Peripheral Blood Mononuclear Cells Research Articles

Association of Human Endogenous Retrovirus-W (HERV-W) Copies with Pemphigus Vulgaris

Pemphigus is classified as a group of chronic, recurrent, and potentially fatal bullous autoimmune diseases that leads to blisters and skin lesions resulting from IgG antibodies and the loss of cellular connections in the epidermis. Human endogenous retrovirus (HERV) sequences and their products (RNA, cytosolic DNA, and proteins) can modulate the immune system and contribute to autoimmunity. The extent to which, HERV-W env copies may be involved in the pathogenesis of pemphigus remains to be elucidated. This study aimed to comparatively evaluate the relative levels of HERV-W env DNA copy numbers in the peripheral blood mononuclear cells (PBMCs) of pemphigus vulgaris patients and healthy controls. Thirty-one pemphigus patients and the corresponding age- and sex-matched healthy controls were included in the study. The relative levels of HERV-W env DNA copy numbers were then evaluated by qPCR using specific primers, in the PBMCs of the patients and controls. Our results indicated that relative levels of HERV-W env DNA copy numbers in the patients were significantly higher than that in the controls (1.67±0.86 vs. 1.17±0.75; p = 0.02). There was also a significant difference between the HERV-W env copies of male and female patients (p = 0.001). Furthermore, there was no relationship between the HERV-W env copy number and disease onset (p = 0.19). According to the obtained data, we could not find any relationship between the HERV-W env copy number and serum Dsg1(p=0.86) and Dsg3 (p=0.76) levels. Our results indicated a positive link between the HERV-W env copies and pathogenesis of pemphigus. The association between clinical severity score and HERV-W env copies in the PBMCs as a biomarker for pemphigus needs further studies.

Mass cytometry reveals atypical immune profile notably impaired maturation of memory CD4 T with Gb3-related CD27 expression in CD4 T cells in Fabry disease.

Fabry disease (FD) is an X-linked disease characterized by an accumulation of glycosphingolipids, notably of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lysoGb3) leading to renal failure, cardiomyopathy, and cerebral strokes. Inflammatory processes are involved in the pathophysiology. We investigated the immunological phenotype of peripheral blood mononuclear cells in Fabry patients depending on the clinical phenotype, treatment, Gb3, and lysoGb3 levels and the presence of anti-drug antibodies (ADA). Leucocytes from 41 male patients and 20 controls were analyzed with mass cytometry using both unsupervised and supervised algorithms. FD patients had an increased expression of CD27 and CD28 in memory CD45- and CD45 + CCR7-CD4 T cells (respectively p < 0.014 and p < 0.02). Percentage of CD45RA-CCR7-CD27 + CD28+ cells in CD4 T cells was correlated with plasma lysoGb3 (r = 0.60; p = 0.0036) and phenotype (p < 0.003). The correlation between Gb3 and CD27 in CD4 T cells almost reached significance (r = 0.33; p = 0.058). There was no immune profile associated with the presence of ADA. Treatment with agalsidase beta was associated with an increased proportion of Natural Killer cells. These findings provide valuable insights for understanding FD, linking Gb3 accumulation to inflammation, and proposing new prognostic biomarkers.

Radioprotective Effects of Thymus Vulgaris L. Essential Oil on Human Peripheral Blood Mononuclear Cells

Purpose: Generated free radicals by ionizing radiations, as powerful cytotoxic agents, can damage DNA and proteins. Thymus vulgaris L (thyme) plant is a rich source of antioxidant phenolic compounds, which makes it a preferable candidate for medical applications. Given this, we set out the present study to investigate the effectiveness of thyme essential oil on Human Peripheral Blood Mononuclear Cells (PBMCs) as a radioprotector agent against ionizing radiations.&#x0D; Materials and Methods: We extracted the thyme essential oil by the conventional Clevenger extraction method. Heparinized peripheral blood samples were also collected from five male volunteers, aged 22-25, without a history of smoking and irradiation. PBMCs were isolated and the maximum nontoxic concentrations (85µg/ml (of thyme essential oil were determined based on the result of the MTT method. In the next step, the PBMCs were cultured in the presence of thyme essential oil before and after X-irradiation with doses of 0.25 and 2.00 Gy.&#x0D; Results: The most radioprotective effect was observed in the dose of 2.00 Gy for thyme-treated cells 24 hours before the irradiation (p-value ≤ 0.001) by a survival enhancement factor of 1.67, compared to the control group.&#x0D; Conclusion: Our results showed that thyme essential oil can be used as an effective radioprotector agent for PBMCs against ionizing radiations. The most radioprotective effect was observed in the presence of thyme essential oil during irradiation.

