Search Results (42)

Interferon signature is one of the key pathogenic pathways in idiopathic inflammatory myopathies (IIMs), particularly in dermatomyositis (DM). The aim of this study was to analyze Siglec-1, an interferon-inducible receptor, on different monocyte subsets in IIM subtypes and investigate its association with disease activity measures. Siglec-1 expression was measured by 8-color flow cytometry in 62 IIM patients and 10 healthy controls (HC). Disease activity was assessed using the International Myositis Assessment and Clinical Studies (IMACS) core set measures. Active DM patients exhibited significantly higher Siglec-1 mean fluorescence intensity (MFI) compared to inactive subgroups and HCs in every monocyte subset. Intermediate monocytes displayed the highest Siglec-1 expression across all groups and the strongest associations between disease activity markers. Siglec-1 expression on monocyte subsets was strongly associated with global, extramuscular global, constitutional, cutaneous, muscular, and gastrointestinal activity measures, but not with pulmonary, skeletal, and cardiac activities in the DM population. The best indicator of DM global disease activity among the examined biomarkers was Siglec-1 MFI on intermediate monocytes. Siglec-1 on intermediate monocytes correlates strongly with organ-specific clinical and biochemical markers of disease activity; therefore, it is a candidate biomarker for monitoring IIM disease activity. Siglec-1 could be useful in patient selection for interferon-targeted treatments. Full article

Background/Objectives: Potency testing of clinical-grade lentiviral vectors (LVVs) is critical to support a drug’s commercial approval. Careful consideration should be paid to the development of a suitable potency test during the drug’s clinical development. We aimed to develop an affordable, quantitative test for our CAR19-LVV, based on a measure of transgene’s functional activity. Methods: Several indicators of functional activity of CAR19-LVV were explored in a co-culture setting of CAR-transduced Jurkat cells and CD19-expressing target cells. The selected assay was further developed and subjected to validation. Assay’s adaptability to other CAR-encoding LVV and autologous CAR-T cell products was also investigated. Results: Measure of CD69 expression on the membrane of Jurkat-CAR-expressing cells is a specific indicator of CAR functionality. Quantification of CD69 in terms of mean fluorescence intensity (MFI), coupled with an intra-assay standard curve calibration, allows for a quantitative assay with high precision, specificity, robustness, linearity and accuracy. The assay has also shown optimal performance for a CARBCMA-LVV product. Importantly, we show that in primary T cells, CD69 expression reflects CAR-T cell cytotoxicity. After adaptation, we have applied a CD69-based potency test, with simultaneous measurement of CAR-T cell cytotoxicity, to autologous CAR-T cell products, demonstrating the assay’s specificity also in this context. Conclusions: We developed a validated, in vitro cell-based potency test, using a quantitative flow-cytometry method, for our CAR19-LVV. The assay is based on the detection of T-cell activation upon CAR binding to antigen, which is a measure of transgene functionality. The assay was easily adapted to another CAR-encoding LVV, targeting a different molecule. Furthermore, the same assay principle can be applied in the context of autologous CAR-T cell products. The quantitative CD69 potency assay shows reduced variability among autologous products compared to the IFNγ assay and allows for simultaneous evaluation of traditional semi-quantitative cytotoxicity, thereby directly evaluating the drug’s mechanism of action (MoA) in the same assay. Full article

