Mononuclear Cell Subpopulations Research Articles

Reduced TRIM expression correlates with anomalous CD4 T cell activation in systemic lupus Erythematosus and its clinical diagnostic potential

ObjectiveThis study seeks to elucidate the expression, function, and clinical relevance of the T cell receptor interacting molecule (TRIM) within circulating CD4+T cell subsets in systemic lupus erythematosus (SLE) patients. MethodsWe assessed TRIM expression across distinct subpopulations of human peripheral blood mononuclear cells (PBMCs) through the analysis of publicly available single-cell RNA sequencing data. In addition, TRIM expression was investigated within CD4+T cell subsets of peripheral blood and spleens in mice. PBMCs were isolated from both SLE patients, healthy controls (HCs) and rheumatoid arthritis (RA) patients with subsequent measurement and comparative analysis of TRIM expression and functional molecules using flow cytometry. To gauge the clinical relevance of TRIM in SLE, correlation and ROC curve analyses were performed. ResultsIn both healthy humans and mice, TRIM was higher expressed within CD4+T cell subsets, especially in naive CD4+T cells. TRIM+ Tregs exhibited lower Helios+ cells and CD45RA-FoxP3hi cells percentages compared to TRIM- Treg cells. TRIM+T cells demonstrated reduced granzyme B and perforin secretion and increased IFN-γ secretion in comparison to TRIM- T cells. Notably, the proportion of TRIM+CD4+T cells was diminished in SLE patients. The downregulation of TRIM+ in CD4+T cells positively correlated with diminished complement C3 and C1q levels and inversely correlated with CRP. The identification of TRIM-associated CD4 T cell subsets aids in distinguishing SLE patients from HCs and those with RA. ConclusionsReduced TRIM expression is linked to abnormal CD4+T cell activation in SLE. TRIM-associated CD4+T cells may be implicated in the pathogenesis of SLE and hold potential for clinical diagnostic purposes.

Mass Cytometry in Food Allergy Research.

Mechanisms underlying food allergy are not well understood. Mass cytometry is a technique that allows the multiple analysis of cell surface markers and intracellular proteins by using the spectrum of rare metal isotopes of different atomic masses without channel overlap. Bioinformatic approaches are implemented to combine and reduce the information of more than 60 parameters to define immune cell subpopulations. To date, mass cytometry has revealed a great heterogeneity in human response to food antigens and that subpopulations of basophils and mononuclear cells might be mechanistically implicated in food allergy. This chapter reviews some fundamentals of mass cytometry and the contributions of this technique in elucidating the immune basis of food allergy, oral tolerance, food desensitization, phenotypes, and the cellular events occurring upon allergen-specific immunotherapy.

Abstract OT2-01-06: Phase II trial of palbociclib plus endocrine therapy followed by combination of pembrolizumab, palbociclib and endocrine therapy in patients with hormone receptor positive metastatic breast cancer

