Comprehensive mapping of immune perturbations associated with aplastic anemia.

BACKGROUND: Aplastic anemia (AA) is an immune-mediated syndrome characterized by bone marrow failure. Therefore, comprehending the cellular profile and cell interactions in affected patients is crucial. METHODS: Human peripheral blood mononuclear cells (PBMCs) were collected from both healthy donors (HDs) and AA patients, and analyzed using multicolor flow cytometry. Utilizing the FlowSOM and t-SNE dimensionality reduction technique, we systematically explored and visualized the major immune cell alterations in AA. This analysis provided a foundation to further investigate the subtypes of cells exhibiting significant changes. RESULTS: Compared to HDs, peripheral blood from patients with AA exhibits a marked reduction in CD56Dim natural killer (NK) cells, which also show diminished functionality. Conversely, an increase in NK-like CD56+ monocytes, which possess compromised functionality. Along with a significant reduction in myeloid-derived suppressor cells (MDSCs), which show recovery post-treatment. Additionally, MDSCs serve as effective clinical markers for distinguishing between acquired aplastic anemia (AAA) and congenital aplastic anemia (CAA). Our comprehensive analysis of correlations among distinct immune cell types revealed significant associations between NKBri cells and CD8+ T cell subsets, as well as between NKDim cells and CD4+ T cells, these results highlight the intricate interactions and correlations within the immune cell network in AA. CONCLUSION: Our study systematically elucidates the pronounced immune dysregulation in patients with AA. The detailed mapping of the immune landscape not only provides crucial insights for basic research but also holds promise for enhancing the accuracy of diagnoses and the effectiveness of timely therapeutic interventions in clinical practice. Consequently, this could potentially reduce the high mortality rate associated with AA.

Defining the immunological compatibility of graphene oxide-loaded PLGA scaffolds for biomedical applications.

Graphene oxide (GO), a carbon-based nanomaterial, presents significant potential across biomedical fields such as bioimaging, drug delivery, biosensors, and phototherapy. This study examines the effects of integrating GO into poly(lactic-co-glycolic acid) (PLGA) scaffolds on human immune cell function. Our results demonstrate that high concentrations of GO reduce the viability of peripheral blood mononuclear cells (PBMCs) following stimulation with anti-CD3 antibody. This reduction extends to T lymphocyte activation, evident from the diminished proliferative response to T cell receptor engagement and impaired differentiation into T helper subsets and regulatory T cells. Interestingly, although GO induces a minimal response in resting monocytes, but it significantly affects both the viability and the differentiation potential of monocytes induced to mature toward M1 pro-inflammatory and M2-like immunoregulatory macrophages. This study seeks to address a critical gap by investigating the in vitro immunomodulatory effects of PLGA scaffolds incorporating various concentrations of GO on primary immune cells, specifically PBMCs isolated from healthy donors. Our findings emphasize the need to optimize the GO to PLGA ratios and scaffold design to advance PLGA-GO-based biomedical applications. STATEMENT OF SIGNIFICANCE: Graphene oxide (GO) holds immense promise for biomedical applications due to its unique properties. However, concerns regarding its potential to trigger adverse immune responses remain. This study addresses this critical gap by investigating the in vitro immunomodulatory effects of PLGA scaffolds incorporating increasing GO concentrations on human peripheral blood mononuclear cells (PBMCs). By elucidating the impact on cell viability, T cell proliferation and differentiation, and the maturation/polarization of antigen-presenting cells, this work offers valuable insights for designing safe and immunologically compatible GO-based biomaterials for future clinical translation.

A novel cystatin in Psoroptes ovis var. cuniculi: molecular characterization, serodiagnostic potential, and its anti-inflammatory property on rabbit peripheral blood mononuclear cells.

