ABSTRACT
During coronary artery bypass grafting (CABG), the surgical procedure, particularly the manipulation of the major arteries of the heart, induces a significant inflammatory state that may compromise platelet function to the extent that platelet transfusion is required. Given stored platelets as a major source of biological mediators, this study investigates the effects of platelet transfusion on the major pro-aggregatory, pro-inflammatory and immunomodulatory markers of platelets. Platelets from 20 patients, 10 who received platelet transfusion and 10 without, were subjected to flow cytometery where P-selectin and CD40 ligand (CD40L) expressions and PAC-1 binding (activation-specific anti GPIIb/GPIIIa antibody) analysed at five-time points of 24 h before surgery, immediately, 2 h, 24 h and 1 week after surgery. Analysis of intra-platelet transforming growth factor-beta-1 (TGF-ß1) was also conducted using western blotting. Patients with platelet transfusion showed increased levels of P-selectin, CD40L and intra-platelet TGF-ß1 2-h after surgery compared to those without transfusion (p < 0.05). PAC-1 binding was increased 24 h after surgery in transfused patients (p < 0.05). Given the significant post-transfusion elevation of platelet TGF-ß1, P-sel/CD40L reduction in transfused patients a week after was of much interest. This study showed for the first time the significant effects of platelet transfusion on the pro-inflammatory, pro-aggeregatory and immunomodulatory state of platelets in CABG patients, which manifested with immediate, midterm and delayed consequences. While the increased pro-inflammatory conditions manifested as an immediate effect of platelet transfusion, the pro-aggregatory circumstances emerged 24 h post-transfusion. A week after surgery, attenuations of pro-inflammatory markers of platelets in transfused patients were shown, which might be due to the immunomodulatory effects of TGF-ß1.
Subject(s)
Blood Platelets , CD40 Ligand , Coronary Artery Bypass , P-Selectin , Platelet Transfusion , Humans , Coronary Artery Bypass/adverse effects , Blood Platelets/metabolism , Male , Female , P-Selectin/blood , P-Selectin/metabolism , Middle Aged , CD40 Ligand/blood , CD40 Ligand/metabolism , Aged , Transforming Growth Factor beta1/blood , Transforming Growth Factor beta1/metabolism , Inflammation/blood , Platelet AggregationABSTRACT
When the membrane protein CD40 ligand (CD40L) on activated T cells binds the receptor CD40 on B-cells, it provides a co-stimulatory signal for B cell activation. Dysregulation of the CD40L:CD40 axis is associated with inflammatory and autoimmune diseases. The presence of soluble CD40L (sCD40L) in plasma is implicated in several diseases, from cardiovascular and autoimmune diseases to different types of cancer, and sCD40L has been suggested as a valuable marker of disease. If sCD40L is to be used as a biomarker, being able to precisely measure and quantify the levels of sCD40L in human blood samples is of utmost importance. We demonstrate the development of a sandwich-type time-resolved immunofluorometric assay for quantification of sCD40L in plasma or serum samples. For this, we generate 29 monoclonal anti-CD40L antibodies, and from these, we select the optimal combination of capture antibody and detection antibody. A number of variables were tested: the influence of the type of sample (comparing 3 different blood collection tubes for serum sampling and 4 different types of tubes for plasma sampling), the influence of freeze-thaw cycles, the influence of sampling time during night and day, and the influence of centrifugation of the samples. We found a very similar level of sCD40L in paired EDTA plasma and serum samples. Out of 100 healthy blood donor samples 61 had a level of sCD40L below the detection level of the assay, whereas the remaining 39 samples had ranging levels of sCD40L from 1.14 to 33.14 ng/mL. In summary, we present a time-resolved immunofluorometric assay based on paired monoclonal antibodies, ensuring high specificity, sensitivity, and homogeneity. The Eu3+-based assay additionally provides consistent assay readouts due to the extended decay time not seen in standard enzyme-linked immunosorbent assays. The assay paves the way for specific and consistent quantification of sCD40L in human plasma and serum samples.
Subject(s)
CD40 Ligand , Humans , CD40 Ligand/blood , Biomarkers/blood , Antibodies, Monoclonal/immunology , Fluoroimmunoassay/methods , Reproducibility of ResultsABSTRACT
Anti-N-methyl-d-aspartate receptor encephalitis (anti-NMDARE) is a B cell- and antibody-mediated autoimmune disease which may be regulated by CD40/CD40L signaling pathway. we enrolled anti-NMDARE patients and measured the serum CD40 and CD40L concentrations. The serum concentration of CD40 was decreased, while CD40L was increased in anti-NMDARE patients compared with that of healthy controls. The concentrations of CD40 and CD40L were both elevated in the acute stage of anti-NMDARE and were reduced during remission. Serum CD40L levels were positively correlated with serum CD40 levels. These results revealed that the CD40/CD40L signaling pathway might contribute to the pathogenesis of anti-NMDARE.
