Functional filter for whole-genome sequencing data identifies HHT and stress-associated non-coding SMAD4 polyadenylation site variants >5 kb from coding DNA.

Despite whole-genome sequencing (WGS), many cases of single-gene disorders remain unsolved, impeding diagnosis and preventative care for people whose disease-causing variants escape detection. Since early WGS data analytic steps prioritize protein-coding sequences, to simultaneously prioritize variants in non-coding regions rich in transcribed and critical regulatory sequences, we developed GROFFFY, an analytic tool that integrates coordinates for regions with experimental evidence of functionality. Applied to WGS data from solved and unsolved hereditary hemorrhagic telangiectasia (HHT) recruits to the 100,000 Genomes Project, GROFFFY-based filtration reduced the mean number of variants/DNA from 4,867,167 to 21,486, without deleting disease-causal variants. In three unsolved cases (two related), GROFFFY identified ultra-rare deletions within the 3' untranslated region (UTR) of the tumor suppressor SMAD4, where germline loss-of-function alleles cause combined HHT and colonic polyposis (MIM: 175050). Sited >5.4 kb distal to coding DNA, the deletions did not modify or generate microRNA binding sites, but instead disrupted the sequence context of the final cleavage and polyadenylation site necessary for protein production: By iFoldRNA, an AAUAAA-adjacent 16-nucleotide deletion brought the cleavage site into inaccessible neighboring secondary structures, while a 4-nucleotide deletion unfolded the downstream RNA polymerase II roadblock. SMAD4 RNA expression differed to control-derived RNA from resting and cycloheximide-stressed peripheral blood mononuclear cells. Patterns predicted the mutational site for an unrelated HHT/polyposis-affected individual, where a complex insertion was subsequently identified. In conclusion, we describe a functional rare variant type that impacts regulatory systems based on RNA polyadenylation. Extension of coding sequence-focused gene panels is required to capture these variants.

An Inflection Point in High-Throughput Proteomics with Orbitrap Astral: Analysis of Biofluids, Cells, and Tissues.

This Technical Note presents a comprehensive proteomics workflow for the new combination of Orbitrap and Astral mass analyzers across biofluids, cells, and tissues. Central to our workflow is the integration of Adaptive Focused Acoustics (AFA) technology for cells and tissue lysis to ensure robust and reproducible sample preparation in a high-throughput manner. Furthermore, we automated the detergent-compatible single-pot, solid-phase-enhanced sample Preparation (SP3) method for protein digestion. The synergy of these advanced methodologies facilitates a robust and high-throughput approach for cell and tissue analysis, an important consideration in translational research. This work disseminates our platform workflow, analyzes the effectiveness, demonstrates the reproducibility of the results, and highlights the potential of these technologies in biomarker discovery and disease pathology. For cells and tissues (heart, liver, lung, and intestine) proteomics analysis by data-independent acquisition mode, identifications exceeding 10,000 proteins can be achieved with a 24 min active gradient. In 200 ng injections of HeLa digest across multiple gradients, an average of more than 80% of proteins have a CV less than 20%, and a 45 min run covers ∼90% of the expressed proteome. This complete workflow allows for large swaths of the proteome to be identified and is compatible with diverse sample types.

Anti-inflammatory and immunoregulatory effects of colistin sulphate on human PBMCs.

In previous studies, CST has been identified as having an immunostimulatory effect on Caenorhabditis elegans and macrophage of rats. Here, we further investigated its immunomodulatory effects on human peripheral blood mononuclear cells (PBMCs). LPS-stimulated PBMCs inflammatory model was established. Flow cytometry was applied to measure phagocytosis of PBMCs. Cytokine mRNA and protein expression levels of LPS-stimulated PBMCs with or without CST were measured by qRT-PCR and ELISA. The transcriptomic profile of CST-treated PBMCs was investigated by RNA-sequencing. Gene Ontology (GO) and Kyoto Encylopedia of Genes and Genomes (KEGG) were applied to find potential signalling pathways. PBMCs showed a significant increase in phagocytic activity at 6 h after being incubated with CST at the concentration of 10 µg/mL. In the presence of LPS, CST maintained and promoted the expression of TNF-α and chemokine CCL24. The content of pro-inflammatory cytokines, such as IL-1ß, IL-6 and IFN-γ, which were released from LPS-stimulated PBMCs, was reduced by CST at 6 h. Anti-inflammatory cytokines, such as IL-4, IL-13 and TGF-ß1, were significantly increased by CST at 24 h. A total of 277 differentially expressed immune-related genes (DEIRGs) were detected and cytokine-cytokine receptor interaction was highly enriched. CST presented obvious anti-inflammatory and immunoregulatory effects in LPS-induced PBMCs inflammatory model not only by improving the ability of PBMCs to clear pathogens but also by decreasing pro-inflammatory cytokines and increasing anti-inflammatory cytokines. And the mechanism may be related to cytokine-cytokine receptor interaction.

