Novel endothelial progenitor cells populations as biomarkers of damage and remission in systemic lupus erythematosus.

INTRODUCTION: Endothelial progenitor cells (EPCs) are essential for maintenance of vascular homeostasis and stability, key processes in the pathogenesis of systemic lupus erythematosus (SLE). However, the role and phenotypic characterization of EPCs populations in SLE have not been completely elucidated. OBJECTIVE: To identify EPCs specific subpopulations in patients with SLE using a novel flow cytometry tool. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from patients with SLE and healthy controls (HC). mRNA and surface protein expression were determined by quantitative PCR (qPCR) and flow cytometry. Clusters identification and characterization were performed using tSNE-CUDA dimensionality reduction algorithms. RESULTS: tSNE-CUDA analysis identified eight different clusters in PBMCs from HC and patients with SLE. Three of these clusters had EPC-like phenotype and the expression was elevated in patients with SLE. Moreover, four SLE-associated subclusters were found mainly expressed in patients with SLE, being only present in patients in remission with SLE and significantly associated with the 2021 Definition of Remission in SLE. Importantly, we also identified specific clusters in SLE patients with organ damage, according to the Systemic Lupus International Collaborating Clinics (SLICC)/American College of Rheumatology damage index (SDI). These clusters showed an EPC-like phenotype, but the expression of angiogenic markers was lower compared to HC or patients without organ damage, suggesting an impaired angiogenic function. CONCLUSION: Our novel approach identified clusters of EPCs in patients with SLE that are associated with remission and damage. Therefore, these clusters might be useful biomarkers to predict disease progression and severity in SLE pathogenesis.

The effects of Staphylococcus aureus protein a (SpA) on the expression of inflammatory cytokines in autoimmune patients and their probable immune response modulation mechanisms.

The recombinant Staphylococcal protein A (SpA) is widely used in biotechnology to purify polyclonal and monoclonal IgG antibodies. At very low concentrations, the highly-purified form of the protein A can down-regulate the activation of human B-lymphocytes and macrophages which are the key cells in determining autoimmune diseases. In the present study, the efficiency of three different forms of protein A, including native full-length SpA, the recombinant full-length SpA, and a recombinant truncated form of SpA on the reduction of 4 inflammatory cytokines, including IL-8, IL-1ß, TNF-α, and IL-6 by peripheral blood mononuclear cell (PBMCs) were studied and compared to an anti-rheumatoid arthritis commercial drug, Enbrel. The recombinant proteins were expressed in E. coli and the native form of SpA was commercially provided. PBMCs were obtained from adult patients with active rheumatoid arthritis (RA) and healthy control donors. Then, the effect of different doses of the three pure forms of SpA in comparison with Enbrel was investigated by analyzing the expression of selected cytokines using ELISA. The results showed that the truncated form of recombinant SpA significantly reduced the expression of cytokines more effectively than the other full-length formulations as well as the commercial drug Enbrel. In silico analysis shows that in the truncated protein, as the radius of gyration increases, the structure of IgG-binding domains become more open and more exposed to IgG. To summarize, our findings indicate that the truncated form of protein A is the most efficient form of SpA as it significantly decreases the secretion of evaluated cytokines from PBMCs in vitro.

Microbe-associated molecular patterns derived from fungi and bacteria promote IgG4 antibody production in patients with type 1 autoimmune pancreatitis.

Enhanced IgG4 antibody (Ab) response is a prominent feature of type 1 autoimmune pancreatitis (AIP). Innate immune responses associated with IgG4 Ab production are poorly defined. We have previously reported that peripheral blood mononuclear cells (PBMCs) isolated from patients with type 1 AIP produce large amounts of IgG4 Abs upon stimulation with bacterial cell wall components. In addition, we showed that activation of plasmacytoid dendritic cells producing interferon (IFN)-α, interleukin (IL)-33, and B cell-activating factor (BAFF) upon sensing intestinal bacteria mediates the development of experimental AIP. In this study, we attempted to clarify the role of innate immunity against fungi in inducing enhanced IgG4 Ab responses in type 1 AIP. PBMCs isolated from healthy controls and patients with type 1 AIP were stimulated with a broad range of bacterial and fungal cell wall components. The concentrations of IgG1, IgG4, and cytokines were measured using enzyme-linked immunosorbent assays. Cell wall components derived from bacteria and fungi induced IgG1 and IgG4 Ab production in patients with type 1 AIP. Various types of microbe-associated molecular pattern motifs enhanced IgG4 Ab production in patients with type 1 AIP compared with the limited motifs in healthy controls. The enhanced IgG1 and IgG4 Ab production that followed in response to bacterial and fungal cell wall components was parallel to that of IFN-α, IFN-γ, IL-10, IL-33, and BAFF. In conclusion, cell wall components derived from fungi as well as bacteria promote IgG4 Ab responses in patients with type 1 AIP.

