Cytometric Bead Array Technology Research Articles

Evaluation of the clinical application value of cytokine expression profiles in the differential diagnosis of prostate cancer

BackgroundThe significance of tumor-secreted cytokines in tumor development has gained substantial attention. Nevertheless, the precise role of tumor-related inflammatory cytokines in prostate cancer (PCa) remains ambiguous.ObjectivesTo gain deeper insights into the inflammatory response in the process of PCa.MethodsA total of 233 cases were collected, including 80 cases of prostate hyperplasia as disease control, 65 cases of postoperative prostate cancer and 36 cases of prostate cancer as PCa group. Additionally, 52 patients undergoing physical examinations during the same period were collected as the healthy control. The levels of 12 inflammatory cytokines in peripheral blood samples were analyzed using flow cytometric bead array technology. The levels of total prostate-specific antigen (TPSA) and free prostate-specific antigen (FPSA) in peripheral blood samples were analyzed using electrochemiluminescence technology.ResultsOur findings revealed significant increases in serum IL-8 levels in PCa group compared to the healthy control group. Additionally, IL-6, IL-10, IFN-γ and IL-12p70 levels were markedly elevated in the PCa group compared to the disease control group (all p < 0.05). Conversely, the level of IL-4, TNF-α, IL-1β, IL-17A and IFN-α were lower in the PCa group compared to those in control group. Following surgery, the concentration of IL-6 decreased; whereas, the concentrations of IL-4, TNF-α, IL-17A, IL-1β, IL-12p70, and IFN-α increased, demonstrating significant differences (p < 0.05). The differential upregulation of IL-6 or downregulation of IL-17A in peripheral blood exhibited diagnostic efficacy in PCa patients. Moreover, we observed a significant increase in IL-17A levels, accompanied by decreased of IL-2, IL-4, IL-10, TNF-a, IFN-γ, IL-1β, and IL-12P70 in patients with distant metastasis.ConclusionThe peripheral blood cytokines are closely associated with the occurrence and development of prostate cancer, especially the serum levels of IL-6 and IL-17A may be useful as potential predictors of PCa diagnosis.

The predictive utility of cytokines, procalcitonin and C-reactive protein among febrile pediatric hematology and oncology patients with severe sepsis or septic shock

Severe sepsis and septic shock are life-threatening for pediatric hematology and oncology patient receiving chemotherapy. Th1/Th2 cytokines, C-reactive protein (CRP), and procalcitonin (PCT) are all thought to be associated with disease severity. The aim of this study was to prospectively verify the utility of Th1/Th2 cytokines and compare them with PCT and CRP in the prediction of adverse outcomes. Data on patients were collected from January 1, 2011, to December 31, 2020. Blood samples were taken for Th1/Th2 cytokine, CRP, and PCT measurements at the initial onset of infection. Severe infection (SI) was defined as severe sepsis or septic shock. Th1/Th2 cytokine levels were determined by using flow cytometric bead array technology. In total, 7,735 febrile episodes were included in this study. For SI prediction, the AUCs of IL-6, IL-10 and TNF-α were 0.814, 0.805 and 0.624, respectively, while IL-6 and IL-10 had high sensitivity and specificity. IL-6 > 220.85 pg/ml and IL-10 > 29.95 pg/ml had high odds ratio (OR) values of approximately 3.5 in the logistic regression. Within the subgroup analysis, for bloodstream infection (BSI) prediction, the AUCs of IL-10 and TNF-α were 0.757 and 0.694, respectively. For multiorgan dysfunction syndrome (MODS) prediction, the AUC of CRP was 0.606. The AUC of PCT for mortality prediction was 0.620. In conclusion, IL-6 and IL-10 provide good predictive value for the diagnosis of SI. For children with SI, IL-10 and TNF-α are associated with BSI, while CRP and PCT are associated with MODS and death, respectively.

