Enzyme-linked Immunosorbent Assay Research Articles

Efficacy of Erythritol Air Polishing for Inducing Initial Healing of Shallow-to-Moderate Periodontal Pockets and Restoring Gingival Crevicular Fluid β-Catenin, 8-Hydroxy-Deoxyguanosine and Total Antioxidant Capacity: A Split-Mouth Randomised Clinical Trial.

To evaluate the efficacy of subgingival erythritol powder air polishing (EPAP) for inducing initial healing of shallow-to-moderate periodontal pockets and its effect on gingival crevicular fluid (GCF) β-catenin, 8-hydroxy-deoxyguanosine (8-OHdG) and total antioxidant capacity (TAC) levels. This randomised, split-mouth clinical trial involved 22 periodontitis patients. Periodontal pockets (4-6 mm) were treated either by scaling and root planing (control) or EPAP followed by subgingival instrumentation 1 month later (test). Clinical periodontal variables were recorded, and GCF samples were collected from the selected sites at baseline, week 4 and week 12 visits. The GCF levels of β-catenin, 8-OHdG and TAC were estimated via enzyme-linked immunosorbent assay (ELISA). Compared with those at baseline, both groups showed non-different significant reductions in clinical variables at week 4 and week 12 after treatment. GCF β-catenin and 8-OHdG levels at both the test and control sites significantly decreased at week 4 and continued to further decrease at week 12. A greater reduction in GCF β-catenin was detected at the control sites at the week 12 visit. Conversely, GCF TAC was only significantly increased at week 12 in both groups, but the difference between the groups was not significant. Using of EPAP has the potential to induce initial healing in shallow-to-moderate pockets as it offers non-different changes in clinical outcomes and GCF biomarkers compared to conventional method. ClinicalTrials.gov identifier: NCT05798871.

Serum Soluble Fibrinogen-Like Protein 2 Estimates Increased T-Helper 2 Cytokine, Elevated Disease Severity, and Unfavorable Outcomes in Allergic Rhinitis Patients.

Soluble fibrinogen-like protein 2 (sFGL2) may be involved in the pathology and progression of allergic rhinitis (AR) through regulating T-helper (Th)2 cell response. This study aimed to explore the ability of sFGL2 to estimate outcomes in AR patients. sFGL2 was detected in the serum sample of 119 AR patients at baseline and 20 healthy controls (HCs) after enrollment by enzyme-linked immunosorbent assay. In AR patients, disease severity was assessed at baseline as well as 4 weeks (W4) and 8 weeks (W8) after enrollment by the total nasal symptom score (TNSS). sFGL2 was higher in AR patients than HCs [median (interquartile range): 100.0 (70.0-154.0) vs 47.0 (31.5-78.5) ng/mL] (P < .001). In AR patients, sFGL2 was positively correlated with TNSS score (P = .002), itching score (P = .037), sneezing score (P = .012), and interleukin (IL)-4 (P = .006) at baseline. TNSS score declined from baseline to W8 in AR patients (P < .001). To further explore the association between sFGL2 levels and treatment outcomes, we compared the sFGL2 levels between patients with TNSS scores at W4 or W8 >4 and those with TNSS scores at W4 or W8 ≤4. It was found that sFGL2 was only increased in AR patients with a TNSS score at W4 >4 versus those with a TNSS score at W4 ≤4 (P = .012). Serum sFGL2 is increased in AR patients, and its high level estimates an increased Th2 cytokine and disease severity as well as poor outcomes.

Development of a latex microsphere-based lateral flow immunoassay for the diagnosis of schistosomiasis japonica.

Zoonotic schistosomiasis, caused by Schistosoma japonicum, is prevalent in China, the Philippines and Indonesia. Rapid point-of-care (POC) diagnostics are attractive and promising tools for evaluating the efficacy of intervention strategies for schistosomiasis control. The diagnostic potential of five recombinant antigens was tested by enzyme-linked immunosorbent assay (ELISA) using sera from individuals with positive Kato-Katz (KK) results for S. japonicum (n = 28) and non-endemic controls (n = 12). A latex microsphere (LM)-based lateral flow immunoassay (LFIA) incorporating the recombinant SjSAP4 (rSjSAP4) was developed for the diagnosis of schistosomiasis japonica. The test conditions including diluent, dilution factor and reaction time, were optimised for the developed LFIA. Under the optimised conditions, serum samples from individuals living in a barangay endemic for S. japonicum (n = 549) and non-endemic controls (n = 50) were tested with the established LFIA cassettes. The results were imaged by a smartphone and analysed by the ImageJ program. The intensity ratio of the test line to the control line (T/C ratio) was calculated for each cassette. ELISA confirmed that rSjSAP4 was the optimal candidate for serological diagnosis of schistosomiasis japonica. Under optimal testing conditions, the developed LFIA strips had a sensitivity of 80.6% and a specificity of 98.0% at a cut-off T/C ratio of 0.1031. Moreover, the results of the LM-based LFIA was positively correlated with those obtained from the rSjSAP4-ELISA (r = 0.8270, 95% CI, 0.7990-0.8514; p < 0.0001). The schistosomiasis prevalence determined by the LFIA strips was about 1.8 times greater than that obtained with the 6-slide KK procedure performed on three stool samples. The developed LFIA represents a POC diagnostic tool that is suitable for onsite screening of human S. japonicum infection with minimal equipment needed. The established immunochromatographic assay complies with most of the WHO's ASSURED criteria for POC diagnostics.

