Key Mediator Research Articles

A Key Mediator and Imaging Target in Alzheimer's Disease: Unlocking the Role of Reactive Astrogliosis Through MAOB.

Astrocytes primarily maintain physiological brain homeostasis. However, under various pathological conditions, they can undergo morphological, transcriptomic, and functional transformations, collectively referred to as reactive astrogliosis. Recent studies have accumulated lines of evidence that reactive astrogliosis plays a crucial role in the pathology of Alzheimer's disease (AD). In particular, monoamine oxidase B, a mitochondrial enzyme mainly expressed in astrocytes, significantly contributes to neuronal dysfunction and neurodegeneration in AD brains. Moreover, it has been reported that reactive astrogliosis precedes other pathological hallmarks such as amyloid-beta plaque deposition and tau tangle formation in AD. Due to the early onset and profound impact of reactive astrocytes on pathology, there have been extensive efforts in the past decade to visualize these cells in the brains of AD patients using positron emission tomography (PET) imaging. In this review, we summarize the recent studies regarding the essential pathological importance of reactive astrocytes in AD and their application as a target for PET imaging.

Ginsenoside Rg3 overcomes tamoxifen resistance through inhibiting glycolysis in breast cancer cells.

Tamoxifen (TAM) resistance poses a significant clinical challenge in human breast cancer and exhibits high heterogeneity among different patients. Rg3, an original ginsenoside known to inhibit tumor growth, has shown potential for enhancing TAM sensitivity in breast cancer cells. However, the specific role and underlying mechanisms of Rg3 in this context remain unclear. Aerobic glycolysis, a metabolic process, has been implicated in chemotherapeutic resistance. In this study, we demonstrate that elevated glycolysis plays a central role in TAM resistance and can be effectively targeted and overcome by Rg3. Mechanistically, we observed upregulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a key mediator of glycolysis, in TAM-resistant MCF-7/TamR and T-47D/TamR cells. Crucially, PFKFB3 is indispensable for the synergistic effect of TAM and Rg3 combination therapy, which suppresses cell proliferation and glycolysis in MCF-7/TamR and T-47D/TamR cells, both in vitro and in vivo. Moreover, overexpression of PFKFB3 in MCF-7 cells mimicked the TAM resistance phenotype. Importantly, combination treatment significantly reduced TAM-resistant MCF-7 cell proliferation in an in vivo model. In conclusion, this study highlights the contribution of Rg3 in enhancing the therapeutic efficacy of TAM in breast cancer, and suggests that targeting TAM-resistant PFKFB3 overexpression may represent a promising strategy to improve the response to combination therapy in breast cancer.

Optimization of a Photobiomodulation Protocol to Improve the Cell Viability, Proliferation and Protein Expression in Osteoblasts and Periodontal Ligament Fibroblasts for Accelerated Orthodontic Treatment.

Numerous pieces of evidence have supported the therapeutic potential of photobiomodulation (PBM) to modulate bone remodeling on mechanically stimulated teeth, proving PBM's ability to be used as a coadjuvant treatment to accelerate orthodontic tooth movement (OTM). However, there are still uncertainty and discourse around the optimal PBM protocols, which hampers its optimal and consolidated clinical applicability. Given the differential expression and metabolic patterns exhibited in the tension and compression sides of orthodontically stressed teeth, it is plausible that different types of irradiation may be applied to each side of the teeth. In this sense, this study aimed to design and implement an optimization protocol to find the most appropriate PBM parameters to stimulate specific bone turnover processes. To this end, three levels of wavelength (655, 810 and 940 nm), two power densities (5 and 10 mW/cm2) and two regimens of single and multiple sessions within three consecutive days were tested. The biological response of osteoblasts and periodontal ligament (PDL) fibroblasts was addressed by monitoring the PBM's impact on the cellular metabolic activity, as well as on key bone remodeling mediators, including alkaline phosphatase (ALP), osteoprotegerin (OPG) and receptor activator of nuclear factor κ-B ligand (RANK-L), each day. The results suggest that daily irradiation of 655 nm delivered at 10 mW/cm2, as well as 810 and 940 nm light at 5 mW/cm2, lead to an increase in ALP and OPG, potentiating bone formation. In addition, irradiation of 810 nm at 5 mW/cm2 delivered for two consecutive days and suspended by the third day promotes a downregulation of OPG expression and a slight non-significant increase in RANK-L expression, being suitable to stimulate bone resorption. Future studies in animal models may clarify the impact of PBM on bone formation and resorption mediators for longer periods and address the possibility of testing different stimulation periodicities. The present in vitro study offers valuable insights into the effectiveness of specific PBM protocols to promote osteogenic and osteoclastogenesis responses and therefore its potential to stimulate bone formation on the tension side and bone resorption on the compression side of orthodontically stressed teeth.