BACKGROUND: The ectoparasite Psoroptes ovis var. cuniculi causes substantial economic losses to the global rabbit industry. Currently, microscopy for identifying Psoroptes mite in skin scrapings, as the "diagnosis gold standard," remains a challenge owing to its poor sensitivity in detecting low-level and/or early stage mite infestations. Additionally, Psoroptes infestations rapidly trigger cutaneous inflammation, thus the mites might produce some molecules to deal with the harmful effects of inflammation for their long-time survival on the host skin, but these molecules are still mostly unknown. METHODS: To seek a sensitive diagnostic method and illuminate the new antiinflammatory molecules, we characterized a novel cystatin of P. ovis var. cuniculi (PsoCys) using bioinformatics and molecular biology methods. RESULTS: The results showed that PsoCys comprised the classical features of the type II cystatin superfamily including an N-terminal glycine residue, a central QXVXG motif, and a C-terminal LW motif. In mixed stages of mites, the transcription level of PsoCys was significantly higher in "fed" mites than in "starved" mites (P < 0.001), and among the different life-cycle stages of "fed" mites, the expression of PsoCys was higher in adult males than in larva, nymph, and adult females (P < 0.001). The established indirect ELISA based on recombinant PsoCys (rPsoCys-iELISA) presented 95.4% sensitivity and 95.7% specificity. The area under the receiver operating characteristic curve (AUC) for this method was 0.991, indicating its excellent diagnostic performance. Moreover, rPsoCys-iELISA had advantages over microscopy for detecting low-level and/or early stage mite infestations (90% versus 40% in artificial infestation cases at 3 weeks post-infestation; 61.9% versus 22.6% in clinical cases). In addition, rPsoCys could inhibit the activity of papain and cathepsin B in vitro, and significantly suppressed mRNA levels of toll-like receptors (TLR 1, 2, 4, and 6) and downstream molecules (NF-κB, p38, MyD88, IL-10, and IFN-γ) in LPS-stimulated rabbit PBMCs, indicating its anti-inflammatory property. CONCLUSIONS: Our findings indicated that PsoCys was a novel type II cystatin of Psoroptes mites, and it served as a potential serological diagnostic antigen for detecting low-level and/or early stage mite infestations, as well as a novel anti-inflammatory molecule of Psoroptes mites.

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.

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.

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.

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.

Development and characterization of dendritic cell internalization and activation assays contributing to the immunogenicity risk evaluation of biotherapeutics.

Introduction: Immunogenicity refers to the ability of a substance, such as a therapeutic drug, to elicit an immune response. While beneficial in vaccine development, undesirable immunogenicity can compromise the safety and efficacy of therapeutic proteins by inducing anti-drug antibodies (ADAs). These ADAs can reduce drug bioavailability and alter pharmacokinetics, necessitating comprehensive immunogenicity risk assessments starting at early stages of drug development. Given the complexity of immunogenicity, an integrated approach is essential, as no single assay can universally recapitulate the immune response leading to the formation of anti-drug antibodies. Methods: To better understand the Dendritic Cell (DC) contribution to immunogenicity, we developed two flow cytometry-based assays: the DC internalization assay and the DC activation assay. Monocyte-derived dendritic cells (moDCs) were generated from peripheral blood mononuclear cells (PBMCs) and differentiated over a five-day period. The internalization assay measured the accumulation rate of therapeutic antibodies within moDCs, while the activation assay assessed the expression of DC activation markers such as CD40, CD80, CD86, CD83, and DC-SIGN (CD209). To characterize these two assays further, we used a set of marketed therapeutic antibodies. Results: The study highlights that moDCs differentiated for 5 days from freshly isolated monocytes were more prone to respond to external stimuli. The internalization assay has been shown to be highly sensitive to the molecule tested, allowing the use of only 4 donors to detect small but significant differences. We also demonstrated that therapeutic antibodies were efficiently taken up by moDCs, with a strong correlation with their peptide presentation on MHC-II. On the other hand, by monitoring DC activation through a limited set of activation markers including CD40, CD83, and DC-SIGN, the DC activation assay has the potential to compare a series of compounds. These two assays provide a more comprehensive understanding of DC function in the context of immunogenicity, highlighting the importance of both internalization and activation processes in ADA development. Discussion: The DC internalization and activation assays described here address key gaps in existing immunogenicity assessment methods by providing specific and reliable measures of DC function. The assays enhance our ability to pre-clinically evaluate the immunogenic potential of biotherapeutics, thereby improving their safety and efficacy. Future work should focus on further validating these assays and integrating them into a holistic immunogenicity risk assessment framework.

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.

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.

Surface Immune Checkpoints as Potential Biomarkers in Physiological Pregnancy and Recurrent Pregnancy Loss.