Abstract Background: The combination of CDK4/6 inhibitors (CDK4/6i) and endocrine therapy (ET) is standard-of-care for patients with hormone receptor positive (HR+) HER2- metastatic breast cancer (MBC). Immune modulatory effects of CDK4/6i are well documented preclinically but poorly understood in the clinical setting. Our previous study combining letrozole, palbociclib and pembrolizumab in patients with HR+ MBC (NCT02778685) showed a promising complete response rate of 31%. Dynamic changes in peripheral blood mononuclear cell (PBMC) subpopulations indicated that palbociclib may increase CD8+ TEMRA (terminally differentiated effector memory cells) and CD4+ TEM (effector memory cells) and enable immune activation. The current cohort 3 was designed to study the immune modulatory effect of palbociclib as an immune-priming agent with a biomarker enriched design. Methods: Women with ECOG 0-1, HR+ HER2- MBC, RECIST 1.1 measurable disease, no prior therapy for MBC were enrolled. Patients with endocrine therapy, including aromatase inhibitor +/- ovarian suppression or fulvestrant, were eligible. A palbociclib + ET lead-in design was used, starting on day -28 followed by combination therapy with pembrolizumab added on C1D1. Peripheral blood and tumor biopsy at baseline and on-treatment were collected to allow in-depth analysis of biomarkers predicting response to the combination. The primary endpoint was to evaluate if the palbociclib potentiated immune responses as a “priming” agent through PBMC analysis and on treatment tumor biopsy. Secondary endpoints included other immune cell subsets and changes that follow the combination with pembrolizumab. With 25 patients, assuming a standard deviation of 0.51 in the relative change in classic monocytes in PBMCs, there is 90% power to detect a relative change of log(C1D1/baseline) of 34.5% with a type I error (two-sided) of 0.05. Results: Between August 2020 and April 2022, 16 patients were enrolled in cohort 3. Currently 11 patients have adverse event (AEs) and 16 patients have response data. Median age was 57 years (39-72). 8/11 (73%) were non-Hispanic white, 1/11 (9%) Hispanic, 1/11 (9%) Asian, and 1/11 (9%) African American. 87% patients had grade 3 AEs, and 30% had grade 4 AEs. Grade 3 AEs were 9/11 (82%) neutropenia, 5/11 (45%) leukopenia, 1/11 (9%) elevated LFTs, and 1/11 (9%) each lymphopenia, hot flashes, febrile neutropenia, and pneumonitis. Grade 4 AEs were 1/11 (9%) lymphopenia. 8/16 (50%) patients achieved a partial response (PR), 5/16 (31%) had stable disease (SD), and 1/16 (6%) had progression of disease (PD) by RECIST 1.1. Additionally, 2/16 (13%) patients were too early to determine best overall response. Response rate (CR+PR) was 50%. PBMCs and tumor microenvironment profiling are ongoing. Conclusion: The combination of palbociclib, pembrolizumab and ET is well tolerated, and a response rate of 50% was identified in HR+ MBC patients who received this combination as front-line therapy. Dynamic changes in peripheral blood mononuclear cells and tumor microenvironment profiling are ongoing. Citation Format: Yuan Yuan, Colt A. Egelston, Weihua Guo, Susan E. Yost, Paul H. Frankel, Christopher Ruel, Mireya Murga, Aileen Tang, Norma Martinez, Daniel Schmolze, Daphne Stewart, James Waisman, Kelly Yap, Joanne Mortimer, Niki Tank. Phase II trial of palbociclib plus endocrine therapy followed by combination of pembrolizumab, palbociclib and endocrine therapy in patients with hormone receptor positive metastatic breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT2-01-06.

CLL-050 Induction of Neutralizing Antibodies in Chronic Lymphocytic Leukemia Patients After SARS-CoV-2 mRNA Vaccination: A Monocentric Experience

Background: SARS-CoV-2 infection increases the clinical risk and mortality of patients with chronic lymphocytic leukemia (CLL). Covid-19 vaccinations have the potential to protect individuals from severe infection, but little is known about the antibody response profile in these patients after vaccination. We compared the anti-receptor binding domain (RBD) and neutralizing antibody responses of 27 patients with CLL to those of healthy donors after mRNA Covid-19 vaccination. Methods: Of the 27 patients enrolled in the study, 21 received the BNT162b2 mRNA vaccine and 6 received the Spikevax mRNA vaccine. Four patients were in clinical remission after treatment, 10 were treatment-naïve, and 13 were under treatment at the time of vaccination. Blood samples were taken before vaccination and after the second dose, with a median time between of 23 days. The levels of anti-RBD IgG, IgM, and IgA were evaluated by ELISA on recombinant RBD; the neutralizing antibody response was evaluated by spike protein inhibition assay (SPIA). We also performed a fluorescence-activated cell sorting study of peripheral blood mononuclear cell subpopulations, a disease burden assessment using blood indicators (lactate dehydrogenase, beta-2-microglobulin, lymphocytic count) and imaging, and an evaluation of IgG levels at the time of vaccination. Results: A comparison of anti-RBD antibody seroconversion (IgG, IgM, and IgA) and neutralizing antibodies present after vaccination between 27 patients with CLL and healthy control subjects indicated that CLL patients had a considerably lower response rate (50% vs. 100%) and lower antibody levels than the healthy controls. We found an inverse correlation between the spleen dimension and lymphonodal area. By stratifying patients according to the presence of neutralizing antibodies after vaccination, we found a larger prevalence of SPIA positives than SPIA negatives in treatment-naïve patients (60%); however, patients undergoing treatment had a much lower percentage of SPIA positives (23.1%). Conclusions: Only around half of vaccinated CLL patients acquire detectable anti-RBD and neutralizing antibodies, according to our findings. Furthermore, we discovered a substantial difference in the rates of detectable anti-SARS-CoV-2 antibodies between patients who were treatment-naïve/in clinical remission and those on CLL-directed treatment. The persistence and burden of disease represent a surrogate of vaccine failure, probably due to the persistence of immune dysfunction.

Analysis of cerebrospinal fluid cells by flow cytometry: Comparison to conventional cytology.