Background: CD79b is a B-cell-specific antigen that is crucial to the B-cell receptor and is considered a key target for treatment in aggressive B-cell lymphomas. Methods: While immunohistochemical studies have shown widespread expression of CD79b in mature B-cell-derived lymphomas, flow cytometry allows for precise measurement and differentiation between surface and intracellular localization. Results: In our comparative analysis, we discovered that CD79b expression percentages and mean fluorescence intensity (MFI) were lower in a group of 127 cases of aggressive B-cell lymphomas compared to a control group of benign reactive hyperplasia. We also observed significant variability in the surface expression of CD79b among lymphoma cases, with 18% showing predominantly intracellular positivity. There was a strong correlation between the surface expression of CD79b and clonal light chains. Notably, primary mediastinal B-cell lymphomas exhibited significantly lower surface CD79b expression compared to other lymphoma subtypes (median 0.8% IQR 0–48.5 vs. 80% IQR 24–97, p = 0.0005). Furthermore, patients over 60 years old and those with a higher Revised International Prognostic Index (R-IPI) had significantly higher CD79b expression, both of which are associated with a significant benefit from adding an anti-CD79b drug conjugate to first-line chemotherapy in diffuse large B-cell lymphomas. Conclusions: In conclusion, the quantitative flow cytometric analysis of CD79b surface expression in aggressive B-cell lymphomas provides clinically relevant information, highlighting its potential usefulness in guiding therapeutic decisions. Full article

Autoimmune diseases represent a severe personal and healthcare problem that seeks novel therapeutic solutions. Mesenchymal stem cells (MSCs) are multipotent cells with interesting cell biology and promising therapeutic potential. The immunoregulatory effects of secretory factors produced by umbilical cord mesenchymal stem cells (UC-MSCs) were assessed on B lymphocytes from 17 patients with systemic lupus erythematosus (SLE), as defined by the 2019 European Alliance of Associations for Rheumatology (EULAR)/American College of Rheumatology (ACR) classification criteria for SLE, and 10 healthy volunteers (HVs). Peripheral blood mononuclear cells (PBMCs) from patients and HVs were cultured in a UC-MSC-conditioned medium (UC-MSCcm) and a control medium. Flow cytometry was used to detect the surface expression of CD80, CD86, BR3, CD40, PD-1, and HLA-DR on CD19+ B cells and assess the percentage of B cells in early and late apoptosis. An enzyme-linked immunosorbent assay (ELISA) quantified the production of BAFF, IDO, and PGE2 in PBMCs and UC-MSCs. Under UC-MSCcm influence, the percentage and mean fluorescence intensity (MFI) of CD19+BR3+ cells were reduced in both SLE patients and HVs. Regarding the effects of the MSC secretome on B cells in lupus patients, we observed a decrease in CD40 MFI and a reduced percentage of CD19+PD-1+ and CD19+HLA-DR+ cells. In contrast, in the B cells of healthy participants, we found an increased percentage of CD19+CD80+ cells and decreased CD80 MFI, along with a decrease in CD40 MFI and the percentage of CD19+PD-1+ cells. The UC-MSCcm had a minimal effect on B-cell apoptosis. The incubation of patients’ PBMCs with the UC-MSCcm increased PGE2 levels compared to the control medium. This study provides new insights into the impact of the MSC secretome on the key molecules involved in B-cell activation and antigen presentation and survival, potentially guiding the development of future SLE treatments. Full article

Tuberculosis (TB) remains one of the leading causes of death among infectious diseases, with 10.6 million new cases and 1.3 million deaths reported in 2022, according to the most recent WHO report. Early studies have shown an expansion of γδ T cells following TB infection in both experimental models and humans, indicating their abundance among lung lymphocytes and suggesting a role in protective immune responses against Mycobacterium tuberculosis (M. tuberculosis) infection. In this study, we hypothesized that distinct subsets of γδ T cells are associated with either protection against or disease progression in TB. To explore this, we applied large-scale scRNA-seq and bulk RNA-seq data integration to define the phenotypic and molecular characteristics of peripheral blood γδ T cells. Our analysis identified five unique γδ T subclusters, each with distinct functional profiles. Notably, we identified a unique cluster significantly enriched in the TCR signaling pathway, with high CD81 expression as a conserved marker. This distinct molecular signature suggests a specialized role for this cluster in immune signaling and regulation of immune response against M. tuberculosis. Flow cytometry confirmed our in silico results, showing that the mean fluorescence intensity (MFI) values of CD81 expression on γδ T cells were significantly increased in individuals with latent TB infection (TBI) compared to those with active TB (ATB). This finding underscores the importance of CD81 and its associated signaling mechanisms in modulating the activity and function of γδ T cells under TBI conditions, providing insights into potential therapeutic targets for TB management. Full article