Interleukin-8 and Interleukin-6 Are Biomarkers of Poor Prognosis in Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma (ESCC) is a common type of cancer characterized by fast progression and high mortality rates, which generally implies a poor prognosis at time of diagnosis. Intricate interaction networks of cytokines produced by resident and inflammatory cells in the tumor microenvironment play crucial roles in ESCC development and metastasis, thus influencing therapy efficiency. As such, cytokines are the most prominent targets for specific therapies and prognostic parameters to predict tumor progression and aggressiveness. In this work, we examined the association between ESCC progression and the systemic levels of inflammatory cytokines to determine their usefulness as diagnostic biomarkers. We analyzed the levels of IL-1β, IL-6, IL-8, IL-10, TNF-α e IL-12p70 in a group of 70 ESCC patients and 70 healthy individuals using Cytometric Bead Array (CBA) technology. We detected increased levels of IL-1β, IL-6, IL-8, and IL-10 in ESCC patients compared to controls. However, multivariate analysis revealed that only IL8 was an independent prognostic factor for ESCC, as were the well-known risk factors: alcohol consumption, tobacco usage, and exposure to pesticides/insecticides. Importantly, patients with low IL-6, IL-8, TNM I/II, or those who underwent surgery had a significantly higher overall survival rate. We also studied cultured Kyse-30 and Kyse-410 cells in mice. We determined that the ESCC cell line Kyse-30 grew more aggressively than the Kyse-410 cell line. This enhanced growth was associated with the recruitment/accumulation of intratumoral polymorphonuclear leukocytes. In conclusion, our data suggest IL-8 as a valuable prognostic factor with potential as a biomarker for ESCC.

Pro-inflammatory cytokine profile is present in the serum of Mexican patients with different stages of diabetic retinopathy secondary to type 2 diabetes

AimIt’s been reported that pro-inflammatory cytokines are elevated in patients with diabetic retinopathy (DR); this may contribute to the pathophysiology of the disease. The aim of this study is to...

Aqueous humour cytokines profiles in eyes with Coats disease and the association with the severity of the disease

BackgroundTo investigate aqueous humour (AH) cytokine profiles in eyes with Coats disease and analyze the association between cytokine concentrations and the severity of the disease.MethodsThe study included 36 patients (36 eyes) with Coats disease and 15 control patients (15 eyes) with congenital cataract. AH samples were obtained preoperatively and the concentrations of 22 different cytokines were measured through Cytometric Bead Array technology. Clinical characteristics of Coats disease, including the extent of retinal exudation and exudative retinal detachment (ERD), were recorded for analysis.ResultsThe concentrations of 8 cytokines (VEGF, IL-6, IL-8, MCP-1, MIP-1α, IP-10, VCAM-1 and ICAM-1) were significantly higher in the Coats disease group than in the control group (all P < 0.002). Except for VCAM-1 and ICAM-1, the concentration of the other cytokines listed above showed a significant increase from stage 2 to stage 3 (all P < 0.05). Meanwhile, the concentrations of VEGF, IL-8, MCP-1 and MIP-1α showed a significant and positive association with the extent of retinal exudation and ERD (all r > 0.4, P < 0.05). Among these, IL-8 showed a strong association with the extent of retinal exudation and ERD (all r > 0.7, P < 0.001). The concentrations of IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-10, IL-12, Fractalkine, RANTES, G-CSF and GM-CSF were very low in both groups.ConclusionsVarious cytokines in the AH, including elevated VEGF, IL-6, IL-8, MCP-1, MIP-1α, IP-10, VCAM-1 and ICAM-1, may be involved in the pathogenesis and progression of Coats disease. Increasing severity of Coats disease is significantly associated with the AH concentrations of VEGF, IL-8, MCP-1 and MIP-1α. Further clinical treatment aimed to reduce vascular leakage and antagonize neovascularization and inflammation may be useful in preventing the progression of Coats disease.

Chimeric antigen receptor T cell therapy can be administered safely under the real-time monitoring of Th1/Th2 cytokine pattern using the cytometric bead array technology for relapsed and refractory acute lymphoblastic leukemia in children

CD19 chimeric antigen receptor T (CD19CAR-T) cell therapy has shown striking response in treating relapsed and refractory B-lineage acute lymphoblastic leukemia (r/r B-ALL). However, side-effects including cytokine release syndrome (CRS) and neurotoxicity can be fatal to patients. In this report, five patients with r/r B-ALL were treated with CD19CAR-T cells. Cytokine release syndrome experienced by four patients who achieved complete remission (CR) with minimal residual disease (MRD) negative. One patient who did not response to the treatment had no CRS. Acute toxicities including fever, hypotension and other neurological toxicities occurred in responding patients within 2 weeks post infusion and managed properly with tocilizumab and/or steroids according to the “real-time” monitoring of a simple 6 Th1/Th2 cytokine pattern. In conclusion, our study demonstrates that CD19CAR-T cell therapy can be safely administered for patients with relapsed and refractory leukemia under the “real-time” monitoring of a simple 6-cytokine pattern.