C-X-C chemokine receptor type 4 promotes tubular cell senescence and renal fibrosis through β-catenin-inhibited fatty acid oxidation.

The prevalence of chronic kidney disease (CKD) is highly increasing. Renal fibrosis is a common pathological feature in various CKD. Previous studies showed tubular cell senescence is highly involved in the pathogenesis of renal fibrosis. However, the inducers of tubular senescence and the underlying mechanisms have not been fully investigated. C-X-C motif chemokine receptor 4 (CXCR4), a G-protein-coupled seven-span transmembrane receptor, increases renal fibrosis and plays an important role in tubular cell injury. Whereas, whether CXCR4 could induce tubular cell senescence and the detailed mechanisms have not studied yet. In this study, we adopted adriamycin nephropathy and 5/6 nephrectomy models, and cultured tubular cell line. Overexpression or knockdown of CXCR4 was obtained by injection of related plasmids. We identified CXCR4 increased in injury tubular cells. CXCR4 was expressed predominantly in renal tubular epithelial cells and co-localized with adipose differentiation-related protein (ADRP) as well as the senescence-related protein P16INK4A . Furthermore, we found overexpression of CXCR4 greatly induced the activation of β-catenin, while knockdown of CXCR4 inhibited it. We also found that CXCR4 inhibited fatty acid oxidation and triggered lipid deposition in tubular cells. To inhibit β-catenin by ICG-001, an inhibitor of β-catenin, could significantly block CXCR4-suppressed fatty acid oxidation. Taken together, our results indicate that CXCR4 is a key mediator in tubular cell senescence and renal fibrosis. CXCR4 promotes tubular cell senescence and renal fibrosis by inducing β-catenin and inhibiting fatty acid metabolism. Our findings provide a new theory for tubular cell injury in renal fibrosis.

Validation of Blunting of Inflammatory Markers in LPS Induced Tissue with SPM Treatment

Background and Hypothesis: Chronic rhinosinusitis (CRS) is defined as persistent inflammation of the mucosa of the nose and paranasal sinuses, either with or without nasal polyps. The pathophysiology of CRS is thought to occur due to a dysfunction of the immune response leading to prolonged NF-kB signaling. Many chronic diseases like CRS have been shown to have chronic NF-kB dysregulation. One hypothesis for the persistent inflammation seen in CRS patients is that they have a less robust pro-resolution response that aids in termination of the NF-kB pathway. In this study, we sought to validate our previous results from nasal polyp tissue using qPCR for key inflammatory mediators, CXCL1, CSF3, and myd88.
 Methods: Human CRS nasal polyp tissue was collected during functional endoscopic sinus surgery to be grown in cell culture. The nasal polyp tissue was grown in 10 ug/ml of LPS to mimic gram-negative conditions commonly seen in CRS. Tissue cDNA was extracted and frozen at – 80° C. Tissue cDNA for control, RvD2, LPS, and LPS+RvD2 was thawed and used to run qPCR for myd88, CXCL1, and CSF3.
 Results: qPCR data was normalized using GAPDH and B-actin. When normalized with GAPDH and B-actin, CSF3 was found to be downregulated with RvD2 exposure, while both myd88 and CXCL1 showed inconsistent results. Downregulation of CSF3 with RvD2 exposure, is consistentwith our hypothesis that RvD2 plays a role in NF-kB resolution.
 Conclusion: Downregulation of the NF-kB pathway can play an important role in reducing the chronic inflammation seen in CRS. CSF3 was one gene target of the NF-kB pathway that was continuously found to be downregulated when nasal polyp tissue was treated with RvD2. Ourfindings demonstrate that when nasal polyp tissue is treated with pro-resolving mediators such as RvD2, at least one or more of the NF-kB-associated genes are downregulated.

Transient suppression of Wnt signaling in poor-quality buffalo oocytes improves their developmental competence.