Molecular-Level Dysregulation of Insulin Pathways and Inflammatory Processes in Peripheral Blood Mononuclear Cells by Circadian Misalignment.

Circadian misalignment due to night work has been associated with an elevated risk for chronic diseases. We investigated the effects of circadian misalignment using shotgun protein profiling of peripheral blood mononuclear cells taken from healthy humans during a constant routine protocol, which was conducted immediately after participants had been subjected to a 3-day simulated night shift schedule or a 3-day simulated day shift schedule. By comparing proteomic profiles between the simulated shift conditions, we identified proteins and pathways that are associated with the effects of circadian misalignment and observed that insulin regulation pathways and inflammation-related proteins displayed markedly different temporal patterns after simulated night shift. Further, by integrating the proteomic profiles with previously assessed metabolomic profiles in a network-based approach, we found key associations between circadian dysregulation of protein-level pathways and metabolites of interest in the context of chronic metabolic diseases. Endogenous circadian rhythms in circulating glucose and insulin differed between the simulated shift conditions. Overall, our results suggest that circadian misalignment is associated with a tug of war between central clock mechanisms controlling insulin secretion and peripheral clock mechanisms regulating insulin sensitivity, which may lead to adverse long-term outcomes such as diabetes and obesity. Our study provides a molecular-level mechanism linking circadian misalignment and adverse long-term health consequences of night work.

SARS-Cov-2 spike induces intestinal barrier dysfunction through the interaction between CEACAM5 and Galectin-9

BackgroundCarcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), as a typical tumor marker, has been found to exert immunomodulatory effects in many diseases. We previously reported the clinical and molecular evidences supporting that SARS-Cov-2 infected the gastrointestinal (GI) tract and found a reduction of CEACAM5 in COVID-19 patients’ feces which associated with gut dysbiosis. Yet the role of CEACAM5 in GI infection is ill-defined.MethodsMice models were established through intraperitoneally injecting with recombinant viral spike-Fc to mimic the intestinal inflammation. We collected duodenum, jejunum, ileum and colon samples after 6h, 2 days, 4 days and 7 days of spike-Fc or control-Fc injection to perform proteomic analysis. Blood was collected from healthy donors and peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation, then CD4+ T cells were isolated with magnetic beads and co-cultured with Caco-2 cells.ResultsIn addition to intestinal CEACAM5, the expression of tight junction and the percent of CD4+ T lymphocytes were significantly decreased in spike-Fc group compared to control (p &amp;lt; 0.05), accompanied with increased level of inflammatory factors. The KEGG analysis revealed differentially expressed proteins were mainly enriched in the coronavirus disease (COVID-19), tight junction, focal adhesion, adherens junction and PI3K-Akt signaling pathway. Protein–protein interaction (PPI) network analysis identified the interaction between CEACAM5 and Galectin-9 that was also verified by molecular docking and co-IP assay. We further confirmed a reduction of CEACAM5 in SARS-CoV-2 spike stimulated enterocytes could promote the expression of Galectin-9 protein in CD4+T cells. Then it gave rise to the increasing release of inflammatory factors and increased apoptosis of CD4+T cells by inhibition of PI3K/AKT/mTOR pathway. Ultimately intestinal barrier dysfunction happened.ConclusionOur results indicated that CEACAM5 overexpression and Galectin-9 knockdown played a protective role in intestinal barrier injury upon spike-Fc stimulation. Collectively, our findings identified firstly that SARS-CoV-2 spike induced intestinal barrier dysfunction through the interaction between CEACAM5 and Galectin-9. The result provides potential therapeutic targets in intestinal barrier dysfunction for treating severe COVID patients.