Due to the genetic diversity between the mother and the fetus, heightened control over the immune system during pregnancy is crucial. Immunological parameters determined by clinicians in women with idiopathic recurrent spontaneous abortion (RSA) include the quantity and activity of Natural Killer (NK) and Natural Killer T (NKT) cells, the quantity of regulatory T lymphocytes, and the ratio of pro-inflammatory cytokines, which indicate imbalances in Th1 and Th2 cell response. The processes are controlled by immune checkpoint proteins (ICPs) expressed on the surface of immune cells. We aim to investigate differences in the expression of ICPs on T cells, T regulatory lymphocytes, NK cells, and NKT cells in peripheral blood samples collected from RSA women, pregnant women, and healthy multiparous women. We aim to discover new insights into the role of ICPs involved in recurrent pregnancy loss. Peripheral blood mononuclear cells (PBMCs) were isolated by gradient centrifugation from blood samples obtained from 10 multiparous women, 20 pregnant women (11-14th week of pregnancy), and 20 RSA women, at maximum of 72 h after miscarriage. The PBMCs were stained for flow cytometry analysis. Standard flow cytometry immunophenotyping of PBMCs was performed using antibodies against classical lymphocyte markers, including CD3, CD4, CD8, CD56, CD25, and CD127. Additionally, ICPs were investigated using antibodies against Programmed Death Protein-1 (PD-1, CD279), T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3, CD366), V-domain Ig suppressor of T cell activation (VISTA), T cell immunoglobulin and ITIM domain (TIGIT), and Lymphocyte activation gene 3 (LAG-3). We observed differences in the surface expression of ICPs in the analyzed subpopulations of lymphocytes between early pregnancy and RSA, after miscarriage, and in women. We noted diminished expression of PD-1 on T lymphocytes (p = 0.0046), T helper cells (CD3CD4 positive cells, p = 0.0165), T cytotoxic cells (CD3CD8 positive cells, p = 0.0046), T regulatory lymphocytes (CD3CD4CD25CD127 low positive cells, p = 0.0106), and NKT cells (CD3CD56/CD16 positive cells, p = 0.0438), as well as LAG-3 on lymphocytes T (p = 0.0225) T helper, p = 0.0426), T cytotoxic cells (p = 0.0458) and Treg (p = 0.0293), and cells from RSA women. Impaired expression of TIM-3 (p = 0.0226) and VISTA (p = 0.0039) on CD8 cytotoxic T and NK (TIM3 p = 0.0482; VISTA p = 0.0118) cells was shown, with an accompanying increased expression of TIGIT (p = 0.0211) on NKT cells. The changes in the expression of surface immune checkpoints indicate their involvement in the regulation of pregnancy. The data might be utilized to develop specific therapies for RSA women based on the modulation of ICP expression.

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.

Mesothelin- and nucleolin-specific T cells from combined short peptides effectively kill triple-negative breast cancer cells.

BACKGROUND: Triple-negative breast cancer (TNBC), known for its aggressiveness and limited treatment options, presents a significant challenge. Adoptive cell transfer, involving the ex vivo generation of antigen-specific T cells from peripheral blood mononuclear cells (PBMCs), emerges as a promising approach. The overexpression of mesothelin (MSLN) and nucleolin (NCL) in TNBC samples underscores their potential as targets for T cell therapy. This study explored the efficacy of multi-peptide pulsing of PBMCs to generate MSLN/NCL-specific T cells targeting MSLN+/NCL+ TNBC cells. METHODS: TNBC patient samples were confirmed for both MSLN and NCL expression via immunohistochemistry. Synthesized MSLN and NCL peptides were combined and administered to activate PBMCs from healthy donors. The cancer-killing ability of the resultant T cells was assessed using crystal violet staining, and their subtypes and cytotoxic cytokines were characterized through flow cytometry and cytokine bead array. RESULTS: Findings showed that 85.3% (127/149) of TNBC cases were positive for either MSLN or NCL, or both; with single positivity rates for MSLN and NCL of 14.1% and 28.9%, respectively. MSLN and NCL peptides, with high binding affinity for HLA-A*02, were combined and introduced to activated PBMCs from healthy donors. The co-pulsed PBMCs significantly induced TEM and TEMRA CD3+/CD8+ T cells and IFN-γ production, compared to single-peptide pulsed or unpulsed conditions. Notably, MSLN/NCL-specific T cells successfully induced cell death in MSLN+/NCL+ MDA-MB-231 cells, releasing key cytotoxic factors such as perforin, granzymes A and B, Fas ligand, IFN-γ, and granulysin. CONCLUSIONS: These findings serve as a proof-of-concept for using multiple immunogenic peptides as a novel therapeutic approach in TNBC patients.

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.

Dynamic changes in immune cells in humanized liver metastasis and subcutaneous xenograft mouse models.