This study compared the results obtained by basic immunophenotyping of cerebrospinal fluid (CSF) cells by flow cytometry (FC) to the results of conventional cytology and evaluated the possibility of detailed analyses of CSF B-cell subpopulations. Samples from 42 patients were examined by conventional cytology (native and/or pre-centrifuged CSF) and FC. The results from 15 patients without evidence of organic neurological disease were used to estimate reference ranges. Pre-centrifugated CSF had significantly higher cell yield on the cytologic slide, but cell subpopulation percentages were altered; the percentage of lymphocytes was significantly higher and monocytes significantly lower compared to both native CSF slides and FC. The percentage of granulocytes was higher in FC compared to cytology. For leukocyte count, the following reference ranges were estimated for Fuchs-Rosenthal chamber (FR) counting and FC, respectively: leukocytes ≤4.7/μL and ≤2.5/μL, lymphocytes ≤4.1/μL and ≤1.8/μL, monocytes ≤1.2/μL and ≤0.9/μL, and granulocytes 0/μL and ≤0.2/μL. The following reference ranges were estimated for basic subpopulations: T-lymphocytes 84.1-100%, B lymphocytes 0.0-1.5%, NK cells 0.0-6.3%, NKT cells 0-9.5%, and CD3+CD4+/CD3+CD8+ 0.8-4.9. Using a volume of 1.2-2.4 mL, the number of B lymphocytes was too low (<20) in samples with ≤2.7 cells/μL in the FR. Even normal CSF samples are amenable to basic mononuclear cell subpopulation analysis by FC. However, analysis of the B-cell subpopulations requires either a larger sample volume or selection of samples with ≥ 3 cells/μL.

Clinical significance of detection of mononuclear phagocyte subsets in blood and bronchoalveolar lavage fluid (BALF) in pulmonary sarcoidosis.

This study aimed to investigate the clinical significance of the detection of mononuclear phagocytes subsets in pulmonary sarcoidosis blood and bronchoalveolar lavage fluid (BALF). For this purpose, a total of 52 patients with pulmonary sarcoidosis were selected as the study group, 52 healthy people served as the "NC Group a" (peripheral blood mononuclear cell control group), 47 patients with chronic cough and no pulmonary sarcoidosis who underwent bronchoscopy were used as "control group b" (alveolar lavage fluid macrophage control group). Fasting peripheral blood and BALF were collected, and flow cytometry was used to detect monocytes and macrophage subpopulations. The monocytes and macrophage subpopulations of the study group were compared before and after treatment. The results showed that the proportion of CD14++CD16- subgroup of patients with pulmonary sarcoidosis was lower than that of healthy people (74.21±4.10% vs 84.32±4.76%); The proportion of CD14++CD16+ subgroups of patients with pulmonary sarcoidosis was higher than that of healthy people (7.42±4.08% vs 3.95±1.94%); The proportion of CD14+CD16++ subgroups of patients with pulmonary sarcoidosis was higher than that of healthy people in the control group, but the difference was not significant. After 2 months of treatment, the proportion of CD14++CD16- subgroups in peripheral blood mononuclear cells increased, and the proportion of CD14++CD16+ subgroups decreased. The proportion of M1 in patients with pulmonary sarcoidosis was lower than that in patients with non-pulmonary nodules (24.32±11.36% vs 47.03±13.86%); the proportion of M2 in patients with pulmonary sarcoidosis was higher than the proportion of M2 in patients with non-pulmonary nodules (75.40±10.23% vs 52.53±12.01%). After treatment, the proportion of M1 of BALF macrophages in patients with pulmonary sarcoidosis was increased (P<0.05), and the proportion of M2 was reduced (P<0.05). In general, detection of changes in peripheral blood mononuclear cell subpopulations and BALF macrophage subpopulations in patients with pulmonary sarcoidosis has certain clinical significance for the treatment.

