Peripheral Blood Mononuclear Cells Research Articles

The clinical significance of signal regulatory protein alpha expression in the immune environment of gastric cancer.

Signal regulatory protein alpha (SIRPα) inhibits phagocytosis by macrophages by interacting with CD47. Despite its known role in various cancers, the clinical significance of SIRPα in gastric cancer (GC) remains unclear. This study aimed to elucidate the clinical implications of SIRPα in GC, exploring its relevance to immunotherapy efficacy and the tumor microenvironment. Two cohorts were studied: a gastrectomy cohort (137 patients) and an immune checkpoint inhibitor (ICI)-treated cohort (19 patients with unresectable advanced GC who received nivolumab). Immunohistochemistry was used to assess SIRPα, CD80, CD163, CD8, and PD-L1 expressions. Kaplan-Meier curves and Cox models were used to analyze the clinical outcomes. In vitro experiments used peripheral blood mononuclear cells and THP-1 macrophage cell lines to examine SIRPα responses to interferon-γ (IFN-γ). In the gastrectomy cohort, high SIRPα expression correlated with advanced tumor invasion, distant metastasis, and poor recurrence-free and overall survival. SIRPα expression was also significantly associated with macrophage and CD8 + T cells infiltration and PD-L1 expression. In the ICI-treated cohort, high SIRPα expression was associated with better overall survival after nivolumab induced. Moreover, in vitro IFN-γ stimulation upregulated SIRPα expression on monocytes in peripheral blood mononuclear cells and THP-1 cells, suggesting high SIRPα expression may reflect an active immune microenvironment. SIRPα expression is not only a poor prognostic factor for GC, possibly through inhibition of the CD47-SIRP⍺ pathway, but may also be involved in the efficacy of ICI therapy in GC.

Autophagy in Mononuclear Cells from Patients with Autoimmune Thyroiditis

Autoimmune thyroiditis (AIT) is a chronic thyroid disorder wherein overstimulated CD4+T lymphocytes activate CD8+ cytotoxic T lymphocytes, thereby inducing Fas-mediated apoptosis of thyrocytes and reducing the hormonal activity of the thyroid gland. Recent studies suggest that autophagy, a process vital for maintaining cellular homeostasis through the degradation of damaged proteins and organelles in autolysosomes, is involved in the pathogenesis of autoimmune diseases. This article examines autophagy in peripheral blood mononuclear cells and the expression of key autophagy proteins (Vps34, p62, and LC3) in patients with AIT. The number of autophagosomes in the cells was tracked and assessed using flow cytometry. The expression of the protein markers was measured by western blotting. It was demonstrated that the levels of Vps34, LC3-II, and p62 increased significantly in the lymphocytes of all patients with AIT. The high level of the autophagosome protein LC3-II correlated with that of the ubiquitin-binding protein p62, which may indicate a disruption in the late stage of autophagy, i.e., in the fusion of autophagosomes with lysosomes. Impaired autophagy promotes excessive accumulation of autophagosomes in the cytoplasm, which, in turn, triggers apoptotic or necrotic cell death. Therefore, understanding the mechanisms of impaired autophagy in lymphocytes could be a promising avenue for slowing and limiting the damage associated with the onset and development of AIT.

The methylation signature of hepatocellular carcinoma trajectory based on pseudotime and chronological time for predicting precancerous patients.

