroptosis in lung cancer cells through Gasdermin E (GSDME) activation, it is unknown whether they can similarly affect skin cells. In this study, we used immunohistochemistry to demonstrate that in acneiform rash, the N-terminus of GSDME (GSDME-N) is predominantly expressed in the basal layer of the follicular epithelium and sebocytes, while it is absent in the interfollicular epidermis. In contrast, in cases of xerosis or secondary eczematous rash, GSDME-N was significantly expressed in the basal layer of the interfollicular epidermis and weakly or partially positive in the follicular epithelium. Bright-field microscopy of HaCaT and SZ95 cells treated with afatinib revealed cell swelling and large bubble formation, while scanning electron microscopy showed a reduction in microvilli and membrane pores formation. Transmission electron microscopy further revealed multiple membrane pores and decreased cytoplasmic density. Importantly, we found that GSDME is cleaved during afatinib-induced pyroptosis via caspase-3 activation. ELISA analysis further confirmed that afatinib-treated cells released elevated levels of HMGB1 and IL-1α. Meanwhile, inhibition of caspase-3 activity or knockdown of GSDME both suppressed afatinib-induced pyroptosis, while GSDME elimination did not affect caspase-3 activation. These results indicate that afatinib-induced pyroptosis in keratinocytes and sebocytes is mediated by the caspase-3/GSDME pathway. Our findings suggest that GSDME-dependent pyroptosis in HaCaT and SZ95 cells contributes to the development of acneiform rash and xerosis, highlighting the need for further investigation into the underlying mechanisms.

Therapeutic role of physalin A in the pathogenesis of Graves’ orbitopathy

Background Graves’ orbitopathy (GO) is an autoimmune condition that causes serious ocular symptoms; its treatment strategies are limited. Physalin A is a phytosterol that has shown various therapeutic properties, including anti-inflammatory and anti-fibrotic effects. In this study, we investigated whether physalin A could inhibit inflammation, fibrosis, hyaluronan (hyaluronic acid) production, and adipogenesis, which are crucial to the pathogenesis of GO. Methods Orbital tissue explants were obtained from patients with GO during orbital decompression surgery and healthy controls. Orbital fibroblasts (OFs) were isolated and treated with different concentrations of physalin A. Using western blot and ELISA analyses, we determined the effects of physalin A on OFs. Results Physalin A treatment suppressed the production of interleukin (IL)-1β-induced prostaglandin E2 (PGE2) and pro-inflammatory molecules, including cyclooxygenase (COX)-2, IL-6, IL-8, and intercellular adhesion molecule (ICAM)-1. We discovered that physalin A attenuated hyaluronan production induced by IL-1β or insulin-like growth factor (IGF)-1. Moreover, physalin A reduced lipid droplet formation and production of peroxisome proliferator activator (PPAR) γ, CCAAT-enhancer-binding protein (C/EBP) α, C/EBP β, sterol regulatory element binding protein (SREBP)-1, leptin, and adiponectin proteins. Physalin A suppressed the phosphorylation of extracellular signal-related kinase (ERK), nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), and suppressor of mothers against decapentaplegic (SMAD) 2 signaling protein. Conclusions Our study suggests that the major mechanisms by which physalin A suppresses GO include reducing inflammation, fibrosis, hyaluronan production, and adipogenesis in OFs. The findings of this study provide evidence of the therapeutic effect of physalin A in GO.

Evaluation of Cytoprotective Effects of Cannabidiol on Neuroinflammation and Neurogenesis Process in Rat Offsprings