Cordycepin ameliorates autoimmunity by promoting STING degradation via autophagy pathway.

Stimulator of interferon response cGAMP interactor 1 (STING), a central hub protein of cyclic GMP-AMP synthase (cGAS)-STING signalling pathway, has a crucial role in regulating type I interferons (IFNs) production and response. Recent studies indicate that excessive activation of STING is strongly associated with autoimmune diseases, including systemic lupus erythematosus (SLE). Searching immunomodulators that negatively regulate STING might greatly contribute to the suppression of autoimmunity. The peripheral blood mononuclear cells (PBMCs) of SLE patients, Hela cells, L929 cells and bone marrow-derived macrophages (BMDMs) from mice were used as in vitro models. While, Trex1 KO mouse autoimmune disease model was used as in vivo model. After treatment with cordycepin, a nucleoside from Cordyceps mushrooms, type I IFNs production and response were determined by western blotting, real-time polymerase chain reaction (PCR), dual-luciferase assay, enzyme-linked immunosorbent assay (ELISA), haematoxylin-eosin staining and RNA-seq. Cordycepin inhibited type I IFNs production and response in human and murine systems following cGAS-STING signalling activation. Importantly, cordycepin markedly attenuates the autoinflammatory and autoimmune responses in Trex1 KO BMDMs and Trex1 KO mice. Furthermore, cordycepin effectively suppressed the production of type I IFNs and interferon-stimulated genes (ISGs) in the PBMCs of SLE patients. Mechanistically, cordycepin promoted STING degradation via autophagy pathway upon DNA stimulation. This study shows that cordycepin promotes STING autophagic degradation to alleviate autoimmunity upon DNA stimulation. Cordycepin might be a potential therapeutic candidate for alleviating aberrant type I IFNs in autoimmune and autoinflammatory diseases.

Longitudinal analysis of serum intestinal fatty acid-binding protein in a patient with non-occlusive mesenteric ischemia following brachial plexus block-induced hypotension: a case study.

Intestinal fatty acid-binding protein (I-FABP) is a promising biomarker for small-bowel ischemia including non-occlusive mesenteric ischemia (NOMI). A 75-year-old woman with diabetic nephropathy sustained a distal radius fracture. Two days later, she underwent a brachial plexus block to facilitate orthopedic surgery, which resulted in hypotension. Despite prompt fluid resuscitation and ephedrine administration, the patient developed abdominal pain. Contrast-enhanced computed tomography revealed hepatic portal venous gas, but no direct evidence of small-bowel ischemia. The gastrointestinal surgery team opted for cautious in-hospital observation overnight. Unfortunately, the patient's condition significantly worsened the following day, prompting an urgent laparotomy. Surgery confirmed ileal segment necrosis, macroscopically characterized by a distinctive geographic pattern. Retrospective analysis of stored serum samples using a human enzyme-linked immunosorbent assay demonstrated that I-FABP levels were moderately elevated (7.2ng/mL) at the initial outpatient visit for the fracture, peaked (17.9ng/mL) at the clinical onset of NOMI, and returned to normal (0.7ng/mL) by postoperative day 2. Serum I-FABP levels correlated with the progression of NOMI, showing potential as an early detection marker. However, the longitudinal analysis of serum I-FABP also highlighted significant challenges of this biomarker, including the influence of renal function and the importance of sampling timing.

Porphyromonas gingivalis-Lipopolysaccharide Induced Gingival Fibroblasts Trained Immunity Sustains Inflammation in Periodontitis.