Early screening of hepatocellular carcinoma (HCC) is strongly recommended for hepatitis B virus (HBV)-infected patients. We aimed to develop and validate a predictive nomogram based on HCC occurrence trajectory for screening precancerous patients with HCC. Peripheral blood mononuclear cells (PBMC) samples from 22 patients with HCC with their precancerous stage (n = 55) and 18 healthy controls were measured using HumanMethylation EPIC BeadChip assay. HCC trajectory was assessed by pseudotime based on TimeAx algorithm and chronological time. The 43 candidate CpG sites were selected from the methylation signature and measured using multiplex bisulfite sequencing in a retrospective cohort of HBV-infected patients (n = 604). A 5-CpG-classifier was built using the LASSO Cox regression model, based on the association between the methylation level of every CpG and the duration from enrollment to HCC occurrence of individual patient. We validated the risk stratification and predictive accuracy of this classifier in both the primary cohort (n = 300) and independent validation cohort (n = 304). Pseudotime and chronological time of HCC trajectory analysis revealed that the PD-1/PD-L1 pathway underwent changes in the precancerous stage. Based on the trajectory of methylation signature, we built a 5-CpG-classifier which remained powerful and independent predictive efficiency after stratified analysis by clinicopathological risk factors in both primary cohort and independent validation cohort. A predicting nomogram including the 5-CpG-classifier was constructed after multivariate analysis. One-year cumulative hazard of HCC in low- and high-risk groups of HBV-infected patients was 3.0% (0.1%-5.8%) and 17.90% (11.00%-24.3%) (P < .0001) in primary cohort, 4.5% (1.20%-7.80%) and 27.3 (18.90-34.90) (P < .0001) in the independent validation cohort. One-year before HCC was a critical period of transitional time when parts of the methylation profile underwent shifting toward HCC like. The nomogram could identify precancerous stage patients with HCC who should be screened for early diagnosis and intervention.

A small-scale preliminary study utilizing mass cytometry to distinguish two forms of arthritis.

Spondyloarthritis (SpA) and rheumatoid arthritis (RA) are hallmarked by immune cell infiltration in synovial joints. Although, in general, different sites are affected, misclassification or delayed diagnosis due to overlapping clinical manifestations is not uncommon. Here, we investigated the diagnostic potential of mass cytometry (MC) in early peripheral SpA (pSpA) and RA patients in a small pilot study. Peripheral blood and synovial fluid mononuclear cells (PBMC and SFMC) of 4 pSpA, 7 RA, and 1 undifferentiated arthritis (UA) patient(s) were evaluated using a 37-marker MC panel. Data were analyzed through Visualyte services, including dimension reduction, clustering, and Cytofast workflow. PBMC data indicated naive CD4 T cell, B cell, and monocyte subsets to be differentially present in RA as compared with SpA. CD4 + Tem cell and NK cell subsets appeared more prominently present in pSpA SFMC. Merged PBMC and SFMC data showed overlapping immune profiles of an UA patient with pSpA patients. These results were in accordance with the formal clinical pSpA diagnosis the UA patient received after this study. Utilizing MC, several differences in immune cell composition in both SFMC and PBMC between RA and pSpA patients were observed. Combining PBMC and SFMC data in an unsupervised analysis resulted in the correct classification of the UA patient as pSpA patient prior to formal clinical pSpA diagnosis. This pilot study provides an example of how deep phenotyping with MC aids in differentiating arthritis patients and offers a rationale to further explore these findings. •Due to overlapping symptoms and the absence of disease-specific biomarkers, the clinical diagnosis of peripheral spondyloarthritis (pSpA) and rheumatoid arthritis (RA) can be difficult. •Visualizing immune cell profiles of peripheral blood (PB) and synovial fluid (SF) by mass cytometry (MC) suggests differences in immune cell composition between pSpA and RA patients. •Based on immune profiles of combined PB and SF data, we could correctly predict the formal clinical pSpA diagnosis of an undifferentiated arthritis (UA) patient received later. •This pilot study gives an example of how MC might contribute to faster clinical diagnosis of pSpA patients in the absence of biomarkers.

Two Disaccharide-Bearing Polyethers, K-41B and K-41Bm, Potently Inhibit HIV-1 via Mechanisms Different from That of Their Precursor Polyether, K-41A