Natural compounds include complex chemical compounds that exist in plants, animals and microbes. Due to their broad spectrum of pharmacological and biochemical actions, they have been widely used to treat multifactorial diseases, including cancer. In addition, their demonstrated neuroprotective properties strongly support their use in the treatment of neurological diseases. The present study investigated the effect of CBD, which can easily cross the placental barrier and is known to have anti-inflammatory effects, on fetal neuroinflammation and neurogenesis in a systemic inflammation model during pregnancy. Herein, 12 weeks adult pregnant rats (n=30) were randomly divided into 5 groups with 6 rats in each group as follows: Control, LPS (lipopolysaccharide, i.p.), LPS+CBD 5mg/kg (i.p.), LPS+CBD10 mg/kg (i.p.) and LPS+CBD30 mg/kg (i.p.). After the injections, blood samples of rats were collected, fetuses and placentas were taken by hysterectomy. Histopathological analysis, immunohistochemical staining, ELISA and immunoblotting analysis were performed to investigate neuroinflammatory and neurogenesis parameters in fetal brain and placenta tissues. Our findings indicated that CBD administration importantly suppressed the inflammatory process in the rat fetal brain by decreasing interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) levels and diminishing nuclear factor kappa B (NF-κB) activation. Moreover, CBD inhibited lipopolysaccharide (LPS)-induced increasing levels of neuroinflammation-associated proteins, including glial fibrillary acidic protein (GFAP), S100B and cAMP-response element binding protein (CREB). These results suggest that CBD usage in pregnancy with inflammation conditions may be an effective therapeutic option for preventing conditions that may cause neuroinflammation in the fetal brain and adversely affect neurogenesis.

Identification and analysis of microplastic aggregation in CAR-T cells

Microplastics (MPs) are increasingly recognized as contaminants present in various environments and are widely acknowledged as potential hazards to the mammalian immune system. In our study of chimeric antigen receptor T cell (CAR-T) therapy, we observed the presence of MP in CAR-T cell products for the first time. It is worth exploring whether MP could enter CAR-T cells and how they might affect CAR-T cells’ functionality. Therefore, we analyzed how MP affected CD19 and BCMA-CAR-T cells. Based on flow cytometry, ELISA, and cytotoxicity analysis of in vitro and in vivo experiments, MP suppressed the activity of CAR-T cells. Subsequent investigation revealed that the exposure of CAR-T cells to varying concentrations of MP resulted in a notable increase in apoptosis, ferroptosis, and exhaustion levels. Furthermore, the hyperactivation of the mTOR signaling pathway in MP-treated CAR-T cells was verified. The partial restoration of CAR-T cell function in MP was achieved by inhibiting the mTOR pathway. MP present a threat to CAR-T cell function due to their role in inducing CAR-T cell apoptosis, ferroptosis, and T-cell exhaustion through the hyperactivation of mTOR signaling pathways.

Identification of fibrinogen as a plasma protein binding partner for lecanemab biosimilar IgG.

Recombinant monoclonal therapeutic antibodies like lecanemab, which target amyloid beta in Alzheimer's disease, offer a promising approach for modifying the disease progression. Due to its relatively short half-life, lecanemab administered as a bi-monthly infusion (typically 10 mg/kg) has a relatively brief half-life. Interaction with abundant plasma proteins binder in the bloodstream can affect pharmacokinetics of drugs, including their half-life. In this study, we investigated potential plasma protein binding (PPB) interaction to lecanemab using lecanemab biosimilar. Lecanemab biosimilar used in this study was based on publicly available sequences. ELISA and western blotting were used to assess lecanemab biosimilar immunoreactivity in the fractions of human plasma obtained through size exclusion chromatography. The binding of lecanemab biosimilar to candidate plasma binders was confirmed by western blotting, ELISA, and surface plasmon resonance analysis. Using a combination of equilibrium dialysis, ELISA, and western blotting in human plasma, we first describe the presence of likely PPB partners to lecanemab biosimilar and then identify fibrinogen as one of them. Utilizing surface plasmon resonance, we confirmed that lecanemab biosimilar does bind to fibrinogen, although with lower affinity than to monomeric amyloid beta. In the context of lecanemab therapy, these results imply that fibrinogen levels could impact the levels of free antibodies in the bloodstream and that fibrinogen might serve as a reservoir for lecanemab. More broadly, these results indicate that PPB may be an important consideration when clinically utilizing therapeutic antibodies in neurodegenerative disease.