To investigate whether trained immunity occurs in gingival fibroblasts (GFs) and its relationship to the persistence of inflammation in periodontitis. Periodontally healthy and inflammatory gingival fibroblasts (HGFs and IGFs) were cultured through continuous adherence subculture of tissue blocks. Trained immunity in HGFs was evaluated via a classic invitro model, with relevant markers assessed via enzyme-linked immunosorbent assay, lactate content assay, glycolytic rate assay, and chromatin immunoprecipitation. A histone methyltransferase blocker and a PI3K inhibitor were added to investigate the mechanisms underlying trained immunity. The relationship between trained immunity and periodontitis was further examined via immunofluorescence staining and chromatin immunoprecipitation on IGFs. Compared with untrained cells, GFs trained with Porphyromonas gingivalis-lipopolysaccharide (P. gingivalis-LPS) exhibited a significant increase in IL-6 and TNF-α secretion, enhanced glycolytic metabolism, and enriched mono-methylation of lysine 4 on histone H3 (H3K4me1) at the enhancer regions of TNF-α and IL-6. The addition of a histone methyltransferase blocker and a PI3K inhibitor greatly reduced trained immunity. Additionally, the response of IGFs to P. gingivalis-LPS stimulation and their epigenetic modifications were similar to those observed in trained HGFs. This study novelly discovered that both P. gingivalis-LPS-stimulated HGFs and IGFs in periodontitis acquired trained immunity. Following P. gingivalis-LPS stimulation, HGFs underwent metabolic and epigenetic changes via the PI3K/AKT pathway, with these epigenetic changes also observed in IGFs. This finding suggests that trained immunity in GFs may be a key mechanism underlying the recurrence and persistence of periodontitis.

Altered eotaxin-1 and interleukin-34 levels in obsessive-compulsive disorder: a case-control observational study in Bangladesh.

Obsessive-compulsive disorder (OCD) is a prevalent mental health condition that impacts daily life. It is thought to be associated with genetic, biological, and structural brain changes, serotonergic abnormalities, altered neuromodulation, and environmental factors. Limited observational studies have examined cytokines in Bangladeshi patients with OCD. This study aimed to assess the levels of eotaxin-1 and interleukin (IL)-34 in individuals with this disorder. This case-control observational study included 58 patients with OCD and 30 healthy controls (HCs) matched for age, sex, and body mass index. The severity of OCD was assessed using the Yale-Brown obsessive-compulsive scale (Y-BOCS). Psychiatrists evaluated participants according to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition. Serum levels of eotaxin-1 and IL-34 were measured using enzyme-linked immunosorbent assay kits. Patients with OCD exhibited significantly higher serum eotaxin-1 levels (121.13±7.84 pg/mL) than HCs (85.52±9.42 pg/mL). Conversely, IL-34 levels were considerably lower in patients than in HCs (119.02±14.53 pg/mL vs. 179.96±27.88 pg/mL). The Cohen d values for eotaxin-1 and IL-34 were 0.55 and -0.48, respectively. Among patients with OCD, a significant positive correlation was found between serum eotaxin-1 level and Y-BOCS score, along with a negative correlation between serum eotaxin-1 and IL-34 levels. The findings suggest that altered eotaxin-1 and IL-34 levels may be associated with OCD. These chemokines and cytokines could serve as primary tools for assessing the risk of OCD, warranting further clinical investigation. This could potentially support more extensive research and the development of diagnostic and therapeutic strategies targeting these pathways.

Elevated IL-6 Expression in Autologous Adipose-Derived Stem Cells Regulates RANKL Mediated Inflammation in Osteoarthritis

Interleukin-6 (IL-6) expression in mesenchymal stem cells (MSCs) has been shown to play a pivotal role in modulating cartilage regeneration and immune responses, particularly in the context of diseases that involve both degenerative processes and inflammation, such as osteoarthritis (OA). However, the precise mechanism through which IL-6 and other immune-regulatory factors influence the therapeutic efficacy of autologous adipose-derived stem cells (ASCs) transplantation in OA treatment remains to be fully elucidated. This study aims to investigate the relationship between IL-6 expression in autologous ASCs isolated from OA patients and their impact on immune modulation, particularly focusing on the regulation of Receptor Activator of Nuclear factor Kappa-Β Ligand (RANKL), a key mediator of immune-driven cartilage degradation in OA. Autologous ASCs were isolated from the stromal vascular fraction (SVF) of adipose tissue obtained from 22 OA patients. The isolated ASCs were cultured and characterized using reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and flow cytometry to the phenotype and immune regulatory factors of MSCs. Based on IL-6 expression levels, ASCs were divided into high and low IL-6 expression groups. These groups were then co-cultured with activated peripheral blood mononuclear cells (PBMCs) to evaluate their immune-modulatory capacity, including the induction of regulatory T cells, inhibition of immune cell proliferation, and regulation of key cytokines, such as interferon-gamma (IFN-γ). Additionally, RANKL expression, a critical factor in osteoclastogenesis and cartilage degradation, was assessed in both ASC groups. High IL-6-expressing ASCs demonstrated a significantly greater capacity to inhibit immune cell proliferation and IFN-γ production compared to their low IL-6-expressing counterparts under co-culture conditions. Moreover, the group of ASCs with high IL-6 expression showed a marked reduction in RANKL expression, suggesting enhanced potential to control osteoclast activity and subsequent cartilage defect in OA. Conclusion: Autologous ASCs with elevated IL-6 expression exhibit enhanced immunomodulatory properties, particularly in regulating over-activated immune response and reducing osteoclastogenesis through RANKL suppression. These findings indicate that selecting ASCs based on IL-6 expression could enhance the therapeutic efficacy of ASC-based treatments for OA by mitigating immune-driven joint inflammation and cartilage degradation, potentially slowing disease progression.