Abstract P69: Immune Landscape of BRCA1/2 -mutated Breast and Ovarian Cancers in an Asian Cohort

Abstract Background: The majority of hereditary breast and ovarian cancers (HBOC) harbor defects in the homologous recombination (HR) DNA repair pathway, such as BRCA1 and BRCA2 mutations, which may influence the response to immune checkpoint blockade (ICB) therapy. Recent clinical trials combining poly-ADP ribose inhibitors and ICB therapy in advanced breast and ovarian cancer have shown promising results. This Asian cohort study set out to determine if immune phenotype, specifically the PD-1/PD-L1 axis, was associated with germline BRCA status in advanced breast and ovarian cancer patients and to develop novel biomarkers of HR deficiency to stratify patients for clinical management. Methods: Tissue microarrays of ovarian tumors and breast tumors were analyzed by multiplex immunohistochemistry (mIHC) to detect the expression of immune markers (CD68, CD8, PD-L1, PD-1) and HR markers (DNA polymerase theta, FANCD2, RAD51, γH2AX, phospho-ATR). The results were used to build a prediction model for BRCA using backward stepwise logistic regression analysis. In addition, we investigated immune infiltration in tumors and performed immune phenotyping on peripheral blood mononuclear cells in a subset of patients. Results: We identified potential BRCA1/2 predictors by mIHC, namely RAD51 and PD-L1 263 for ovarian tumors and FANCD2 and phospho-ATR for breast tumors. The area under the curve was 0.770 (95% confidence interval [CI]: 0.564, 0.977) in ovarian cancer patients and 0.841 (95% CI: 0.682, 0.999) in breast cancer patients. In addition, the proportion of tumor-infiltrating CD68+PD-L1+ macrophages was significantly upregulated in BRCA1-mutated tumors, while the levels of PD-1 in circulating central memory CD8+ T cells were higher in breast cancer patients with germline BRCA2 mutations, compared with non-BRCA. Conclusion: This study provides evidence of the association of the PD-1/PD-L1 axis with germline BRCA status in HBOC, which may aid clinical management. Citation Format: Siao Ting Chong, Felicia Wee, Jiang Feng Ye, Craig Joseph, Xinru Lim, Zhen Wei Neo, Justina Nadia Lee, Nur Diana Binte Ishak, Ming Ren Toh, Ee Ling Chew, Claresta Yeo, Sock Hoai Chan, Jeffrey Lim, Joe Yeong, Joanne Ngeow. Immune Landscape of BRCA1/2 -mutated Breast and Ovarian Cancers in an Asian Cohort [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P69.

Establishment of a humanized mouse model using steady‐state peripheral blood‐derived hematopoietic stem and progenitor cells facilitates screening of cancer‐targeted T‐cell repertoires

AbstractBackgroundCancer‐targeted T‐cell receptor T (TCR‐T) cells hold promise in treating cancers such as hematological malignancies and breast cancers. However, approaches to obtain cancer‐reactive TCR‐T cells have been unsuccessful.MethodsHere, we developed a novel strategy to screen for cancer‐targeted TCR‐T cells using a special humanized mouse model with person‐specific immune fingerprints. Rare steady‐state circulating hematopoietic stem and progenitor cells were expanded via three‐dimensional culture of steady‐state peripheral blood mononuclear cells, and then the expanded cells were applied to establish humanized mice. The human immune system was evaluated according to the kinetics of dendritic cells, monocytes, T‐cell subsets, and cytokines. To fully stimulate the immune response and to obtain B‐cell precursor NAML‐6‐ and triple‐negative breast cancer MDA‐MB‐231‐targeted TCR‐T cells, we used the inactivated cells above to treat humanized mice twice a day every 7 days. Then, human T cells were processed for TCR β‐chain (TRB) sequencing analysis. After the repertoires had been constructed, features such as the fraction, diversity, and immune signature were investigated.ResultsThe results demonstrated an increase in diversity and clonality of T cells after treatment. The preferential usage and features of TRBV, TRBJ, and the V–J combination were also changed. The stress also induced highly clonal expansion. Tumor burden and survival analysis demonstrated that stress induction could significantly inhibit the growth of subsequently transfused live tumor cells and prolong the survival of the humanized mice.ConclusionsWe constructed a personalized humanized mouse model to screen cancer‐targeted TCR‐T pools. Our platform provides an effective source of cancer‐targeted TCR‐T cells and allows for the design of patient‐specific engineered T cells. It therefore has the potential to greatly benefit cancer treatment.