The secreted aspartic peptidases (Saps) of Candida albicans play crucial roles in various steps of fungal–host interactions. Using a flow cytometry approach, this study investigated the expression of Saps1–3 antigens after (i) incubation with soluble proteins, (ii) interaction with mammalian cells, and (iii) infection in immunosuppressed BALB/c mice. Supplementation strategies involving increasing concentrations of bovine serum albumin (BSA) added to yeast carbon base (YCB) medium as the sole nitrogenous source revealed a positive and significant correlation between BSA concentration and both the growth rate and the percentage of fluorescent cells (%FC) labeled with anti-Saps1–3 antibodies. Supplementing the YCB medium with various soluble proteins significantly modulated the expression of Saps1–3 antigens in C. albicans. Specifically, immunoglobulin G, gelatin, and total bovine/human sera significantly reduced the %FC, while laminin, human serum albumin, fibrinogen, hemoglobin, and mucin considerably increased the %FC compared to BSA. Furthermore, co-cultivating C. albicans yeasts with either live epithelial or macrophage cells induced the expression of Saps1–3 antigens in 78% (mean fluorescence intensity [MFI] = 152.1) and 82.7% (MFI = 178.2) of the yeast cells, respectively, compared to BSA, which resulted in 29.3% fluorescent cells (MFI = 50.9). Lastly, the yeasts recovered from the kidneys of infected immunosuppressed mice demonstrated a 4.8-fold increase in the production of Saps1–3 antigens (MFI = 246.6) compared to BSA, with 95.5% of yeasts labeled with anti-Saps1–3 antibodies. Altogether, these results demonstrated the positive modulation of Saps’ expression in C. albicans by various key host proteinaceous components, as well as by in vitro and in vivo host challenges. Full article

Background: The goal of this study was to compare mitochondrial activity in cumulus cells (CCs) between young and advancing-aged women, the factors that affect mitochondrial activity, and their association with blastocyst quality. Materials and methods: This prospective study included 80 infertile women who underwent ICSI between May and October 2023. Participants were divided into two groups: older and younger than 38. The oocyte mitochondrial activity from CCs was evaluated using MitoTracker, and the mean fluorescence intensity (MFI) was also evaluated. Results: The univariate and multivariate analyses revealed a significant difference in the MFI between the woman ≥ 38 age group and the lower age group (162.68 ± 79.87 vs. 228.39 ± 121.38; p-value = 0.005; 95%CI 19.97, 111.45). The factors that affected the MFI were women ≥ 38 years of age (p-value = 0.005; 95%CI −111.45, −19.91), total gonadotropin dosages (p-value = 0.006; 95%CI −0.08, 0.01), and gonadotropin-releasing hormone agonist (GnRHa) triggering (p-value = 0.006; 95%CI 36.46, 210.06). However, only women aged ≥38 years remained statistically significant after a multivariable regression analysis (p-value = 0.014; 95%CI −121.00, −14.30). In addition, only male age (mean age ± SD = 38.26 ± 5.13) was associated with high blastocyst quality in univariate and mixed multivariate analyses (OR 0.91; 95%CI 0.56, 3.04). The chemical pregnancy rate was not significantly different between the two age groups (34.5% vs. 56.7%; p-value = 0.162; 95%CI 0.2, 1.30). Conclusion: Advancing age decreased mitochondrial activity in CCs but did not affect blastocyst quality. By contrast, male age may be a predictor of high-grade blastocyst quality. Full article