The number of regulatory B cells is increased in mice with collagen-induced arthritis

The aim of this study is to investigate changes in regulatory B cells (Bregs) and the expression of related cytokines such as interleukin-10 (IL-10) and transforming growth factor (TGF)-β in a mouse model of collagen-induced arthritis (CIA). A total 20 DBA/1 mice (6-8 weeks old) were randomly divided into control and CIA disease groups. For the CIA disease group, animals were injected intradermally with chicken collagen type II and complete Freund's adjuvant. The calculated arthritis index score of the CIA group was significantly higher than that in control group. Hematoxylin and eosin staining showed tumid synovial cells with irregular arrangement and obvious hyperplasia, with a high degree of inflammatory cell infiltration in CIA model group. Cytometric bead array technology and quantitative RT-PCR indicated that the levels of IL-10 and TGF-β in serum, and synovial cells were significantly increased in the CIA group. The proportion of Bregs in the spleen of the CIA group was significantly increased compared to the control group. In conclusion, our findings demonstrate that the number of Bregs and the expression of TGF-β and IL-10 are enhanced in mice with CIA.

Serum Apolipoprotein E and Other Inflammatory Markers Can Identify Non-Responding Patients to a Dendritic Cell Vaccine

BACKGROUND: Despite the majority of patients do not gain any benefit from dendritic cells (DC) vaccines, this approach has occasionally given rise to dramatic responses in melanoma. Biomarkers are crucial to identify which patients are more likely to respond. We looked for correlations between pre- or post- vaccination biomarkers and clinical outcomes to DC therapy in a cohort of patients with stage IV melanoma receiving a vaccine with autologous ex-vivo expanded DCs pulsed with allogeneic tumor cell lysate. METHODS: Serial serum samples were collected at baseline, week 4 and 12 and they were analyzed for a panel of different inflammatory markers using cytometric bead array technology and ELISA. RESULTS: Twenty-one patients were evaluable for response. Patients were separated into responders and non-responders based on clinical benefit. Responders were defined as patients who achieved a complete response, partial response or stable disease the latter lasting for at least 6 months. Responders (N = 9) showed a significantly longer Progression-free Survival (PFS; HR 0.23; 95% CI 0.08–062; P < .001) and Overall Survival (OS; HR 0.22; 95% CI 0.08–0.59; P < .001). The clinical non-responder phenotype correlated with an elevated pre-vaccination level of cytokines associated with inflammation compared to clinical responders (Apolipoprotein C111; IL-12 p40; MiP1α; Stem Cell Factor and TNFα). Apolipoprotein E (ApoE) was also significantly elevated in the pre-vaccine sera of the clinically non-responding group and in addition it was found to correlate with outcomes. Patients with increased levels of ApoE had a significantly shorter PFS (HR 3.02; 95% CI 1.09–8.35; P = .015) and OS (HR 2.40; 95% CI 0.9–6.3; P = .034). CONCLUSION: Our findings support the notion that treating the inflammatory background may have an impact on clinical outcome for patients receiving immunotherapy. A larger study is needed to confirm the significance of ApoE as a predictive biomarker for response to DC vaccines.

Effective Chimeric Antigen Receptor T Cell Therapy for Relapsed and Refractory Acute Lymphoblastic Leukemia in Children Can be Administered Safely Under the Real-Time Monitoring of Th1/Th2 Cytokine Patterns Measured By the Cytometric Bead Array Technology