A unique human cord blood CD8+CD45RA+CD27+CD161+ T-cell subset identified by flow cytometric data analysis using Seurat.

Advances in single-cell level analytical techniques, especially cytometric approaches, have led to profound innovation in biomedical research, particularly in the field of clinical immunology. This has resulted in an expansion of high-dimensional data, posing great challenges for comprehensive and unbiased analysis. Conventional manual analysis is thus becoming untenable to handle these challenges. Furthermore, most newly developed computational methods lack flexibility and interoperability, hampering their accessibility and usability. Here, we adapted Seurat, an R package originally developed for single-cell RNA sequencing (scRNA-seq) analysis, for high-dimensional flow cytometric data analysis. Based on a 20-marker antibody panel and analyses of T-cell profiles in both adult blood and cord blood (CB), we showcased the robust capacity of Seurat in flow cytometric data analysis, which was further validated by Spectre, another high-dimensional cytometric data analysis package, and conventional manual analysis. Importantly, we identified a unique CD8+ T-cell population defined as CD8+CD45RA+CD27+CD161+ T cell that was predominantly present in CB. We characterised its IFN-γ-producing and potential cytotoxic properties using flow cytometry experiments and scRNA-seq analysis from a published dataset. Collectively, we identified a unique human CB CD8+CD45RA+CD27+CD161+ T-cell subset and demonstrated that Seurat, a widely used package for scRNA-seq analysis, possesses great potential to be repurposed for cytometric data analysis. This facilitates an unbiased and thorough interpretation of complicated high-dimensional data using a single analytical pipeline and opens a novel avenue for data-driven investigation in clinical immunology.

Identification of NETs formation and the renoprotective effect of degraded NETs in lupus nephritis.

To explore molecular biomarkers associated with the pathophysiology and therapy of lupus nephritis (LN), we conducted a joint analysis of transcriptomic data from 40 PBMCs (GSE81622) and 21 kidney samples (GSE112943) from the GEO database using bioinformatics. 976 and 2427 differentially expressed genes (DEGs) were identified in PBMCs and renal tissues. Seven and two functional modules closely related to LN were identified. Further enrichment analysis revealed that the neutrophil activation pathway was highly active in both PBMC and kidney. Subsequently, 16 core genes closely associated with LN were verified by PPI screening and qPCR. In vitro cell models and MRL/lpr mouse models confirmed that the abnormal expression of these core genes was closely linked to neutrophil extracellular traps (NETs) generated by neutrophil activation, while degradation of NETs led to down-regulation of core gene expression, thereby improving pathological symptoms of LN. Therefore, identification of SLE patients exhibiting abnormal expression patterns for these core genes may serve as a useful indicator for kidney involvement. Additionally, targeting neutrophils to modulate their activation levels and inhibit aberrant expression of these genes represents a potential therapeutic strategy for treating LN.

Interleukin-27 Promotes Divergent Effects on HIV-1 Infection in Peripheral Blood Mononuclear Cells through BST-2/Tetherin.