Previous investigators have used various anti-HLA-F monoclonal antibodies (mAbs) to demonstrate that the tissue distribution of HLA-F is highly restricted. Notably, these mAbs differed in their immunodiagnostic capabilities. Specifically, mAbs Fpep1.1 and FG1 detected HLA-F intracellularly in B cells but not on the cell surface, whereas mAb 3D11 detected HLA-F on the cell surface. The presence of HLA-F on T cells was recognized by mAb FG1 but not by mAb Fpep1.1. mAb 3D11 detected HLA-F on the cell surface of activated B cells and on peripheral blood lymphocytes, but not on the normal cells. Importantly, mAb 3D11 revealed that HLA-F exists as a heavy chain (HC) monomer, rather than as an HC associated with B2m. Although these mAbs are believed to be specific to HLA-F, their monospecificity has not been formally established, which is critical for immunodiagnostic and therapeutic purposes. Previously, we investigated the diversity of HLA class I reactivities of anti-HLA-E mAbs using HLA-I coated multiplex bead assays on a Luminex platform. We reported that more than 80% of the HLA-E mAbs were cross-reactive with other HLA-I molecules, with exceptionally few truly HLA-E-monospecific mAbs. In the present investigation, we generated IgG mAbs against HCs of HLA-F in Balb/C mice and examined the cross-reactivity of anti-HLA-F mAbs with other HLA-I alleles using a multiplex bead assay on the Luminex platform. Beads coated with an array of HLA homo- and heterodimers of different HLA-Ia (HLA-A, HLA-B, and HLA-C) and Ib (HLA-E, HLA-F, and HLA-G) alleles were used to examine the binding of the anti-HLA-F mAbs. Only two mAbs were HLA-F monospecific, and five were HLA-Ib restricted. Several anti-HLA-F mAbs cross-reacted with HLA-E (n = 4), HLA-G (n = 3), HLA-Ia alleles (n = 9), HLA-G and HLA-Ia (n = 2), and HLA-Ib and HLA-Ia (n = 6). This monospecificity and polyreactivity were corroborated by the presence of HLA-F monospecific and HLA-I-shared sequences. This study emphasizes the need to monitor the mono-specificity of HLA-F for reliable immunodiagnostics and passive immunotherapy. Full article

Two semi-quantitative, Luminex-based, single-antigen bead (SAB) assays are available to detect anti-HLA antibodies and evaluate their reactivity with complement binding. Sera from 97 patients with positive panel reactive antibody tests (>5%) were analyzed with two SAB tests, Immucor (IC) and One-Lambda (OL), for anti-HLA antibody detection and the evaluation of their complement-binding capacity. IC detected 1608/8148 (mean fluorescent intensity (MFI) 4195 (1995–11,272)) and 1136/7275 (MFI 6706 (2647–13,184)) positive anti-HLA class I and II specificities, respectively. Accordingly, OL detected 1942/8148 (MFI 6185 (2855–12,099)) and 1247/7275 (MFI 9498 (3630–17,702)) positive anti-HLA class I and II specificities, respectively. For the IC assay, 428/1608 (MFI 13,900 (9540–17,999)) and 409/1136 (MFI 11,832 (7128–16,531)) positive class I and II specificities bound C3d, respectively. Similarly, OL detected 485/1942 (MFI 15,452 (9369–23,095)) and 298/1247 (MFI18,852 (14,415–24,707)) C1q-binding class I and II specificities. OL was more sensitive in detecting class I and II anti-HLA antibodies than IC was, although there was no significant difference in the number of class II specificities per case. MFI was higher for complement vs. non-complement-binding anti-HLA antibodies in both assays. Both methods were equal in detecting complement-binding anti-HLA class I antibodies, whereas the C3d assay was more sensitive in detecting complement-binding anti-HLA class II antibodies. Full article

We investigated the IL-6 receptor (IL-6R) expression on the surface of T cells isolated from peripheral blood mononuclear cells (PBMCs) of microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA) patients and measured the serum soluble IL-6R (sIL-6R) levels in these patients. Sera and PBMCs were obtained from 51 patients with MPA (n = 32) and GPA (n = 19), with 25 patients having active disease (defined as a Birmingham Vasculitis Activity Score [BVAS] ≥ 5). The median age of patients was 67.0 years, and 52.9% were women. Serum IL-6 levels were significantly correlated with the BVAS (r = 0.384); however, IL-6R expression on the surface of T cells did not significantly differ based on disease activity. Meanwhile, IL-6R expression on the surface of stimulated CD4+ (median mean fluorescence intensity [MFI] 588.0 vs. 1314.8; p < 0.001), CD4+CD25+ (MFI 853.3 vs. 1527.3; p < 0.001), and CD4+CD45RO+ (MFI 679.5 vs. 1241.5; p < 0.001) T cells was significantly reduced compared with unstimulated conditions. Conversely, patients with active disease exhibited a significantly higher median serum sIL-6R level than those with inactive disease (38.1 ng/mL vs. 34.7 ng/mL; p = 0.029). These results imply that the trans-signalling IL-6 pathway may be more activated than the classical signalling pathway in patients with MPA and GPA, suggesting the therapeutic potential of targeting sIL-6R. Full article