Background and Objectives: Chimeric antigen receptor modified T (CAR-T) cell therapy has emerged as a promising strategy for the treatment of relapsed and refractory hematological malignancies recently. CD19 chimeric antigen receptor T (CD19CAR-T) cell therapy is a representative case showing promising results in treating relapsed and refractory B-lineage acute lymphoblastic leukemia (r/r B-ALL). However, challenges remain due to the concurrent severe side-effects such as cytokine release syndrome (CRS) or neurological toxicities which may cause death if they are not properly managed. So, how to safely use the CAR-T cell therapy in patients has drawn a lot of concern and attention worldwide. The objective of this study was to investigate the cytokine patterns to early identify and monitor the severe side-effects of cytokine release syndrome (CRS) and neurotoxicity caused by the therapy so that the therapy can be safely administered. Patients and Method: From December 2015 to March 2017, five patients with r/r B-ALL treated with CD19CAR-T cells were enrolled in this study. The Th1/Th2 cytokines including Interleukin (IL)-2, IL-4, IL6, IL-10, Tumor necrosis factor (TNF) and Interferon-gamma (IFN-γ ) in the serum and cerebral spinal fluid (CSF) were quickly measured by cytometric bead array (CBA) technology which allowed the result being available within 5 hours after the samples taken. The test were repeated more than once a day during the therapy. Results: 5 pediatric patients (2 boys and 3 girls) with r/r B-ALL aged 4 months -12 years old were treated by CD19CAR-T cell therapy. Four patients achieved complete remission (80% of CR rate) with minimal residual diseases (MRD) negative detected by flow cytometry within 2 weeks. One patient did not respond to the therapy due to the poor quality of his T cells. Five to 10 times of cytokine measurements were carried out for the five patients. Two types of cytokine patterns were identified: (1) very high levels of IL-6, IFN-γ with moderately elevated IL-10 indicated the CAR-T therapy effective pattern; (2) IL-6 slightly - severely elevated IL-6 level without concurrent elevation of IFN-γ represented a CAR-T cell therapy ineffective pattern but rather indicating a current infection. All the four responding patients presented the CAR-T therapy effective cytokine pattern, while the non-responding patient presented the CAR-T therapy ineffective cytokine pattern. We also analyzed the relationship between the cytokine levels and the severity of the side-effects and found that they were highly correlated. The cytokine levels in the CSF of two patients with seizure were also measured and we found that the elevation of the cytokine pattern in the CSF was paralleled to those in their serum but CNS-leukemia and CAR-T cells in their CSFs were not detected, indicating that the neurotoxicity concurrently presented in these two patients might be caused by the cytokines in the serum leaked into the CSF through the blood brain barrier. Extremely high level of cytokines occurred at 12 hours after CAR-T cell infusion in a patient was identified immediately and treated timely in onn-ICU setting to avoid the fatal consequence. Acute toxicities including fever, hypotension and other neurological toxicities occurred in responding patients within 2 weeks post infusion and managed properly with tocilizumab and/or steroids according to the “real-time” monitoring of this simple 6-Th1/Th2 cytokine patterns at the non-ICU setting. In conclusion, our study demonstrates that CD19CAR-T cell therapy can be administered safely for patients with relapsed and refractory leukemia under the “real-time” monitoring of a simple 6-cytokine pattern. DisclosuresNo relevant conflicts of interest to declare.

Aging, rather than Parkinson's disease, affects the responsiveness of PBMCs to the immunosuppression of bone marrow mesenchymal stem cells.

Whether aging or Parkinson's disease (PD) affects the responses of peripheral blood mononuclear cells (PBMCs) to immunosuppression by bone marrow-derived mesenchymal stem cell (BM-MSCs) and which cytokines are more effective in inducing BM-MSCs to be immunosuppressive remains to be elucidated. PBMCs were isolated from healthy young (age 26–35), healthy middle-aged (age 56–60) and middle-aged PD-affected individuals. All the recruits were male. The mitogen-stimulated PBMCs and proinflammatory cytokine-pretreated BM-MSCs were co-cultured. The PBMC proliferation was measured using Cell Counting Kit-8, while the cytokine secretion was assayed by cytometric bead array technology. The immunosuppressive ability of BM-MSCs was confirmed in young healthy, middle-aged healthy and middle-aged PD-affected individuals. Among the three groups, the PBMC proliferation and cytokine secretion of the young healthy group were suppressed more significantly compared with those of the middle-aged healthy and middle-aged PD-affected group. No significant differences were identified in the PBMC proliferation and cytokine secretion between the patients with PD and the middle-aged healthy subjects. Interferon (IFN)-γ synergized with tumor necrosis factor (TNF)-α, interleukin (IL)-1α or IL-1β was more effective than either one alone, and the combinations of IFN-γ + IL-1α and IFN-γ + IL-1β were more effective than IFN-γ + TNF-α in inducing BM-MSCs to inhibit PBMC proliferation. The results of the present study suggested that aging, rather than PD, affects the response of PBMCs toward the suppression of BM-MSC, at least in middle-aged males. Patients with PD aged 56–60 remain eligible for anti-inflammatory BM-MSC-based therapy. Treatment of BM-MSCs with IFN-γ + IL-1α or IFN-γ + IL-1β prior to transplantation may result in improved immunosuppressive effects.