Interleukin-27 (IL-27) is able to inhibit HIV-1 replication in peripheral blood mononuclear cells (PBMCs), macrophages, and dendritic cells. Here, we identify that IL-27 can produce opposing effects on HIV-1 replication in PBMCs and that the HIV-1 restriction factor BST-2/Tetherin is involved in both inhibitory and enhancing effects on HIV-1 infection induced by IL-27. IL-27 inhibited HIV-1 replication when added to cells 2 h after infection, promoting the prototypical BST-2/Tetherin-induced virion accumulation at the cell membrane of HIV-1-infected PBMCs. BST-2/Tetherin gene expression was significantly upregulated in the IL-27-treated PBMCs, with a simultaneous increase in the number of BST-2/Tetherin+ cells. The silencing of BST-2/Tetherin diminished the anti-HIV-1 effect of IL-27. In contrast, IL-27 increased HIV-1 production when added to infected cells 4 days after infection. This enhancing effect was prevented by BST-2/Tetherin gene knockdown, which also permitted IL-27 to function again as an HIV-1 inhibitory factor. These contrasting roles of IL-27 were associated with the dynamic of viral production, since the IL-27-mediated enhancement of virus replication was prevented by antiretroviral treatment of infected cells, as well as by keeping cells under agitation to avoid cell-to-cell contact. Likewise, inhibition of CD11a, an integrin associated with HIV-1 cell-to-cell transmission, abrogated the IL-27 enhancement of HIV-1 production. Our findings illustrate the complexity of the HIV-1-host interactions and may impact the potential therapeutic use of IL-27 and other soluble mediators that induce BST-2/Tetherin expression for HIV-1 infection. IMPORTANCE Here, we describe new findings related to the ability of the cytokine IL-27 to regulate the growth of HIV-1 in CD4+ T lymphocytes. IL-27 has long been considered a potent inhibitor of HIV-1 replication, a notion based on several reports showing that this cytokine controls HIV-1 infection in peripheral blood mononuclear cells (PBMCs), monocyte-derived macrophages, and dendritic cells. However, our present results are contrary to the current knowledge that IL-27 acts only as a powerful downregulator of HIV-1 replication. We observed that IL-27 can either prevent or enhance viral growth in PBMCs, an outcome dependent on when this cytokine is added to the infected cells. We detected that the increase of HIV-1 dissemination is due to enhanced cell-to-cell transmission with the involvement of the interferon-induced HIV-1 restriction factor BST-2/Tetherin and CD11a (LFA-1), an integrin that participates in formation of virological synapse.

B cell immune response to human bocaviruses.

BACKGROUND: Human bocaviruses (HBoVs) have been demonstrated in respiratory and gastrointestinal infections; however, the immune response to them has not been studied in detail. In this study, we investigated the B cell immune responses to HBoV1 and HBoV2, representing two different species of bocaviruses in humans. METHODS: We analyzed the effects of stimulations with HBoV1 and 2 virus-like particles (VLPs) and of co-stimulation with HBoV1-rhinovirus (RV) on cells of the immune system by flow cytometry, transcriptomics, and luminometric immune assays. RESULTS: Human B cells, and particularly B regulatory cells (Breg cells), showed an increased immune response to HBoV1-VLPs stimulation. These immune responses were also supported by increased IL-1RA and PDL1 expressions in IL-10+ B cells from peripheral blood mononuclear cells (PBMCs) stimulated with HBoV1-VLPs. In addition, increased levels of IL-10 and IL-1RA were determined in the supernatants of PBMCs following HBoV1-VLPs stimulation. HBoV1-VLPs and RV co-stimulation increased the IL-10+ B cell population. Transcriptome analysis by next-generation RNA sequencing showed an increased expression of IL-10 signalling and Breg cell markers in PBMCs stimulated with HBoV1-VLPs. Furthermore, TGF-ß and chemoattractants MIP-1α, MIP-1ß and IP10 protein levels were high in the supernatants of PBMCs stimulated with HBoV1-VLPs. CONCLUSIONS: The findings demonstrate that in Breg cells, IL-10 signalling pathways, and anti-inflammatory activity are induced by HBoV1, which can explain the often mild nature of the disease. In addition, the immune regulatory response induced by HBoV1-VLPs may indicate a potential immunomodulatory role of HBoV1 on the immune system and may represent an immune regulatory strategy.

Standardized high-dimensional spectral cytometry protocol and panels for whole blood immune phenotyping in clinical and translational studies.

Flow cytometry is the method of choice for immunophenotyping in the context of clinical, translational, and systems immunology studies. Among the latter, the Milieu Intérieur (MI) project aims at defining the boundaries of a healthy immune response to identify determinants of immune response variation. MI used immunophenotyping of a 1000 healthy donor cohort by flow cytometry as a principal outcome for immune variance at steady state. New generation spectral cytometers now enable high-dimensional immune cell characterization from small sample volumes. Therefore, for the MI 10-year follow up study, we have developed two high-dimensional spectral flow cytometry panels for deep characterization of innate and adaptive whole blood immune cells (35 and 34 fluorescent markers, respectively). We have standardized the protocol for sample handling, staining, acquisition, and data analysis. This approach enables the reproducible quantification of over 182 immune cell phenotypes at a single site. We have applied the protocol to discern minor differences between healthy and patient samples and validated its value for application in immunomonitoring studies. Our protocol is currently used for characterization of the impact of age and environmental factors on peripheral blood immune phenotypes of >400 donors from the initial MI cohort.