The screening of novel antiviral agents from marine microorganisms is an important strategy for new drug development. Our previous study found that polyether K-41A and its analog K-41Am, derived from a marine Streptomyces strain, exhibit anti-HIV activity by suppressing the activities of HIV-1 reverse transcriptase (RT) and its integrase (IN). Among the K-41A derivatives, two disaccharide-bearing polyethers—K-41B and K-41Bm—were found to have potent anti-HIV-1IIIB activity in vitro. This study aimed to clarify whether K-41B and K-41Bm have inhibitory effects on different HIV-1 strains or whether these two derivatives have mechanisms of action different from that of their precursor, K-41A. An anti-HIV-1 assay indicated that K-41B and K-41Bm have potent anti-HIV-1BaL activity, with low 50% inhibitory concentrations (IC50s) (0.076 and 0.208 μM, respectively) and high selective indexes (SIs) (58.829 and 31.938, respectively) in the peripheral blood mononuclear cell (PBMC)-HIV-1BaL system. The time-of-addition (TOA) assay indicated that K-41B and K-41Bm may exert antiviral effects by activating multiple stages of HIV-1 replication. A cell protection assay indicated that the pretreatment of cells with K-41B or K-41Bm has almost no inhibitory effect on HIV-1 infection. A virus inactivation assay indicated that pretreatment of the virus with K-41B or K-41Bm inhibits HIV-1 infection by 60%. A cell–cell fusion assay showed that K-41B and K-41Bm blocked the cell fusion mediated by viral envelope proteins. The HIV-1 key enzyme experiment also indicated that both compounds have certain inhibitory effects on HIV-1 IN. Furthermore, molecular docking showed that K-41B and K-41Bm interact with several viral and host proteins, including HIV-1 IN, an envelope protein (gp120), a transmembrane protein (gp41), and cell surface receptors (CD4, CCR5, and CXCR4). Overall, in addition to having a similar anti-HIV-1 mechanism of inhibiting HIV-1 IN like the precursor polyether K-41A, the disaccharide-bearing polyether derivatives K-41B and K-41Bm may also inhibit viral entry. This suggests that they display anti-HIV-1 mechanisms that are different from those of their precursor polyethers.

Novel Immunological Markers of Intestinal Impairment Indicative of HIV-1 Status and/or Subclinical Atherosclerosis.

Antiretroviral therapy (ART) controls HIV-1 replication in people with HIV-1 (PWH), but immunological restauration at mucosal barrier surfaces is not achieved. This fuels microbial translocation, chronic immune activation, and increased comorbidities, including cardiovascular disease (CVD). Here, we sought to identify novel markers of mucosal barrier impairment in the blood to predict the HIV and/or CVD status. Flow cytometry was used to characterize CD326/EpCAM + intestinal epithelial cells (IEC); CD4 + T-cells; CD8 + and CD4 + intraepithelial lymphocytes (IELs); and subsets of CD4 + T-cells expressing Th17 (CCR6) and gut-homing (Itgβ7) markers. To this aim, we collected peripheral blood mononuclear cells (PBMCs) from 42 ART-treated PWH (HIV + ) and 40 uninfected participants (HIV - ) from the Canadian HIV and Aging Cohort Study (CHACS). Both groups were categorized based on the presence of coronary atherosclerotic plaques measured by CT scan angiography as total plaque volume (TPV, mm 3 ). Our findings associate the HIV-1 status with increased frequencies of circulating CD326 + IEC; CD326 + CD4 + T-cells with activated (CD69 + HLA-DR + ) and gut-homing (ItgαE + CCR6 + CCR9 + ) phenotypes, CCR6 + Itgβ7 - CD4 + T-cells; and decreased frequencies of CD8 + IELs. Logistic regression analyses confirmed the predictive capacity of the above cellular markers regarding HIV status. Spearman correlation revealed a positive correlation between TPV and CCR6 + Itgβ7 - and CCR6 + Itgβ7 + CD4 + T-cell frequencies.Together, these results highlighted significant immune dysregulation and persistent mucosal barrier alterations despite effective viral suppression by ART and linked the abundance of CCR6 + Itgβ7 + and CCR6 + Itgβ7 - CD4 + T-cells to increased atherosclerotic plaque burden. Thus, strategies targeting the gut-immune axis restoration may reduce CVD onset and improve long-term health outcomes in PWH.

Inborn Error of WAS Presenting with SARS-CoV-2-Related Multisystem Inflammatory Syndrome in Children.

Multisystem inflammatory syndrome in children (MIS-C) has been reported in patients with inborn errors of immunity (IEI), providing insights into disease pathogenesis. Here, we present the first case of MIS-C in a child affected by Wiskott-Aldrich syndrome (WAS) gene mutation, elucidating underlying predisposing factors and the involved inflammatory pathways. Genetic analysis revealed a frameshift truncating variant in the WAS gene, resulting in WAS protein expression between mild and severe forms, despite a clinical phenotype resembling X-linked thrombocytopenia (XLT). IL-1β secretion by LPS-stimulated peripheral blood mononuclear cells from patient during MIS-C was lower compared to healthy subjects but increased during follow-up. Conversely, the percentage of ASC (apoptosis-associated speck-like protein containing a CARD) specks in the patient's circulating monocytes during the acute phase was higher than in healthy subjects. The type I interferon (IFN) signature during MIS-C was normal, in contrast to the raised IFN signature measured far from the acute event. This case supports the association of IEI with MIS-C, potentially linked to delayed immune responses to SARS-CoV-2. The XLT phenotype underlies a subclinical immunodysregulation involving the NLRP3 inflammasome and the type-I IFN response.