Regnase-1 regulates inflammation in T cells of ankylosing spondylitis through the TRAF6.

The study aimed to investigate the regulatory role of Regnase-1 in ankylosing spondylitis (AS) inflammation. We collected 10ml peripheral venous blood and epidemiological data from 45 AS patients and 45 healthy controls and performed enzyme-linked immunosorbent assay (ELISA) experiments to measure the levels of inflammatory cytokines. Then CD3 + T lymphocytes were isolated by magnetic bead sorting method, and the transcriptional levels of Regnase-1 and TNF receptor-associated factor 6 (TRAF6) were detected by real-time quantitative PCR (qRT-PCR). The regnase-1 knockdown human T lymphocyte leukemia cell (Jurkat T) model was constructed by small interfering RNA (siRNA) technology. Then PCR and Western blot were used to detect the transcription level and protein level of downstream genes. Co-immunoprecipitation was used to verify the interaction between Regnase-1 and TRAF6. Regnase-1 and TRAF6 transcription levels were down-regulated and positively correlated with each other in T cells from AS patients. The ROC curve analysis indicates that both Regnase-1 and TRAF6 possess diagnostic capabilities, with Regnase-1 demonstrating a particularly high area under the curve (AUC) of 0.876 (95% CI: 0.789-0.936). Subgroup analysis shows NSAIDs boost Regnase-1 and TRAF6 transcription while reducing IL23 and IL17 levels. The results of cell experiments showed that si-Regnase-1 significantly reduced the mRNA and protein levels of TRAF6 in Jurkat T cells and increased the expression level of inflammatory gene TNF-α. Co-immunoprecipitation assay further verified the binding between the two proteins. Regnase-1 may participate in the chronic inflammatory process of AS by regulating TNF-α through TRAF6.

Quantitative Assessment of Bony Fish Tropomyosin Using a Monoclonal Antibody Competitive ELISA.

Bony fish is one of the big-nine allergenic foods in the US. This study established a monoclonal antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of bony fish tropomyosin (TM), a fish allergen. Immunoassay (Western blot and ELISA) was performed to characterize anti-TM mAb8F5 (target configuration, immunoaffinity, and species selectivity). Atlantic cod (Gadus morhua) tropomyosin (CTM) and mAb8F5 were used for assay development. The immunosignal was captured by two light sources, i.e., colorimetry (cELISA) and luminescence (lELISA). During assay validation, cELISA had a wider working range (1-250 ng/μL) than lELISA (0.5-62.5 ng/μL). However, lELISA showed better sensitivity because of its lower half-maximal inhibitory concentration (IC50, 8.8 ng/μL) than cELISA (37.1 ng/μL). The optimal cELISA was applied to measure CTM-equivalents in 21 bony fish samples (0.03-4.2 mg/g) and laboratory-spiked samples (recovery rates of CTM-spiked shrimp samples ranged from 104.7 to 120.8%). lELISA was used to quantify 5.25 μg CTM residues on four food contact surfaces (3.4-6.9 μg from CTM residues and 3.2-4.8 μg from Atlantic cod were detected). This immunoassay has the potential to surveil undeclared allergenic bony fish residues and validate the hygiene of food contact surfaces.