SNHG15 is a negative regulator of inflammation by mediating TRAF2 ubiquitination in stroke-induced immunosuppression

BackgroundAbnormal expression of long noncoding RNAs (lncRNAs) has been reported in the acute stage of acute ischemic stroke (AIS). This study aimed to explore differential lncRNA expression in the subpopulations of peripheral blood mononuclear cells (PBMCs) from AIS patients and further evaluate its underlying mechanisms in stroke-induced immunosuppression.MethodsWe reanalyzed lncRNA microarray data and investigated abnormally expressed lncRNAs in the subpopulations of PBMCs by magnetic cell sorting and real-time quantitative PCR. The potential mechanism of small nucleolar RNA host gene 15 (SNHG15) was explored through in vitro and in vivo approaches.ResultsThe stroke-induced SNHG15 acted as a checkpoint to inhibit peripheral inflammatory responses. Functional studies showed that SNHG15 promoted M2 macrophage polarization. Mechanistically, SNHG15 expression was dysregulated through the Janus kinase (JAK)-signal transducer and activator of transcription 6 (STAT6) signaling pathway. SNHG15, localized in the cytoplasm, interfered with K63-linked ubiquitination of tumor necrosis factor receptor-associated factor 2 and thereby repressed the activation of mitogen-activated protein kinase and nuclear factor kappa-B signaling pathways and prevented the production of proinflammatory cytokines. Administration of an adenovirus targeting SNHG15 improved stroke-induced immunosuppression in mice.ConclusionsThis study identified SNHG15 as a negative regulator of inflammation in stroke-induced immunosuppression, suggesting it as a novel biomarker and therapeutic target in stroke-associated infection.Trial registration ClinicalTrials.gov NCT04175691. Registered November 25, 2019, https://www.clinicaltrials.gov/ct2/show/NCT04175691.

Kinetics of Mononuclear Cell Subpopulations in the Peripheral Blood of Patients with Giant Cell Arteritis During the Acute Phase of the Disease: The Role of Steroids.

Background/Aim:Giant cell arteritis (GCA) represents the most prevalent form of systemic vasculitis in the elderly, primarily affecting the temporal artery, the extracranial branches of carotid arteries, and the aorta. GCA is a highly heterogeneous disease in terms of clinical and histological findings, pathophysiology, and treatment selection strategies. The disease is highly responsive to glucocorticosteroids (GCs), but almost half of patients may relapse following GCs tapering. The main hypothesis of GCA pathogenesis includes altered immune responses and changes in the vascular microenvironment, leading to a dynamic interplay between innate and adaptive immunity. The aim of this study is to explore the effect of GCs on the phenotype of peripheral mononuclear cell subpopulations and on the major inflammatory molecules detected in the peripheral blood of patients during the acute phase of the disease.Methods:Patient PBMCs will be studied using Cytometry by time of flight (CyTOF). Following the CyTOF analysis, Luminex Assay will be performed on the same patient samples to identify the kinetics of the most prominent inflammatory mediators correlating with the subpopulations detected. Patient population consists of 8 patients with GCA, 6 with polymyalgia rheumatica, as disease control group and 5 healthy controls (sex and age matched) at 3 time points: disease diagnosis, 48 and 96 hours after treatment administration.Conclusion:The identification of potential alterations in cell subpopulations and the kinetics of inflammatory mediators are expected to lead to the production of new knowledge regarding the role of corticosteroids in the phase of acute inflammatory response.

Potential PD-L1 expressing cytotoxic T-cell immunopathology in Alzheimer disease.

Growing evidence suggested that adaptive immunity involved Alzheimer's disease (AD). Peripheral blood mononuclear cell (PBMC) subpopulations and expression of immune checkpoints such as programmed death-ligand 1(PD-L1) and programmed cell death protein-1 (PD-1) could play roles in Alzheimer disease. The interaction of PD-L1/PD-1 lead PD-1 expressing T cells to attenuate T cell-activating signals. Recent studies had demonstrated the blockade of PD-L1/PD-1 axis modified brain pathology and restored cognitive performance in tauopathy murine model. However similar human experiment suggested controversial results in AD patients. To observe the changes of PBMC subpopulations and PD-L1/PD-1 expression in AD and healthy controls, we utilized flow cytometry, and applied t-distributed stochastic neighbor embedding (t-SNE) algorithm as a new approach to analyze results. AD patients (n=16) with different disease severity (CDR= 1 - 2) and healthy controls (n=16) were enrolled. Expression levels of PD-L1/PD-1 in AD patients and healthy controls were derived by flow cytometric analysis parameters. Moreover, a dimensionality reduction algorithm for visualized t-SNE, was implemented to analyze the PD-L1/PD-1 expressing cell clusters between AD patients and healthy controls. We found no significant PD-1 expression intensity difference between cytotoxic T lymphocytes (CTL) of AD patients and healthy controls. In AD patients, subpopulation of CTL expressing PD-L1 was elevated by 85.53%. Further, PD-L1 expressing CTL clusters between AD patients and healthy controls were compared. We observed that densities of PD-L1high CTL clusters are significantly higher than the healthy controls. Upregulation of PD-L1 and PD-L1 expressing CTL were increased in peripheral blood of AD patients, linking to newly found strong correlation between the level of PD-L1 positive CTL and markers which indicated negative immune climate. Abnormal adaptive immune response of CTL might associate with concurrent viral infection, intracellular bacterial infection, and underlying cancer in AD patients. Our results suggested a novel strategy to prevent AD progression with regulation of PD-L1 expressing CTL, and notation of underlying intracellular infection and malignancy for which could relate to dementia progression.