Identification of autophagy-related signatures in doxorubicin-induced cardiotoxicity.

PURPOSE: Doxorubicin is an antibiotic drug used clinically to treat infectious diseases and tumors. Unfortunately, it is cardiotoxic. Autophagy is a cellular self-decomposition process that is essential for maintaining homeostasis in the internal environment. Accordingly, the present study was proposed to characterize the autophagy-related signatures of doxorubicin-induced cardiotoxicity. METHODS: Datasets related to doxorubicin-induced cardiotoxicity were retrieved by searching the GEO database and differentially expressed genes (DEGs) were identified. DEGs were taken to intersect with autophagy-related genes to obtain autophagy-related signatures, and Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and protein-protein interaction (PPI) network were performed on them. Further, construction of miRNA-hub gene networks and identification of target drugs to reveal potential molecular mechanisms and therapeutic strategies. Animal models of doxorubicin-induced cardiotoxicity were constructed to validate differences in gene expression for autophagy-related signatures. RESULTS: PBMC and heart samples from the GSE37260 dataset were selected for analysis. There were 995 and 2357 DEGs in PBMC and heart samples, respectively, and they had 23 intersecting genes with autophagy-related genes. RT-qPCR confirmed the differential expression of 23 intersecting genes in doxorubicin-induced cardiotoxicity animal models in general agreement with the bioinformatics results. An autophagy-related signatures consisting of 23 intersecting genes is involved in mediating processes and pathways such as autophagy, oxidative stress, apoptosis, protein ubiquitination and phosphorylation. Moreover, Akt1, Hif1a and Mapk3 are hub genes in autophagy-associated signatures and their upstream miRNAs are mainly rno-miR-1188-5p, rno-miR-150-3p and rno-miR-326-3p, and their drugs are mainly CHEMBL55802, Carboxyamidotriazole and 3-methyladenine. CONCLUSION: This study identifies for the first-time autophagy-related signatures in doxorubicin's cardiotoxicity, which could provide potential molecular mechanisms and therapeutic strategies for doxorubicin-induced cardiotoxicity.

Aging-related biomarkers for the diagnosis of Parkinson's disease based on bioinformatics analysis and machine learning.

Parkinson's disease (PD) is a multifactorial disease that lacks reliable biomarkers for its diagnosis. It is now clear that aging is the greatest risk factor for developing PD. Therefore, it is necessary to identify novel biomarkers associated with aging in PD. In this study, we downloaded aging-related genes from the Human Ageing Gene Database. To screen and verify biomarkers for PD, we used whole-blood RNA-Seq data from 11 PD patients and 13 healthy control (HC) subjects as a training dataset and three datasets retrieved from the Gene Expression Omnibus (GEO) database as validation datasets. Using the limma package in R, 1435 differentially expressed genes (DEGs) were found in the training dataset. Of these genes, 29 genes were found to occur in both DEGs and 307 aging-related genes. By using machine learning algorithms (LASSO, RF, SVM, and RR), Venn diagrams, and LASSO regression, four of these genes were determined to be potential PD biomarkers; these were further validated in external validation datasets and by qRT-PCR in the peripheral blood mononuclear cells (PBMCs) of 10 PD patients and 10 HC subjects. Based on the biomarkers, a diagnostic model was developed that had reliable predictive ability for PD. Two of the identified biomarkers demonstrated a meaningful correlation with immune cell infiltration status in the PD patients and HC subjects. In conclusion, four aging-related genes were identified as robust diagnostic biomarkers and may serve as potential targets for PD therapeutics.

The influence of 4G/5G polymorphism in the plasminogen-activator-inhibitor-1 promoter on COVID-19 severity and endothelial dysfunction.

