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Aim: Oral candidiasis is often complicated by other oral diseases, and long‐term follow‐up is essential because oral candidiasis often recurs. Failure to provide appropriate treatment for oral candidiasis will lead to persistence of the fungal cell in the oral cavity and increase the risk of severe oral candidiasis and recurrence of infection. We hypothesized that various inflammatory cytokines are involved in the pathogenesis and progression of oral candidiasis. Methods: We measured cytokine levels in the saliva samples of candida‐negative and candida‐positive patients. Results: Saliva interleukin (IL)‐17 concentrations were significantly lower in patients with oral candidiasis than in healthy individuals. Saliva IL‐17A concentrations were significantly lower in candida‐positive patients than in candida‐negative patients. However, the levels of tumor necrosis factor‐α, IL‐2, IL‐4, IL‐5, and IL‐6 showed no significant differences between the two groups of patients. Conclusion: Saliva IL‐17A levels were significantly lower in patients with severe oral candidiasis, providing a potentially highly useful indicator for the pathogenesis and progression of severe oral candidiasis. [ABSTRACT FROM AUTHOR]

Yunxia Tang,1,2,&ast; Chenjie Ma,1,2,&ast; Huali Sun,3 Siyuan Yang,1,2 Fengting Yu,1,2 Xingwang Li,1,2 Linghang Wang1,2 1Beijing Ditan Hospital, Capital Medical University, Beijing, People’s Republic of China; 2Clinical Center for HIV/AIDS, Capital Medical University, Beijing, People’s Republic of China; 3Department of Infectious Diseases, The Affiliated Hospital of Qingdao University, Qingdao, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Linghang WangBeijing Di Tan Hospital, Capital Medical University, Beijing, People’s Republic of ChinaTel/Fax +86-10- 84323103Email linghang.wang@ccmu.edu.cnPurpose: Human brucellosis is the most common bacterial zoonosis globally that poses a severe health threat. Despite the availability of antibiotic therapy for human brucellosis, its tendencies of chronicity and persistence may lead to severe debilitating and disabling conditions in patients. Comprehensive understanding of the immune response in brucellosis will be helpful in improving the treatment strategies. In this study, we measured serum levels of T helper cell type 1 (Th1), Th2, and Th17 cytokines in patients with acute brucellosis before and after treatment.Patients and Methods: Overall, 30 patients with acute brucellosis from the Beijing Di Tan Hospital and 26 healthy controls were enrolled in this study. All the patients received a 6-week therapy regimen comprising ceftriaxone, doxycycline, and rifampicin. Serum samples were collected from patients with acute Brucella infection and healthy controls before and after treatment. Serum seven cytokine levels of Th1 (IL-2, IFN-γ, and TNF-α), Th2 (IL-4, IL-6, IL-10), and Th17 (IL-17A) were measured using cytometric bead array.Results: In patients with acute infection, the IL-2, IFN-γ, and IL-10 levels were significantly increased compared with those in healthy controls (P < 0.001). After treatment, IL-2, IFN-γ, and IL-10 levels significantly decreased (P < 0.05) and the TNF-α level significantly increased compared with the corresponding baseline levels and those in healthy controls (P < 0.05). Furthermore, the IFN-γ, IL-4, and IL-10 levels were higher in patients after treatment than in healthy controls (P < 0.05). IL-2 and IL-6 levels exhibited a positive correlation with the C-reactive protein (CRP) level in acute brucellosis (P < 0.05).Conclusion: Levels of most serum Th1 and Th2 cytokines were simultaneously increased in acute infection, followed by reduction in the corresponding cytokine levels and residual cytokine response during treatment. This residual immune response could represent a therapeutic opportunity that may improve the long-term clinical outcomes in patients with acute brucellosis after treatment.Keywords: brucellosis, acute infection, Th1, Th2, cytometric bead array, cytokines