Role of the mononuclear cell infiltrate in Graves' orbitopathy (GO): results of a large cohort study.

The extent to which mononuclear cells and TSH-receptor autoantibodies (TRAb) contribute to Graves' orbitopathy (GO) is not completely defined. Here we investigated the relationship between the immunohistochemical phenotype of orbital infiltrating cells and GO features in a large number of patients. We conducted an observational cohort study in 76 consecutive patients with GO (16 men and 60 women) who underwent orbital decompression over a period of 18 consecutive months. An ophthalmological evaluation was performed in all patients, as well as immunohistochemistry for CD3, CD4, CD8, CD56 (T-cell markers), CD25 (T and B-cell marker), CD20, CD19 (B-cell markers), and CD138 (plasmacell marker) in specimens collected at decompressive surgery. Having established cutoff values for each marker, cell infiltrates were found in 60 patients (78.9%; CD3: 39.4%, CD4 55.2%, CD8 50%, CD56: 0%, CD25: 28.9%, CD20: 51.3%, CD19: 25%, CD138: 26.3%). Eleven (14.4%) stained exclusively for CD138 (plasmacells). Patients with CD4-positive mononuclear cells had a significantly greater GO clinical activity score (CAS) (mean difference 1.07, 95% CI - 0.33 to - 1.82, P = 0.004 by univariate, P = 0.05 by multivariate analysis). CAS as well as the remaining GO features were not affected significantly by the mononuclear cell subpopulations in multivariate analyses. Mononuclear cell infiltrates are present in the majority of GO patients, with a small percentage represented exclusively by plasmacells. CD4 cells exert a major role on GO activity. These findings may represent a further advancement in the comprehension of GO pathogenesis.

Circulating mitochondrial DNA (mtDNA) variants to predict metastatic progression of rectal cancer.

e16132 Background: High rates of systemic failure after curative-intent therapy in locally advanced rectal cancer (LARC) call for new diagnostic tools to improve risk-based treatment stratification. We hypothesized that blood mtDNA polymorphisms, generated by the high mtDNA mutation rate when the mitochondrial biogenesis is mobilized during clonal immune cell activation, correlate with systemic immune control of LARC dissemination and thus can be used as diagnostic marker. Methods: 41 LARC patients given neoadjuvant (chemo)radiotherapy had DNA extracted from whole blood (WB) sampled at the time of diagnosis, for high-throughput full-length mtDNA sequencing followed by annotation of mtDNA variants using the curated reference genome (mitomap.org). The composition of peripheral blood mononuclear cell (PBMC) subpopulations was analyzed by high-dimensional single-cell mass cytometry. Metastatic events were recorded over 24-60 months of follow-up after completion of the multimodal treatment. Results: The total number of WB-mtDNA variants was strikingly different across the study cases but without correlation to patient’s age, body mass index, or metformin use, which may affect mitochondrial function, or to the disease extension at presentation or the local tumor response to the neoadjuvant therapy. All patients had microsatellite-stable tumor. The higher the number of variants in MT-RNR2, encoding one of the two ribosomal subunits of mitochondria, the better distant metastasis-free survival (DMFS). For each patient, two MT-RNR2 sites were homoplasmic for either the normal or a deletion (3105AC &gt; A, 3106CN &gt; C) variant, categorizing the patients into two groups that comprised the same cases for both mtDNA sites. Nine of a total of 10 DMFS events occurred during the 18 first months of follow-up in one of the patient groups ( n = 23). The single event in the other group ( n = 18) appeared in a patient who had declined primary tumor surgery. In the former group, a low number of variants in the ribosomal subunit MT-RNR1 identified all patients who had metastatic progression. These patients had reduced PBMC frequency of CD8+ (cytolytic) T cells that were positive for CXCR4 ( p = 0.001), a chemokine receptor that promotes their homing to tumor sites. Conclusions: The WB-ribosomal mtDNA polymorphisms reflected systemic immune cells endowed with cytolytic and tumor-homing properties. The actual mtDNA variants were single-base alterations and might thus be analyzed by PCR and developed as a feasible assay for metastatic risk assessment in rectal cancer. Clinical trial information: 01816607 .