Introduction: Plasminogen activator inhibitor-1 (PAI-1) is linked to thrombosis and endothelial dysfunction in severe COVID-19. The +43 G>A PAI-1 and 4G/5G promoter polymorphism can influence PAI-1 expression. The 4G5G PAI-1 promoter gene polymorphism constitutes the 4G4G, 4G5G, and 5G5G genotypes. However, the impact of PAI-1 polymorphisms on disease severity or endothelial dysfunction remains unclear. Methods: Clinical data, sera, and peripheral blood mononuclear cells (PBMCs) of COVID-19 patients were studied. Results: Comorbidities and clinical biomarkers did not correlate with genotypes in either polymorphism. However, differences between fibrinolytic factors and interleukin-1ß (IL-1ß) were identified in genotypes of the 4G/5G but not the 43 G>A PAI polymorphism. Patients with the 4G4G genotype of the 4G/5G polymorphism showed high circulating PAI-1, mainly complexed with plasminogen activators, and low IL-1ß and plasmin levels, indicating suppressed fibrinolysis. NFκB was upregulated in PBMCs of COVID-19 patients with the 4G4G genotype. Discussion: Mechanistically, IL-1ß enhanced PAI-1 expression in 4G4G endothelial cells, preventing the generation of plasmin and cleavage products like angiostatin, soluble uPAR, and VCAM1. We identified inflammation-induced endothelial dysfunction coupled with fibrinolytic system overactivation as a risk factor for patients with the 5G5G genotype.

HIV-1 controllers exhibit an enhanced antiretroviral innate state characterised by overexpression of p21 and MCPIP1 and silencing of ERVK-6 RNA expression.

BACKGROUND: Human immunodeficiency virus (HIV)-1 infection can activate the expression of human endogenous retroviruses (HERVs), particularly HERV-K (HML-2). HIV controllers (HICs) are rare people living with HIV (PLWHs) who naturally control HIV-1 replication and overexpress some cellular restriction factors that negatively regulate the LTR-driven transcription of HIV-1 proviruses. OBJECTIVES: To understand the ability of HICs to control the expression of endogenous retroviruses. METHODS: We measured endogenous retrovirus type K6 (ERVK-6) RNA expression in peripheral blood mononuclear cells (PBMCs) of HICs (n = 23), antiretroviral (ART)-suppressed subjects (n = 8), and HIV-1-negative (NEG) individuals (n = 10) and correlated the transcript expression of ERVK-6 with multiple HIV-1 cellular restriction factors. FINDINGS: Our study revealed that ERVK-6 RNA expression in PBMCs from HICs was significantly downregulated compared with that in both the ART and NEG control groups. Moreover, we detected that ERVK-6 RNA levels in PBMCs across all groups were negatively correlated with the expression levels of p21 and MCPIP1, two cellular restriction factors that limit the activation of macrophages and T cells by downregulating the activity of NF-kB. MAIN CONCLUSIONS: These findings support the hypothesis that HICs activate innate antiviral mechanisms that may simultaneously downregulate the transcription of both exogenous (HIV-1) and endogenous (ERVK-6) retroviruses. Future studies with larger cohorts should be performed to confirm this hypothesis and to explore the role of p21 and MCPIP1 in regulating HERV-K expression in physiological and pathological conditions.

Assessing the effects of ex vivo hormonal exposure on oxidative responses in equine leukocytes: A preliminary study.

Breed differences exist between horses and ponies in circulating concentrations of several hormones, notably ACTH and insulin. These hormones regulate stress and metabolic responses, but in other species, they also impact leukocyte oxidant responses. The effects of these hormones on equine leukocytes have not been evaluated to date. If equine leukocytes are similarly regulated, breed differences in increased plasma hormone concentrations or altered sensitivity to them at the leukocyte level could result in breed-related differences in oxidant responses or oxidative status. The objective of this study was therefore to determine the effects of ex vivo exposure to adrenocorticotropic hormone (ACTH), α-melanocyte stimulating hormone (α-MSH), insulin, or leptin on reactive oxygen species (ROS) production from leukocytes isolated from horses and ponies. We hypothesized that ACTH, α-MSH, insulin, and leptin would alter oxidant responses from equine leukocytes in a breed specific manner. Blood was collected from 10 apparently healthy Quarter horses and seven Welsh ponies for isolation of neutrophils and peripheral blood mononuclear cells (PBMCs) via density gradient centrifugation. Cells were incubated with media (negative control), microbial antigens (positive control), or ACTH, α-MSH, leptin, or insulin for two hours. Induced ROS production was quantified with a previously validated fluorometric assay. Data was compared within groups by comparing a stimulant within a group (horses or ponies) to baseline, between groups by comparing horse response to pony response, and among stimulants using one- and two-way, repeated measures ANOVA (P<0.05). There was no significant effect of breed on basal, microbial-induced, or hormone-induced ROS production from neutrophils (P=0.465) or PBMCs (P=0.749), but in neutrophils, a significant interaction between breed and stimulant was present (P=0.037). ROS production from PBMCs from horses after hormone exposure did not differ from cells exposed to media only (P=0.1520-0.8180). Similarly, neither leptin nor insulin exposure significantly induced ROS production from PBMCs from ponies (P= 0.2645 and 0.4678 respectively), but exposure to ACTH or α-MSH induced a significant increase in ROS production (P=0.0441 and 0.0440 respectively) compared to unstimulated cells. Hormones that vary in availability among breeds may induce ex vivo pro-oxidant responses in equine leukocytes, but specific effects are breed-, leukocyte type-, and hormone-dependent. Breed differences in hormonally induced leukocyte ROS production may warrant further investigation in the context of circulating oxidative stress and how this might relate to future disease risk.