Complement activation by HLA antibodies is a key component of immune-mediated graft injury. We examined the clinical outcomes of kidney transplant recipients with complement-fixing de novo donor-specific antibodies (dnDSA) who were followed in our center. The C1q-binding ability was retrospectively assessed in 69 patients with dnDSA and mean fluorescence intensity (MFI) values > 2000 out of the 1325 kidney transplant recipients who were screened for DSA between 2015 and 2019. Luminex IgG single antigen beads (SAB)and C1q-SAB assays (One Lambda) were used. C1q-binding dnDSA was identified in 32/69 (46.4%) of the patients. Significantly higher MFI values were observed in C1q-positive DSA (18,978 versus 5840, p < 0.001). Renal graft biopsies were performed in 43 of the kidney transplant recipients (62.3%) with allograft dysfunction. Antibody-mediated rejection (ABMR) was detected in 29/43 (67.4%) of the patients. The incidence of ABMR was similar among patients with C1q-binding and non-C1q-binding DSA (51.7% vs. 48.3%, p = 0.523). Graft loss occurred in 30/69 (43.5%) of the patients at a median time of 82.5 months (IQR 45–135) from DSA detection. C1q-binding DSA was present in more patients who experienced graft loss (53.1% vs. 35.1%, p = 0.152). Higher MFI values and inferior clinical outcomes occurred in most of the kidney transplant recipients with C1q-binding dnDSA. Full article

Background: Intraplaque hemorrhage (IPH) is a hallmark of atherosclerotic plaque instability. Biliverdin reductase B (BLVRB) is enriched in plasma and plaques from patients with symptomatic carotid atherosclerosis and functionally associated with IPH. Objective: We explored the biomarker potential of plasma BLVRB through (1) its correlation with IPH in carotid plaques assessed by magnetic resonance imaging (MRI), and with recurrent ischemic stroke, and (2) its use for monitoring pharmacotherapy targeting IPH in a preclinical setting. Methods: Plasma BLVRB levels were measured in patients with symptomatic carotid atherosclerosis from the PARISK study (n = 177, 5 year follow-up) with and without IPH as indicated by MRI. Plasma BLVRB levels were also measured in a mouse vein graft model of IPH at baseline and following antiangiogenic therapy targeting vascular endothelial growth factor receptor 2 (VEGFR-2). Results: Plasma BLVRB levels were significantly higher in patients with IPH (737.32 ± 693.21 vs. 520.94 ± 499.43 mean fluorescent intensity (MFI), p = 0.033), but had no association with baseline clinical and biological parameters. Plasma BLVRB levels were also significantly higher in patients who developed recurrent ischemic stroke (1099.34 ± 928.49 vs. 582.07 ± 545.34 MFI, HR = 1.600, CI [1.092–2.344]; p = 0.016). Plasma BLVRB levels were significantly reduced following prevention of IPH by anti-VEGFR-2 therapy in mouse vein grafts (1189 ± 258.73 vs. 1752 ± 366.84 MFI; p = 0.004). Conclusions: Plasma BLVRB was associated with IPH and increased risk of recurrent ischemic stroke in patients with symptomatic low- to moderate-grade carotid stenosis, indicating the capacity to monitor the efficacy of IPH-preventive pharmacotherapy in an animal model. Together, these results suggest the utility of plasma BLVRB as a biomarker for atherosclerotic plaque instability. Full article