One of the most evolutionary conserved communication systems, the Wnt signaling pathway is a major gene regulatory pathway that affects the developmental competence of oocytes and regulates most embryonic developmental processes. The present study was undertaken to modulate the canonical Wnt (Wingless/integration) signaling pathway in the poor-quality (colorless cytoplasm after Brilliant Cresyl Blue staining, BCB-) buffalo cumulus-oocyte complexes (COCs) to improve their in vitro maturation (IVM) and embryo production (IVEP) rates. The expression of key Wnt pathway genes was initially assessed in the good (blue cytoplasm after Brilliant Cresyl Blue staining, BCB+) and poor quality (BCB-) buffalo COCs to establish a differential activity of the Wnt pathway. The BCB- COCs were supplemented with the Wnt pathway inhibitor, Dickkopf-related protein 1 (DKK1) and later subjected to IVM and IVEP along with the BCB+ and BCB- controls. The cumulus expansion index (CEI), rate of nuclear maturation (mean percentage of oocytes in the MII stage) and embryo production, and the expression of developmentally important genes were evaluated to assess the effect of Wnt pathway inhibition on the development competence of these poor-quality oocytes. The Wnt pathway genes exhibited a significantly higher expression (p < 0.05) in the poor-quality BCB- oocytes compared to the good-quality BCB+ oocytes during the early maturation stages. The supplementation of BCB- COCs with 100 ng/mL DKK1 effectively inhibited the expression of the key mediators of the Wnt pathway (β-catenin and dishevelled homolog 1, DVL1). DKK1 supplemented BCB- COCs exhibited significantly improved cytoplasmic and nuclear maturation indices, development rates and significantly elevated expression (p < 0.05) of genes implicated in germinal vesicle breakdown (GVBD) and embryonic genome activation (EGA) vis-à-vis BCB- control COCs. These data indicate that inhibition of the Wnt pathway during the initial course of oocyte maturation can improve the development competence of poor-quality buffalo oocytes.

Intra-articular injection of stigmasterol-loaded nanoparticles reduce pain and inhibit the inflammation and joint destruction in osteoarthritis rat model: A pilot study.

Stigmasterol, a plant-derived sterol, sharing structural similarity with cholesterol, has demonstrated anti-osteoarthritis (OA) properties, attributed to its antioxidant and anti-inflammatory capabilities. Given that OA often arises in weight bearing or overused joints, prolonged localized treatment effectively targets inflammatory aspects of the disease. This research explored the impact of stigmasterol-loaded nanoparticles delivered via intra-articular injections in an OA rat model. Employing mesoporous silica nanomaterials (MSNs) combined with β-cyclodextrin (β-CD) as a vehicle, stigmasterol was loaded in conjunction with tannic acid, forming stigmasterol/β-CD-MSNs to facilitate a sustained stigmasterol release. The study employed RAW 264.7 cells to examine the in vitro cytotoxicity and anti-inflammatory effect of stigmasterol/β-CD-MSNs. For in vivo experimentation, we used healthy control rats and monosodium iodoacetate (MIA)-induced OA rats, separated into five groups, varying the injection substances. In vitro findings indicated that stigmasterol/β-CD-MSNs suppressed the mRNA expression of key pro-inflammatory mediators such as interleukin-6, tumor necrosis factor-α, and matrix metalloproteinase-3 in a dose-dependent manner. In vivo experiments revealed a substantial decrease in the mRNA levels of pro-inflammatory factors in the stigmasterol(50µg)/β-CD-MSN group compared to the others. Macroscopic, radiographic, and histological evaluations established that intra-articular injections of stigmasterol/β-CD-MSNs inhibited cartilage degeneration and subchondral bone deterioration. Therefore, in a chemically induced OA rat model, intra-articular stigmasterol delivery was associated with reduction in both local and systemic inflammatory responses, alongside a slowdown in joint degradation and arthritic progression.

Abstract A028: Alcohol induces DNA damage and genomic instability in MCF-7 breast cancer cells and primary mammary tissues

Abstract Alcohol consumption has been associated with increased breast cancer risk. Previous studies indicated that alcohol-induced DNA damage plays a central role in carcinogenesis. However, the underlying molecular mechanisms remain unclear. This study aimed to examine the effect of alcohol exposure on key DNA damage/repair pathways in both in vitro and in vivo settings. When MCF-7 breast cancer cells were exposed to 0.1 ~ 0.8% alcohol, the treatment induced concentration-dependent DNA damage, as indicated by the increased 8-OHdG (8-hydroxy 2 deoxyguanosine) production and p-H2AX foci formation. Signal transduction analysis showed that alcohol induced DNA damage signals, including p53, p21, E2F-1 and CHK1, and the markers in DNA repair pathways, including XLF, Rad50 and DNA-PK. p53 knockdown in MCF-7 cells resulted in enhanced DNA damage and DNA repair responses, which underscores the role of p53 in alcohol-induced cellular regulation. We also demonstrated that alcohol induces sister chromatid exchanges (SCEs) in MCF-7 cells, and ALDH2 knockout enhanced alcohol induced SCEs, which suggests that alcohol-induced acetaldehyde (AA) is a key mediator in this process. Moreover, we examined the DNA damage markers in mammary tissues of FVB/N mice exposed to alcohol and showed that alcohol exposure in vivo induced similar patterns of DNA damage and repair responses. Taken together, our data highlights the contribution of alcohol-induced DNA damage in cellular injury and the consequent tumorigenesis, which provide fundamental support for further studies on alcohol-induced DNA damage and carcinogenesis. Citation Format: Xiaohe Yang. Alcohol induces DNA damage and genomic instability in MCF-7 breast cancer cells and primary mammary tissues [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A028.