The Circulating CDC42 Expression in Sepsis: Relation to Disease Susceptibility, Inflammation, Multiple Organ Dysfunctions and Mortality Risk.

OBJECTIVE: Cell division cycle 42 (CDC42) modulates inflammation and multiple organ dysfunction by regulating T-cell differentiation and macrophage polarization. This research intended to explore the association of blood CDC42 expression with septic risk, multi-organ dysfunctions, and mortality. METHODS: 145 sepsis patients and 50 health controls were recruited, then CDC42 expression in peripheral blood mononuclear cell (PBMC) from them was measured by RT-qPCR. RESULTS: CDC42 was decreased in sepsis patients versus health controls (P<0.001); meanwhile, the receiver operating characteristic (ROC) curve showed that CDC42 had a certain value to predict sepsis risk with an area under the curve (AUC) (95% confidence interval (CI): 0.797 (0.725-0.869). Furthermore, CDC42 was negatively correlated with C-reactive protein (P<0.001), tumor necrosis factor-alpha (P<0.001) and interleukin-17A (P<0.001) but less with interleukin-6 (P=0.056). Moreover, CDC42 was negatively related to the SOFA score (P<0.001) and its several subscales (respiratory system, liver, cardiovascular, and renal system) (P<0.05). Furthermore, CDC42 was lower in septic deaths versus survivors (P<0.001); meanwhile, the ROC curve exhibited a certain ability of CDC42 in estimating 28-day mortality with an AUC (95%CI) of 0.766 (0.676-0.855). CONCLUSION: Circulating CDC42 exhibits potency to be a prognostic biomarker reflecting multi-organ dysfunctions and higher mortality risk in sepsis.

Comparison of Cytokine Responses (IL-21, IL-12, IL-13) in Chlamydia pneumoniae-Stimulated PBMC in Asthma and Non-Asthma.

OBJECTIVE: Chlamydia pneumoniae (C. pneumoniae) is a gram-negative intracellular bacterium that causes respiratory infections in humans. C. pneumoniae is responsible for cell activation and production of cytokines that may contribute to inflammatory responses in asthma. Cell-mediated immune responses are important for protective immunity; however, these responses may be impaired in asthma. In this study, we examined cytokine responses (IL-21, IL-12, IL-13) responsible for T helper (Th)1 versus Th2 responses in C. pneumoniae-stimulated PBMC from subjects with or without asthma. These cytokines could be potential biomarkers in the evaluation of past C. pneumoniae infection. METHODS: Peripheral blood mononuclear cells (PBMC) (1×106/mL) from stable adult asthmatic (N=6) and non-asthmatic subjects (N=6) were infected +/- C. pneumoniae TW-183 at a multiplicity of infection (MOI)=0.1, using dose responses (1:10, 1:100), and cultured 48 hrs. Cytokine responses (Interleukin (IL)-21, IL-12, IL-13) were measured in supernatants (ELISA). RESULTS: Cytokine responses (mean differences: unstimulated-stimulated cells) were significant for IL-12 (1:10, 1:100) (P=0.0005, 0.0005) but not for IL-21 or IL-13 (Wilcoxon signed-rank test). Cytokine levels were higher in asthmatic subjects for IL-13 (mean differences: non-asthma-asthma) (unstimulated, 1:10, 1:100) (-210±167, -140±113, -89±59, respectively) (P=0.05, 0.05, 0.05, respectively) compared with non-asthma. However, IL-21 and IL-12 responses were similar in both groups. When subjects were stratified according to C. pneumoniae IgG antibody status, no significant differences in cytokine responses were observed. CONCLUSION: Differential cytokine patterns in subjects with or without asthma may suggest a mechanism for the development of persistent infection with C. pneumoniae.