Subject(s)
Anti-N-Methyl-D-Aspartate Receptor Encephalitis/blood , Anti-N-Methyl-D-Aspartate Receptor Encephalitis/immunology , CD40 Antigens/blood , CD40 Ligand/blood , Adolescent , Adult , CD40 Antigens/immunology , CD40 Ligand/immunology , Child , Female , Humans , Male , Middle Aged , Young AdultABSTRACT
Infection with SARS-CoV-2, the causative agent of COVID-19, causes mild to moderate disease in most patients but carries a risk of morbidity and mortality. Seriously affected individuals manifest disorders of hemostasis and a cytokine storm, but it is not understood how these manifestations of severe COVID-19 are linked. Here, we showed that the SARS-CoV-2 spike protein engaged the CD42b receptor to activate platelets via 2 distinct signaling pathways and promoted platelet-monocyte communication through the engagement of P selectin/PGSL-1 and CD40L/CD40, which led to proinflammatory cytokine production by monocytes. These results explain why hypercoagulation, monocyte activation, and a cytokine storm are correlated in patients severely affected by COVID-19 and suggest a potential target for therapeutic intervention.
Subject(s)
Blood Platelets/physiology , COVID-19/blood , Inflammation/blood , Monocytes/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/physiology , Blood Platelets/metabolism , CD40 Antigens/blood , CD40 Ligand/blood , Cell Communication , Cytokine Release Syndrome , Cytokines , HEK293 Cells , Humans , P-Selectin/bloodABSTRACT
Background: Oral immunotherapy (OIT) is an emerging treatment for cow's milk protein (CMP) allergy in children. The mechanisms driving tolerance following OIT are not well understood. Regulatory T cells (TREG) cells are key inhibitors of allergic responses and promoters of allergen-specific tolerance. In an exploratory study, we sought to detect induction of allergen-specific TREG in a cohort of subjects undergoing OIT. Methods: Pediatric patients with a history of allergic reaction to cow's milk and a positive Skin Pick Test (SPT) and/or CMP-specific IgE >0.35 kU, as well as a positive oral challenge to CMP underwent OIT with escalating doses of milk and were followed for up to 6 months. At specific milestones during the dose escalation and maintenance phases, casein-specific CD4+ T cells were expanded from patient blood by culturing unfractionated PBMCs with casein in vitro. The CD4+ T cell phenotypes were quantified by flow cytometry. Results: Our culture system induced activated casein-specific FOXP3+Helios+ TREG cells and FOXP3- TEFF cells, discriminated by expression of CD137 (4-1BB) and CD154 (CD40L) respectively. The frequency of casein-specific TREG cells increased significantly with escalating doses of milk during OIT while casein-specific TEFF cell frequencies remained constant. Moreover, expanded casein-specific TREG cells expressed higher levels of FOXP3 compared to polyclonal TREG cells, suggesting a more robust TREG phenotype. The induction of casein-specific TREG cells increased with successful CMP desensitization and correlated with increased frequencies of casein-specific Th1 cells among OIT subjects. The level of casein-specific TREG cells negatively correlated with the time required to reach the maintenance phase of desensitization. Conclusions: Overall, effective CMP-OIT successfully promoted the expansion of casein-specific, functionally-stable FOXP3+ TREG cells while mitigating Th2 responses in children receiving OIT. Our exploratory study proposes that an in vitro TREG response to casein may correlate with the time to reach maintenance in CMP-OIT.