OMIP-102: 50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells.

We report the development of an optimized 50-color spectral flow cytometry panel designed for the in-depth analysis of the immune system in human blood and tissues, with the goal of maximizing the amount of information that can be collected using currently available flow cytometry platforms. We established and tested this panel using peripheral blood mononuclear cells (PBMCs), but included CD45 to enable its future use for the analysis of human tissue samples. The panel contains lineage markers for all major immune cell subsets, and an extensive set of phenotyping markers focused on the activation and differentiation status of the T cell and dendritic cell (DC) compartment. We outline the biological insight that can be gained from the simultaneous measurement of such a large number of proteins and propose that this approach provides a unique opportunity for the comprehensive exploration of the immune status in human samples with a limited number of cells. Of note, we tested the panel to be compatible with cell sorting for further downstream applications. Furthermore, to facilitate the wide-spread implementation of such a panel across different cohorts and samples, we established a trimmed-down 45-color version which can be used with different spectral cytometry platforms. Finally, to generate this panel, we utilized not only existing panel design guidelines, but also developed new metrics to systematically identify the optimal combination of 50 fluorochromes and evaluate fluorochrome-specific resolution in the context of a 50-color unmixing matrix.

Evaluation of the expression of fibrosis-related genes as non-invasive diagnostic biomarkers for cirrhotic HCV-infected patients.

Liver cirrhosis is a condition with high mortality that poses a significant health and economic burden worldwide. The clinical characteristics of liver cirrhosis are complex and varied. Therefore, the evaluation of immune infiltration-involved genes incirrhosis has become mandatory in liver disease research, not only to identify the potential biomarkers but also to provide important insights into the underlying mechanisms of the disease. In this study, we aimed to investigate the expression profile of cytokine genes in peripheral blood mononuclear cells (PBMCs) of HCV patients and identify the gene expression signature associated with advanced cirrhosis. A cross-sectional study of 90 HCV genotype 4 patients, including no fibrosis patients (F0, n = 24), fibrotic patients (F1-F3, n = 36), and cirrhotic patients (F4, n = 30) has been conducted. The expression of cytokine genes was analyzed by quantitative real-time PCR in the subjects' PBMCs, and the serum level of TGFß2 was measured by ELISA. Our findings showed that the expression level of the TGIF1 transcript was lower in cirrhotic and fibrotic patients compared to no fibrosis patients (p = 0.046 and 0.022, respectively). Also, there was an upregulation of the TGFß1 gene in cirrhotic patients relative to fibrotic patients (p = 0.015). Additionally, the cirrhotic patients had higher expression levels of the TGF-ß2 transcript and elevated levels of the TGF-ß2 protein than patients with no cirrhosis or fibrosis. According to the ROC analysis, TGFß1, TGIF1 transcripts, and TGFß2 protein have a good discriminatory performance in distinguishing between cirrhotic, fibrotic, and non-fibrotic patients. Our results suggested that the expression of TGIF1, TGF-ß1, and TGF-ß2 genes in PBMCs may provide a valuable tool for identifying patients with advanced cirrhosis and that TGF-ß and TGIF1 may be potential biomarkers for cirrhosis. These findings may have implications for the diagnosis and treatment of cirrhosis in HCV patients.

The roles of SFKs in the regulation of proinflammatory cytokines and NLRP3 in familial mediterranean fever patients.

Familial Mediterranean Fever (FMF) is caused by mutations in pyrin, a protein produced in innate immune cells that regulates the development of interleukin (IL)-1ß by interacting with caspase-1 and other components of inflammasomes. Although overexpression of proinflammatory cytokines have been observed in FMF patients, no studies have been conducted on the role of Src family kinases (SFKs). The purpose of this study was to examine the impact of SFKs on the modulation of IL-1ß, IL-6, IL-8, TNF-α, and NLRP3 inflammasome in patients with FMF. The study included 20 FMF patients and 20 controls. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation. Protein expression levels of SFKs members were measured by western blot. The effect of lipopolysaccharide-induced (LPS) activation and PP2- induced inhibition of SFKs on NLRP3 and IL-1ß, IL 6, IL-8, TNF-α were examined by western blot and flow cytometry respectively. Patients with FMF have considerably greater levels of Lck expression. In addition, patients had a substantially greater basal level of NLRP3 than the control group (*p = 0.016). Most importantly, the levels of IL-1 ß were elevated with LPS stimulation and reduced with PP2 inhibition in FMF patients. These results suggest that SFKs are effective in regulation of IL-1 ß in FMF patients.