Shared and unique transcriptomic signature genes and pathways among biopsy, peripheral blood mononuclear cells and bronchoalveolar lavage samples in IPF patients revealed using comparative meta-transcriptome analysis.

Introduction: Idiopathic pulmonary fibrosis (IPF) affects the tissue surrounding the alveoli and occurs when the lung tissue becomes thick and stiff for unknown reasons. Clinical findings are fairly well settled, but the molecular mechanisms of IPF are still poorly known. Materials and Methods: To further our understanding, we collected publicly available transcriptome dataset from IPF cohorts, grouped them according to sampling method [bronchoalveolar lavage (BAL), biopsy, blood], and performed comparative meta-transcriptome study to (I) unravel key pathways (II), set out differences in discovered genes, pathways, and functional annotation with respect to the sampling method, and (III) find biomarkers for early diagnosis. Result: The resulting lists are also compared with DisGeNet reported genes, earlier work, and Kyoto encyclopedia of genes and genomes (KEGG) pathways. Several pathways are shared among BAL and biopsy samples while blood samples point to alternative pathways, indicating the noise in information obtained from these samples. Conclusions: Common to all sampling methods, interleukin-10 pathway and extracellular signaling pathways are pointed as further targets.

Improving Reliability of Immunological Assays by Defining Minimal Criteria for Cell Fitness.

Human PBMC-based assays are often used as biomarkers for the diagnosis and prognosis of disease, as well as for the prediction and tracking of response to biological therapeutics. However, the development and use of PBMC-based biomarker assays is often limited by poor reproducibility. Complex immunological assays can be further complicated by variation in cell handling before analysis, especially when using cryopreserved cells. Variation in postthaw viability is further increased if PBMC isolation and cryopreservation are done more than a few hours after collection. There is currently a lack of evidence-based standards for the minimal PBMC viability or "fitness" required to ensure the integrity and reproducibility of immune cell-based assays. In this study, we use an "induced fail" approach to examine the effect of thawed human PBMC fitness on four flow cytometry-based assays. We found that cell permeability-based viability stains at the time of thawing did not accurately quantify cell fitness, whereas a combined measurement of metabolic activity and early apoptosis markers did. Investigation of the impact of different types and levels of damage on PBMC-based assays revealed that only when cells were >60-70% live and apoptosis negative did biomarker values cease to be determined by cell fitness rather than the inherent biology of the cells. These data show that, to reproducibly measure immunological biomarkers using cryopreserved PBMCs, minimal acceptable standards for cell fitness should be incorporated into the assay protocol.

Preservation of functionality, immunophenotype, and recovery of HIV RNA from PBMCs cryopreserved for more than 20 years.

Background: Many research laboratories have long-term repositories of cryopreserved peripheral blood mononuclear cells (PBMC), which are costly to maintain but are of uncertain utility for immunological studies after decades in storage. This study investigated preservation of cell surface phenotypes and in-vitro functional capacity of PBMC from viraemic HIV+ patients and healthy seronegative control subjects, after more than 20 years of cryopreservation. Methods: PBMC were assessed by 18-colour flow cytometry for major lymphocyte subsets within T, B, NK, and dendritic cells and monocytes. Markers of T-cell differentiation and activation were compared with original immunophenotyping performed in 1995/1996 on fresh blood at the time of collection. Functionality of PBMC was assessed by culture with influenza antigen or polyclonal T-cell activation, to measure upregulation of activation-induced CD25 and CD134 (OX40) on CD4 T cells and cytokine production at day 2, and proliferative CD25+ CD4 blasts at day 7. RNA was extracted from cultures containing proliferating CD4+ blast cells, and intracellular HIV RNA was measured using short amplicons for both the Double R and pol region pi code assays, whereas long 4-kbp amplicons were sequenced. Results: All major lymphocyte and T-cell subpopulations were conserved after long-term cryostorage, except for decreased proportions of activated CD38+HLA-DR+ CD4 and CD8 T cells in PBMC from HIV+ patients. Otherwise, differences in T-cell subpopulations between recent and long-term cryopreserved PBMC primarily reflected donor age-associated or HIV infection-associated effects on phenotypes. Proportions of naïve, memory, and effector subsets of T cells from thawed PBMC correlated with results from the original flow cytometric analysis of respective fresh blood samples. Antigen-specific and polyclonal T-cell responses were readily detected in cryopreserved PBMC from HIV+ patients and healthy control donors. Intracellular HIV RNA quantitation by pi code assay correlated with original plasma viral RNA load results. Full-length intracellular and supernatant-derived amplicons were generated from 5/12 donors, and sequences were ≥80% wild-type, consistent with replication competence. Conclusions: This unique study provides strong rationale and validity for using well-maintained biorepositories to support immunovirological research even decades after collection.