Associations of cytokine concentrations in aqueous humour with retinal vascular abnormalities and exudation in Coats’ disease

To investigate the associations of cytokine concentrations in aqueous humour with the severity of retinal vascular abnormalities, exudation and fibrosis in patients with Coats' disease. Aqueous humour samples were collected in 23 paediatric patients (23 eyes) with Coats' disease and six age-matched control patients (six eyes) with congenital cataract in this cross-sectional, case-control study. Through Cytometric Bead Array technology, six angiogenic, inflammatory and fibrotic cytokines were measured for their concentrations in aqueous humour. Ophthalmologic characteristics including retinal vessel abnormalities, exudation and fibrosis of Coats' disease were also clinically evaluated for analysis. The aqueous levels of vascular endothelial growth factor (VEGF) (p=0.006) and monocyte chemoattractant protein-1 (MCP-1) (p<0.001) were significantly higher in the Coats' disease group than in the control group. The concentrations of angiogenin were peaked in eyes with first-grade vessels tortuosity (p<0.001), and also positively correlated with the severity of retinal capillary abnormalities (r=0.910, p<0.001). The concentrations of MCP-1 (r=0.966, p<0.001) and VEGF (r=0. 765, p=0.002) were significantly correlated with the extent of retinal exudation. The aqueous humour transforming growth factor-β (TGFβ) concentrations were higher in eyes with retinal fibrosis than in non-fibrotic eyes with Coats' disease (p=0.004). In Coats' disease, angiogenin may act as a potential biomarker for retinal vascular abnormalities. The concentrations of VEGF and MCP-1 may positively correlate with the severity of retinal exudation.

In vitro immunomodulatory activity and in vivo anti-inflammatory and analgesic potential with gastroprotective effect of the Mediterranean red alga Laurencia obtusa

Context: Red algae have been recognized as a rich natural source of compounds possessing interesting biological and pharmacological activities.Objective: This work investigates anti-inflammatory, analgesic and gastroprotective activities of MeOH/CH2Cl2 crude extract and its fractions F1 (50% MeOH) and F2 (80% MeOH) from the whole alga plant Laurencia obtusa Hudson (Rhodomelaceae).Materials and methods: Anti-inflammatory activity was evaluated in vitro using cytometric bead array (CBA) technology to follow up the secretion of tumour necrosis factor alpha (TNF-α) in lipopolysaccharide activated THP-1 monocytic cells at doses of 10–250 μg/mL and in vivo using carrageenan-induced paw oedema in Wistar rats at doses of 25, 50, 100 and 200 mg/kg. Crude extract and fractions were tested at the doses of 25, 50, 100 and 200 mg/kg for peripheral and central analgesic activity by acetic acid-induced writhing test and hot-plate method, respectively, in Swiss albino mice. Gastroprotective activity was evaluated using HCl/ethanol-induced gastric ulcer test in rats at doses of 25, 50, 100 and 200 mg/kg.Results: Crude extract, F1 and F2 showed an interesting inhibition of TNF-α secretion with IC50 values of 25, 52 and 24 μg/mL, respectively, and a significant anti-inflammatory activity in vivo (p < 0.01), 3 h after carrageenan injection, the oedema inhibition was 55.37%, 52.18% and 62.86%, respectively, at the dose of 100 mg/kg. Furthermore, they showed a significant peripheral analgesic activity with 53.79%, 55.92% and 57.37% (p < 0.01) of writhing inhibition, respectively. However, no significant activity was found in the hot-plate test. An interesting gastroprotective effect was observed with crude extract and its fractions F1 and F2 with a gastric ulcer inhibition of 65.48%, 77.42% and 81.29%, respectively, at the dose of 50 mg/kg.Discussion and conclusion: These results suggest that L. obtusa might be used as a potential source of natural anti-inflammatory and analgesic agents with gastroprotective effect.