Interleukin 21, plasminogen activator inhibitor 1 and fractalkine improve risk stratification in elderly patients with pulmonary embolism

Abstract Background The importance of clinical parameters for predicting short-term mortality in hemodynamically stable patients with pulmonary embolism (PE) is acknowledged within the Pulmonary Embolism Severity Index (PESI) Score. Little is known about the value of novel biomarkers in this context. Given the pathophysiology of PE, markers related to inflammation and thrombosis, such as interleukin 21 (IL-21), fractalkine and plasminogen activator inhibitor 1 (PAI1) are of particular interest. Purpose To identify new protein biomarkers that improve 90d mortality prediction compared to the PESI score in elderly PE patients. Methods 1003 patients with venous thromboembolism aged ≥65 years were recruited between 2009 and 2011 from 9 hospitals in Switzerland (SWITCO65+ cohort). Among these, 778 patients were excluded due to missing data, deep venous thrombosis only, blood sample taken &amp;gt;1 day after diagnosis, no initial anticoagulation, systolic blood pressure &amp;lt;90 mmHg, withdrawal within 1 day or denying further use of data; 225 patients were prospectively included. Outcome was independently adjudicated up to 1 year after the index event. To identify candidate proteins, untargeted proteomic analyses (OLINK) were conducted in cases with 90d all-cause mortality (n=20) matched 1:2 (age, sex) to controls without events (n=40). 26 candidate proteins were then validated in all 225 PE patients using ELISA and Mesoscale analyses. Using ROC analyses, the most promising proteins to predict death within 90d were identified within this cohort. Optimal biomarker cut-off levels were derived using the Youden Index and survival analyses were computed. To evaluate the predictive value of these biomarkers, hazard ratios (HR) were calculated after adjusting for age, sex, and BMI. AUC-ROC analyses were performed to assess the biomarkers’ value for risk prediction compared to the PESI score. Results Baseline characteristics (Figure 1) did not differ between patients who died within 90d and those who did not, except for median PESI score (108 [95 – 125] vs. 87 [79 – 101], p&amp;lt;0.001). Optimal biomarker cut-off values were: IL-21: 56.17pg/ml, PAI1: 36.82pg/ml, fractalkine: 66’515pg/ml. Increased levels of IL-21 and PAI1 were associated with increased mortality (HR IL-21 = 1.5 [95% CI: 0.99 - 2.79], HR PAI1 = 2.6 [95% CI: 1.7 – 4.8]), whereas patients with elevated fractalkine levels were at lower risk to die within 90d (HR fractalkine = 0.37 [95% CI: 0.16 - 0.60]). For prediction of 90d mortality, the three assessed biomarkers showed a higher AUC than the PESI score. Combining the PESI score with the biomarkers did not further improve the AUC (Figure 2). Conclusions The protein biomarkers IL-21, PAI1 and fractalkine improve prediction of 90d mortality in elderly, hemodynamically stable PE patients compared to the PESI score. These biomarkers highlight the contribution of inflammation and thrombosis in the pathophysiology of PE and warrant further investigation.Baseline CharacteristicsROC Curves for Biomarkers and PESI Score

Codon and Signal Peptide Optimization to Enhance Therapeutic Antibody Production from CHO Cells.

A simple and rapid method for reproducible transient expression experiments to quickly identify the best conditions before the development of a stable CHO cell line is described, using adalimumab as a model antibody. Firstly, the signal peptide (SP) is optimized using different in silico programs, of which those having a D score of more than 0.85 will be selected. A list of useful SPs is provided. Next, guidelines for selecting the codon optimization algorithm is provided based on aCAI value of more than 0.95. After that, the codon optimized genes of the heavy chain (HC) and light chain (LC) of an antibody with different SPs are engineered into an expression vector and the two recombinant plasmids are co-transfected into a CHO cell line of interest. The antibody titer, specific productivity (Qp), and viable cell density (VCD) are then checked by ELISA and flow cytometry analysis. The best SP pairs can then be used for the development of stable CHO cell lines.