Evaluation of Sézary cell marker expression and cell death behaviour upon in vitro treatment by flow cytometry in Sézary syndrome patients.

The diagnosis of Sézary syndrome (SS) relies on the identification of blood Sézary cells (SC) by different markers via flow cytometry. Treatment of SS is challenging since its pathogenesis is characterized by cell death resistance rather than hyperproliferation. In this study, we establish an integrated approach that considers both the expression of SC markers and sensitivity to cell death both spontaneously and upon in vitro treatment. Peripheral blood mononuclear cells were isolated from 20 SS patients and analysed for the SC markers CD7 and CD26 loss as well as CD158k and PD1 gain. The cells were then treated with different established and experimental therapies in vitro and cell death was measured. Spontaneous and therapeutically induced cell death were measured and correlated to cellular marker profiles. In the marker-positive cells, spontaneous cell death sensitivity was reduced. Different treatments in vitro managed to specifically induce cell death in the putative CTCL cell populations. Interestingly, a repeated analysis after 3 months of treatment revealed the CTCL cell death sensitivity to be restored by therapy. We propose this novel integrated approach comprising the evaluation of SC marker expression and analysis of cell death sensitivity upon treatment that can also enable a better therapy stratification.

SEMA6B induces macrophage-mediated inflammation and hepatocyte apoptosis in hepatitis B virus-related acute-on-chronic liver failure.

Rationale: Patients with hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) have a high short-term mortality rate. Semaphorin-6B (SEMA6B) plays a crucial role in the pathogenesis of HBV-ACLF, but its molecular basis remains unclear. This study aimed to elucidate the mechanisms of SEMA6B in HBV-ACLF progression. Methods: A total of 321 subjects with HBV-ACLF, liver cirrhosis (LC), chronic hepatitis B (CHB), and normal controls (NC) from a prospective multicenter cohort were studied. 84 subjects (HBV-ACLF, n = 50; LC, n = 10; CHB, n = 10; NC, n = 14) among them underwent mRNA sequencing using peripheral blood mononuclear cells (PBMCs) to clarify the mechanisms of SEMA6B in HBV-ACLF. These mechanisms were validated through in vitro studies with hepatocytes and macrophages, as well as in vivo using SEMA6B knockout mice and mice treated with synthetic SEMA6B siRNA. Results: Transcriptome analysis of PBMCs showed that SEMA6B was among the most differentially expressed genes when comparing patients with HBV-ACLF to those with LC, CHB, or NC. ROC analysis demonstrated the reliable diagnostic value of SEMA6B for HBV-ACLF in both the sequencing cohort and an external validation cohort (AUROC = 0.9788 and 0.9026, respectively). SEMA6B levels were significantly higher in the HBV-ACLF patients, especially in non-survivors, with high expression mainly observed in macrophages and hepatocytes in liver tissue. Genes significantly associated with highly expressed SEMA6B were enriched in inflammation and apoptosis pathways in HBV-ACLF non-survivors. Overexpression of SEMA6B in macrophages activated systemic inflammatory responses, while its overexpression in hepatocytes inhibited proliferation through G0/G1 cell cycle arrest and induced apoptosis. Knocking out SEMA6B rescued mice with liver failure by improving liver functions, reducing inflammatory responses, and decreasing hepatocyte apoptosis. Transcriptome analysis of liver tissue showed that SEMA6B knockout significantly ameliorated the liver failure signature, significantly downregulating inflammation-related pathways. Importantly, therapeutic delivery of synthetic SEMA6B siRNA also improved liver function, and reduced both inflammation and hepatocyte apoptosis in mice with liver failure. Conclusion: SEMA6B, a potential diagnostic biomarker for HBV-ACLF, exacerbates liver failure through macrophage-mediated systemic inflammation and hepatocyte apoptosis. These findings highlight SEMA6B as a promising early treatment target for HBV-ACLF patients.