Background: Nasal mucus is proving to be a useful means by which to study the pathogenesis of chronic rhinosinusitis (CRS). Given the increase in publications examining nasal mucus and the lack of a review on this topic, we will focus on this noninvasive approach to studying CRS. Particular attention will be drawn towards inflammatory cytokines and biomarkers and their influence on disease severity. Methods: A literature review of papers published in English pertaining to nasal mucus was performed using the PubMed database. The search utilized combinations of the following keywords: sinusitis, polyps, sample collection, nasal mucus, or nasal secretion. Studies solely on acute or bacterial sinusitis, allergic rhinitis, or cystic fibrosis were not included. Results: A wide variety of materials and methods have been used to collect nasal mucus. Numerous assay types have been performed with the most common being ELISA, cytometric bead array, and proteomics. Most studies have focused on examining the levels of Th1/Th2 cytokines along with chemokines associated with type 2 immunity. Other factors identified include growth factors, senescence-associated proteins, complement, and antimicrobial defenses have also been identified. Nasal mucus cytokines have proven useful in cluster analysis and predicting postoperative improvement in Sino-nasal Outcome Test (SNOT-22) scores. One limitation of the use of nasal mucus is that some studies have suggested that nasal mucus does not always reflect the tissue microenvironment. Conclusions: Nasal mucus represents a critical tool by which to examine the sinonasal microenvironment in a noninvasive manner. Unlike studies of tissue, it can be utilized in both surgically and medically managed patients and avoids the trauma of biopsies. However, studies are still needed to determine the most effective method for nasal mucus collection. Studies should also take care to confirm that nasal mucus markers do, in fact, reflect the levels of the product studied in the tissue. [ABSTRACT FROM AUTHOR]

Photodynamic Therapy (PDT) is a non-invasive treatment that has shown its usefulness in eliminating low grade squamous intraepithelial lesion (LSIL) and Human Papilloma Virus (HPV) infection. The response to PDT is variable, how patient´s immunology works during human PDT is unclear and therefore, the main of this work was to characterize the local immune response during treatment with PDT in patients with LSIL and / or persistent HPV infection. Two groups divided into Complete Response and Non Response patients to PDT, which were treated with a scheme of 12% δ-aminolevulinic acid (ALA), 2 irradiations 48 h apart at a total energy dose of 200 J/cm2, their local immune response were analyzed by Cytometric Bead Array (CBA) and spectral Cytometry before and after each irradiation. Cytokine mostly expressed previously PDT was IL-8 in all groups. PDT stimulates the increase in IL-6 and IL-8 concentrations at 48 h, being greater in the group of responding patients as well as IL-6 and IL-10 proving the host immune stimulus to remit diagnosis. Complete Response group has a higher percentage of total CD45+ cells before treatment than Non Response. A decrement pattern of CD8+ were observed on Non Response group during treatment. An increase tendency was observed on CD14+ and HLADR-ABC+ cells in both response groups. PDT is able to stimulate immune response, being important to treatment effectiveness increasing innate immune cells and their activation, such as monocytes, cytotoxic lymphocytes. A multiple HPV infection could be related to a less expression of cytokines during PDT.

Foodborne diseases caused by foodborne pathogenic microorganisms have long been a severe public health problem. Timely and accurate detection of food-borne microorganisms can minimize their harm and enable the development of efficient control strategies. In this study, we establish a multiplex nucleic acid detection method based on recombinase-aided amplification reaction on a suspension microarray (referred to as SC-RAA) to identify and quantify microorganisms. The experimental results confirmed that this approach has an outstanding sensitivity, with a limit of detection (LOD) of 0.8 fM and a linear range of 0.8 fM–80 pM, spanning five orders of magnitude. Moreover, the prepared method also showed exquisite specificity, which the amplified signal can be detected only when the target nucleic acid is present. The recovery rate of the SC-RAA assay was 82.2%–113.4%, suggesting that the SC-RAA assay is performing well and accurately measuring the concentration of the substance in the sample. In addition, the consistency analysis showed that the results of the SC-RAA detection were also in good agreement with those of traditional qPCR detection (R2 > 0.95). These results highlight the potential of the SC-RAA assay to be applied as a multiplex and sensitive DNA detection platform for identifying and quantifying foodborne pathogenic microorganisms. [ABSTRACT FROM AUTHOR]