Collagen-polyurethane-dextran hydrogels enhance wound healing by inhibiting inflammation and promoting collagen fibrillogenesis.

Diabetic foot ulcers are a serious complication of uncontrolled diabetes, emphasizing the need to develop wound healing strategies that are not only effective but also biocompatible, biodegradable, and safe. We aimed to create biomatrices composed of semi-interpenetrated polymer networks of collagen, polyurethane, and dextran, to enhance the wound healing process. The hydrogels were extensively characterized by various analytical techniques, including analysis of their structure, crystallinity, thermal properties, gelation process, reticulation, degradation, cell proliferation, and healing properties, among others. Semi-interpenetrated hydrogels containing dextran at levels of 10%, 20%, and 30% exhibited porous interconnections between collagen fibers and entrapped dextran granules, with a remarkable crosslinking index of up to 94% promoted by hydrogen bonds. These hydrogels showed significant improvements in mechanical properties, swelling, and resistance to proteolytic and hydrolytic degradation. After 24 h, there was a significant increase in the viability of several cell types, including RAW 264.7 cells, human peripheral blood mononuclear cells, and dermal fibroblasts. In addition, these hydrogels demonstrated an increased release of interleukin-10 and transforming growth factor-beta1 while inhibiting the release of monocyte chemotactic protein-1 and tumor necrosis factor-alpha after 72 h. Furthermore, these hydrogels accelerated the wound healing process in diabetic rats after topical application. Notably, the biomaterial with 20% dextran (D20) facilitated wound closure in only 21 days. These results highlight the potential of the D20 hydrogel, which exhibits physicochemical and biological properties that enhance wound healing by inhibiting inflammation and fibrillogenesis while remaining safe for application to the skin.

Assessing the Feasibility and Efficacy of Pre-Sleep Dim Light Therapy for Adults with Insomnia: A Pilot Study

Background: Insomnia is increasingly recognized for its marked impact on public health and is often associated with various adverse health outcomes, including cardiovascular diseases and mental health disorders. The aim of this study was to investigate the efficacy of pre-sleep dim light therapy (LT) as a non-pharmacological intervention for insomnia in adults, assessing its influence on sleep parameters and circadian rhythms. Methods: A randomized, open-label, two-arm clinical trial was conducted over two weeks with 40 participants aged 20–60 years, all of whom had sleep disorders (CRIS, KCT0008501). They were allocated into control and LT groups. The LT group received exposure to warm-colored light, minimizing the blue spectrum, before bedtime. The study combined subjective evaluation via validated, sleep-related questionnaires, objective sleep assessments via actigraphy, and molecular analyses of circadian clock gene expression in peripheral blood mononuclear cells. Baseline characteristics between the two groups were compared using an independent t-test for continuous variables and the chi-squared test for categorical variables. Within-group differences were assessed using the paired t-test. Changes between groups were analyzed using linear regression, adjusting for each baseline value and body mass index. The patterns of changes in sleep parameters were calculated using a linear mixed model. Results: The LT group exhibited significant improvements in sleep quality (difference in difference [95% CI]; −2.00 [−3.58, −0.43], and sleep efficiency (LT: 84.98 vs. control: 82.11, p = 0.032), and an advanced Dim Light Melatonin Onset compared to the control group (approximately 30 min). Molecular analysis indicated a significant reduction in CRY1 gene expression after LT, suggesting an influence on circadian signals for sleep regulation. Conclusions: This study provides evidence for the efficacy of LT in improving sleep quality and circadian rhythm alignment in adults with insomnia. Despite limitations, such as a small sample size and short study duration, the results underscore the potential of LT as a viable non-pharmacological approach for insomnia. Future research should expand on these results with larger and more diverse cohorts followed over a longer period to validate and further elucidate the value of LT in sleep medicine. Trial registration: The trial was registered with the Clinical Research Information Service (KCT0008501).