HDC downregulation induced by chronic stress promotes ovarian cancer progression via the IL-6/STAT3/S100A9 pathway

ObjectiveThis study aimed to investigate the underlying mechanism of chronic stress promoting ovarian cancer growth comorbid with depression and evaluate the potential role of histamine (HIS) in treating this comorbidity.MethodsChronic unpredictable mild stress (CUMS) was used to establish a comorbid mouse model of ovarian cancer and depression. The behavioral phenotypes were assessed using the sucrose preference test (SPT), tail suspension test (TST), forced swimming test (FST), and open field test (OFT). Ovarian cancer growth was monitored by tracking the tumor volume and weight. Histidine decarboxylase (HDC) expression in the tumor tissue was analyzed using Western blot and qRT-PCR techniques. The serum levels of inflammatory factors (IL-6 and IL-17A), stress hormones (norepinephrine, NE and cortisol, and COR), histamine, and 5-hydroxytryptamine (5-HT) were detected by enzyme-linked immunosorbent assay (ELISA). In vitro experiments were conducted to explore the direct impacts of stress hormones on A2780 and ES-2 ovarian cancer cell lines, as well as the modulation of these effects by histamine. HDC knockdown and overexpression approaches were used to study its regulatory role in the IL-6/STAT3/S100A9 signaling pathway.ResultsChronic stress not only induced depressive behaviors but also accelerated ovarian cancer growth in mice by downregulating HDC expression in tumors, whereas exogenous HIS treatment alleviated depressive symptoms, suppressed cancer growth, and countered the decreased levels of HIS and increased levels of IL-6, IL-17A, NE, COR, and 5-HT induced by CUMS. Furthermore, HIS positively modulated the immune response by increasing the populations of CD3+T and CD8+ T cells and reducing IL-17A secretion. In vitro experiments revealed that stress hormones downregulated HDC expression, consequently promoting cancer cell proliferation, migration, and invasion via the IL-6/STAT3/S100A9 pathway. Knockdown of HDC activated this pathway, whereas HDC overexpression inhibited its activation.ConclusionChronic stress leads to the downregulation of HDC expression, thereby facilitating the progression of ovarian cancer through the IL-6/STAT3/S100A9 pathway. HIS might serve as a potential molecule for treating the comorbidities of ovarian cancer and depression.

Characterisation and serodiagnostic evaluation of a recombinant 22.6-kDa tegument protein of Schistosoma spindale.

Schistosomosis in animals due to Schistosoma spindale significantly burdens India's livestock economy because of high prevalence and morbidity and is mostly underdiagnosed from the lack of sensitive tools for field-level detection. This study aimed to clone, express the 22.6-kDa tegument protein of S. spindale (rSs22.6kDa) and to utilise it in a dot enzyme-linked immunosorbent assay for serodiagnosis. RNA was extracted from adult worms recovered from the mesenteries of slaughtered cattle to amplify the gene encoding the 22.6-kDa protein. In silico analysis revealed the protein's secondary structure, consisting of 190 amino acids forming alpha helices (47.89%), extended strands (17.37%), beta turns (8.95%), and random coils (25.79%), with α helices and β sheets in the tertiary structure. Two conserved domains were noted: an EF-hand domain at the N-terminus and a dynein light-chain domain at the C-terminus. Phylogenetic studies positioned the S. spindale sequence as a sister clade to Schistosoma haematobium and Schistosoma bovis. The gene was cloned into a pJET vector and transformed into Escherichia coli Top 10 cells, with expression achieved using a pET28b vector, BL21 E. coli cells, and induction with 0.6 mM isopropyl-β-d-thiogalactopyranoside. The protein's soluble fraction was purified using nickel-chelating affinity chromatography, confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting, identifying a distinct immunodominant 22.6-kDa protein. The diagnostic utility was validated using a dot enzyme-linked immunosorbent assay which demonstrated a of sensitivity of 89.47% and specificity of 100%. The study records for the first time the prokaryotic expression and evaluation of the 22.6-kDa tegumental protein of S. spindale, highlighting its potential as a diagnostic antigen for seroprevalence studies in bovine intestinal schistosomosis.

Assessment of the Monocyte Subpopulations and M1/M2 Macrophage Ratio in Concentrated Bone Marrow Aspirate.