Food-borne diseases are widespread; one of the reasons is contamination with staphylococcal pathogens that can produce enterotoxins of the protein, causing numerous diseases in humans. The long-term stability of the toxins requires that the methods for foodstuff quality control be refined to prevent contamination with staphylococcal enterotoxins. We developed a bead-based multiplex assay for the simultaneous detection of three staphylococcal enterotoxins (staphylococcal enterotoxin A (SEA), staphylococcal enterotoxin B (SEB), staphylococcal enterotoxin H (SEH)) by flow cytometry. For detection, we used functional beads coupled with monoclonal capture antibodies, biotinylated monoclonal detection antibodies, and streptavidin conjugated to phycoerythrin. The linear ranges of detection of SEA, SEB, and SEH were 0.02–20 ng/mL, 0.2–30 ng/mL, and 0.2–20 ng/mL, respectively. Each toxin was specifically detected without cross-reactions. An efficient use of the developed method for the analysis of foodstuffs was reported. [ABSTRACT FROM AUTHOR]

Mpox is a zoonotic disease caused by mpox virus (MPXV) infection. Since May 2022, there has been a marked increase in human mpox cases in different regions. Rash, fever, and sore throat are typical signs of mpox. However, other viruses, such as the B virus (BV), herpes simplex virus types 1 (HSV-1), herpes simplex virus types 2 (HSV-2), and varicella zoster virus (VZV), can also infect people and cause comparable symptoms. Therefore, clinical symptoms and signs alone make distinguishing MPXV from these viruses difficult. In this study, we combined suspension microarray technology with recombinase-aided amplification technology (RAA) to establish a high-throughput, sensitive, and quantitative method for detecting MPXV and other viruses that can cause similar symptoms. The experimental results confirmed that the technique has outstanding sensitivity, with a minimum detection limit (LOD) of 0.1 fM and a linear range of 0.3 fM to 20 pM, spanning five orders of magnitude. The approach also exhibits exquisite selectivity, as the amplified signal can only be detected when the target virus nucleic acid is present. Additionally, serum recoveries ranging from 80.52% to 119.09% suggest that the detection outcomes are generally considered reliable. Moreover, the time required for detection using this high-throughput method is very short. After DNA extraction, the detection signal amplified by isothermal amplification on the bead array can be obtained in just 1 h. Our research introduces a new technique that utilizes suspension microarray technology and isothermal amplification to create a high-throughput nucleic acid assay. This innovative method offers multiple benefits compared to current techniques, such as being cost-effective, time-efficient, highly sensitive, and having high throughput capabilities. Furthermore, the assay is applicable not only for detecting MPXV and viruses with similar symptoms, but also for clinical diagnostics, food safety, and environmental monitoring, rendering it an effective tool for screening harmful microorganisms. [Display omitted] • A high throughput, sensitive, and quantitative method for detecting mpox virus and related viruses. • A time-saving DNA detection method providing results within an hour post-extraction. • A labor-saving nucleic acid detection method aiding automated instrument development. [ABSTRACT FROM AUTHOR]

Accurate and sensitive quantification of a specific class of mycotoxins at trace levels in complex matrices with greener approaches is of significant importance. In this study, a green and economical protocol of magnetic microspheres-based cytometric bead array (CBA) assay on indirect competitive principle was developed for sensitive and rapid detection of ochratoxin A (OTA) in malts with a small number of standard and sample solutions. The protocol included the competition of OTA in malt samples and that covalently coupled on the surface of microspheres with its monoclonal antibodies, the separation and aggregation of the magnetic microspheres, and the fluorescence detection of fluorescein isothiocyanate labeled goat anti-mouse immunoglobulin G probes. The magnetic microspheres-based CBA assay allowed for ultralow limit of detection (0.025 μg kg −1 ) for OTA and showed higher sensitivity compared with the common polystyrene beads-based CBA method. This is the first report on the magnetic microspheres-based CBA assay by using a simple and easy-to-operate magnetic separator for highly sensitive and rapid detection of OTA in complex malt samples. By consuming less solvent, time and cost, as well as fewer standard and samples, the developed green protocol expressed high potential for one-site real-time detection of trace components in complex matrices. [ABSTRACT FROM AUTHOR]

We aimed to investigate the immunologic effects of vitamin D replacement in RRMS patients. In a controlled single center study, patients deficient in 25-hydroxyvitamin D (serum level < 25 ng/ml) received 10,000 IU/week cholecalciferol for 3 months. Sufficient vitamin D patients (serum level > 35 ng/ml) were followed for the same period. Assessments were performed at baseline and at 3 months. 25-hydroxyvitamin D levels increased significantly from baseline to month-3 in the deficient group after treatment and remained stable in the sufficient group. We observed a decreased interferon-γ (IFNγ) secretion by CD4 + T cells in vitamin D deficient group but not in the sufficient group, and a negative correlation between baseline serum vitamin D and IFNγ production. There was no change in the frequency of T helper or regulatory T cell subsets in either group. Increasing serum levels of 25-hydroxyvitamin D are associated with decreased production of IFNγ by CD4 + T cells. [ABSTRACT FROM AUTHOR]