Vitamin D treatment distinctly modulates cytokine production by peripheral blood mononuclear cells among patients with chronic cardiac and indeterminate clinical forms of Chagas disease.

INTRODUCTION: Chagas disease is caused by the protozoan Trypanosoma cruzi and is clinically divided into acute and chronic phases. Chronic Chagas cardiomyopathy is the most studied manifestation of the disease. Vitamin D deficiency has been suggested as a risk factor for cardiovascular disease. No studies demonstrate the action of this hormone in the cells of patients with chronic Chagas heart disease. OBJECTIVE: To evaluate the in vitro immunomodulatory effect of vitamin D on peripheral blood mononuclear cells of patients with the different chronic clinical forms of Chagas disease. Evaluating vitamin D's in vitro effect on blood cells by producing cytokines. METHODS: Thirteen patients of the undetermined form (IND), 13 of the mild cardiac form (CARD1) and 14 of the severe cardiac form (CARD2) of Chagas disease, and 12 with idiopathic heart disease (CARDid) were included. The cells obtained from peripheral blood were treated in vitro with vitamin D (1 × 10-7 M) for 24 h and cytokines were dosed in the culture supernatant. RESULTS: Although it was not possible to demonstrate statistically significant differences between the groups studied, our data showed that the cells treated with vitamin D modify (p < .05) the production of interferon-γ (IFN-γ) (decrease in IND), tumor necrosis factor-α (TNF-α) (decreased in CARD1 and CARDid), interleukin (IL)-6 (increased in all groups), and IL-10 (decreased in CARD1, CARD2, and CARDid) when compared to untreated cells. CONCLUSION: In vitro treatment with vitamin D distinctly modulated the production of cytokines by mononuclear cells of peripheral blood among patients with chronic and indeterminate cardiac clinical forms of Chagas disease.

Manual Therapy Improves Fibromyalgia Symptoms by Downregulating SIK1.

Fibromyalgia (FM), classified by ICD-11 with code MG30.0, is a chronic debilitating disease characterized by widespread pain, fatigue, cognitive impairment, sleep, and intestinal alterations, among others. FM affects a large proportion of the worldwide population, with increased prevalence among women. The lack of understanding of its etiology and pathophysiology hampers the development of effective treatments. Our group had developed a manual therapy (MT) pressure-controlled custom manual protocol on FM showing hyperalgesia/allodynia, fatigue, and patient's quality of life benefits in a cohort of 38 FM cases (NCT04174300). With the aim of understanding the therapeutic molecular mechanisms triggered by MT, this study interrogated Peripheral Blood Mononuclear Cell (PBMC) transcriptomes from FM participants in this clinical trial using whole RNA sequencing (RNAseq) and reverse transcription followed by quantitative Polymerase Chain Reaction (RT-qPCR) technologies. The results show that the salt-induced kinase SIK1 gene was consistently downregulated by MT in FM, correlating with improvement of patient symptoms. In addition, this study compared the findings in a non-FM control cohort subjected to the same MT protocol, evidencing that those changes in SIK1 expression with MT only occurred in individuals with FM. This positions SIK1 as a potential biomarker to monitor response to MT and as a therapeutic target of FM, which will be further explored by continuation studies.

Internalization of extracellular vesicles of cancer patients by peripheral blood mononuclear cells during polychemotherapy: connection with neurotoxicity.

Extracellular vesicles (EVs), exhibiting their functional activity after internalization by recipient cells, are involved in the pathogenesis of drug-induced polyneuropathy (DIPN), a common complication of antitumor therapy. In this work, the internalization of EVs obtained from colorectal cancer patients undergoing polychemotherapy and its relationship with neurotoxicity were assessed using a model system of mononuclear leukocytes. Circulating EVs were isolated from 8 colorectal cancer patients who received antitumor therapy according to the FOLFOX or XELOX regimens before the start of chemotherapy (point 1) and after 3-4 courses (point 2). Mononuclear leukocytes of a healthy donor served as a cellular model system for EV internalization in vitro. EV internalization was assessed using fluorescence microscopy. It was shown that internalization of EVs obtained from colorectal cancer patients with high neurotoxicity was higher than in the group with low neurotoxicity. The ability of CD11b-positive (CD11b⁺) and CD11b-negative (CD11b⁻) mononuclear leukocytes of a healthy donor to internalize EVs obtained from patients before and after chemotherapy did not reveal significant differences. A direct relationship was found between the relative number of CD11b⁻ cells with internalized EVs and the integral index of neurotoxicity according to the NRS scale at the peak of its manifestation (point 2) (r=0.675, p.