Barcoding of viable peripheral blood mononuclear cells with selenium and tellurium isotopes for mass cytometry experiments.

Barcoding viable cells combined with pooled sample staining is an effective technique that eliminates batch effects from serial cell staining and facilitates uninterrupted data acquisition. We describe three novel and isotopically pure selenium-containing compounds (SeMals) that are useful cellular labeling tools. The maleimide-functionalized selenophenes (76SeMal, 77SeMal, and 78SeMal) covalently react with cellular sulfhydryl groups and uniquely label cell samples. The SeMal reagents label viable and paraformaldehyde-fixed peripheral blood mononuclear cells (PBMC), are well resolved by the mass cytometer, and have little spill into adjacent channels. They appear non-toxic to viable cells at working concentrations. We used SeMal reagents in combination with four isotopically pure tellurium maleimide reagents (124TeMal, 126TeMal, 128TeMal, and 130TeMal) to label 21 individual PBMC samples with unique combinations of selenium and tellurium isotopes (seven donors with three replicates using a 7 isotope pick 2 combinatorial schema). The individually barcoded samples were pooled, stained with an antibody cocktail as a pool, and acquired on the mass cytometer as a single suspension. The single-cell data were de-barcoded into separate sample-specific files after data acquisition, enabling an uninterrupted instrument run. Each donor sample retained its unique phenotypic profile with excellent replicate reproducibility. Unlike current live cell barcoding methods, this approach does not require antibodies to surface markers, allowing for the labeling of all cells regardless of surface antigen expression. Additionally, since selenium and tellurium isotopes are not currently utilized in CyTOF antibody panels, this method expands barcoding options and frees up commonly used isotopes for more detailed cell profiling.

Dimethyl fumarate ameliorates chronic graft-versus-host disease by inhibiting Tfh differentiation via Nrf2.

Chronic graft-versus-host disease (cGVHD), characterized by chronic tissue inflammation and fibrosis involving multiple organs, remains a major complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Dimethyl fumarate (DMF) is an anti-inflammatory drug approved for the treatment of multiple sclerosis and psoriasis. We previously reported that DMF effectively inhibits acute GVHD (aGVHD) while preserving the graft-versus-leukemia effect. However, the role of DMF in cGVHD progression remains unknown. Here, we found that DMF administration significantly suppresses follicular helper T cell (Tfh) differentiation, and germinal center formation and alleviates disease severity in different murine cGVHD models. Mechanistically, DMF treatment downregulates IL-21 transcription by activation of Nrf2, thus orchestrating Tfh-related gene programs both in mice and humans. The inhibitory role of DMF on Tfh cell differentiation was diminished in Nrf2 deficient T cells. Importantly, the therapeutic potential of DMF in clinical cGVHD has been validated in human data whereby DMF effectively reduces IL-21 production and Tfh cell generation in peripheral blood mononuclear cells from active cGVHD patients and further attenuates xenograft GVHD. Collectively, our findings reveal that DMF potently inhibits cGVHD development by repressing Tfh cell differentiation via Nrf2, paving the way for the treatment of cGVHD in the clinic.