Anti-inflammatory, analgesic activities and gastro-protective effects of the phenolic contents of the red alga, Laurencia obtusa

IntroductionThe present study was conducted to evaluate anti-inflammatory, analgesic and gastro-protective activities of methanol/dichloromethane extract and its semi-purified fractions from the red alga Laurencia obtusa. MethodologyAnti-inflammatory activity was evaluated in vitro using cytometric bead array technology to follow up the secretion of TNF-α in lipopolysaccharide activated THP-1 cells and in vivo using carrageenan-induced rat paw edema test. Edema was induced with 1% carrageenan after 30min of intra-peritoneal treatments. Paw thickness was checked at t=1, 2, 3, 4 and 5h. Analgesic activity was carried out using acetic acid-induced writhing test in mice. 1% of acetic acid was administrated intraperitoneally at the dose of 10mL/kg, 30min after treatment and the number of writhing was recorded during 30min. Gastro-protective effect was determined using HCl/EtOH induced gastric ulcers in rats. 10mL/kg of HCl/ethanol (60% ethanol in 150mM HCl) was orally administrated to all groups after treatment. 1h later, stomachs were examined for the presence of lesions. ResultsCrude extract and ethanol fraction showed an excellent inhibition of TNF-α secretion and a significant decrease in paw thickness (P<0.05), 3h after carrageenan injection. Ethanol fraction also showed a significant (P<0.05) analgesic activity and an excellent decrease of gastric damages. DiscussionThese activities, observed especially with the crude extract and the ethanol fraction may be related to their phytochemical composition. ConclusionThis study indicates that the red alga L. obtusa may be useful for developing potential therapeutic substances with anti-inflammatory, analgesic and gastro-protective activities.

Constitutively high-level expression of TGFβ isoforms in cord blood and its relationship to perinatal findings

BackgroundThe clinical significance of TGFβ isoforms in cord blood is not well understood. MethodsWe obtained cord blood samples from 37 term infants and 85 preterm infants who were born in several clinical settings. The serum levels of 3 TGFβ isoforms and of the other 17 cytokines in cord blood were investigated using cytometric bead array technology. ResultsVery high levels of TGFβ1 and TGFβ2 isoforms compared to the level of other cytokines were found; mean levels were 44,180 and 1871pg/mL, respectively. The levels of all 3 isoforms of TGFβ were significantly correlated with birth weight, and the levels of TGFβ1 and TGFβ3 were correlated with gestational age. The levels of TGFβ1 and β2 isoforms were strongly correlated with each other, but not with levels of other cytokines. The levels of TGFβ1 and TGFβ2 were significantly higher in male infants and significantly lower in infants with fetal growth restriction. The prevalence of chronic lung disease was related to a low level of TGFβ1, and that of patent ductus arteriosus was related to a high level of TGFβ1 in preterm infants. ConclusionsTGFβ1 and TGFβ2 appeared to play a significant role in physiological and pathological conditions in the fetus. TGFβ isoform levels appear to be regulated independently of those of other cytokines and do not appear to be influenced by inflammation in the fetal period. The role of TGFβ3 in cord blood and the postnatal chronological changes of the TGFβ isoforms should be investigated in the future.

Cerebrospinal fluid Th1/Th2 cytokine profiles in children with enterovirus 71‐associated meningoencephalitis

Enterovirus 71 (EV71) infection can cause severe neurological complications including meningoencephalitis (ME) in some patients with hand, foot and mouth disease (HFMD). However, to date no studies have reported changes in cytokine concentrations and their correlations with clinical variables in patients with ME following EV71 infection. In this study, responses of Th1/Th2 cytokine, including IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ, in cerebrospinal fluid (CSF) from patients with EV71-related HFMD with ME and patients with febrile convulsions (FC) were analyzed using cytometric bead array technology. It was found that CSF IL-6 and IFN-γ concentrations were significantly higher in patients with EV71-related ME than in those with FC. Additionally, both CSF IL-6 and IFN-γ concentrations were correlated with CSF cytology, fever duration and duration of hospital stay. More interestingly, a positive correlation between CSF IL-6 and IFN-γ concentrations was observed. Finally, receiver operating characteristic analysis revealed that when a cutoff value of 9.40 pg/mL was set for IL-6, the sensitivity and specificity were 84.5% and 85.5%, respectively, for discriminating EV71-related ME from FC. In conclusion, IL-6 and IFN-γ may be associated with EV71-induced neuropathology.