Programmed Death 1 and Cytotoxic T-Lymphocyte-Associated Protein 4 Gene Expression in Peripheral Blood Mononuclear Cells Can Serve as Prognostic Biomarkers for Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a highly aggressive form of liver cancer with poor prognosis. The lack of reliable biomarkers for early detection and accurate diagnosis and prognosis poses a significant challenge to its effective clinical management. In this study, we investigated the diagnostic and prognostic potential of programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expression in peripheral blood mononuclear cells (PBMCs) in HCC. PD-1 and CTLA-4 gene expression was analyzed comparatively using PBMCs collected from HCC patients and healthy individuals. The results revealed higher PD-1 gene expression levels in patients with multifocal tumors, lymphatic invasion, or distant metastasis than those in their control counterparts. However, conventional serum biomarkers of liver function do not exhibit similar correlations. In conclusion, PD-1 gene expression is associated with OS and PFS and CTLA-4 gene expression is associated with OS, whereas the serum biomarkers analyzed in this study show no significant correlation with survival in HCC. Hence, PD-1 and CTLA-4 expressed in PBMCs are considered potential prognostic biomarkers for patients with HCC that can facilitate prediction of malignancy, response to currently available HCC treatments, and overall survival.

Monocytic myeloid-derived suppressor cells as an immune indicator of early diagnosis and prognosis in patients with sepsis

BackgroundImmunosuppression is a leading cause of septic death. Therefore, it is necessary to search for biomarkers that can evaluate the immune status of patients with sepsis. We assessed the diagnostic and prognostic value of low-density neutrophils (LDNs) and myeloid-derived suppressor cells (MDSCs) subsets in the peripheral blood mononuclear cells (PBMCs) of patients with sepsis.MethodsLDNs and MDSC subsets were compared among 52 inpatients with sepsis, 33 inpatients with infection, and 32 healthy controls to investigate their potential as immune indicators of sepsis. The percentages of LDNs, monocytic MDSCs (M-MDSCs), and polymorphonuclear MDSCs (PMN-MDSCs) in PBMCs were analyzed. Sequential organ failure assessment (SOFA) scores, C-reactive protein (CRP), and procalcitonin (PCT) levels were measured concurrently.ResultsThe percentages of LDNs and MDSC subsets were significantly increased in infection and sepsis as compared to control. MDSCs performed similarly to CRP and PCT in diagnosing infection or sepsis. LDNs and MDSC subsets positively correlated with PCT and CRP levels and showed an upward trend with the number of dysfunctional organs and SOFA score. Non-survivors had elevated M-MDSCs compared with that of patients who survived sepsis within 28 days after enrollment.ConclusionsMDSCs show potential as a diagnostic biomarker comparable to CRP and PCT, in infection and sepsis, even in distinguishing sepsis from infection. M-MDSCs show potential as a prognostic biomarker of sepsis and may be useful to predict 28-day hospital mortality in patients with sepsis.

Concordant B and T Cell Heterogeneity Inferred from the Multiomic Landscape of Peripheral Blood Mononuclear Cells in a Crohn's Disease Cohort.

Crohn's disease is characterized by inflammation in the gastrointestinal tract due to a combination of genetic, immune, and environmental factors. Transcriptomic and epigenomic profiling of intestinal tissue of Crohn's disease patients have revealed valuable insights into pathology, however have not been conducted jointly on less invasive peripheral blood mononuclear cells (PBMCs). Furthermore, the heterogeneous responses to treatments among individuals with Crohn's disease imply hidden diversity of pathological mechanisms. We employed single nucleus multiomic analysis, integrating both snRNA-seq and snATAC-seq of PBMCs with a variety of open source bioinformatics applications. Our findings reveal a diverse range of transcriptional signatures among individuals, highlighting the heterogeneity in PBMC profiles. Nevertheless, striking concordance between three heterogeneous groups was observed across B cells and T cells. Differential gene regulatory mechanisms partially explain these profiles, notably including a signature involving TGFß signaling in two individuals with Crohn's disease. A mutation mapped to a transcription factor binding site within a differentially accessible peak associated with the expression of this pathway, with implications for a personalized approach to understanding disease pathology. This study highlights how multiomic analysis can reveal common regulatory mechanisms that underlie heterogeneity of PBMC profiles, one of which may be specific to inflammatory disease.