Abstract A002: Genotype-directed synthetic cytotoxicity of ATR inhibition with radiotherapy

Abstract The DNA damage response is crucial for the efficacy of radiotherapy (RT), prompting efforts to target this pathway with radiosensitizers to improve outcomes. Early clinical trials evaluating this approach, however, have failed to yield evidence of benefit in unselected populations. Here we demonstrate that a genotype-directed combination of DNA repair inhibition and RT can unleash the significant therapeutic benefit of this approach in pre-clinical models and present first experience from a Phase I/II trial (NCT05566574) testing this concept. Using CRISPR/Cas9, we created isogenic murine models (Atm-/- and WT) in 4T1 breast and CT26 colorectal cancer models, and then studied the synergy between camonsertib and RT across genotypes. In vitro, camonsertib radio-sensitized both Atm-/- and WT models, but was more potent and synergistic in Atm-/- models. In the orthotopically implanted 4T1 model, the combination of camonsertib (30 mg/kg, 3 days on; 4 days off) with RT (20Gy x 1) significantly delayed tumor growth in Atm-/- tumors compared to WT (p &amp;lt; 0.001; n=10 mice per group). Overall survival (OS) also improved in mice with Atm-/- tumors (20% alive at 150 days) vs. WT (0% alive; p &amp;lt; 0.001; log-rank test). Similar results were observed using other RT dose-fractionations (4Gy x 5 daily, 6Gy x 3, 8Gy x 2) with camonsertib. In the flank-implanted CT26 model, Atm-/- tumors had markedly improved response to combination therapy with 7/8 mice achieving a complete response (CR) compared to only 1/8 WT tumors (p&amp;lt;0.001). OS was also significantly improved with 87% of mice with CT26 Atm-/- tumors cured compared to 0% of CT26 WT tumors (p &amp;lt; 0.001). To evaluate the genotype-specificity for synergy from combination therapy, we performed similar experiments in a 4T1 Brca1-/- model, a key mediator of homologous recombination, and observed an absence of synergy, consistent with our hypothesis. Analysis of the microenvironment of 4T1 Atm-/- treated with combination therapy by flow cytometry did not reveal significant increases in CD4 or CD8 T-cells compared to vehicle nor increases in activated CD8 T-cells (Ki67+, PD1+, or GzmB+) suggesting the therapeutic benefit was primarily mediated by cell intrinsic effects. Similarly, re-challenge experiments in cured mice did not identify strong evidence of immunologic memory, again suggesting a modest immunologic role for efficacy of combination therapy in this setting. Based on these results, we initiated a Phase I/II study of camonsertib with RT (4Gy x 5) in patients with metastatic cancer that require palliative RT and have mutations in ATM (pathogenic or variant of unknown significance). The first two patients with pathogenic ATM mutant tumors enrolled in the Phase I portion of the study have demonstrated metabolic CR to combination therapy on PET scan. Overall, our findings suggest that genotype-directed concurrent radio-sensitization could represent a novel approach to develop more effective combinatorial synthetic cytotoxic RT-based treatments. Further research to test this hypothesis is ongoing. Citation Format: Victor Ng, Sonali Sinha, Ardijana Novaj, Jennifer Ma, Niamh McDermott, Ana Longhini, Rui Gardner, Ezra Rosen, Simon Powell, Diana Mandelker, Atif Khan, Jeremy Setton, Anne Roulston, Stephen Morris, Maria Koehler, Nancy Lee, Jorge Reis-Filho, Nadeem Riaz. Genotype-directed synthetic cytotoxicity of ATR inhibition with radiotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A002.