The purpose of this study was to determine the M1/M2 macrophage ratio in concentrated bone marrow aspirate (cBMA) in patients undergoing surgical intervention augmented with cBMA for osteochondral lesions of the talus (OLTs). Samples of peripheral blood (PB), bone marrow aspirate (BMA), and cBMA were collected during the procedure. The samples were analyzed by automated cell counting and multicolor fluorescence-activated cell sorting with specific antibodies recognizing monocytes (CD14+ CD16+) and the M1 (CD86+) and M2 (CD163+CD206+) populations within that monocyte population. Cytokine concentrations within the samples were evaluated with enzyme-linked immunosorbent assay (ELISA). The composition of cBMA was compared between 2 commercially available BMA concentration systems. Thirty-eight patients with a mean age of 43.2 ± 10.1 years old undergoing a surgical procedure for the treatment of OLTs involving the use of cBMA were included. cBMA had a mean fold increase of 4.7 for all white blood cells, 6.1 for monocytes, 7.9 for lymphocytes, 2.4 for neutrophils, and 9.6 for platelets when compared to BMA. The mean M1/M2 ratio for PB, BMA, and cBMA was 15.2 ± 12.0, 20.8 ± 13.3, and 22.1 ± 16.0, respectively. There was a statistically significant higher concentration of interleukin-1 receptor antagonist (IL-1Ra) in the cBMA sample (8243.3 ± 14,837.4 pg/mL) compared to both BMA (3143.0 ± 2218.5 pg/mL) and PB (1847.5 ± 1520.4 pg/mL) samples. The IL-1Ra/IL-1β ratio for PB, BMA, and cBMA was 790.6 ± 581.9, 764.7 ± 675.2, and 235.7 ± 192.1, respectively. There was no difference in the cBMA M1/M2 ratio (19.0 ± 11.1 vs 24.0 ± 18.3) between the Magellan (Isto Biologics, Hopkinton, Massachusetts) and Angel systems (Arthrex Inc, Naples, Florida). This prospective study found that the M1/M2 ratio in cBMA was 22.1 ± 16.0, with significant patient to patient variation observed. Overall, there was no statistically significant difference in the M1/M2 ratio across PB, BMA, and cBMA samples. This is the first study to characterize the macrophage subpopulation within cBMA, which may have significant clinical implications in future studies.

Osimertinib exacerbates immune checkpoint inhibitor-related severe adverse events by activating the IL-6/JAK/STAT3 pathway in macrophages.

The combination of epithelial growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) and immune checkpoint inhibitors (ICIs) leads to an increased incidence of severe immune-related adverse events (irAEs). However, the mechanisms underlying macrophages in irAEs have not been elucidated. An osimertinib and ICI-induced irAE mouse model was constructed. Lung micro-CT scans were used to assess the degree of inflammatory infiltration. Hematoxylin-eosin staining was used to analyze the histopathologic inflammatory infiltration in mouse liver and lung tissues. Flow cytometry was used to detect the percentages of T cells, NK cells, and macrophages and the expression of EGFR. Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum interleukin (IL)-6, alanine transaminase (ALT), ferritin, and tumor necrosis factor (TNF)-α levels. Total RNA extracted from mouse liver macrophages was analyzed by RNA-seq. Simple Western blot analysis was used to detect the IL-6/JAK/STAT3 pathway activation state. Osimertinib combined with ICIs upregulated EGFR expression on macrophages with increased serum IL-6, ALT, and ferritin levels. RNA-seq and simple Western blot analysis of mouse liver macrophages confirmed that that the IL-6/JAK/STAT3 pathway was activated in the combination treatment group. Ruxolitinib blocked the IL-6/JAK/STAT3 pathway and significantly decreased the serum IL-6, ALT, and ferritin levels in the combination treatment group. An osimertinib and ICI-induced irAE mouse model was constructed that showed osimertinib combined with ICIs inhibited EGFR phosphorylation and activated the IL-6/JAK/STAT3 signaling pathway in mouse liver macrophages, which led to the release of relevant cytokines.

Chondrogenic commitment of human umbilical cord blood and umbilical cord-derived mesenchymal stem cells induced by the supernatant of chondrocytes: A comparison study.

Native cartilage has low capacity for regeneration because it has very few progenitor cells. Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) and human umbilical cord-derived MSCs (hUC-MSCs) have been employed as promising sources of stem cells for cartilage injury repair. Reproduction of hyaline cartilage from MSCs remains a challenging endeavor. The paracrine factors secreted by chondrocytes possess the capability to induce chondrogenesis from MSCs. The conditioned medium derived from chondrocytes was utilized to induce chondrogenic differentiation of hUCB-MSCs and hUC-MSCs. The expression levels of collagen type I alpha 1 chain (Col1a1), collagen type II alpha 1 chain (Col2a1), and SRY-box transcription factor 9 (SOX9) were assessed through quantitative real-time polymerase chain reaction (qRT-PCR), Western blot (WB), and immunofluorescence (IF) assays. To elucidate the mechanism of differentiation, the concentration of transforming growth factor-β1 (TGF-β1) in the conditioned medium of chondrocytes was quantified using enzyme-linked immunosorbent assay (ELISA). Meanwhile, the viability of cells was assessed using Cell Counting Kit-8 (CCK-8) assays. The expression levels of Col2a1 and SOX9 were found to be higher in induced hUC-MSCs compared to those in induced hUCB-MSCs. The conditioned medium of chondrocytes contained TGF-β1. The CCK-8 assays revealed that the proliferation rate of hUC-MSCs was significantly higher compared to that of hUCB-MSCs. The chondrogenic potential and proliferation capacity of hUC-MSCs surpass those of hUCB-MSCs, thereby establishing hUC-MSCs as a superior source of seed cells for cartilage tissue engineering.