Subject(s)
Caseins/immunology , Desensitization, Immunologic/methods , Milk Hypersensitivity/immunology , Milk Hypersensitivity/therapy , T-Lymphocytes, Regulatory/immunology , Administration, Oral , Adolescent , Allergens/administration & dosage , Animals , CD40 Ligand/blood , Cattle , Child , Cohort Studies , Female , Forkhead Transcription Factors/blood , Humans , In Vitro Techniques , Male , Milk Hypersensitivity/blood , T-Lymphocytes, Regulatory/classification , Th2 Cells/immunology , Time Factors , Tumor Necrosis Factor Receptor Superfamily, Member 9/bloodABSTRACT
In organ transplantation, human leukocyte antigen (HLA)-mismatch grafts not only induce the activation of cellular mediated immune response but also the development of chronic antibody-mediated rejection due to the donor-specific anti-HLA antibody (DSA) produced by B cells and plasma cells interacting with the graft endothelium. Significant improvement in long-term survival after transplantation can be expected if antibody-mediated rejection due to the DSA can be overcome. However, the mechanism of producing or controlling the DSA remains to be elucidated. In recent decades, "humanized" mouse models have been widely used for the basic research of human immune systems, but a humanized mouse model to analyze the mechanism of DSA production has not been established yet. Thus, we aimed to create a humanized mouse using a severe immunodeficiency mouse (NSG mouse) administered with human peripheral blood mononuclear cells (PBMCs). Initially, we detected a very low level of human total-IgG and no anti-HLA antibodies (Abs) in these mice. In our next attempt, we mixed PBMCs of various HLA antigenic combinations with or without regulatory T cells and preconditioned them by culturing on feeder cells stably transfected with human CD40 ligand (h-CD40L) alone or with h-CD40L and human B cell activating factor (h-BAFF). They were subsequently co-cultured with the corresponding irradiated stimulator PBMCs, and all cells were administered into naïve NSG mice. Although all three humanized models had sufficient human total-IgG and anti-HLA antibody production, allospecific anti-HLA Ab production was prominently suppressed whereas non-specific anti-HLA Abs were sufficiently detected. Therefore, this novel humanized mouse model might be useful for analyzing the mechanism of anti-allogeneic human B cell tolerance induction.
Subject(s)
Graft Rejection/immunology , HLA Antigens/analysis , HLA Antigens/immunology , Animals , CD40 Ligand/blood , CD40 Ligand/immunology , Female , Graft Survival/immunology , Histocompatibility Antigens Class II , Humans , Immunity, Cellular/immunology , Isoantibodies/blood , Kidney Transplantation/methods , Leukocytes, Mononuclear/immunology , Male , Mice , Mice, Inbred NOD , Mice, SCID , Models, Animal , Organ Transplantation/methods , Tissue DonorsABSTRACT
Sepsis-associated disseminated intravascular coagulation (DIC) is related to marked hemostatic changes such as transient thrombocytopenia secondary to the endogenous activation and consumption of platelets. This study measured markers of platelet function in 103 adult ICU patients with clinically established sepsis-associated DIC to determine the biomarker association with disease severity. Patients were categorized as having no DIC, nonovert DIC, or overt DIC using the International Society of Thrombosis and Hemostasis scoring system. Plasma levels of CD40L, platelet factor 4 (PF4), platelet-derived microparticles, and microparticle-associated tissue factor were quantified. Markers of platelet activation were significantly elevated in patients with DIC compared to healthy individuals. This increase was independent of platelet count. Levels of PF4 differed based on the severity of DIC and differentiated nonsurvivors and survivors. These findings suggest that the markers of platelet activation in DIC may not be regulated by the number of circulating platelets and may be independent of the factors leading to their consumption.
Subject(s)
Blood Platelets/metabolism , Disseminated Intravascular Coagulation/diagnosis , Platelet Activation , Platelet Factor 4/blood , Sepsis/complications , Adult , Aged , Biomarkers/blood , CD40 Ligand/blood , Case-Control Studies , Disseminated Intravascular Coagulation/blood , Disseminated Intravascular Coagulation/etiology , Disseminated Intravascular Coagulation/mortality , Female , Humans , Male , Middle Aged , Predictive Value of Tests , Prognosis , Risk Factors , Sepsis/blood , Sepsis/diagnosis , Sepsis/mortality , Severity of Illness Index , Thromboplastin/metabolism , Young AdultABSTRACT
BACKGROUND: Soluble CD40 ligand (sCD40L) exhibits proinflammatory and procoagulant effects. Recent data indicated that sCD40L plays a significant role in septic patients. The aim of the present study was to determine sCD40L changes in surgical patients without sepsis (SWS) and surgical sepsis patients (SS) during the first 3 days after intensive care unit (ICU) admission and to observe the association between sCD40L and mortality. METHODS: Time changes in sCD40L levels were assessed for 3 days after ICU admission in 49 patients with SS and compared with those in 19 SWS patients. Serum sCD40L concentration was detected by ELISA. Survival at 28 days served as the endpoint. RESULTS: SS had significantly higher sCD40L levels than SWS and control patients. We observed an association between sCD40L levels ≥1028.75 pg/mL at day 2 and 28-day mortality (odds ratio = 7.888; 95% confidence interval = 1.758 to 35.395; P = 0.007). We could not discover any significant differences in sex, presence of septic shock, site of infection, length of stay in the ICU, PaO2/FiO2 ratio, incidence of AKI, ARDS, or type of surgery between nonsurvivors and survivors. CONCLUSIONS: Septic patients show persistently higher circulating sCD40L levels in the first 3 days after ICU admission, and serum sCD40L levels are associated with the mortality of patients with sepsis. Thus, serum sCD40L may be used as a reliable biomarker and therapeutic target in sepsis.