Formononetin inhibits IgE by huPlasma/PBMCs and mast cells/basophil activation via JAK/STAT/PI3-Akt pathways.

Rationale: Food allergy is a prevalent disease in the U.S., affecting nearly 30 million people. The primary management strategy for this condition is food avoidance, as limited treatment options are available. The elevation of pathologic IgE and over-reactive mast cells/basophils is a central factor in food allergy anaphylaxis. This study aims to comprehensively evaluate the potential therapeutic mechanisms of a small molecule compound called formononetin in regulating IgE and mast cell activation. Methods: In this study, we determined the inhibitory effect of formononetin on the production of human IgE from peripheral blood mononuclear cells of food-allergic patients using ELISA. We also measured formononetin's effect on preventing mast cell degranulation in RBL-2H3 and KU812 cells using beta-hexosaminidase assay. To identify potential targets of formononetin in IgE-mediated diseases, mast cell disorders, and food allergies, we utilized computational modeling to analyze mechanistic targets of formononetin from various databases, including SEA, Swiss Target Prediction, PubChem, Gene Cards, and Mala Cards. We generated a KEGG pathway, Gene Ontology, and Compound Target Pathway Disease Network using these targets. Finally, we used qRT-PCR to measure the gene expression of selected targets in KU812 and U266 cell lines. Results: Formononetin significantly decreased IgE production in IgE-producing human myeloma cells and PBMCs from food-allergic patients in a dose-dependent manner without cytotoxicity. Formononetin decreased beta-hexosaminidase release in RBL-2H3 cells and KU812 cells. Formononetin regulates 25 targets in food allergy, 51 in IgE diseases, and 19 in mast cell diseases. KEGG pathway and gene ontology analysis of targets showed that formononetin regulated disease pathways, primary immunodeficiency, Epstein-Barr Virus, and pathways in cancer. The biological processes regulated by formononetin include B cell proliferation, differentiation, immune response, and activation processes. Compound target pathway disease network identified NFKB1, NFKBIA, STAT1, STAT3, CCND1, TP53, TYK2, and CASP8 as the top targets regulated at a high degree by formononetin. TP53, STAT3, PTPRC, IL2, and CD19 were identified as the proteins mostly targeted by formononetin. qPCR validated genes of Formononetin molecular targets of IgE regulation in U266 cells and KU812 cells. In U266 cells, formononetin was found to significantly increase the gene expression of NFKBIA, TP53, and BCL-2 while decreasing the gene expression of BTK TYK, CASP8, STAT3, CCND1, STAT1, NFKB1, IL7R. In basophils KU812 cells, formononetin significantly increased the gene expression of NFKBIA, TP53, and BCL-2 while decreasing the gene expression of BTK, TYK, CASP8, STAT3, CCND1, STAT1, NFKB1, IL7R. Conclusion: These findings comprehensively present formononetin's mechanisms in regulating IgE production in plasma cells and degranulation in mast cells.

RAB13 regulates macrophage polarization in sepsis.

To select the core target (RAB13) in sepsis patients' peripheral blood and investigate its molecular functions and possible mechanisms. The peripheral blood of sepsis patients (n = 21) and healthy individuals (n = 9) within 24 h after admission were collected for RNA-seq, and differential gene screening was performed by iDEP online analysis software (P < 0.01; log2FC ≥ 2) and enrichment analysis, the potential core target RAB13 was screened out. The association between RAB13 expression and sepsis severity was explored using multiple datasets in the GEO database, and survival analysis was conducted. Subsequently, peripheral blood mononuclear cells (PBMCs) from sepsis and control groups were isolated, and 10 × single-cell sequencing was used to identify the main RAB13-expressing cell types. Finally, LPS was used to stimulate THP1 cells to construct a sepsis model to explore the function and possible mechanism of RAB13. We found that RAB13 was a potential core target, and RAB13 expression level was positively associated with sepsis severity and negatively correlated with survival based on multiple public datasets. A single-cell sequencing indicated that RAB13 is predominantly localized in monocytes. Cell experiments validated that RAB13 is highly expressed in sepsis, and the knockdown of RAB13 promotes the polarization of macrophages towards the M2 phenotype. This mechanism may be associated with the ECM-receptor interaction signaling pathway. The upregulation of RAB13 in sepsis patients promotes the polarization of M2-like macrophages and correlates positively with the severity of sepsis.