Exploring expression levels of the cGAS-STING pathway genes in peripheral blood mononuclear cells of spinal tuberculosis patients.

BACKGROUND: This study aimed to investigate the differential expression levels of the cGAS-STING pathway in peripheral blood mononuclear cells (PBMCs) of spinal tuberculosis (TB) patients with different progression and its feasibility as a diagnostic marker. METHODS: Peripheral blood and medical records of 25 patients with spinal TB and 10 healthy individuals, were prospectively collected and analyzed. PBMCs and serum were extracted from peripheral blood and the expression levels of the cGAS-STING pathway in PBMCs were measured by real-time PCR (RT-PCR) and serum interferon ß (IFN-ß) expression levels were measured by enzyme-linked immunosorbent assay (ELISA). The expression of Interferon regulatory Factor 3 (IRF3) in PBMCs was measured using western blot. Statistical analysis was performed using the SPSS 26.0 statistical package. RESULTS: The results showed that the expression level of the TANK-binding kinase 1 (TBK1) and IRF3 was significantly higher in PBMCs (P < 0.05), in patients with active lesions than in patients with stable lesions. The serum concentration of IFN-ß was significantly higher in patients with active lesions (P = 0.028). Compared with healthy individuals, the expression level of the cGAS-STING pathway was elevated in PBMCs of TB patients (P < 0.05), and the difference in the expression level of IFN-ß was not statistically significant (P > 0.05), and the serum IFN-ß concentration was elevated (P < 0.05). The calculated AUC values for TBK1 and IRF3 in PBMCs, IFN-ß in serum and erythrocyte sedimentation rate (ESR) to distinguish between patients with active and stable lesions were 0.732, 0.714, 0.839, and 0.714 respectively. CONCLUSIONS: The expression level of TBK1 and IRF3 in PBMCs, and IFN-ß in the serum of patients with spinal TB is positively correlated with disease activity. TBK1 has higher specificity and IFN-ß in serum has higher sensitivity when used to differentiate between patients with active and stable lesions.

A C-type lectin induces NLRP3 inflammasome activation via TLR4 interaction in human peripheral blood mononuclear cells.

Lectins are a large group of proteins found in many snake venoms. BjcuL is a C-type lectin from Bothrops jararacussu snake venom that does not present cytotoxicity action on human peripheral blood mononuclear cells (PBMCs) at concentrations of 5 and 10 µg/mL. BjcuL demonstrates an immunomodulatory role in PBMCs with the production of pro- and anti-inflammatory cytokines (IL-2, IL-10, IFN-γ, IL-6, TNF-α, and IL-17) in addition to stimulate T cells to produce reactive oxygen species (ROS) that could play a role in the acute inflammatory reaction observed in the victims. Inflammasomes are an essential arm in cells of innate immunity to detect and sense a range of endogenous or exogenous, sterile, or infectious stimuli to elicit cellular responses and effector mechanisms. NLRP3 inflammasome is a significant target for this study, because the lectin is responsible for leukocyte activation stimulating the release of inflammatory mediators, which results in dynamic cellular responses to remove the detrimental process to the body in snakebites. Thus, this study aimed to investigate how isolated BjcuL from B. jararacussu venom affects NLRP3 inflammasome activation on PBMCs. For this, the cells were isolated by density gradient and incubated with BjcuL at different periods and concentrations for the evaluation of the activation of the NLRP3 inflammasome through gene and protein expressions of ASC, CASPASE-1, and NLRP3 by RT-qPCR, Western blot, and immunofluorescence, as well as the participation of Toll-like receptor 4 (TLR4) and ROS in the IL-1ß production, a product resultant of the NLRP3 inflammasome activation. Herein, BjcuL interacts with TLR4 as demonstrated by in vitro and in silico studies and induces cytokines release via NF-κB signaling. By genic and protein expression assays, BjcuL activates NLRP3 inflammasome, and the pharmacological modulation with LPS-RS, an antagonist of TLR4; LPS-SM, an agonist of TLR4; MCC950, a specific NLRP3 inhibitor, and rotenone, an inhibitor of mitochondrial ROS, confirmed the participation of TLR4 and ROS in the NLRP3 inflammasome activation and IL-1ß liberation. The effects of BjcuL on the regulation and activation of the NLRP3 inflammasome complex via TLR4 activation with ROS participation may be determinant for the development of the inflammatory local effects seen in snakebite victims. In addition, in silico together with in vitro studies provide information that may be useful in the rational design of TLR agonists as well as new adjuvants for immunomodulatory therapy.