Subject(s)
Biomarkers/blood , CD40 Ligand/blood , Sepsis/blood , Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Prospective StudiesABSTRACT
Purpose: To evaluate potential host biomarkers detectable in QuantiFERON supernatants as diagnostic candidates for ocular tuberculosis (OTB).Methods: We investigated 47 host markers in QuantiFERON supernatants from 92 individuals with uveitis using the Luminex platform. We evaluated the potential of individual and combined biomarkers to distinguish between patients with possible, probable, and no OTB.Results: Differences were observed in median concentrations of several biomarkers including IL-13, IFN-γ, IFN-α2, and IL-1ß, in individuals with OTB versus no OTB regardless of HIV status. Individuals with probable and possible OTB only differed regarding GM-CSF. We identified a four-marker biosignature (CD40 L, IL-33, IFN-γ, and SAP) which diagnosed OTB with an area under the ROC curve of 0.80, sensitivity = 56.3% and specificity = 90.0%.Conclusion: This represents the first attempt at screening QuantiFERON supernatants for biomarkers to diagnose OTB. We identified candidate biosignatures which may aid in diagnosing OTB in both HIV positive and negative patients.
Subject(s)
Biomarkers/blood , Tuberculosis, Ocular/diagnosis , Uveitis/diagnosis , Adult , Antigens, Bacterial/immunology , CD40 Ligand/blood , Female , HIV Infections/complications , Humans , Interferon-gamma/blood , Interferon-gamma Release Tests , Interleukin-33/blood , Male , Middle Aged , Mycobacterium tuberculosis/immunology , ROC Curve , Sensitivity and Specificity , Serum Amyloid P-Component/metabolism , Tuberculosis, Ocular/blood , Uveitis/bloodABSTRACT
Prolonged use of dual antiplatelet therapy (DAPT) post-percutaneous coronary intervention (PCI) has been shown to reduce the risk of major adverse cardiovascular events (MACE), but with increased bleeding. It remains unknown whether biomarkers of platelet activation may be useful for identifying patients at increased risk of MACE. The DAPT study was a randomized trial of 12 versus 30 months of DAPT in patients who underwent PCI. Serum biomarkers [myeloid-related protein (MRP)-8/14, P-selectin, soluble CD-40 ligand (sCD40L)] were assessed in 1399 patients early post-PCI. On-treatment platelet reactivity index (PRI) using VASP phosphorylation was assessed in 443 patients randomized to continued DAPT at 1 year. MACE was defined as CV death, MI, or ischemic stroke. Multivariable models were adjusted for baseline characteristics, index event, and stent type. A stepwise increase in the risk of MACE was observed with increasing tertiles of both MRP-8/14 and P-selectin (p-trend = 0.04 for both). After multivariable adjustment, the adjusted HR (95% CI) for MACE in patients in the top tertile was 1.94 (1.14-3.30) for MRP-8/14 and 1.62 (0.99-2.64) for P-selectin. In contrast, baseline sCD40L was not associated with CV risk. Among patients randomized to continued DAPT, higher on-treatment platelet reactivity was not significantly associated with risk of MACE (p-trend = 0.32; adj-HR T3 vs. T1 1.54, 95% CI 0.20-12.18) or bleeding (P-trend = 0.17; adj-HR 0.25, 95% CI 0.05-1.21). MRP-8/14 and soluble P-selectin may be useful for identifying patients at increased risk of MACE after PCI. The utility of on-treatment platelet function testing requires further study.Clinical Trial Registration https://www.clinicaltrials.gov . Unique identifier NCT00977938.