GRP78 expression in peripheral blood mononuclear cells is a new predictive marker for the benefit of taxanes in breast cancer neoadjuvant treatment

BackgroundBreast cancer treatment is tailored to the specific cancer subtype. Often, systemic treatment is given prior to surgery. Chemotherapy induces significant endoplasmic reticulum (ER) stress-mediated cell death and upregulation of 78-kDa glucose-regulated protein (GRP78). We hypothesized that chemotherapy induces ER stress not only in the tumor tissue but also in immune cells, which may affect the response to anti-cancer treatment.MethodsWe determined the surface expression of GRP78 on 15 different peripheral blood mononuclear cell (PBMC) subpopulations in 20 breast cancer patients at three time points of the neoadjuvant treatment, i.e., at baseline, after anthracycline treatment, and after taxanes treatment. For this purpose, we performed flow cytometric analyses and analyzed the data using ANOVA and the Tukey test. Serum cytokine levels were also evaluated, and their levels were correlated with response to treatment using the t-test after log transformation and Mann-Whitney U Wilcoxon W test.ResultsA significant increase in GRP78 expression in PBMCs was documented during the taxane phase, only in patients who achieved pathological complete response (pCR). GRP78-positive clones correlated with increased serum levels of interferon gamma (IFNγ).ConclusionsThe presence of GRP78-positive clones in certain PBMC subpopulations in pCR patients suggests a dynamic interaction between ER stress and immune responsiveness. The correlation of GRP78-positive clones with increased levels of IFNγ supports the idea that GRP78 expression in PBMCs might serve as a new predictive marker to identify the possible benefits of taxanes in the neoadjuvant setting.

Coronary Progenitor Cells and Soluble Biomarkers in Cardiovascular Prognosis after Coronary Angioplasty

Major adverse cardiovascular events (MACEs) negatively impact the cardiovascular prognosis of patients undergoing coronary angioplasty due to coronary ischemic injury. The extent of coronary damage and the mechanisms of vascular repair are factors influencing the future development of MACEs. Intrinsic vascular features like the plaque characteristics and coronary artery complexity have demonstrated prognostic information for MACEs. However, the use of intracoronary circulating biomarkers has been postulated as a convenient method for the early identification and prognosis of MACEs, as they more closely reflect dynamic mechanisms involving coronary damage and repair. Determination of coronary circulating biomarkers during angioplasty, such as the number of subpopulations of mononuclear progenitor cells (MPCs) as well as the concentration of soluble molecules reflecting inflammation, cell adhesion, and repair, allows for assessment of future developments and the prognosis of MACEs 6 months post coronary angioplasty. This method is highlighted by its translational nature and better performance than peripheral blood circulating biomarkers regarding prediction of MACEs and its effect on the cardiovascular prognosis, which may be applied for risk stratification of patients with coronary artery disease undergoing angioplasty.

Expression of membrane-bound human leucocyte antigen-G in systemic sclerosis and systemic lupus erythematosus

Human leucocyte antigen-G (HLA-G) is a nonclassical class I major histocompatibility complex (MHC) molecule characterized by complex immunoregulatory and tolerogenic functions. Membrane-bound HLA-G is expressed on the surface of different cell populations in both physiological and pathological conditions. Systemic sclerosis (SSc) is a multisystem autoimmune disease characterized by widespread tissue fibrosis, vascular lesions and immunological alterations. Systemic lupus erythematosus is the prototypic systemic autoimmune disease affecting virtually any organ system, such as skin, joints, central nervous system, or kidneys. In SSc and SLE patients, the membrane expression of HLA-G on monocytes (0.88 ± 1.54 and 0.43 ± 0.75, respectively), CD4+ (0.42 ± 0.78 and 0.63 ± 0.48, respectively), CD8+ (2.65 ± 3.47 and 1.29 ± 1.34, respectively) and CD4+ CD8+ double-positive cells (13.87 ± 15.97 and 3.79 ± 3.11, respectively) was significantly higher than in healthy controls (0.12 ± 0.07; 0.01 ± 0.01; 0.14 ± 0.20 and 0.32 ± 0.38, respectively) (p < 0.0001). Our results show that in SSc and SLE the membrane expression of HLA-G by different subpopulations of peripheral blood mononuclear cells (PBMC) is increased, suggesting a potential role of HLA-G molecules in the complex immunological pathogenesis of these two autoimmune disorders.