Sex-Specific Variations in the mRNA Levels of Candidate Genes in Peripheral Blood Mononuclear Cells from Patients with Diabetes: A Multistep Study.

BACKGROUND: Type 2 diabetes (T2D) is one of the most prevalent diseases that also show sexual dimorphism in many different aspects. OBJECTIVES: This study aimed to distinguish the mRNA expression of genes in peripheral blood mononuclear cells (PBMCs) in men or women with T2D using a multistep analysis. METHODS: A total of 95 patients with T2D were compared based on their sex in terms of clinical variables and mRNA expression in their PBMCs. RESULTS: Men with T2D had lower LDLC, HDLC, and HbA1c values in their blood, but greater creatinine levels. In men with T2D, TLR4, CCR2, NOX2, and p67phox mRNA expression was greater, but IL6 and NF-κB mRNA expression was lesser in PBMCs. There was a link between fasting plasma glucose (FPG), triglycerides, and hs-CRP, as well as COX1 mRNA in men with T2D. In women with T2D, FPG was associated with the mRNA expression of THBS1 and p67phox, as well as triglycerides and HDLC levels. We found the exclusive effect of FPG on HDLC, HbA1c, as well as p67phox mRNA in PBMCs of women with T2D. Analysis revealed the exclusive effect of FPG on hs-CRP and PAFR mRNA in PBMCs of men with T2D. FPG was shown to be associated with body mass index, hs-CRP, triglycerides, and COX1 mRNA in men with T2D, and with serum triglycerides, THSB1, and p67phox mRNA in women with T2D, according to network analysis. HbA1c was linked with NF-κB mRNA in women with T2D. CONCLUSIONS: Using a multistep analysis, it was shown that network analysis outperformed traditional analytic techniques in identifying sex-specific alterations in mRNA gene expression in PBMCs of T2D patients. The development of sex-specific therapeutic approaches may result from an understanding of these disparities.

Ultrastructural Characterization of PBMCs and Extracellular Vesicles in Multiple Sclerosis: A Pilot Study.

Growing evidence identifies extracellular vesicles (EVs) as important cell-to-cell signal transducers in autoimmune disorders, including multiple sclerosis (MS). If the etiology of MS still remains unknown, its molecular physiology has been well studied, indicating peripheral blood mononuclear cells (PBMCs) as the main pathologically relevant contributors to the disease and to neuroinflammation. Recently, several studies have suggested the involvement of EVs as key mediators of neuroimmune crosstalk in central nervous system (CNS) autoimmunity. To assess the role of EVs in MS, we applied electron microscopy (EM) techniques and Western blot analysis to study the morphology and content of plasma-derived EVs as well as the ultrastructure of PBMCs, considering four MS patients and four healthy controls. Through its exploratory nature, our study was able to detect significant differences between groups. Pseudopods and large vesicles were more numerous at the plasmalemma interface of cases, as were endoplasmic vesicles, resulting in an activated aspect of the PBMCs. Moreover, PBMCs from MS patients also showed an increased number of multivesicular bodies within the cytoplasm and amorphous material around the vesicles. In addition, we observed a high number of plasma-membrane-covered extensions, with multiple associated large vesicles and numerous autophagosomal vacuoles containing undigested cytoplasmic material. Finally, the study of EV cargo evidenced a number of dysregulated molecules in MS patients, including GANAB, IFI35, Cortactin, Septin 2, Cofilin 1, and ARHGDIA, that serve as inflammatory signals in a context of altered vesicular dynamics. We concluded that EM coupled with Western blot analysis applied to PBMCs and vesiculation can enhance our knowledge in the physiopathology of MS.