Subject(s)
Calgranulin A/blood , Calgranulin B/blood , Coronary Restenosis , Hemorrhage , P-Selectin/blood , Percutaneous Coronary Intervention/adverse effects , Biomarkers/blood , CD40 Ligand/blood , Coronary Restenosis/blood , Coronary Restenosis/etiology , Coronary Restenosis/prevention & control , Drug Monitoring/methods , Dual Anti-Platelet Therapy/adverse effects , Dual Anti-Platelet Therapy/methods , Duration of Therapy , Female , Hemorrhage/blood , Hemorrhage/chemically induced , Hemorrhage/prevention & control , Humans , Male , Middle Aged , Percutaneous Coronary Intervention/instrumentation , Percutaneous Coronary Intervention/methods , Platelet Function Tests/methods , Reproducibility of Results , Risk Assessment/methodsABSTRACT
The aim of this study (NCT04343053) is to investigate the relationship between platelet activation, myocardial injury, and mortality in patients affected by Coronavirus disease 2019 (COVID-19). Fifty-four patients with respiratory failure due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were enrolled as cases. Eleven patients with the same clinical presentation, but negative for SARS-CoV-2 infection, were included as controls. Blood samples were collected at three different time points (inclusion [T1], after 7 ± 2 days [T2] and 14 ± 2 days [T3]). Platelet aggregation by light transmittance aggregometry and the circulating levels of soluble CD40 ligand (sCD40L) and P-selectin were measured. Platelet biomarkers did not differ between cases and controls, except for sCD40L which was higher in COVID-19 patients (p = .003). In COVID-19 patients, P-selectin and sCD40L levels decreased from T1 to T3 and were higher in cases requiring admission to intensive care unit (p = .004 and p = .008, respectively). Patients with myocardial injury (37%), as well as those who died (30%), had higher values of all biomarkers of platelet activation (p < .05 for all). Myocardial injury was an independent predictor of mortality. In COVID-19 patients admitted to hospital for respiratory failure, heightened platelet activation is associated with severity of illness, myocardial injury, and mortality.ClinicalTrials.gov number: NCT04343053.
Subject(s)
Blood Platelets/metabolism , COVID-19 , Heart Injuries , Myocardium , Respiratory Insufficiency , SARS-CoV-2/metabolism , Aged , Aged, 80 and over , Biomarkers/blood , CD40 Ligand/blood , COVID-19/blood , COVID-19/mortality , COVID-19/pathology , Female , Heart Injuries/blood , Heart Injuries/mortality , Heart Injuries/pathology , Heart Injuries/virology , Humans , Male , Middle Aged , Myocardium/metabolism , Myocardium/pathology , P-Selectin/blood , Platelet Aggregation , Respiratory Insufficiency/blood , Respiratory Insufficiency/mortality , Respiratory Insufficiency/pathology , Respiratory Insufficiency/virologyABSTRACT
CXCL12, also known as stromal cell-derived factor-1, is a chemokine classified into CXC families, which exerts its function by binding to specific receptors called CXCR4 and CXCR7. Human platelets express CXCR4 and CXCR7 on the plasma membrane. It has been reported that CXCL12 potentiates to induce platelet aggregation in cooperation with agonists including collagen. However, the precise roles and mechanisms of CXCL12 in human platelet activation are not fully elucidated. In the present study, we investigated the effect of simultaneous stimulation with low doses of collagen and CXCL12 on the activation of human platelets. The simultaneous stimulation with collagen and CXCL12 induced the secretion of platelet-derived growth factor (PDGF)-AB and the release of soluble CD40 ligand (sCD40L) from human platelets in addition to their aggregation, despite the fact that the simultaneous stimulation with thrombin receptor-activating peptide (TRAP) or adenosine diphosphate (ADP), and CXCL12 had little effects on the platelet aggregation. The agonist of Glycoprotein (GP) â ¥ convulxin and CXCL12 also induced platelet aggregation synergistically. The monoclonal antibody against CXCR4 but not CXCR7 suppressed the platelet aggregation induced by simultaneous stimulation with collagen and CXCL12. The phosphorylation of p38 mitogen-activated protein kinase (MAPK), but not p44/p42 MAPK, was induced by the simultaneous stimulation. In addition, the simultaneous stimulation with collagen and CXCL12 induced the phosphorylation of HSP27 and the subsequent release of phosphorylated-HSP27 from human platelets. SB203580, a specific inhibitor of p38 MAPK, attenuated the platelet aggregation, the phosphorylation of p38 MAPK and HSP27, the PDGF-AB secretion, the sCD40L release and the phosphorylated-HSP27 release induced by the simultaneous stimulation with collagen and CXCL12. These results strongly suggest that collagen and CXCL12 in low doses synergistically act to induce PDGF-AB secretion, sCD40L release and phosphorylated-HSP27 release from activated human platelets via p38 MAPK activation.