Effectiveness of bacterial infection-related cytokine profile (BIRCP) determination for monitoring pathogen infections in children with hemopathy in the bone marrow inhibition phase.

Serum cytokine profiles were analyzed before and after infection in children with hemopathy in the bone marrow inhibition phase to explore the utility of cytokine variations for detecting infections. Serum Th1/Th2 cytokine levels, including tumor necrosis factor, interleukin (IL)-2, IL-4, IL-6, IL-10, and interferon, were quantitatively determined by cytometric bead array technology in 480 cases (230 children) of children with hemopathy in the bone marrow inhibition phase with signs of infection, such as fever, and without, to establish baseline and affected levels for comparison with healthy control children. We used the cytokine profile of infected, blood culture-positive children to establish a bacterial infection-related cytokine profile (BIRCP) for predicting infections by pathogens in blood culture-negative children. Overall, 82.9% of children with Gram-negative bacterial infections were accompanied by marked increases of IL-6 and IL-10 levels [>10 times (means ± SD)], whereas only a mild increase of IL-6 levels occurred in Gram-positive bacteria-infected children [>2 times (means ± SD)] and only a mild increase of IFN-γ levels occurred in fungal culture-positive children [>2 times (means ± SD)]. Gram-positive bacterial and fungal infections did not cause a marked increase in IL-6 or IL- 10 levels. The effective rate (86.05%, N = 43) of infectious cases predicted by BIRCP was significantly higher than that obtained using traditional methods for selecting antibiotics based on clinical indications (65.45%, N = 55, P < 0.05). In summary, BIRCP can be used to predict the infections by pathogens in children with hemopathy and to select appropriate antibiotics.

Quantification of multiple cytokines and chemokines using cytometric bead arrays.

Quantitative suspension array technology allows the simultaneous measurement of different cytokines and chemokines in small sample volumes. The possibility of measuring multiple variables is important for discovery of biomarkers of pathogenesis or protection in complex diseases as well as measurement of antigen-specific cellular responses. Measurements can be made in biological specimens, such as plasma or serum, cell culture supernatants, and others. This technology is based on a capture-detection sandwich-type assay using fluorescent microspheres analyzable by Luminex instruments or flow cytometers. The complexity and cost of producing highly multiplexed cytokine/chemokine in-house assays make them especially apt for commercial production. There are several commercial kits available that vary in absolute cytokine concentration, sensitivity, reproducibility, and cost. This chapter gives an overview of cytometric bead array technology, introduces some of the kits, and provides detailed information about the one that performed well in a comparative study (Cytokine Human Magnetic 30-Plex Panel from Life Technologies™).

Association between Mediators in the Tear Fluid and the Severity of Keratoconus

Purpose: To study the association between different types of mediators in the tear fluid and topographic indices characterizing the severity of keratoconus (KC). Methods: In this study, nonstimulated tear fluid samples were collected from 14 eyes of 11 patients with KC. The following indices were measured by corneal topography: maximum K value, average K value, Klyce/Maeda keratoconus index (KCI), Smolek/Klyce keratoconus severity index, opposite sector index, center/surround index, keratoconus prediction index and standard deviation of corneal power. The concentrations of interleukin (IL)-6, IL-13, CXCL8 (IL-8), chemokine (C-C motif) ligand 5 (CCL5, regulated and normal T cell expressed and secreted), matrix metalloproteinase-9 (MMP-9), MMP-13, tissue inhibitor of metalloproteinase-1, nerve growth factor (NGF) and epidermal growth factor were measured by cytometric bead array technology. Release of mediators was calculated from their concentrations and the volume of tears collected over 2 min. Results: Significant positive associations were found between CCL5, MMP-13 and NGF and several topographic indices. Significant negative correlations were found between IL-6 and KCI. Age-dependent associations were observed between IL-13, CXCL8, CCL5 and MMP-13 and the topographic data. Conclusion: Several correlations were observed between the mediators and the topographic indices, suggesting possible roles in the pathophysiology of KC. Our data indicate that some mediators have different effects on the severity of disease in an age-dependent manner.