Diabetic Glycation of Human Serum Albumin Affects Its Immunogenicity

Advanced glycation end-products (AGEs) are products of a non-enzymatic reaction between amino acids and reducing sugars. Glycated human serum albumin (HSA) increases in diabetics as a consequence of elevated blood glucose levels and glycating metabolites like methylglyoxal (MGO). The impact of different types of glycation on the immunomodulatory properties of HSA is poorly understood and is studied here. HSA was glycated with D-glucose, MGO, or glyoxylic acid (CML). Glycation-related biochemical changes were characterized using various biochemical methods. The binding of differentially glycated HSA to AGE receptors was determined with inhibition ELISAs, and the impact on inflammatory markers in macrophage cell line THP-1 and adherent monocytes isolated from human peripheral blood mononuclear cells (PBMCs) was studied. All glycation methods led to unique AGE profiles and had a distinct impact on protein structure. Glycation resulted in increased binding of HSA to the AGE receptors, with MGO modification showing the highest binding, followed by glucose and, lastly, CML. Additionally, modification of HSA with MGO led to the increased expression of pro-inflammatory markers in THP-1 macrophages and enhanced phosphorylation of NF-κB p65. The same pattern, although less prominent, was observed for HSA glycated with glucose and CML, respectively. An increase in pro-inflammatory markers was also observed in PBMC-derived monocytes exposed to all glycated forms of HSA, although HSA–CML led to a significantly higher inflammatory response. In conclusion, the type of HSA glycation impacts immune functional readouts with potential relevance for diabetes.

Single-cell analysis uncovers liver susceptibility to pancreatic cancer metastasis via myeloid cell characterization

The liver is the predominant metastatic site for diverse cancers, including pancreatic and colorectal cancers (CRC), etc. The high incidence of hepatic metastasis of pancreatic cancer is an important reason for its refractory and high mortality. Therefore, it is important to understand how metastatic pancreatic cancer affects the hepatic tumor immune microenvironment (TME) in patients. Here, we characterized the TME of liver metastases unique to pancreatic cancer by comparing them with CRC liver metastases. We integrated two single-cell RNA-seq (scRNA-seq) datasets including tumor samples of pancreatic cancer liver metastasis (P-LM), colorectal cancer liver metastasis (C-LM), primary pancreatic cancer (PP), primary colorectal cancer (PC), as well as samples of peripheral blood mono-nuclear cells (PBMC), adjacent normal pancreatic tissues (NPT), to better characterize the heterogeneities of the microenvironment of two kinds of liver metastases. We next performed comparative analysis on cellular compositions between P-LM and C-LM, found that Mph_SPP1, a subset of macrophages associated with angiogenesis and tumor invasion, was more enriched in the P-LM group, indicating this kind of macrophages provide a TME niche more vulnerable for pancreatic cancers. Analysis of the developmental trajectory implied that Mph_SPP1 may progressively be furnished with increased expression of genes regulating endothelium. Cell–cell communications analysis revealed that Mph_SPP1 potentially interacts with endothelial cells in P-LM via FN1/SPP1-ITGAV/ITGB1, implying this macrophage subset may construct an immunosuppressive TME for pancreatic cancer by regulating endothelial cells. We also found that Mph_SPP1 has a prognostic value in pancreatic adenocarcinoma that is not present in colon adenocarcinoma or rectum adenocarcinoma. This study provides a new perspective for understanding the characteristics of the hepatic TME in patients with liver metastatic cancer. And it provides a subset of macrophages specifically associated with the liver metastasis of pancreatic cancer, and its detection and intervention have potential value for preventing the metastasis of pancreatic cancer to the liver.

Propionibacterium freudenreichii Prevents Food Allergy in Mice via the Surface Layer Protein SlpB.

The prevalence of food allergies has increased in recent decades in industrialized developed countries. Defects are influenced by environmental factors in early life, including early colonizers of the human gut microbiota. Therapeutic solutions are limited, and the lack of efficient treatments has led to the search for new treatments, including biotherapies. Promising results from this search suggest that immunomodulatory probiotic bacteria, in particular, may yield new biotherapeutic or preventive strategies to address the increasing burden of food allergies. In this context, we investigated the potential impact of Propionibacterium freudenreichii CIRM-BIA129, a recognized immunomodulatory probiotic bacterium, on food allergy development in a murine model. Preventive effects of this probiotic were evaluated in the context of an induced wheat gliadin allergy. Following sensitization using gliadins, clinical and immunological parameters were monitored following an oral challenge with wheat gliadin. When consumed orally, P. freudenreichii CIRM-BIA129 prevented induced wheat gliadin allergy. Probiotic administration favored the differentiation of Treg cells at the expense of Th2 cells in mice. Notably, P. freudenreichii CIRM-BIA129 ΔslpB, which contains a mutation in the slpB gene encoding a key surface protein involved in adhesion and immunomodulation, failed to induce the same phenotype. Accordingly, the wild-type probiotic stimulated IL-10 production by human peripheral blood mononuclear cells, while the mutant did not. Altogether, these results indicate that the P. freudenreichii CIRM-BIA129 strain can mitigate the food allergic response through its immunomodulatory effects mediated by the surface layer protein SlpB. This finding provides new perspectives for biotherapies aimed at managing the increased prevalence of food allergy.