Subject(s)
Blood Platelets/drug effects , Chemokine CXCL12/pharmacology , Collagen/pharmacology , Platelet Activation/drug effects , Blood Platelets/cytology , Blood Platelets/metabolism , CD40 Ligand/blood , Healthy Volunteers , Heat-Shock Proteins/blood , Humans , Molecular Chaperones/blood , Platelet-Derived Growth Factor/metabolism , p38 Mitogen-Activated Protein Kinases/bloodABSTRACT
The pathogenesis of psoriatic arthritis (PsA) involves several pathways, including the CD40/CD40L signaling which promotes the release of multiple cytokines. Transmembrane CD40L is also released in soluble form (sCD40L) and phosphodiesterase 4 (PDE4) seems to be involved in its cleavage. We aimed to investigate whether apremilast, a PDE4 inhibitor, could modify circulating levels of sCD40L in PsA patients, and the possible associations of these changes with clinical response. Consecutive PsA patients starting apremilast in routine clinical practice were prospectively observed. Disease Activity of Psoriatic Arthritis (DAPSA), Psoriasis Area Severity Index (PASI), Leeds Enthesitis Score (LEI) and serum samples were collected at baseline and at 6 months. Samples were run in a Bio-Plex ProTM plate for sCD40L. To investigate the association of sCD40L level with DAPSA based minor response, low disease activity (LDA) and/or remission at 6 months of treatment, multivariate logistic regression models with backward selection (P < 0·05) were built. We studied 27 patients (16 of 27 women, 59·6%) with PsA and mean age [± standard deviation (s.d.)] of 58·4 ± 10 years. A significant reduction of the mean values of DAPSA, LEI and PASI was detected at 6 months. Mean serum levels of sCD40L decreased from baseline 5364 ± 2025 pg/ml to 4412 ± 2629 at 6 months (P = 0·01). Baseline DAPSA [odds ratio (OR) = 0·80, 95% confidence interval (CI) = 0·65-0·98] and sCD40L (OR = 1·001, 95% CI = 1·0001-1·0027) were independently associated with DAPSA LDA/remission at 6 months. In PsA patients, sCD40L levels decrease upon apremilast treatment and might predict short-term clinical response to apremilast.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Arthritis, Psoriatic/drug therapy , Biomarkers, Pharmacological/blood , CD40 Antigens/metabolism , CD40 Ligand/blood , Phosphodiesterase 4 Inhibitors/therapeutic use , Thalidomide/analogs & derivatives , Aged , Animals , Arthritis, Psoriatic/diagnosis , Female , Humans , Male , Mice , Middle Aged , Prospective Studies , Signal Transduction , Thalidomide/therapeutic useABSTRACT
Flow cytometric quantification of CD154+ mould specific T-cells in antigen-stimulated peripheral blood mononuclear cells (PBMCs) or whole blood has been described as a supportive biomarker to diagnose invasive mould infections and to monitor therapeutic outcomes. As patients at risk frequently receive immunosuppressive and antifungal medication, this study compared the matrix-dependent impact of representative drugs on CD154+ T-cell detection rates. PBMCs and whole blood samples from healthy adults were pre-treated with therapeutic concentrations of liposomal amphotericin B, voriconazole, posaconazole, cyclosporine A (CsA) or prednisolone. Samples were then stimulated with an Aspergillus fumigatus lysate or a viral antigen cocktail (CPI) and assessed for CD154+ T-helper cell frequencies. Specific T-cell detection rates and technical assay properties remained largely unaffected by exposure of both matrices to the studied antifungals. By contrast, CsA and prednisolone pre-treatment of isolated PBMCs and whole blood adversely impacted specific T-cell detection rates and caused elevated inter-replicate variation. Unexpectedly, the whole blood-based protocol that uses additional α-CD49d co-stimulation was less susceptible to CsA and prednisolone despite prolonged drug exposure in the test tube. Accordingly, addition of α-CD49d during PBMC stimulation partially attenuated the impact of immunosuppressive drugs on test performance. Translating these results into the clinical setting, false-negative results of CD154+ antigen-specific T-cell quantification need to be considered in patients receiving T-cell-active immunosuppressive medication. Optimized co-stimulation regimes with α-CD49d could contribute to an improved feasibility of functional T-cell assays in immunocompromised patient populations.