Targeted RNAseq Revealed the Gene Expression Signature of Ferroptosis-Related Processes Associated with Disease Severity in Patients with Multiple Sclerosis.

Detrimental molecular processes in multiple sclerosis (MS) lead to the cellular accumulation of lipid peroxidation products and iron in the CNS, which represents the main driving force for ferroptosis. Ferroptosis is an iron-dependent form of regulated cell death, with proposed roles in neurodegeneration, oligodendrocyte loss and neuroinflammation in the pathogenesis of MS. Ferroptosis-related gene expression signature and molecular markers, which could reflect MS severity and progression, are currently understudied in humans. To tackle these challenges, we have applied a curated approach to create and experimentally analyze a comprehensive panel of ferroptosis-related genes covering a wide range of biological processes associated with ferroptosis. We performed the first ferroptosis-related targeted RNAseq on PBMCs from highly distinctive MS phenotype groups: mild relapsing-remitting (RR) (n = 24) and severe secondary progressive (SP) (n = 24), along with protein detection of GPX4 and products of lipid peroxidation (MDA and 4-HNE). Out of 138 genes, 26 were differentially expressed genes (DEGs), indicating changes in both pro- and anti-ferroptotic genes, representing a molecular signature associated with MS severity. The top three DEGs, as non-core ferroptosis genes, CDKN1A, MAP1B and EGLN2, were replicated by qPCR to validate findings in independent patient groups (16 RR and 16 SP MS). Co-expression and interactions of DEGs were presented as additional valuable assets for deeper understanding of molecular mechanisms and key targets related to MS severity. Our study integrates a wide genetic signature and biochemical markers related to ferroptosis in easily obtainable PBMCs of MS patients with clinical data and disease severity, thus providing novel molecular markers which can complement disease-related changes in the brain and undergo further research as potential therapeutic targets.

Effects of Anti-Fibrotic Drugs on Transcriptome of Peripheral Blood Mononuclear Cells in Idiopathic Pulmonary Fibrosis.

Two anti-fibrotic drugs, pirfenidone (PFD) and nintedanib (NTD), are currently used to treat idiopathic pulmonary fibrosis (IPF). Peripheral blood mononuclear cells (PBMCs) are immunocompetent cells that could orchestrate cell-cell interactions associated with IPF pathogenesis. We employed RNA sequencing to examine the transcriptome signature in the bulk PBMCs of patients with IPF and the effects of anti-fibrotic drugs on these signatures. Differentially expressed genes (DEGs) between "patients with IPF and healthy controls" and "before and after anti-fibrotic treatment" were analyzed. Enrichment analysis suggested that fatty acid elongation interferes with TGF-ß/Smad signaling and the production of oxidative stress since treatment with NTD upregulates the fatty acid elongation enzymes ELOVL6. Treatment with PFD downregulates COL1A1, which produces wound-healing collagens because activated monocyte-derived macrophages participate in the production of collagen, type I, and alpha 1 during tissue damage. Plasminogen activator inhibitor-1 (PAI-1) regulates wound healing by inhibiting plasmin-mediated matrix metalloproteinase activation, and the inhibition of PAI-1 activity attenuates lung fibrosis. DEG analysis suggested that both the PFD and NTD upregulate SERPINE1, which regulates PAI-1 activity. This study embraces a novel approach by using RNA sequencing to examine PBMCs in IPF, potentially revealing systemic biomarkers or pathways that could be targeted for therapy.

Pulsed Hyperoxia Acts on Plasmatic Advanced Glycation End Products and Advanced Oxidation Protein Products and Modulates Mitochondrial Biogenesis in Human Peripheral Blood Mononuclear Cells: A Pilot Study on the "Normobaric Oxygen Paradox".