Bactericidal Permeability-Increasing Protein (BPI) Inhibits Mycobacterium tuberculosis Growth

Bactericidal permeability-increasing protein (BPI) is a multifunctional cationic protein produced by neutrophils, eosinophils, fibroblasts, and macrophages with antibacterial anti-inflammatory properties. In the context of Gram-negative infection, BPI kills bacteria, neutralizes the endotoxic activity of lipopolysaccharides (LPSs), and, thus, avoids immune hyperactivation. Interestingly, BPI increases in patients with Gram-positive meningitis, interacts with lipopeptides and lipoteichoic acids of Gram-positive bacteria, and significantly enhances the immune response in peripheral blood mononuclear cells. We evaluated the antimycobacterial and immunoregulatory properties of BPI in human macrophages infected with Mycobacterium tuberculosis. Our results showed that recombinant BPI entered macrophages, significantly reduced the intracellular growth of M. tuberculosis, and inhibited the production of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-α). Furthermore, BPI decreased bacterial growth directly in vitro. These data suggest that BPI has direct and indirect bactericidal effects inhibiting bacterial growth and potentiating the immune response in human macrophages and support that this new protein’s broad-spectrum antibacterial activity has the potential for fighting tuberculosis.

Effect of PCV-2 Vaccination on Cytokines Gene Expression Profile in Wild Boar Peripheral Blood Mononuclear Cells after Stimulation with Mycobacteria Antigens

Wild boar (Sus scrofa) is a common wild ungulate known as the most important reservoir of tuberculosis (TB) in Spain. The severity of TB lesions in this species and the high prevalence of porcine circovirus type 2 (PCV-2) have been related. PCV-2 is ubiquitous in swine populations, being usual for the free-living ones the contact with this agent. Recent studies found a correlation between a decrease of generalised TB prevalence in wild boar populations and the PCV-2-vaccination. The aim of this study was to find out if PCV-2 vaccination modulates the gene expression of cytokines from immune cells after its exposition with mycobacterial antigens using an in vitro methodology. A total of 46 wild boars from a PCV-2 infection endemic area were blood-sampled before and after the PCV-2 vaccination of 22 of them. Peripheral blood mononuclear cells (PBMC) were obtained and isolated from these samples. Aliquots of the cells were in vitro cultured and respectively stimulated with PPDa, PPDb, and a mitogen. A complete analysis of the gene expression of cytokines from the cultured PBMC was carried out. Also, Mycobacterium bovis and PCV-2 contacts were revealed by ELISA and/or qPCR. The results demonstrated that the animals which have had contact with PCV-2 and had been vaccinated, manifested a significant decrease in gene expression of proinflammatory cytokines, like interleukin 1 beta, interleukin 6, and tumour necrosis factor-alpha, possibly related with the severity of TB lesions, and also a significant decrease of interleukin 10, a key cytokine. In conclusion, in case of possible infection or contact events with the virus, PCV-2 vaccination could be an effective measure to reduce the TB severity in wild boar populations, which could decrease the intra and interspecies transmission of TB.

Activated and Naïve Allogenic Human Placental Mesenchymal Stromal Cells Exert an Immunomodulatory Effect on Hidradenitis Suppurativa Patient Peripheral Blood Mononuclear Cells.