Subject(s)
Antifungal Agents/administration & dosage , Aspergillosis , CD40 Ligand/blood , Immunosuppressive Agents/administration & dosage , T-Lymphocytes/immunology , Aspergillosis/diagnosis , Aspergillosis/drug therapy , Aspergillosis/microbiology , Aspergillus fumigatus/immunology , Biomarkers/blood , Flow Cytometry , Humans , T-Lymphocytes/cytologyABSTRACT
OBJECTIVE: We examined whether maternal serum cytokine profiles of mothers with early-onset fetal growth restriction (FGR) were associated with delivery within 2 weeks after sampling during the third trimester. STUDY DESIGN: This exploratory prospective cross-sectional study included a total of 20 singleton fetuses with early-onset FGR and 31 healthy controls. Maternal serum samples during the early third trimester were analyzed for 23 cytokines. RESULTS: Of 20 fetuses with early-onset FGR, 14 had delivery within 2 weeks after sampling. Multivariate analysis revealed that maternal serum concentrations of soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) and soluble CD40 ligand (sCD40L) were independently associated with delivery within 2 weeks in early-onset FGR. Among cases of early-onset FGR, concentrations of almost all maternal serum cytokines were similar. Maternal serum sVEGFR-1 concentrations were high when delivery occurred within 2 weeks. Maternal serum sCD40L concentrations were elicited only in cases in which delivery within 2 weeks occurred due to fetal deterioration. CONCLUSION: We identified two biomarkers, one specific for FGR and the other dependent on severity, that were significant components of angiogenic activities and inflammation factors. Imbalances in serum protein expression may have a substantial effect on the pathogenesis of FGR.
Subject(s)
CD40 Ligand/blood , Cesarean Section , Cytokines/blood , Fetal Growth Retardation/blood , Labor, Induced , Pre-Eclampsia/blood , Vascular Endothelial Growth Factor Receptor-1/blood , Adult , Biomarkers/blood , Birth Weight , Case-Control Studies , Cross-Sectional Studies , Elective Surgical Procedures , Endoglin/blood , Female , Heparin-binding EGF-like Growth Factor/blood , Humans , Infant, Newborn , Infant, Small for Gestational Age , Leptin/blood , Male , Multivariate Analysis , Placenta Growth Factor/blood , Pregnancy , Pregnancy Trimester, Third , Premature Birth , Time Factors , Vascular Endothelial Growth Factor A/bloodABSTRACT
BACKGROUND/AIMS: The interaction of CD40 ligand (CD40L) and CD40 triggers the induction of pro-inflammatory cytokines. It has been proposed that vitamin D deficiency might be an important factor, which causes or aggregates the autoimmune situations. The aim of the present study was to assess the effect of vitamin D on CD40L gene expression in patients with ulcerative colitis (UC). MATERIALS AND METHODS: Ninety mild-to-moderate UC patients were randomized to receive a single injection of 7.5 mg cholecalciferol or 1 mL normal saline. At baseline and 90 days following the intervention, RNA samples from whole blood were obtained. Fold changes in CD40L mRNA expression were determined for each patient using the 2-ΔΔCq method. The data were analyzed. RESULTS: The serum levels of vitamin D and calcium increased only in the vitamin D group (p<0.05). Relative to baseline values, the CD40L gene expression fold change was significantly lower in the vitamin D group compared with the placebo group (median±interquartile range: 0.34±0.30 vs 0.43±1.20, respectively, p=0.016). CONCLUSION: The results of this study showed that vitamin D administration in mild-to-moderate UC patients led to the downregulation of the CD40L gene, which is an essential part of inflammatory pathways.
Subject(s)
CD40 Ligand/blood , Cholecalciferol/administration & dosage , Colitis, Ulcerative/genetics , Vitamin D Deficiency/drug therapy , Vitamin D Deficiency/genetics , Adult , Calcium/blood , Colitis, Ulcerative/blood , Cytokines/blood , Double-Blind Method , Down-Regulation/drug effects , Female , Gene Expression/drug effects , Humans , Injections, Subcutaneous , Male , RNA/blood , Treatment Outcome , Vitamin D/blood , Vitamin D Deficiency/bloodSubject(s)
Amoxicillin-Potassium Clavulanate Combination/adverse effects , CD40 Ligand/blood , Drug Hypersensitivity , Adolescent , Amoxicillin-Potassium Clavulanate Combination/administration & dosage , Child , Child, Preschool , Drug Hypersensitivity/blood , Drug Hypersensitivity/diagnosis , Female , Humans , MaleABSTRACT
BACKGROUND AND OBJECTIVES: Transfusion-related acute lung injury (TRALI) is a life-threatening complication of transfusion and is one of leading causes of transfusion-associated fatalities. However, the pathogenesis of TRALI is still unclear. Soluble CD40 ligand (sCD40L) is a proinflammatory cytokine that accumulates during blood component storage and is involved in transfusion reactions. The objective of this study was to establish a clinically relevant TRALI animal model and to evaluate the role of sCD40L in TRALI. MATERIALS AND METHODS: Rats' red-blood-cell (RBC) suspensions were prepared, and the quality of RBC was evaluated. A trauma-haemorrhage-transfusion strategy was applied to build the animal model. Lung oedema was evaluated by histopathology examination, total bronchoalveolar lavage fluid (BALF) protein concentration, Evans blue dye (EBD) leakage and inflammatory cytokines. The sCD40L concentrations were measured. RESULTS: Storage lesions of RBCs gradually increased over time. Obvious histological evidence of lung injury of rats transfused with a 35-day RBC was observed. The total BALF protein concentration, EBD leakage, inflammatory cytokines concentration were increased significantly in the Day 35 group. The sCD40L concentration increased significantly in the storage RBC suspension over time but was slightly elevated in rat plasma. CONCLUSIONS: These findings indicated successful establishment of a TRALI animal model with trauma-haemorrhage-transfusion, in which sCD40L may play a minor role in the development of TRALI.