Abstract IA017: Drugging the DNA damage response (DDR) in the Clinic: Going beyond the approved PARP inhibitors

Abstract Genomic instability is a key hallmark of cancer that arises owing to defects in the DNA damage response (DDR) and/or increased replication stress. These alterations promote the clonal evolution of cancer cells via the accumulation of driver aberrations, including gene copy-number changes, rearrangements, and mutations; however, these same defects also create vulnerabilities that are relatively specific to cancer cells, which could potentially be exploited to increase the therapeutic index of anticancer treatments and thereby improve patient outcomes. The discovery that BRCA-mutant cancer cells are exquisitely sensitive to inhibition of poly(ADP-ribose) polymerase has ushered in a new era of research on biomarker-driven synthetic lethal treatment strategies for different cancers. The therapeutic landscape of antitumor agents targeting the DDR has rapidly expanded to include inhibitors of other key mediators of DNA repair and replication, such as ATR, ATM, CHK1, WEE1, PKMYT1 and PARG. Efforts to optimize these therapies are ongoing across a range of cancers, involving the development of predictive biomarker assays of response (beyond BRCA mutations), assessment of the mechanisms underlying intrinsic and acquired resistance, and evaluation of rational combinations to improve patient outcomes and widen the application of DDR agents. Citation Format: Timothy A. Yap. Drugging the DNA damage response (DDR) in the Clinic: Going beyond the approved PARP inhibitors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr IA017.

Extracellular vesicles in the Chronic Myeloid Leukemia scenario: an update about the shuttling of disease markers and therapeutic molecules.

Extracellular vesicles (EVs) are various sets of cell-derived membranous structures containing lipids, nucleic acids, and proteins secreted by both eukaryotic and prokaryotic cells. It is now well recognized that EVs are key intercellular communication mediators, allowing the functional transfer of bioactive chemicals from one cell to another in both healthy and pathological pathways. It is evident that the condition of the producer cells heavily influences the composition of EVs. Hence, phenotypic changes in the parent cells are mirrored in the design of the secreted EVs. As a result, EVs have been investigated for a wide range of medicinal and diagnostic uses in different hematological diseases. EVs have only recently been studied in the context of Chronic Myeloid Leukemia (CML), a blood malignancy defined by the chromosomal rearrangement t(9;22) and the fusion gene BCR-ABL1. The findings range from the impact on pathogenesis to the possible use of EVs as medicinal chemical carriers. This review aims to provide for the first time an update on our understanding of EVs as carriers of CML biomarkers for minimal residual disease monitoring, therapy response, and its management, as well as the limited reports on the use of EVs as therapeutic shuttles for innovative treatment approaches.

A Novel Protozoa Parasite-Derived Protein Adjuvant Is Effective in Immunization with Cancer Cells to Activate the Cancer-Specific Protective Immunity and Inhibit the Cancer Growth in a Murine Model of Colorectal Cancer.

Cancer-specific CD8+ cytotoxic T cells play important roles in preventing cancer growth, and IFN-γ, in addition to IL-12 and type I interferon, is critical for activating CD8+ cytotoxic T cells. We recently identified the capability of the amino-terminus region of dense granule protein 6 (GRA6Nt) of Toxoplasma gondii, an intracellular protozoan parasite, to activate IFN-γ production of microglia, a tissue-resident macrophage population. Therefore, in the present study, we examined whether recombinant GRA6Nt protein (rGRA6Nt) functions as an effective adjuvant to potently activate cancer-specific protective immunity using a murine model of MC38 colorectal cancer (CRC). When mice were immunized with non-replicable (either treated with mitomycin C or irradiated by X-ray) MC38 CRC cells in combination with rGRA6Nt adjuvant and received a challenge implantation of replication-capable MC38 tumor cells, those mice markedly inhibited the growth of the implanted tumors in association with a two-fold increase in CD8+ T cell density within the tumors. In addition, CD8+ T cells of the immunized mice secreted significantly increased amounts of granzyme B, a key mediator of the cytotoxic activity of CD8+ T cells, and IFN-γ in response to MC38 CRC cells in vitro when compared to the T cells from unimmunized mice. Notably, the protective effects of the immunization were specific to MC38 CRC cells, as the immunized mice did not exhibit a significantly inhibited growth of EL4 lymphoma tumors. These results indicate that rGRA6Nt is a novel and effective protein adjuvant when used in immunizations with non-replicable cancer cells to potently activate the protective immunity specifically against the cancer cells employed in the immunization.

An Examination of the Effect of Aspirin and Salicylic Acid on Soluble Fms-like Tyrosine Kinase-1 Release from Human Placental Trophoblasts.