Preclinical drug efficacy and tumor microenvironment (TME) investigations often utilize humanized xenograft mouse models, yet these models typically fall short in replicating the intricate TME. We developed a humanized liver metastasis (LM) model by transplanting human peripheral blood mononuclear cells (PBMCs) and assessed it against the conventional subcutaneous (SC) xenograft model, focusing on immune cell dynamics post-transplantation and immunotherapy response. NOD-scid IL2Rgammanull(NSG) were inoculated with PBMCs to create humanized models. We induced SC and LM models using HCT116 cells, to investigate and compare the distributions and transformations of immune cell subsets, respectively. Both models were subjected to anti-PD-L1 therapy, followed by an analysis the TME analysis. The LM model demonstrated enhanced central tumor infiltration by tumor-infiltrating lymphocytes (TILs) compared to the peripheral pattern of SC model. TIL subpopulations in the LM model showed a progressive increase, contrasting with an initial rise and subsequent decline in the SC model. Post-anti-PD-L1 therapy, the LM model exhibited a significant rise in central and effector memory T cells, a response absents in the SC model. Our study highlights differential TME responses between SC and LM models and introduces a robust humanized LM model that swiftly indicates the potential efficacy of immunotherapies. These insights could streamline the preclinical evaluation of TME-targeting immunotherapeutic agents.

Cloning and Functional Characterization of Novel Human Neutralizing Anti-IFN-α and Anti-IFN-ß Antibodies.

Type I IFNs play a pivotal role in immune response modulation, yet dysregulation is implicated in various disorders. Therefore, it is crucial to develop tools that facilitate the understanding of their mechanism of action and enable the development of more effective anti-IFN therapeutic strategies. In this study, we isolated, cloned, and characterized anti-IFN-α and anti-IFN-ß Abs from PBMCs of individuals treated with IFN-α or IFN-ß, harboring confirmed neutralizing Abs. Clones AH07856 and AH07857 were identified as neutralizing anti-IFN-α-specific with inhibition against IFN-α2a, -α2b, and -αK subtypes. Clones AH07859 and AH07866 were identified as neutralizing anti-IFN-ß1a-specific signaling and able to block lipopolysaccharide or S100 calcium-binding protein A14-induced IFN-ß signaling effects. Cloned Abs bind rhesus but not murine IFNs. The specificity of inhibition between IFN-α and IFN-ß suggests potential for diverse research and clinical applications.

Dexmedetomidine induces IL-10 secretion by B lymphocytes in the peripheral blood of patients with hepatocellular carcinoma.

BACKGROUND/AIM: To investigate the distribution of subpopulations of peripheral blood B lymphocytes in individuals with hepatocellular carcinoma (HCC), and to evaluate the effect of dexmedetomidine (DEX) on B lymphocyte differentiation in patients with HCC in vitro. METHODS: Peripheral blood mononuclear cells (PBMCs) were collected from the HCC group and the healthy group, and the distribution of peripheral blood B-lymphocyte subpopulations in the two groups was examined by Flow Cytometry (FCM). B lymphocytes extracted from the peripheral blood of the HCC group were divided into D0, D1, D2 and D4 groups according to the different dose of DEX in the culture medium (0 µM, 1 µM, 2 µM and 4 µM). After 72 h of in vitro culture, FCM was used to detect differences in the percentage of apoptotic B lymphocytes and the percentage of B lymphocytes that can express interleukin 10(IL-10) and transforming growth factor-ß (TGF-ß) in each group. RESULTS: In contrast to the healthy group, the HCC group exhibited a statistically significant increase in the proportion of CD19 + CD73 + B lymphocyte subpopulation (P<0.05). In the in vitro culture experiment, the differences in apoptosis of B lymphocytes and the percentage of TGF-ß expression in each group were not statistically significant; When compared to the control group, there was a significant increase in the percentage of B lymphocytes expressing IL-10 across the D1, D2, and D4 groups (P<0.05). CONCLUSION: The peripheral blood of HCC patients is characterized by an elevated presence of CD19 + CD73 + B lymphocyte subpopulations; DEX may have an immunosuppressive effect by promoting IL-10 secretion from peripheral blood B lymphocytes of HCC patients.

RORα negatively regulates BCG-induced trained immunity.