This pilot study aimed to evaluate the immunomodulatory effect of placental mesenchymal stem/stromal cells (MSCs) on peripheral blood mononuclear cells (PBMCs) from patients with hidradenitis suppurativa (HS). Blood samples were collected from 3 healthy and 3 patients with HS. Isolated PBMCs were stained with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with phorbol 12-myristate 13-acetate (PMA)/Ionomycin solution. The PBMCs of patients with HS were co-cultured with naïve MSCs (n-MSCs), activated with tumor necrosis factor (TNF)-α (10 ng/mL) and interferon (IFN)-γ (10 ng/mL) MSCs (a-MSCs), or adalimumab (30 μg/mL). The division index (proliferation inhibition) of PBMCs was analyzed by flow cytometry using the Proliferation Modeling tool after 5 days of coculture. The relative inflammatory gene expression dynamics and cytokine secretion were quantified in triplicate using real-time polymerase chain reaction (PCR) and Luminex assays. PBMCs from the HS control group showed statistically significant increases in interleukin (IL)-6 and IFN-γ cytokine concentrations and IL-17A gene expression when compared with healthy subjects. Statistically significant reduction of the division index was found in the a-MSCs group (P = 0.04). Also, the Luminex assay revealed significantly reduced proinflammatory cytokine concentrations of IL-9 (P = 0.022) and IL-17A (P = 0.022) in the a-MSCs group with the same trend of numerical lowering in n-MSCs group when compared to HS control. The results of real-time PCR revealed a numerical increase in the expression of the IL-1β, IL-36α, and TNF-α genes in both the a-MSCs and n-MSCs groups compared with the HS control. In conclusion, our findings suggest that MSCs can effectively curb PBMCs proliferation and suppress the production of inflammatory cytokines. Moreover, the preactivation of MSCs with IFN-γ and TNF-α before use can enhance their therapeutic effectiveness. Nevertheless, a larger sample size is imperative to validate these results.

Levels of Lysozyme and SLPI in Bronchoalveolar Lavage: Exploring Their Role in Interstitial Lung Disease

Interstitial lung diseases (ILDs) are characterized by inflammation or fibrosis of the pulmonary parenchyma. Despite the involvement of immune cells and soluble mediators in pulmonary fibrosis, the influence of antimicrobial peptides (AMPs) remains underexplored. These effector molecules display a range of activities, which include immunomodulation and wound repair. Here, we investigate the role of AMPs in the development of fibrosis in ILD. We compare the concentration of different AMPs and different cytokines in 46 fibrotic (F-ILD) and 17 non-fibrotic (NF-ILD) patients by ELISA and using peripheral blood mononuclear cells from in vitro stimulation in the presence of lysozyme or secretory leukocyte protease inhibitor (SLPI) from 10 healthy donors. We observed that bronchoalveolar lavage (BAL) levels of AMPs were decreased in F-ILD patients (lysozyme: p &lt; 0.001; SLPI: p &lt; 0.001; LL-37: p &lt; 0.001; lactoferrin: p = 0.47) and were negatively correlated with levels of TGF-β (lysozyme: p = 0.02; SLPI: p &lt; 0.001) and IL-17 (lysozyme: p &lt; 0.001; SLPI: p &lt; 0.001). We observed that lysozyme increased the percentage of CD86+ macrophages (p &lt; 0.001) and the production of TNF-α (p &lt; 0.001). We showed that lysozyme and SLPI were associated with clinical parameters (lysozyme: p &lt; 0.001; SLPI: p &lt; 0.001) and disease progression (lysozyme: p &lt; 0.001; SLPI: p = 0.01). These results suggest that AMPs may play an important role in the anti-fibrotic response, regulating the effect of pro-fibrotic cytokines. In addition, levels of lysozyme in BAL may be a potential biomarker to predict the progression in F-ILD patients.

Zinc Ionophore Pyrithione Mimics CD28 Costimulatory Signal in CD3 Activated T Cells

Zinc is an essential trace element that plays a crucial role in T cell immunity. During T cell activation, zinc is not only structurally important, but zinc signals can also act as a second messenger. This research investigates zinc signals in T cell activation and their function in T helper cell 1 differentiation. For this purpose, peripheral blood mononuclear cells were activated via the T cell receptor-CD3 complex, and via CD28 as a costimulatory signal. Fast and long-term changes in intracellular zinc and calcium were monitored by flow cytometry. Further, interferon (IFN)-γ was analyzed to investigate the differentiation into T helper 1 cells. We show that fast zinc fluxes are induced via CD3. Also, the intracellular zinc concentration dramatically increases 72 h after anti-CD3 and anti-CD28 stimulation, which goes along with the high release of IFN-γ. Interestingly, we found that zinc signals can function as a costimulatory signal for T helper cell 1 differentiation when T cells are activated only via CD3. These results demonstrate the importance of zinc signaling alongside calcium signaling in T cell differentiation.