Generation of the TSHSUi002-A induced pluripotent stem cell line from a patient with Peutz-Jeghers syndrome carring STK11 gene mutation

We report the derivation of an induced pluripotent stem cell (iPSC) line, designated TSHSUi002-A, from a patient with Peutz-Jeghers syndrome carrying a heterozygous c.909C>G mutation in the STK11 gene. The iPSCs were generated through the reprogramming of peripheral blood mononuclear cells using a non-integrating method involving episomal vectors expressing OCT4, SOX2, KLF4, BCL-XL, and c-MYC. These iPSCs exhibit pluripotency markers, are capable of differentiating into cells of all three germ layers in vitro, and maintain a normal karyotype.

Exosomal LncRNA and CircRNA Regulate Peripheral Blood Mononuclear Cell Function through a Competitive Endogenous RNA Mechanism in Allergic Rhinitis.

Peripheral blood mononuclear cells (PBMCs) dysfunction is involved in the pathogenesis and progression of allergic rhinitis (AR). This study aims to investigate the competing endogenous RNA (ceRNA) networks in PBMCs influenced by differentially expressed long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) found in plasma exosomes induced by AR. All subjects were from the Affiliated Taizhou People's Hospital of Nanjing Medical University. Differential expression of mRNAs in PBMCs and lncRNAs/circRNAs in plasma exosomes was analyzed using high-throughput sequencing. Differentially expressed lncRNAs and circRNAs that target mRNAs were identified using bioinformatics methods. The predicted target mRNAs were intersected with the differentially expressed mRNAs in PBMCs to construct ceRNA networks. The subcellular localizations of lncRNAs and circRNAs within the ceRNA networks were determined using RNA fluorescence in situ hybridization or bioinformatics methods. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of differentially expressed mRNAs in PBMCs were conducted using the clusterProfiler R package. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to validate the expression levels of each molecule within the constructed ceRNA networks in clinical samples, along with receiver operating characteristic (ROC) curve analysis to assess diagnostic value. Further validation was performed using in vitro cultured PBMCs and dual-luciferase reporter assays. Five differentially expressed circRNAs and 31 differentially expressed lncRNAs were identified in exosomes. In PBMCs, 130 differentially expressed mRNAs were identified. Six ceRNA networks were constructed, affecting PBMCs chemorepellent activity, JAK-STAT signaling pathway, and other functions or pathways. The expression level of ENST00000650850 in plasma exosomes was significantly lower in AR patients, suggesting its potential diagnostic value. The expression level of ENST00000650850 in plasma exosomes was positively correlated with the expression levels of ENST00000650850 and IL6 mRNA in PBMCs. PBMCs from healthy individuals were stimulated with plasma exosomes isolated from AR patients, leading to a reduction in IL6R mRNA expression levels in the PBMCs. Differentially expressed lncRNA (ENST00000650850) in plasma-derived exosomes of AR patients may regulate IL6R mRNA expression in PBMCs via miR-6747-3p, thereby influencing PBMC function and contributing to the pathogenesis and progression of AR.

HDL Cholesterol-Associated Shifts in the Expression of Preselected Genes Reveal both Pro-Atherogenic and Atheroprotective Effects of HDL in Coronary Artery Disease.