Trained immunity is a long-lasting change in the responsiveness of innate immune cells, leading to a stronger response upon an unrelated secondary challenge. Epigenetic, transcriptional, and metabolic reprogramming contribute to the development of trained immunity. By investigating the impact of gene variants on trained immunity responses after Bacillus Calmette-Guérin (BCG) vaccination, we identified a strong association between polymorphisms in the RORA gene and BCG-induced trained immunity in PBMCs isolated from healthy human donors. RORα, encoded by the RORA gene in humans, is a nuclear receptor and a transcription factor, regulating genes involved in circadian rhythm, inflammation, cholesterol, and lipid metabolism. We found that natural RORα agonists in the circulation negatively correlate with the strength of trained immunity responses after BCG vaccination. Moreover, pharmacological inhibition of RORα in human PBMCs led to higher cytokine production capacity and boosted trained immunity induction by BCG. Blocking RORα activity also resulted in morphological changes and increased ROS and lactate production of BCG-trained cells. Blocking lactate dehydrogenase A (LDHA) and glycolysis with sodium oxamate reduced the cytokine production capacity of cells trained with a combination of BCG and the RORα agonist. In conclusion, this study highlights the potential role of RORα in trained immunity, and its impact on human vaccination and diseases should be further investigated.

Genomic insights into pediatric intestinal inflammatory and eosinophilic disorders using single-cell RNA-sequencing.

Introduction: Chronic inflammation of the gastrointestinal tissues underlies gastrointestinal inflammatory disorders, leading to tissue damage and a constellation of painful and debilitating symptoms. These disorders include inflammatory bowel diseases (Crohn's disease and ulcerative colitis), and eosinophilic disorders (eosinophilic esophagitis and eosinophilic duodenitis). Gastrointestinal inflammatory disorders can often present with overlapping symptoms necessitating the use of invasive procedures to give an accurate diagnosis. Methods: This study used peripheral blood mononuclear cells from individuals with Crohn's disease, ulcerative colitis, eosinophilic esophagitis, and eosinophilic duodenitis to better understand the alterations to the transcriptome of individuals with these diseases and identify potential markers of active inflammation within the peripheral blood of patients that may be useful in diagnosis. Single-cell RNA-sequencing was performed on peripheral blood mononuclear cells isolated from the blood samples of pediatric patients diagnosed with gastrointestinal disorders, including Crohn's disease, ulcerative colitis, eosinophilic esophagitis, eosinophilic duodenitis, and controls with histologically healthy gastrointestinal tracts. Results: We identified 730 (FDR < 0.05) differentially expressed genes between individuals with gastrointestinal disorders and controls across eight immune cell types. Discussion: There were common patterns among GI disorders, such as the widespread upregulation of MTRNR2L8 across cell types, and many differentially expressed genes showed distinct patterns of dysregulation among the different gastrointestinal diseases compared to controls, including upregulation of XIST across cell types among individuals with ulcerative colitis and upregulation of Th2-associated genes in eosinophilic disorders. These findings indicate both overlapping and distinct alterations to the transcriptome of individuals with gastrointestinal disorders compared to controls, which provide insight as to which genes may be useful as markers for disease in the peripheral blood of patients.

Immunological insights: assessing immune parameters in medical professionals exposed to SARS-CoV-2.

BACKGROUND: The immunological background responsible for the severe course of COVID-19 and the immune factors that protect against SARS-CoV-2 infection are still unclear. The aim of this study was to investigate immune system status in persons with high exposure to SARS-CoV-2 infection. METHODS: Seventy-one persons employed in the observation and infectious diseases unit were qualified for the study between November 2020 and October 2021. Symptomatic COVID-19 was diagnosed in 35 persons. Anti-SARS-CoV-2 antibodies were also found in 8 persons. Peripheral blood mononuclear cells subpopulations were analyzed by flow cytometry, and the concentrations of cytokines and anti-SARS-CoV-2 antibodies were determined by ELISA. RESULTS: The percentages of cytotoxic T lymphocytes (CTLs), CD28+ and T helper (Th) cells with invariant T-cell receptors were significantly higher in persons with symptomatic COVID-19 than in those who did not develop COVID-19' symptoms. Conversely, symptomatic COVID-19 persons had significantly lower percentages of: a) CTLs in the late stage of activation (CD8+/CD95+), b) NK cells, c) regulatory-like Th cells (CD4+/CTLA-4+), and d) Th17-like cells (CD4+/CD161+) compared to asymptomatic COVID-19' persons. Additionally, persons with anti-SARS-CoV-2 antibodies had a significantly higher lymphocyte count and IL-6 concentration than persons without these antibodies. CONCLUSION: Numerous lymphocyte populations are permanently altered by SARS-CoV-2 infection. High percentages of both populations: NK cells-as a part of the non-specific response, and T helper cells' as those regulating the immune response, could protect against the acute COVID-19 symptoms development. Understanding the immune background of COVID-19 may improve the prevention of this disease by identifying people at risk of a severe course of infection. TRIAL REGISTRATION: This is a retrospective observational study without a trial registration number.