Determination of IL-23 receptor expression and gene polymorphism (rs1884444) in Iranian patients with ankylosing spondylitis

BackgroundThrough investigating genetic variations, it has been demonstrated that single nucleotide polymorphisms (SNPs) in the IL-23 receptor (IL23R) gene have a critical role in the pathophysiology of ankylosing spondylitis (AS). Here, we investigated whether the IL23R variant (rs1884444) is associated with AS in the Iranian population.Methods and materialIn this research, we analyzed rs1884444 in a group of 425 patients with AS and 400 matched controls. For DNA extraction, the phenol/chloroform technique was utilized. Peripheral blood mononuclear cells (PBMCs) were obtained from the whole blood of 39 patients and 43 healthy controls and total RNA was extracted. Genotyping was performed by amplification-refractory mutation system (ARMS)–PCR method. Afterward, the expression level of IL23R was analyzed by the real-time quantitative (Q)-PCR method.ResultsWe observed no significant association between the distribution of alleles and genotypes of rs1884444 and susceptibility to AS. In addition, the expression level of IL23R did not differ between PBMCs from AS patients compared to the control group (P = 0.167). Furthermore, the relative expression level of IL23R was positively correlated with the BASDAI (P < 0.01) and BASFI (P < 0.05) scores of the patients.ConclusionIt appears that IL23R polymorphism (rs1884444) and the level of gene expression might not contribute to the susceptibility to AS in the Iranian population. The correlation of IL23R expression with the level of BASDAI and BASFI scores in patients may be due to the role of the IL-23/IL-23R signaling cascade in inflammation and exert a critical role in the development of AS.

Autologous human preclinical modeling of melanoma interpatient clinical responses to immunotherapeutics

BackgroundDespite recent advances in immunotherapy, a substantial population of late-stage melanoma patients still fail to achieve sustained clinical benefit. Lack of translational preclinical models continues to be a major challenge...

The Expression of MAFB Gene in Circulating Monocytes Is Related to Chronic Inflammatory Status in T2DM Patients.

Immune cell-mediated chronic inflammation is one of the causes of type 2 diabetes mellitus (T2DM). Therefore, identifying inflammatory markers in circulating immune cells is highly important for predicting insulin resistance (IR) and the occurrence of T2DM. In this study, we discovered that differentially expressed genes (DEGs) in peripheral blood mononuclear cells (PBMCs) from T2DM patients were associated with innate immunity and chronic inflammatory responses through bulk transcriptome sequencing (bulk RNA-seq). Gene integration analysis revealed that nine DEGs were upregulated, and receiver operating characteristic (ROC) curve analysis revealed that V-maf musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB), a candidate biomarker, has a certain predictive value for T2DM. In population-based cohort studies, we found that MAFB expression was significantly upregulated in the PBMCs of T2DM patients and was significantly correlated with homeostasis model assessment of IR (HOMA-IR), tumor necrosis factor-α (TNF-α), adiponectin (Adipoq), etc. We further evaluated the sensitivity and specificity of MAFB and other clinical parameters for predicting and diagnosing T2DM and found that MAFB expression in PBMCs had a positive effect on the prediction and diagnosis of T2DM. Finally, single-cell RNA sequencing (scRNA-seq) analysis revealed that the increase in MAFB expression was mainly in nonclassical monocytes. Our results suggest that increased MAFB expression in circulating monocytes may mediate chronic inflammatory status in patients with T2DM. Therefore, MAFB gene expression in circulating monocytes has certain clinical significance for predicting and assisting in the diagnosis of T2DM.