Ochratoxin A (OTA), the most harmful and abundant ochratoxin, is chemically stable and commonly existed in foodstuffs. In this work, upconversion luminescent-magnetic microbeads (UCLMMs) -based cytometric bead array for OTA detection with a less reagent consumption and high sensitivity has been established and optimized. In UCLMMs, upconversion nanocrystals (UCNs) for optical code present a weak background noise and no spectral cross talk between the encoding signals and target labels under two excitation conditions to improve detection sensitivity. While the superparamagnetic FeO nanoparticles (FeO NPs) aim for rapid analysis. The results show that the developed method has a sensitivity of 9.553 ppt below HPLC with a 50-μL sample and can be completed in <2 h with good accuracy and high reproducibility. Therefore, different colors of UCLMMs will become a promising assay platform for multiple mycotoxins after further improvement. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor (TNF)-α is a key cytokine in the pathogenesis of dengue virus infection, and its accurate detection in several types of human samples is critical. The enzyme-linked immunosorbent assay (ELISA) is the gold standard for the detection of TNF-α, but multiplexed bead-based assays such as cytometric bead array (CBA) are now frequently used. Here, using ELISA and two CBAs commercially available, we measured TNF-α concentrations in plasma and serum from children with acute dengue virus infection and healthy controls. To evaluate the detection efficiency and factors affecting it, spiked recovery and immune complex dissociation assays were also performed. The levels of TNF-α evaluated by ELISA in paired serum and plasma samples from children with dengue positively correlated (rho = 0.99, p < 0.0001). Children with dengue had higher levels of plasma TNF-α than those of healthy children ( p = 0.004). The ELISA detected TNF-α in a higher number of plasma samples than the CBA ( p < 0.0001), and both methods only correlated when TNF-α was evaluated in buffer-based solutions but not in plasma, indicating the presence of a factor interfering with the detection of TNF-α in plasma. The recovery of several types of human recombinant TNF-α was dramatically decreased in plasma but not in tissue culture media ( p ≤ 0.01), and this effect was similar in the plasma obtained from the children with dengue or the healthy controls. The dissociation of immune complexes did not improve TNF-α recovery. Dilution of the plasma samples increased the recovery of TNF-α, but at high concentrations of the cytokine. In short, plasma affects the efficiency of TNF-α detection, and this effect should be considered in the measurement of this cytokine. [ABSTRACT FROM AUTHOR]

Purpose: Bronchiolitis obliterans (BO) is an inadequately researched disease in terms of lung function as well as inflammatory profile. The short-term variation of these parameters has not been investigated. Therefore, the objective of this study was the investigation of lung function, sputum cells and cytokine profiles in BO at two visits within of four to six weeks. Methods: Twenty patients with BO (median age = 14.6, range 8.3-24.3) performed lung function tests, airway reversibility testing and induction of sputum within four to six weeks. The cell composition in the sputum was analysed and cytokine levels of IL-1ß, IL-6 and IL-8 were determined by cytometric bead array analysis. The short-term variation was then statistically quantified and compared to that of twenty-two healthy controls. Furthermore, we compared data on short-term variation of lung function and airway inflammation with a previous investigation in these patients 10-15 months earlier. Results: Patients with BO showed minimal variation of lung function (VCmax, FVC, FEV1, FEV1/VC, MEF25 and RV/TLC) and the inflammatory cell profile. The lung function data were significantly lower for FVC, FEV1, the Tiffeneau index and MEF25 compared to the control group, whereas RV/TLC was significantly increased. Analysis of the BO sputum cells showed a consistent neutrophil inflammation. The levels of inflammatory cytokines IL-1ß, IL-6 and IL-8 had a great variability. Conclusions: The short-term variability of sputum neutrophilia and lung function is low in BO patients. This finding should be considered to identify successful treatment in the individual patient and could be used as endpoints for future BO-related studies. [ABSTRACT FROM AUTHOR]

Chemokines recruit and activate leukocytes, assisting granuloma formation. Herein, we evaluated plasma chemokines in patients with active tuberculosis (ATB) and after completing treatment (TTB) and compared them to BCG-vaccinated healthy controls (HC). Levels of chemokines were measured by cytometric bead array. Levels of CXCL8, CXCL9 and CXCL10 were higher in ATB patients compared to HC, but they decreased in TTB. Levels of CCL2 and CCL5 in ATB patients were similar to those observed in HC. Thus, the high levels of CXC-chemokines detected during ATB, which can modulate the trafficking of immune cells from the periphery to the site of infection, were reversed by anti-mycobacterial treatment.