Modulation of 8-Oxoguanine DNA Glycosylase 1 (OGG1) Alleviated Anemia Severity and Excessive Cytokines Release during Plasmodium berghei Malaria in Mice

Background: The interplay of OGG1, 8-Oxoguanine, and oxidative stress triggers the exaggerated release of cytokines during malaria, which worsens the outcome of the disease. We aimed to investigate the involvement of OGG1 in malaria and assess the effect of modulating its activity on the cytokine environment and anemia during P. berghei malaria in mice. Methods: Plasmodium berghei ANKA infection in ICR mice was used as a malaria model. OGG1 concentration and oxidative stress levels in P. berghei-infected mice and their control counterparts were assessed during malaria using enzyme-linked immunosorbent assay. OGG1 activity in malaria mice was modulated using treatment with TH5487 and O8-OGG1 inhibitors. The effects of modulating OGG1 activity using OGG1 inhibitors on cytokine release and anemia during P. berghei malaria infection were assessed by cytometric bead array and measurement of total normal red blood cell count respectively. Results: The plasma OGG1 level was significantly upregulated and positively correlated with parasitemia during P. berghei malaria in mice. Modulation of OGG1 ameliorated malaria severity by improving the total normal RBC count in TH5487 and O8-treated mice. Modulation of OGG1 with TH5487 caused significant reductions in serum levels of TNF-α, IFN-γ, IL-6, and IL-10. Similarly, OGG1 modulation activity using an O8-OGG1 inhibitor caused a significant reduction in serum levels of TNF-α, IL-2, IL-6, and IL-10. Conclusion: The findings indicate the involvement of OGG1 in the P. berghei malaria infection. OGG1 inhibition by TH5487 and O8-OGG1 inhibitors suppressed excessive cytokine release, and this may represent a novel therapeutic strategy for ameliorating the severity of malaria infection.

. Diagnostic value of HMGB-1 and Acetylcholinesterase in Assessing the Prognosis of Patients with Acute Pancreatitis

Aim: Acute pancreatitis (AP) is a disorder of tissue digestion caused by abnormal activation of pancreatic enzymes, which may lead to multi-organ failure and ultimately death as the disease progresses. How to quickly and accurately evaluate the progression of AP has always been a hotspot and difficulty in clinical research. Methods: Sixty-four AP patients admitted to our hospital from August 2022 to June 2023 and 60 concurrent healthy people were selected as the research subjects, with AP patients as the observation group and healthy people as the control group. Using kits from Wuhan Fine Biotechnology Co., Ltd., an enzyme-linked immunosorbent assay (ELISA) was carried out to measure HMGB-1 and AChE levels. In addition, an automatic biochemical analyzer was utilized to quantify the levels of nutritional proteins albumin (ALB), transferrin (TRF), and total protein (TP) in the observation group. Results: Compared with the control group, the HMGB-1 in the observation group was higher while the AChE was lower (P&lt;0.05). ROC curve analysis showed that when HMGB-1 was less than 7.25 mg/L, the sensitivity and specificity for diagnosing AP were 89.06% and 51.67%, respectively (P&lt;0.05); when AChE was less than 6.18 U/L, the sensitivity for diagnosing AP was 43.75% and the specificity was 90.00% (P&lt;0.05). Using the joint detection Log(P)=-1.736+(0.645×HMGB-1)+(-0.454×AChE), it was found that HMGB-1+AChE had a sensitivity of 48.44% and a specificity of 88.33% for the diagnosis of AP (P&lt;0.05, cut-off&gt;0.639,) Conclusion: In this study, we found elevated HMGB-1 expression in AP and reduced acetylcholinesterase (AChE), suggesting that both of them may be involved in the occurrence and development of AP. Meanwhile, the two demonstrated excellent diagnostic efficacy in the onset and poor prognosis of AP, and were closely related to AP progression, confirming their potential as new clinical indicators for AP. Due to the self-digestion by pancreatic enzymes, the nutritional metabolism of AP patients is disrupted, so protein loss is also a focus of attention. In this study, we also found a negative correlation of nutritional protein levels with HMGB-1 in AP patients and a positive correlation between them with AChE, further supporting the relationship between the two and AP progression. The findings can lay a reliable foundation for future research on the diagnosis and treatment of AP based on HMGB-1 and AChE.