Subject(s)
CD40 Ligand/blood , Transfusion-Related Acute Lung Injury/pathology , Animals , Blood Safety/standards , Disease Models, Animal , Erythrocytes/pathology , Male , Rats , Rats, Inbred Lew , Transfusion-Related Acute Lung Injury/blood , Transfusion-Related Acute Lung Injury/etiologyABSTRACT
BACKGROUND AND OBJECTIVES: The prognosis of colorectal cancer (CRC) has improved in the last decades, however, a lower overall survival persists in the elderly. The understanding of immunity changes in the elderly with CRC will allow the emergence of new treatments with higher response rates. 4-1BB and CD40L, an immune checkpoint stimulator, play an important role in T-cell responses and platelets. Our aim was to characterize the soluble levels of CD40L and 4-1BB in CRC elderly patients. METHODS: A cross-sectional study was performed in 41 patients with CRC and 35 healthy elderly controls. Patients with CRC were divided into three groups according to staging: 13 patients with advanced tumor restricted to the organ (stages II); 16 patients with lymph node metastasis (stage III); and 12 patients with distant metastasis (stage IV). RESULTS: There were higher levels of soluble s4-1BB and sCD40L in CRC elderly stage II patients when compared with healthy controls (P = .0009 and P < .0001, respectively), stage III patients (P = .008 and P < .0001, respectively) and stage IV patients (P = .007 and P < .0001, respectively). CONCLUSIONS: We concluded that sCD40L and s4-1BB molecules may be prognostic biomarkers, since the reduction in plasma levels of these molecules was associated with disease progression.
Subject(s)
CD40 Ligand/blood , Colorectal Neoplasms/mortality , Tumor Necrosis Factor Receptor Superfamily, Member 9/blood , Aged , Biomarkers, Tumor/blood , Case-Control Studies , Colorectal Neoplasms/blood , Colorectal Neoplasms/pathology , Cross-Sectional Studies , Female , Humans , Lymphatic Metastasis , Male , Neoplasm MetastasisABSTRACT
BACKGROUND: To investigate the relationship between changes in circulating soluble CD40 ligand (sCD40L) levels and the presence and severity of type 2 diabetic retinopathy (DR). SUBJECTS AND METHODS: sCD40L plasma concentrations were measured in 205 type 2 diabetes (T2DM) patients without DR (DWR; n=50) and with DR (n=155), the latter subdivided into non-proliferative diabetic retinopathy [NPDR; n=98 (63.2%)], or proliferative retinopathy [PDR; n=57 (36.8%)] patients. RESULTS: Receiver operating characteristic analysis provided good discriminatory power for sCD40L as predictor of DR presence, with high sensitivity and specificity. Categorizing DWR and DR patients into sCD40L quartiles, based on sCD40L concentrations in T2DM without DR, demonstrated statistically significant gradual increase in DR risk with increasing sCD40L levels. sCD40L levels were significantly higher in DR compared to DWR patients. Plasma sCD40L levels differed significantly according to DR severity, and correlated with diabetes duration, dyslipedimea, nephropathy, and presence of DR, but not with gender, age, SBP, DBP, FPG, HbA1c, T2DM medications. Linear regression analysis confirmed the association of increased sCD40L levels with DR, independent of others parameters; mean plasma sCD40L levels differing significantly according to DR severity. CONCLUSION: Plasma sCD40L levels were positively associated with DR. The significant finding here is that sCD40L levels can be predictors of DR severity.