Soluble CD23 measurement by CBA: A convenient and reliable quantification method in chronic lymphocytic leukemia

The soluble form of the transmembrane glycoprotein CD23 corresponding to the low-affinity receptor for the immunoglobulin E (sCD23) is found in the serum of patients with chronic lymphocytic leukemia (CLL). In this disease, an increase in sCD23 level is predictive of poor prognosis at diagnosis as well as during clinical outcome. Quantification of sCD23 is classically performed by enzyme-linked immunosorbent assay (ELISA), a method not routinely used in hematology laboratories. Our aim was to apply cytometric bead array (CBA) technology to measure sCD23 levels. We tested 420 serum samples, 360 from patients and 60 from healthy volunteers. We selected three pairs of monoclonal antibodies recognizing the CD23 molecule that were tested in various conditions of temperature, centrifugation, washing, or chemical supplementation. Satisfactory performances in terms of repeatability (CV: 5%) and reproducibility (CV: 6%) were obtained with the selected pair of antibodies, with a threshold of positivity at 6 ng/mL. CBA and ELISA techniques were correlated with a Spearman coefficient at 0.99. The reproducibility and reliability of the sCD23 CBA assay were confirmed, with a Spearman coefficient at 0.99 in a series of 23 CLL patients and 13 controls tested in two laboratories equipped with different cytometers and using different lots of CBA reagents. Data obtained with serum and plasma samples were correlated with a Spearman coefficient at 0.99. Our study validates a simple method that allows the clinicians to benefit from an indicator of prognosis at the diagnosis as well as a marker of the evolution of CLL disease.

Alterations of Tear Mediators in Patients with Keratoconus after Corneal Crosslinking Associate with Corneal Changes

Keratoconus (KC) is the most common primary corneal ectatic disease which has considerable importance in public health. Corneal collagen crosslinking (CXL) is a procedure to mitigate progression of KC and reduce demand for corneal transplantation. Although studies have proven the efficacy of CXL regarding corneal shape, none have investigated the effects of CXL on tear biomarkers which are useful tools to understand molecular mechanisms behind CXL. Our purpose was to determine the effect of CXL on tear mediators in patients with KC and analyze associations with corneal changes. Tear samples were collected pre-CXL from 26 eyes of 23 patients and during a 12-month follow-up. The mediators’ concentration was measured by Cytometric Bead Array technology. Corneal topography parameters measured by Scheimpflug Camera included: Thinnest-corneal-thickness (ThCT), keratometry values (K1, K2), Radii-Minimum (Rmin), Keratoconus-Index (KI), Center-KI (CKI), Index-of-Height Asymmetry (IHA) and Index-of-Surface Variance (ISV). At baseline, KI was correlated negatively with chemokine (C-C motif) ligand 5 (CCL5) (p=0.015) and matrix metalloproteinase (MMP)-13 (p=0.007). At day 4, interleukin (IL)-6 and IL-8 increased, while IL-13, IL-17A, interferon (IFN)-γ, CCL5, MMP-13, epidermal growth factor (EGF), nerve growth factor (NGF) and plasminogen activator inhibitor (PAI-1) decreased significantly compared to pre-CXL concentrations (p≤0.02). At 6 months tissue plasminogen activator (t-PA) increased (p=0.02), while at 12 months Rmin increased (p≤0.004), and IL-6 and CXCL8 (p=0.005 and p=0.047) as well as K1, ISV and KI decreased. After 6 months CKI and ISV showed significant associations with IL-17A; CKI with IL-13 and ThCT with IL-13 (p≤0.02), while at 12 months there were reverse associations between ThCT and IL-6, IL-13, INFγ, CCL5 and PAI-1 (p≤0.02). Alterations of mediators in tear fluid after CXL associate with topographic changes highlight the fact that many mediators are involved in the complex mechanisms after CXL. Further studies on biomarkers to investigate the efficacy of CXL are needed.