Antitumoral and Immunomodulatory Effect of Mahonia aquifolium Extracts.

The prodrug potential of Mahonia aquifolium, a plant used for centuries in traditional medicine, recently gained visibility in the literature, and the activity of several active compounds isolated from its extracts was studied on biologic systems in vitro and in vivo. Whereas the antioxidative and antitumor activities of M. aquifolium-derived compounds were studied at some extent, there are very few data about their outcome on the immune system and tumor cells. To elucidate the M. aquifolium potential immunomodulatory and antiproliferative effects, the bark, leaf, flower, green fruit, and ripe fruit extracts from the plant were tested on peripheral blood mononuclear cells and tumor cells. The extracts exert fine-tuned control on the immune response, by modulating the CD25 lymphocyte activation pathway, the interleukin-10 signaling, and the tumor necrosis-alpha secretion in four distinct human peripheral blood mononuclear cell (PBMC) subpopulations. M. aquifolium extracts exhibit a moderate cytotoxicity and changes in the signaling pathways linked to cell adhesion, proliferation, migration, and apoptosis of the tumor cells. These results open perspectives to further investigation of the M. aquifolium extract prodrug potential.

Treatment with mononuclear cell populations improves post-infarction cardiac function but does not reduce arrhythmia susceptibility.

BackgroundClinical and experimental data give evidence that transplantation of stem and progenitor cells in myocardial infarction could be beneficial, although the underlying mechanism has remained elusive. Ventricular tachyarrhythmia is the most frequent and potentially lethal complication of myocardial infarction, but the impact of mono nuclear cells on the incidence of ventricular arrhythmia is still not clear.ObjectiveWe aimed to characterize the influence of splenic mononuclear cell populations on ventricular arrhythmia after myocardial infarction.MethodsWe assessed electrical vulnerability in vivo in mice with left ventricular cryoinfarction 14 days after injury and intramyocardial injection of specific subpopulations of mononuclear cells (MNCs) (CD11b-positive cells, Sca-1-positive cells, early endothelial progenitor cells (eEPCs)). As positive control group we used embryonic cardiomyocytes (eCMs). Epicardial mapping was performed for analysing conduction velocities in the border zone. Left ventricular function was quantified by echocardiography and left heart catheterization.ResultsIn vivo pacing protocols induced ventricular tachycardia (VT) in 30% of non-infarcted mice. In contrast, monomorphic or polymorphic VT could be evoked in 94% of infarcted and vehicle-injected mice (p<0.01). Only transplantation of eCMs prevented post-infarction VT and improved conduction velocities in the border zone in accordance to increased expression of connexin 43. Cryoinfarction resulted in a broad aggravation of left ventricular function. All transplanted cell types augmented left ventricular function to a similar extent.ConclusionsTransplantation of different MNC populations after myocardial infarction improves left ventricular function similar to effects of eCMs. Prevention of inducible ventricular arrhythmia is only seen after transplantation of eCMs.

Phenotypic and functional stability of leukocytes from human peripheral blood samples: considerations for the design of immunological studies

BackgroundHuman peripheral blood mononuclear cells (PBMCs) are extensively used for research of immune cell functions, identification of biomarkers and development of diagnostics and therapeutics for human diseases, among others. The assumption that “old blood samples” are not appropriate for isolation of PBMCs for functional assays has been a dogma in the scientific community. However, partial data on the impact of time after phlebotomy on the quality and stability of human PBMCs preparations impairs the design of studies in which time-controlled blood sampling is challenging such as field studies involving multiple sampling centers/sites. In this study, we evaluated the effect of time after phlebotomy over a 24 h time course, on the stability of human blood leukocytes used for immunological analyses. Blood samples from eight healthy adult volunteers were obtained and divided into four aliquots, each of which was left in gentle agitation at room temperature (24 °C) for 2 h (control), 7 h, 12 h and 24 h post phlebotomy. All samples at each time point were independently processed for quantification of mononuclear cell subpopulations, cellular viability, gene expression and cytokine secretion.ResultsA 24 h time delay in blood sample processing did not affect the viability of PBMCs. However, a significantly lower frequency of CD3+ T cells (p < 0.05) and increased LPS-induced CXCL10 secretion were observed at 12 h post-phlebotomy. Alterations in TNFα, CCL8, CCR2 and CXCL10 gene expression were found as early as 7 h after blood sample procurement.ConclusionsThese data reveal previously unrecognized early time-points for sample processing control, and provide an assay-specific time reference for the design of studies that involve immunological analyses of human blood samples.