Low-dose aspirin (LDA) is efficacious in preventing preeclampsia, but its mechanism of action is unclear. Conflicting evidence suggests that it may inhibit placental trophoblast release of soluble fms-like tyrosine kinase-1 (sFlt1), a key mediator of preeclampsia. We examined whether, and at what concentrations, aspirin and its principal metabolite, salicylic acid, modulate sFlt1 release and/or expression in trophoblasts. Human trophoblast lines BeWo and HTR-8/SVneo were cultured; BeWo cells were also treated with 1% oxygen vs. normoxia to mimic hypoxia in preeclamptic placentas. Cells were treated with aspirin or salicylic acid vs. vehicle for 24 h at concentrations relevant to LDA and at higher concentrations. Protein concentrations (ELISA) and mRNA expression (RT-PCR) of sFlt1 were determined. Under normoxia, LDA-relevant concentrations of aspirin (10-50 µmol/L) or salicylic acid (20-100 µmol/L) had no significant effect on sFlt1 protein release or mRNA expression in BeWo cells. However, inhibition was observed at higher concentrations (1 mmol/L for aspirin and ≥200 μmol/L for salicylic acid). Hypoxia enhanced sFlt1 protein release and mRNA expression in BeWo cells, but these responses were not significantly affected by either aspirin or salicylic acid at LDA concentrations. Similarly, neither drug altered sFlt1 protein secretion or mRNA expression in normoxic HTR-8/SVneo cells at LDA concentrations. We suggest that direct modulation of trophoblast release or expression of sFlt1 is unlikely to be a mechanism underlying the clinical efficacy of LDA in preeclampsia.

Inhibition of xanthine oxidase by allopurinol suppresses HMGB1 secretion and ameliorates experimental asthma

BackgroundExtracellular high mobility group box 1 (HMGB1) is a key mediator in driving allergic airway inflammation and contributes to asthma. Yet, mechanism of HMGB1 secretion in asthma is poorly defined. Pulmonary metabolic dysfunction is recently recognized as a driver of respiratory pathology. However, the altered metabolic signatures and the roles of metabolic to allergic airway inflammation remain unclear. MethodsMale C57BL/6 J mice were sensitized and challenged with toluene diisocyanate (TDI) to generate a chemically induced asthma model. Pulmonary untargeted metabolomics was employed. According to results, mice were orally administered allopurinol, a xanthine oxidase (XO) inhibitor. Human bronchial epithelial cells (16HBE) were stimulated by TDI-human serum albumin (HSA). ResultsWe identified the purine metabolism was the most enriched pathway in TDI-exposed lungs, corresponding to the increase of xanthine and uric acid, products of purine degradation mediated by XO. Inhibition of XO by allopurinol ameliorates TDI-induced oxidative stress and DNA damage, mixed granulocytic airway inflammation and Th1, Th2 and Th17 immunology as well as HMGB1 acetylation and secretion. Mechanistically, HMGB1 acetylation was caused by decreased activation of the NAD+-sirtuin 1 (SIRT1) axis triggered by hyperactivation of the DNA damage sensor poly (ADP-ribose)-polymerase 1 (PARP-1). This was rescued by allopurinol, PARP-1 inhibitor or supplementation with NAD+ precursor in a SIRT1-dependent manner. Meanwhile, allopurinol attenuated Nrf2 defect due to SIRT1 inactivation to help ROS scavenge. ConclusionsWe demonstrated a novel regulation of HMGB1 acetylation and secretion by purine metabolism that is critical for asthma onset. Allopurinol may have therapeutic potential in patients with asthma.

GB12-09, a bispecific antibody targeting IL4Rα and IL31Rα for atopic dermatitis therapy.

Atopic dermatitis (AD) is a chronic inflammatory skin condition characterized by dysregulated immune responses. The key mediators of AD pathogenesis are T helper 2 (TH2) cells and TH2 cytokines. Targeting interleukin 4 (IL4), IL13 or IL31 has become a pivotal focus in both research and clinical treatments for AD. However, the need remains pressing for the development of a more effective and safer therapy, as the current approaches often yield low response rates and adverse effects. In response to this challenge, we have engineered a immunoglobulin G-single-chain fragment variable (scFv) format bispecific antibody (Ab) designed to concurrently target IL4R and IL31R. Our innovative design involved sequence optimization of VL-VH and the introduction of disulfide bond (VH44-VL100) within the IL31Rα Ab scFv region to stabilize the scFv structure. Our bispecific Ab efficiently inhibited the IL4/IL13/IL31 signaling pathways in vitro and reduced serum immunoglobulin E and IL31 levels in vivo. Consequently, this intervention led to improved inflammation profiles and notable amelioration of AD symptoms. This research highlighted a novel approach to AD therapy by employing bispecific Ab targeting IL4Rα and IL31Rα with potent efficacy.