Discovery of Novel MyD88 Inhibitor A5S to Alleviate Acute Lung Injury with Favorable Drug-like Properties.

Myeloid differentiation primary response 88 (MyD88) plays a central role in inflammatory responses and diseases. However, only a few inhibitors of MyD88 with some limits have been reported currently. Herein, we identified a lead compound (L7) through virtual screening and synthesized twenty-seven L7 derivatives. An optimal compound (A5) was determined through enzyme-linked immunosorbent assay (ELISA), 2,5-diphenyl-2H-tetrazolium bromide (MTT), and biolayer interferometry (BLI) assay. The potent isomer A5S showed a high MyD88 binding ability and exerted an anti-inflammatory effect through the NF-κB/MAPK pathway. A5S had good stability and safety, showed the highest distribution concentration in the lungs, and exhibited good therapeutic effects on LPS-induced and sepsis-induced ALI mouse models. Most importantly, A5S showed advantages in PK properties, and was identified as a promising MyD88 inhibitor with favorable drug-like properties, compared to the only approved MyD88 inhibitor, TJ-M2010-5, which is currently undergoing a Phase I study, and our previously reported MyD88 inhibitors LM8.

Calcitriol Concentration in the Early Phase of Myocardial Infarction and Its Relation to Left Ventricular Ejection Fraction

Vitamin D deficiency is one of the most common metabolic disorders in the European population. A low level of 25-OH vitamin D3 is related to an elevated risk of myocardial infarction (MI). The aim of our study was to examine the relationship between calcidiol and calcitriol serum concentration and left ventricular ejection fraction early after interventional treatment for acute coronary syndrome. A total of 80 patients diagnosed with MI, who underwent primary percutaneous coronary intervention, were included in the study. Blood samples for calcidiol, calcitriol, and vitamin D-binding protein were obtained 24 h after primary PCI and were measured using an enzyme-linked immunosorbent assay. Only 9% of patients had a proper level of 25-OHD3 in the serum (30–80 ng/mL). A total of 16% of patients revealed a suboptimal concentration of 25-OHD3 (20–30 ng/mL), and in 75% of patients, the concentration of 25-OHD3 was lower than 20 ng/mL. Moreover, patients with left ventricle ejection fraction of &lt; 40% had significantly lower concentrations of calcidiol and calcitriol. A low calcitriol serum concentration affects post-MI left ventricle ejection fraction early after myocardial infarction onset. It seems that 1.25(OH)D3 may contribute to acute myocardial infarction; however, there are insufficient clinical trials related to this topic, and the available evidence is mainly from in vitro studies. We hope these preliminary reports will provide a better understanding of post-MI.

The role of inflammasome dysregulation in obstructive and non-obstructive azoospermia: a comparative molecular analysis of blood, tissue, and seminal plasma

BackgroundTo address knowledge gaps, this study aimed to investigate the involvement of inflammasomes in the etiology of azoospermia. This study focused on the gene expression of key inflammasome components, including NLR family pyrin domain containing 3 (NLRP-3), CASPASE-1, Interleukin-1β (IL-1β), Interleukin-18 (IL-18), NLR family CARD domain-containing protein 4/ice protease-activating factor (NLRC-4/IPAF), and Absent in melanoma 2 (AIM-2).MethodsWe analyzed gene expression in blood and testicular tissue from patients with obstructive azoospermia (OA) and non-obstructive azoospermia (NOA). Additionally, we compared IL-1β and IL-18 expression levels in seminal plasma samples using the Enzyme-Linked Immunosorbent Assay (ELISA) method. For comparison, blood samples from normospermic (NS) individuals were also genetically evaluated.ResultsOur results indicated significantly higher gene expression of inflammasome components in NOA patients than those in OA patients either in blood or in testicular tissue. Both azoospermic groups exhibited higher mRNA levels of inflammasome genes comparing with those from blood samples of NS men. Seminal plasma samples showed significantly increased levels of IL-1β and IL-18 in NOA patients compared to men with OA. The ROC curve analysis indicated strong and significant predictive power of IL-18, AIM-2 and NLRC-4/IPAF gene expression profiles between NOA vs. NS patients and NOA vs. OA.ConclusionsOur findings highlight the role of hidden chronic inflammation in azoospermia, particularly within the NOA group. This study provides a foundation for further detailed research, which could aid in the development of diagnostic panels to differentiate between various azoospermic groups.