The "normobaric oxygen paradox" (NOP) describes the response to the return to normoxia after a hyperoxic event, sensed by tissues as an oxygen shortage, up-regulating redox-sensitive transcription factors. We have previously characterized the time trend of oxygen-sensitive transcription factors in human PBMCs, in which the return to normoxia after 30% oxygen is sensed as a hypoxic trigger, characterized by hypoxia-induced factor (HIF-1) activation. On the contrary, 100% and 140% oxygen induce a shift toward an oxidative stress response, characterized by NRF2 and NF-kB activation in the first 24 h post exposure. Herein, we investigate whether this paradigm triggers Advanced Glycation End products (AGEs) and Advanced Oxidation Protein Products (AOPPs) as circulating biomarkers of oxidative stress. Secondly, we studied if mitochondrial biogenesis was involved to link the cellular response to oxidative stress in human PBMCs. Our results show that AGEs and AOPPs increase in a different manner according to oxygen dose. Mitochondrial levels of peroxiredoxin (PRX3) supported the cellular response to oxidative stress and increased at 24 h after mild hyperoxia, MH (30% O2), and high hyperoxia, HH (100% O2), while during very high hyperoxia, VHH (140% O2), the activation was significantly high only at 3 h after oxygen exposure. Mitochondrial biogenesis was activated through nuclear translocation of PGC-1α in all the experimental conditions. However, the consequent release of nuclear Mitochondrial Transcription Factor A (TFAM) was observed only after MH exposure. Conversely, HH and VHH are associated with a progressive loss of NOP response in the ability to induce TFAM expression despite a nuclear translocation of PGC-1α also occurring in these conditions. This study confirms that pulsed high oxygen treatment elicits specific cellular responses, according to its partial pressure and time of administration, and further emphasizes the importance of targeting the use of oxygen to activate specific effects on the whole organism.

Characteristics of peripheral blood mononuclear cells and potential related molecular mechanisms in patients with autoimmune hepatitis: a single-cell RNA sequencing analysis.

Autoimmune hepatitis (AIH) is an immune disorder characterized by hypergammaglobulinemia, autoantibodies, and chronic active hepatitis on liver histology. However, immune cell population characteristics in AIH patients remain poorly understood. This study was designed to analyze peripheral blood mononuclear cell (PBMC) characteristics in AIH through single-cell RNA sequencing (scRNA-seq) and explore potential AIH-related molecular mechanisms. We generated 3690 and 3511 single-cell transcriptomes of PBMCs pooled from 4 healthy controls (HCs) and 4 AIH patients, respectively, by scRNA-seq. These pooled PBMC transcriptomes were used for cell cluster identification and differentially expressed gene (DEG) identification. GO functional enrichment analysis was performed on the DEGs to determine the most active AIH immune cell biological functions. Although the PCA-based uniform manifold approximation and projection (UMAP) algorithm was used to cluster cells with similar expression patterns in the two samples, 87 up- and 12 downregulated DEGs were retained in monocytes and 101 up- and 15 downregulated DEGs were retained in NK cells from AIH PBMCs. Moreover, enriched GO terms in the PBMC-derived monocyte and NK cell clusters were related mainly to antigen processing and presentation, IFN-γ-mediated signaling, and neutrophil degranulation and activation. These potential molecular mechanisms may be important targets for AIH treatment.

Molecular Analysis and Ex Vivo Infectivity of Seronegative Occult Hepatitis C Virus: A Study in Single Haemodialysis Centre.

Background: In occult hepatitis C virus infection (OCI), hepatitis C virus ribonucleic acid (HCV RNA) is detectable in peripheral blood mononuclear cells (PBMCs) but is not evident in serum or plasma. Understanding of OCI in patients with seronegative anti-HCV antibodies is limited. Methods: In this study, six HCV isolates from haemodialysis (HD) patients with seronegative OCI were identified by molecular assays and phylogenetic analysis. The virus infectivity was assessed ex vivo using a primary naïve PBMC culture system. HCV isolates obtained from the PBMCs of 10 patients with chronic HCV infection (CCI) were characterised concurrently and used as positive controls in the cell culture. Results: Sequence analysis of the 5' untranslated region (UTR) and non-structural 5B (NS5B) region revealed that HCV genotype 3 was the most prevalent virus type in both the OCI and CCI groups. One of the occult HCV isolates was identified as a mixed type. The mean viral load (log10 RNA copies/106 cells) in the PBMC samples of the OCI group (M = 3.4, SD = 0.7) was lower than that of the CCI group (M = 4.6, SD = 1.7). Upon culture, de novo OCI-HCV replicates were detected in five out of six naïve PBMC cultures. Analysis of the replicates showed a single guanine addition in the domain III of 5'-UTR but the overall molecular structure was retained. Conclusion: Seronegative OCI is an active form of infection that replicates at a low level in PBMCs. Seronegative OCI may share the same route of transmission as CCI. The retained viral competency may have an implication for its persistence.