B-cell precursor acute lyphoblastic leukemia (ALL) is a common pediatric malignancy and patients may have significant benefits from monoclonal antibodies therapy with increased survival rates. Positive CD20 expression is identified in about half of these patients and its presence may serve as a prognostic factor in disease evolution. We performed a retrospective study including 114 patients diagnosed with B-ALL and evaluated the expression of CD20 through flow cytometry at diagnosis and on day 15. Additional immunophenotypic analyses as well as cytogenetic and molecular genetic analyses were also performed. We observed an increase in the mean fluorescence intensity (MFI) of CD20 between diagnosis—1.9 (1.2–3.26) and day 15: 6.17 (2.14–27.4), (p < 0.0001). Furthermore, we assessed that both diagnosis and day 15 CD20 MFI had an impact on RFS and OS, respectively, for cut-off values of >8.08 at diagnosis and >28.65 at day 15. In conclusion, CD20 expression appears to be a poor prognostic feature of B-ALL in pediatric patients. In this study, stratification of the outcome by the intensity of CD20 has implications concerning the allocation to rituximab-based chemotherapy and may offer new, potentially useful information for pediatric patients with B-ALL. Full article

Post-transplant antihuman leukocyte antigen donor-specific antibodies (anti-HLA DSAs) monitoring in kidney transplant recipients remains unclear and is currently under investigation. The pathogenicity of anti-HLA DSAs is determined by antibody classes, specificity, mean fluorescent intensity (MFI), C1q-binding capacity, and IgG subclasses. The aim of this study was to investigate the association of circulating DSAs and their characteristics with renal allograft long-term outcomes. The study included 108 consecutive patients from our transplant center who underwent kidney allograft biopsy between November 2018 and November 2020, 3 to 24 months after kidney transplantation. At the time of biopsy, patients’ sera were collected for analysis of anti-HLA DSAs. Patients were followed for a median time of 39.0 months (Q1–Q3, 29.8–45.0). Detection of anti-HLA DSAs at the time of biopsy (HR = 5.133, 95% CI 2.150–12.253, p = 0.0002) and their C1q-binding capacity (HR = 14.639, 95% CI 5.320–40.283, p ≤ 0.0001) were independent predictors of the composite of sustained 30% reduction from estimated glomerular filtration rate or death-censored graft failure. Identification of anti-HLA DSAs and their C1q-binding capacity could be useful in identifying kidney transplant recipients at risk for inferior renal allograft function and graft failure. Analysis of C1q is noninvasive, accessible, and should be considered in clinical practice in post-transplant monitoring. Full article

Background: Desensitization strategies improve access to transplantation in highly sensitized kidney transplant candidates. Tocilizumab could be a valuable addition to more traditional desensitization regimens. We investigated the effect of tocilizumab as an add-on therapy to our standard of care (SoC) desensitization strategy based on rituximab and apheresis. Methods: In this study, we prospectively included highly sensitized patients to receive monthly tocilizumab infusions for 6 months before our SoC regimen (Toci + SoC group). We compared the reductions in the mean fluorescent intensity (MFI) rebound at post-transplantation and kidney function at 1-year post-transplantation to patients treated by SoC (based on apheresis and two doses of rituximab). Results: Twenty-six patients were included in the SoC group; seven in the Toci + SoC group. Reductions in pre-transplantation MFI were similar between groups. At 1-year post-transplantation, there was no absolute difference in overall MFI rebounds, including donor-specific antibodies. Toci + SoC helped lower the rebound of antibodies with more elevated baseline MFIs. Graft function and survival rates were similar at one-year post-transplantation (median eGFR 62.8 vs. 65.6 mL/min/1.73 m² for SoC and Toci + SoC, respectively). Conclusions: Tocilizumab as an add-on to SoC desensitization may help control the post-transplantation rebound of antibodies with elevated baseline MFIs. However, reductions in pre-transplantation MFIs were similar with or without tocilizumab. Further studies are needed to validate this pilot study. Full article