Evaluating microRNA-499 expression and serum IL-17, ACCP and PADI4 cytokine levels in rheumatoid arthritis patients undergoing anti-TNF alpha therapy

Introduction: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterized by joint inflammation and damage. While anti-TNF-alpha therapies like infliximab have revolutionized RA, treatment response varies among patients. Identifying reliable biomarkers for treatment response is crucial for optimizing therapy and patient outcomes. MicroRNA-499 has been implicated in autoimmune diseases, and IL-17 is a key mediator in RA pathogenesis. Additionally, PADI4 and ACCP are linked to RA autoimmunity. This study aimed to investigate the potential of microRNA-499 expression, serum levels of IL-17, PADI4 and ACCP as biomarkers for predicting and monitoring treatment response in RA patients undergoing anti-TNF-alpha (infliximab) therapy. Methodology: In this case-control research, blood samples from 100 Iraqi patients with RA and 100 controls were collected. RNA was extracted and the levels of miRNA499 were quantified using real-time PCR. The serum IL-17, PADI4 and ACCP levels were determined using ELISA. Patients were sub-grouped as responders and non-responders based on their responses to anti-TNF-alpha (infliximab) treatment. Results: The mean age of the patients in this study was 46.22 years. The occurrence of RA was higher in age group (31-45) years. There was a significant association between RA, smoking and BMI. miRNA-499 expression was significantly higher in RA patients versus controls (1.1 folds in patients compared to 0.52 in control group). The serum levels of IL-17, PADI4 and ACCP was significantly higher in RA patients than controls (54.3ng/L, 2.52 ng/ml and 2.15 U/ml) vs (40.35ng/ L, 1.78 ng/ml and 1.06 U/ml) respectively. Subdividing of patients showed that miRNA-499 expression and serum IL-17, PADI4 and ACCP to be significantly higher in non-responders patients than responders. Conclusion: MicroRNA-499 expression, serum levels of IL-17, PADI4 and ACCP were found to be significantly higher in RA patients compared to control group as well as in non-responding than responding patients.

Thrombin Priming Promotes the Neuroprotective Effects of Human Wharton's Jelly-Derived Mesenchymal Stem Cells Via the HGF/AKT/STAT3 Signaling Pathway.

Mesenchymal stem cells (MSCs) directly differentiate into neurons and endothelial cells after transplantation, and their secretome has considerable potential for treating brain injuries. Previous studies have suggested that the effects of MSCs priming with exposure to hypoxia, cytokines, growth factors, or chemical agents could optimize the paracrine potency and therapeutic potential of MSCs. Studies have suggested that thrombin-primed Wharton's Jelly-derived mesenchymal stem cells (Th.WJ-MSCs) significantly enhance the neuroprotective beneficial effects of naive MSCs in brain injury such as hypoxic-ischemic brain injury (HIE) and intraventricular hemorrhage (IVH). This study aimed to characterize WJ-MSCs in terms of stem cell markers, differentiation, cell proliferation, and paracrine factors by comparing naive and Th.WJ-MSCs. We demonstrated that compared with naive MSCs, Th.MSCs significantly enhanced the neuroprotective effects in vitro. Moreover, we identified differentially expressed proteins in the conditioned media of naive and Th.WJ-MSCs by liquid chromatography-tandem mass spectrometry analysis. Secretome analysis of the conditioned medium of WJ-MSCs revealed that such neuroprotective effects were mediated by paracrine effects with secretomes of Th.WJ-MSCs, and hepatocyte growth factor was identified as a key paracrine mediator. These results can be applied further in the preclinical and clinical development of effective and safe cell therapeutics for brain injuries such as HIE and IVH.

Engagement with mHealth Alcohol Interventions: User Perspectives on an App or Chatbot-Delivered Program to Reduce Drinking.

Research suggests participant engagement is a key mediator of mHealth alcohol interventions' effectiveness in reducing alcohol consumption among users. Understanding the features that promote engagement is critical to maximizing the effectiveness of mHealth-delivered alcohol interventions. The purpose of this study was to identify facilitators and barriers to mHealth alcohol intervention utilization among hazardous-drinking participants who were randomized to use either an app (Step Away) or Artificial Intelligence (AI) chatbot-based intervention for reducing drinking (the Step Away chatbot). We conducted semi-structured interviews from December 2019 to January 2020 with 20 participants who used the app or chatbot for three months, identifying common facilitators and barriers to use. Participants of both interventions reported that tracking their drinking, receiving feedback about their drinking, feeling held accountable, notifications about high-risk drinking times, and reminders to track their drinking promoted continued engagement. Positivity, personalization, gaining insight into their drinking, and daily tips were stronger facilitator themes among bot users, indicating these may be strengths of the AI chatbot-based intervention when compared to a user-directed app. While tracking drinking was a theme among both groups, it was more salient among app users, potentially due to the option to quickly track drinks in the app that was not present with the conversational chatbot. Notification glitches, technology glitches, and difficulty with tracking drinking data were usage barriers for both groups. Lengthy setup processes were a stronger barrier for app users. Repetitiveness of the bot conversation, receipt of non-tailored daily tips, and inability to self-navigate to desired content were reported as barriers by bot users. To maximize engagement with AI interventions, future developers should include tracking to reinforce behavior change self-monitoring and be mindful of repetitive conversations, lengthy setup, and pathways that limit self-directed navigation.