The associations of high-density lipoprotein (HDL) level and functionality with lipid metabolism, inflammation, and innate immunity in coronary artery disease (CAD) remain controversial. The differential expression of a set of genes related to HDL metabolism (24 genes) and atherogenesis (41 genes) in peripheral blood mononuclear cells (PBMC) from CAD and control patients with varied HDL cholesterol (HDL-C) levels was compared. 76 male patients 40-60 years old with CAD diagnosed by angiography and 63 control patients were divided into three groups with low, normal (1.0-1.4 mM), and increased HDL-C levels. Transcript levels were measured by real-time PCR. The differentially expressed genes (DEGs) and associated metabolic pathways were analyzed for three groups, with prevalent CAD as an outcome. The common feature was the increased odds ratio values for liver X receptor (LXR) gene expression for three patient groups. CAD patients with low HDL-C possessed 24 DEGs with lower expression of genes involved in cholesterol efflux, and down-regulated SREBF1 and ABCG1 are suggested as gene signatures. CAD patients with normal HDL-C possessed nine DEGs with down-regulated ITGAM and ALB as gene signatures. CAD patients with increased HDL-C possessed 19 DEGs with down-regulated APOA1 and HMGCR as gene signatures. With gene expression signatures, one standard deviation higher average gene expressions were associated with 5.1-, 48.8-, and 38.9-fold fewer CAD cases for three patient groups. As HDL-C increased in CAD patients, the expression of ABCG1, CUBN, and HDLBP genes increased, while the expression of HMGCR and NPC2 genes, involved in cholesterol synthesis and trafficking, decreased. The expression of CD14, CD36, S100A8, S100A9, S100A12, TLR5, TLR8, and VEGFA genes, involved in angiogenesis and inflammation mainly via nuclear factor-κB (NF-κB), decreased. The increased accumulation of cholesteryl ester in PBMC from patients with low HDL-C was suggested. This assumption contrasts with the suggested accumulation of free cholesterol in PBMC from patients with increased HDL-C, concomitant with suppression of cholesterol synthesis and traffic to the plasma membrane, and with an inflammatory state controlled by depressed CD36-mediated and upregulated apoE-mediated immunometabolic signaling. Gene signatures may be used for the diagnosis, prognosis, and treatment of CAD in dependence on HDL-C levels.

Human blood cell transcriptomics unveils dynamic systemic immune modulation along colorectal cancer progression

BackgroundColorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide. CRC deaths can be reduced with prevention and early diagnosis. Circulating tumor DNA-based liquid biopsies, are emerging tools...

Generation and characterization of two induced pluripotent stem cell lines (ICGi052-A and ICGi052-B) from a patient with frontotemporal dementia with parkinsonism-17 associated with the pathological variant c.2013T&gt;G in the MAPT gene

Frontotemporal dementia with parkinsonism-17 is a neurodegenerative disease characterised by pathological aggregation of the tau protein with the formation of neurofibrillary tangles and subsequent neuronal death. The inherited form of frontotemporal dementia can be caused by mutations in several genes, including the MAPT gene on chromosome 17, which encodes the tau protein. As there are currently no medically approved treatments for frontotemporal dementia, there is an urgent need for research using in vitro cell models to understand the molecular genetic mechanisms that lead to the development of the disease, to identify targets for therapeutic intervention and to test potential drugs to prevent neuronal death. Analysis of exome sequencing data from a 46-year-old patient with a clinical diagnosis of Parkinson’s disease revealed the presence of the pathological variant c.2013T&gt;G (rs63750756) in the MAPT gene, which is associated with frontotemporal dementia with parkinsonism-17. By reprogramming the patient’s peripheral blood mononuclear cells, we obtained induced pluripotent stem cells (iPSCs). Two iPSC lines were characterised in detail. Reprogramming was performed by transfection with non-integrating episomal vectors expressing the OCT4, SOX2, KLF4, LIN28, L-MYC and mp53DD proteins. The iPSC lines ICGi052-A and ICGi052-B proliferate stably, form colonies with a morphology characteristic of human pluripotent cells, have a normal diploid karyotype (46,XX), express endogenous alkaline phosphatase and pluripotency markers (OCT4, NANOG, SSEA-4 and TRA-1-60) and are able to differentiate into derivatives of three germ layers: ento-, ecto- and mesoderm. The iPSC lines obtained and characterised in detail in this work represent a unique tool for studying the molecular genetic mechanisms of the pathogenesis of frontotemporal dementia with parkinsonism-17, as well as for testing potential drugs in vitro.

Pre-clinical development of a tolerogenic peptide from glutamate decarboxylase as a candidate for antigen-specific immunotherapy in type 1 diabetes.