Effects of ruminal pH on gene expression in the rumen epithelium, peripheral blood mononuclear cell subpopulations, and blood metabolites from Holstein calves during weaning transition.

We investigated the relationships between ruminal pH, gene expression in the rumen epithelium (RE), peripheral blood mononuclear cell subpopulations, and blood metabolites in Holstein calves during weaning transition. Calves (Weaning group, n=7) were assigned to one of two groups, and fed calf starter with forage (Forage group, n=3) or without forage (Starter group, n=4). Ruminal pH was measured continuously. Samples were collected at −1, 0, 1, and 3 weeks (blood and rumen fluid) or 3 weeks (rumen epithelium) after weaning. In the Weaning group, ruminal pH increased, and several blood metabolites increased (blood urea nitrogen [BUN], beta-hydroxybutyrate [BHB], and gamma-glutamyl transferase [GGT]) or decreased (total cholesterol [T-CHO] and phospholipid) after weaning. Ruminal pH was positively correlated with CD8+CD45R− cell populations and blood metabolites (BUN, glucose, and BHB) and negatively correlated with GGT activity. The 24 hr mean ruminal pH was higher in the Forage group during weaning transition, and toll-like receptor 4 mediated signaling pathway was activated in the Starter group at 3 weeks post-weaning. The number of CD8+CD45R− cells tended to be higher, and several blood metabolites (glucose, triglycerides, T-CHO, and phospholipid) were higher in the Forage group after weaning. Calves with higher ruminal pH also showed a greater energy metabolism status simultaneously with lesser hepatic disturbance enzymes in the peripheral blood. The results of our study indicate that serum GGT activity may be a plausible biomarker for predicting ruminal acidosis in Holstein calves during weaning transition.

Frequency of bovine viral diarrhea virus detected in subpopulations of peripheral blood mononuclear cells in persistently infected animals and health outcome

Bovine viral diarrhea viruses (BVDV) cause acute and persistent infections. Acute infection results in generalized immunosuppression characterized by a decrease in circulating lymphocytes as a result of depletion of CD4+ and CD8 + T cell populations. Persistent infection with BVDV is the result of immune tolerance and is generally not associated with lymphocytopenia. The health outcome of persistently infected (PI) calves varies widely; some die of mucosal disease, some succumb to ill thrift and others appear normal and survive to adulthood. Detection of BVDV at the single lymphoid cell level is important to the study of subpopulations of peripheral blood mononuclear cells (PBMC) during BVDV infections, however there are few methods available for the detection and quantification of BVDV at this level. To circumvent this difficulty, a novel flow cytometry-based PrimeFlow RNA assay using in-situ detection of BVDV was developed. This assay was used to evaluate differences in viral distribution within subpopulations of PBMC over time in PI calves carrying one of two different species of BVDV (type 1 and type 2). Calves were sampled at 3 different time points approximately one month apart. During the course of the study, a subset of the calves died from ill thrift. Mucosal disease was not indicated in any of the deaths. Using RNA probes specific for the BVDV Npro-Erns coding regions for each respective virus, BVDV RNA was detected in all PBMC of PI that appeared clinically healthy. Calves that succumbed to ill thrift were found to have no or little virus in T cells. The clearance of virus from T cells suggests a breakdown in immune tolerance in these calves. This is the first report of a pattern observed in the viral load in the T cell subpopulations and survival in PI calves.

Quasispecies Changes with Distinctive Point Mutations in the Hepatitis C Virus Internal Ribosome Entry Site (IRES) Derived from PBMCs and Plasma.

The 5' untranslated region (UTR) of the hepatitis C virus (HCV) genome contains the internal ribosome entry site (IRES), a highly conserved RNA structure essential for cap-independent translation of the viral polyprotein. HCV, apart from the liver, is thought to be associated with lymphocyte subpopulations of peripheral blood mononuclear cells (PBMCs), in lymph nodes and brain tissue. In this study, RT-PCR, cloning, and sequence analysis were employed to investigate the quasispecies nature of the 5'UTR following extraction of viral RNA from PBMCs and plasma of HCV infected individuals. The nucleotide variation between IRES-derived sequences from PBMCs and plasma indicated the existence of polymorphic sites within the IRES. HCV isolates had divergent variants with unique mutations particularly at positions 107, 204, and 243 of the IRES. Most of the PBMC-derived sequences contained an A-A-A variant at these positions. The mutations associated with the IRESes suggested the presence of unique quasispecies populations in PBMCs compared with plasma.