Regulation of Precipitation on Soil Dissolved Organic Matter in Perturbed Mangrove Ecosystems

Carbon sinks in mangrove soils play a critical role in climate change mitigation globally. Soil dissolved organic matter (DOM) is a major form of labile organic matter and influences carbon cycling in wetland ecosystems. However, the factors regulating DOM pools in mangrove soils on a regional scale are not well understood. Here, we used a novel approach to assess soil DOM dynamics and its environmental drivers in mangrove wetlands on a national scale in China. Soil samples were collected from 43 sampling sites and distributed in mangroves across the coastline of China. DOM extracted from mangrove soils was characterized by fluorescence spectroscopy. We estimated that dissolved organic carbon (DOC) content ranged from 0.20 ± 0.02 g/kg to 3.85 ± 0.09 g/kg and nationally averaged 0.73 ± 0.07 g/kg in mangrove soils. Soil DOM was composed of humic-like substances, including humic acid-like (53.46 ± 8.74%) and fulvic acid-like (46.54 ± 8.74%). DOM pools in mangrove soils were identified to be perturbed by terrestrial inputs. Besides, the coupling interactions of environmental controls on the soil DOM pool were validated by establishing the structural equation modeling (SEM). We found that precipitation is the most important driver, which controlled directly the inputs and outputs of DOM pools. It also indirectly influenced DOM pools by regulating soil parameters through the cascade reactions. Cu, salinity, and clay are key mediators among soil parameters for precipitation affecting DOM pools. Precipitation influences soil DOC negatively and CDOM and FDOM positively markedly. The results provide novel insights into the labile carbon pool in mangrove soils and are beneficial for improving the assessment frameworks in the blue carbon ecosystems.

Reciprocal interactions between malignant cells and macrophages enhance cancer stemness and M2 polarization in HBV-associated hepatocellular carcinoma.

Background: The tumor microenvironment of cancers has emerged as a crucial component in regulating cancer stemness and plays a pivotal role in cell-cell communication. However, the specific mechanisms underlying these phenomena remain poorly understood. Methods: We performed the single-cell RNA sequencing (scRNA-seq) on nine HBV-associated hepatocellular carcinoma (HCC) patients. The heterogeneity of the malignant cells in pathway functions, transcription factors (TFs) regulation, overall survival, stemness, as well as ligand-receptor-based intercellular communication with macrophages were characterized. The aggressive and stemness feature for the target tumor subclone was validated by the conduction of in vitro assays including sphere formation, proliferation, Annexin V apoptosis, flow cytometry, siRNA library screening assays, and multiple in vivo preclinical mouse models including mouse hepatoma cell and human HCC cell xenograft models with subcutaneous or orthotopic injection. Results: Our analysis yielded a comprehensive atlas of 31,664 cells, revealing a diverse array of malignant cell subpopulations. Notably, we identified a stemness-related subclone of HCC cells with concurrent upregulation of CD24, CD47, and ICAM1 expression that correlated with poorer overall survival. Functional characterization both in vitro and in vivo validated S100A11 as one of the top downstream mediators for tumor initiation and stemness maintenance of this subclone. Further investigation of cell-cell communication within the tumor microenvironment revealed a propensity for bi-directional crosstalk between this stemness-related subclone and tumor-associated macrophages (TAMs). Co-culture study showed that this interaction resulted in the maintenance of the expression of cancer stem cell markers and driving M2-like TAM polarization towards a pro-tumorigenic niche. We also consolidated an inverse relationship between the proportions of TAMs and tumor-infiltrating T cells. Conclusions: Our study highlighted the critical role of stemness-related cancer cell populations in driving an immunosuppressive tumor microenvironment and identified the S100A11 gene as a key mediator for stemness maintenance in HCC. Moreover, our study provides support that the maintenance of cancer stemness is more attributed to M2 polarization than the recruitment of the TAMs.