Dysregulation and loss of immune tolerance towards pancreatic β-cell autoantigens are features of type 1 diabetes (T1D). Until recently, life-long insulin injection was the only approved treatment for T1D, and this does not address the underlying disease pathology. Antigen-specific immunotherapy (ASI) seeks to restore tolerance and holds potential as a new therapeutic strategy for treating autoimmune diseases with well characterised antigens. Peptide ASI using processing independent CD4+ T-cell epitopes (PIPs) shows promising results in several autoimmune diseases. Here we successfully applied the principles of PIP design to the T1D autoantigen glutamate decarboxylase 65 (GAD65). Peptides spanning GAD65 predicted to be pan-HLA-DR binding were selected. Peptide P10 displayed enriched responses in peripheral blood mononuclear cells from people with T1D. The minimal epitope of the P10 peptide was fine mapped using T-cell hybridomas generated from HLA-DRB1*04:01 transgenic mice. This minimal epitope, P10Sol, was demonstrated to induce tolerance to the parent peptide in HLA-DRB1*04:01 transgenic mice using a novel activation-induced marker assay. Finally, we show that GAD65 P10Sol PIP is recognised by CD4+ T-cells from people with T1D who possess a range of HLA-DR alleles and can, therefore, be defined as a pan-DR binding peptide with therapeutic potential.

MicroRNAs as Biomarkers for Uveitis in Juvenile Idiopathic Arthritis

ABSTRACT Purpose Uveitis associated with juvenile idiopathic arthritis (JIA-U) is a clinically silent vision-impairing disease. Early detection and aggressive treatment are crucial for optimal visual outcome. Alterations in levels of microRNAs (miRNAs) are characteristic of autoimmune diseases. The present clinical study sought to explore the expression of miRNAs in JIA-U and their potential role as a predictive biomarker. Methods MiRNA expression profiling was performed on peripheral blood mononuclear cells derived from pediatric patients with JIA, JIA-U, or other types of uveitis using the high-throughput small-RNA sequencing (on Next Generation Sequencing (NGS)). Patient- and disease-related data were retrieved from the medical files. Main outcome measure was the differential expression of miRNAs among the groups. Results The cohort included 35 patients; 20 children with JIA-U (8 with active disease), 10 with JIA without ocular involvement, and 5 with other types of uveitis (4 with active disease). Mean age was 8.6 years; 83% were female. Nineteen patients (54%) received immunomodulatory treatment. The expression of miR-4485-3p was significantly increased in patients with JIA-U compared to patients with JIA alone (p < 0.05), with no difference between patients with active or inactive uveitis. The expression in patients with uveitis of other etiologies was similar to the expression in JIA-U patients. Conclusions This study demonstrates a differential expression profile of a specific miRNA in JIA patients with and without uveitis. If verified in larger studies, the findings may assist to identify JIA patients at risk to develop uveitis and to improve early detection of disease activity.

Clonal hematopoiesis of indeterminate potential: a risk factor for future exacerbation in patients with COPD

BackgroundThere is limited data regarding the clinical significance of clonal hematopoiesis of indeterminate potential (CHIP) in patients with COPD. We aimed to evaluate whether CHIP could serve as a biomarker for the long-term prognosis of patients with COPD.MethodsNext-generation sequencing was used to detect CHIP in peripheral blood mononuclear cell samples. Participants with COPD were enrolled in a prospective observational cohort between January 2013 and December 2023. We investigated the association of CHIP status with the risk of subsequent acute exacerbation and longitudinal changes in lung function, by using negative binomial regression and linear mixed regression models, respectively.ResultsAmong 125 patients with COPD, the CHIP-positive group was 32 (25.6%). Higher smoking intensity and CAT score were significantly associated with increased risk of positive CHIP status, especially in current smokers. Positive CHIP of specific genes was significantly associated with a higher risk of subsequent mild and total acute exacerbation. Positive CHIP of ASXL transcriptional regulator 1 (ASXL1) had a lower risk of subsequent total acute exacerbation (aOR, 0.19; 95% CI, 0.04–0.95). The CHIP status did not show a statistically significant association with longitudinal changes in lung function.ConclusionsIn patients with COPD, CHIP positivity was significantly associated with an increased risk of subsequent acute exacerbations, but not with longitudinal changes in lung function.