Despite extensive research into inflammatory diseases to date, no drugs with favourable safety profiles are available for treatment. Euphorbia hirta (E. hirta) is a tree that is locally used as a traditional medicine in Africa and Australia to treat numerous diseases such as hypertension, antipyretic and anti-inflammatory activities. The aim of the present study was to determine the potential anti-arthritic effects of E. hirta in mouse models of adjuvant induced arthritis (AIA). We treated BALB/c mice with (p.o.) E. hirta (25, 50, 100, and 200 mg/kg) daily (13 days) beginning at the onset of AIA. We examined the effect of E. hirta on key mediators of arthritic-inflammation, including pro-inflammatory (IL-2, IFN-γ, and TNF-α) and anti-inflammatory (IL-4 and IL-5) cytokines, T-cell activation markers (CD25/CD69), and co-stimulatory molecules (CD80/CD86). We also examined the inflammatory mediators (PGE2 and LTB4) response. E. hirta-treated mice showed a substantial reduction in the levels of pro-inflammatory cytokines, down regulated cell activation markers and co-stimulatory molecules, and up regulated anti-inflammatory cytokines . E. hirta decreased the levels of inflammatory-mediators in AIA animals. Supplementation with an E. hirta extract may be a promising treatment for arthritic and inflammatory diseases. [ABSTRACT FROM AUTHOR]

Lymphoma-associated hemophagocytic syndrome (LAHS) is a serious disorder, and its early diagnosis and treatment with appropriate chemotherapy are very important. However, reliable markers for early diagnosis of LAHS have not been identified. We screened serum cytokines using a newly introduced assay system, cytometric bead array (CBA), and identified interferon-inducible protein 10 (IP-10)/CXCL10 and monokine induced by interferon gamma (MIG)/CXCL9 as useful markers. Serum concentrations of IP-10 and MIG at the time of LAHS diagnosis were greater than 500 and 5,000 pg/ml, respectively. The sensitivity and specificity for LAHS diagnosis were 100 and 95 %, respectively, when we set the above values as the cut-off levels. Serum levels of these two chemokines were already elevated at the time of admission and significantly decreased after successful treatment, indicating their usefulness for both the diagnosis and therapeutic outcomes for LAHS. IP-10 and MIG were also useful in distinguishing severe from moderate/mild LAHS, and B-cell-type LAHS from T-cell/natural killer cell-type LAHS. Furthermore, IP-10 and MIG were of use to distinguish LAHS from sepsis in patients with hematologic malignancies. Rapid measurement of IP-10 and MIG by CBA appeared to be important for early diagnosis and treatment of LAHS. [ABSTRACT FROM AUTHOR]

Bead-based multiplex assays (BBMAs) are applicable for high throughput, simultaneous detection of multiple analytes in solution (from several to 50–500 analytes within a single, small sample volume). Currently, few assays are commercially available for veterinary applications, but they are available to identify and measure various cytokines, growth factors and their receptors, inflammatory proteins, kinases and inhibitors, neurobiology proteins, and pathogens and antibodies in human beings, nonhuman primates, and rodent species. In veterinary medicine, various nucleic acid and protein-coupled beads can be used in, or for the development of, antigen and antibody BBMAs, with the advantage that more data can be collected using approximately the same amount of labor as used for other antigen and antibody assays. Veterinary-related BBMAs could be used for detection of pathogens, genotyping, measurement of hormone levels, and in disease surveillance and vaccine assessment. It will be important to evaluate whether BBMAs are “fit for purpose,” how costs and efficiencies compare between assays, which assays are published or commercially available for specific veterinary applications, and what procedures are involved in the development of the assays. It is expected that many veterinary-related BBMAs will be published and/or become commercially available in the next few years. The current review summarizes the BBMA technology and some of the currently available BBMAs developed for veterinary settings. Some of the human diagnostic BBMAs are also described, providing an example of possible templates for future development of new veterinary-related BBMAs. [ABSTRACT FROM PUBLISHER]

Abstract: HIV infection causes the dysregulation of cytokine production. A cytokinomics approach employing cytometric bead array (CBA) technology, flow cytometry and multivariate analysis was applied to the investigation of HIV-induced T helper cell type 1 (Th1), Th2 and Th17 cytokine changes in the sera of treatment naive individuals. Stepwise linear discriminant analysis (LDA) and logistic regression identified interleukin (IL)-6 to be discriminatory for HIV infection with 74.6% and 71.2% of the cases correctly classified. Analysis of variance (ANOVA) confirmed IL-6 and IL-10 concentrations to be significantly (p =0.001 and p =0.025) different between the groups. A scatter plot of the log IL-6 and IL-10 concentrations for the groups largely overlapped, with improved differentiation where patients were advancing to the acquired immunodeficiency syndrome (AIDS). IL-17A levels were higher than other cytokines but did not significantly distinguish the groups suggesting that the HIV− and HIV+ individuals had similar immune profiles. This possibility was supported by other clinical indicators. Taken together, the measured cytokines (IL-6, 10 and 17) have potential prognostic value. [Copyright &y& Elsevier]

Abstract: Lipopolysaccharide (LPS) has been widely used as a model of immune challenge in pigs as it induces the immediate synthesis of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6, which trigger the production of the acute phase proteins (APPs) C-reactive protein (CRP), haptoglobin (Hp) and pig-Major Acute Phase Protein (pig-MAP). To measure secreted proteins in porcine plasma, specific and sensitive Enzyme-Linked Immuno Sorbent Assays (ELISAs) are well-suited to perform single parameter analysis, yet this approach is time-consuming and expensive for multi-parameter analyses. During the last decade, multiplex bead-based flow cytometry has been increasingly applied as it offers the opportunity to estimate protein ratios in a small sample volume. Cytometric bead array (CBA) is a flow cytometric application using a diversity of beads with unique fluorescence intensities, covalently coupled to a capture antibody for each protein of interest. Detection antibodies, either directly or indirectly conjugated to a fluorochrome, are added to accomplish the desired sandwich format. The aim of the present study was to develop a CBA 3-plex assay for the major pro-inflammatory cytokines TNF-α, IL-1β and IL-6, and an additional CBA 2-plex assay for the major APPs, CRP and pig-MAP, in porcine plasma. Results were compared to commercial ELISA kits. For the CBA 3-plex assay, the limits of detection (LODs) varied between 0.005 and 0.363ng/mL, the intra- and inter-assay coefficients of variation were <10% and <16%, respectively. For TNF-α, IL-1β, IL-6 and pig-MAP, CBA time-concentration profiles similar to those obtained with commercial ELISAs were observed. In conclusion, the novel validated CBA 3-plex assay provides a fast and economical screening tool for determination of pro-inflammatory cytokine profiles in limited porcine plasma volumes. This tool will be applied to study the immunomodulatory properties of drugs in a porcine LPS inflammation model. This study also demonstrated the applicability of CBA for measurement of APPs in pigs, although a different combination than pig-MAP with CRP is recommended. [Copyright &y& Elsevier]

Rapid and accurate detection of BCR-ABL fusion proteins is vital for the diagnosis and classification of leukemias, especially chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). Recently, the cytometric bead array (CBA) system has been used to identify BCR-ABL proteins in leukemic cell lysates. To evaluate the accuracy of this new technique, we tested 70 patients with hematological diseases using the CBA system, cytogenetic analysis, fluorescence in situ hybridization (FISH) and real-time quantitative polymerase chain reaction (RQ-PCR). Most of the results obtained by the CBA system were concordant with those by cytogenetic analysis, FISH and RQ-PCR. In the CBA system, dilution experiments with positive control samples revealed sensitivities of at least 1%. The statistical analysis revealed that the mean fluorescence intensity (MFI) values of the BCR-ABL fusion proteins from mononuclear cells (MNCs) were higher than those from WBC or BM samples. The MFI values of cells were not dramatically reduced after 24 hours of storage at room temperature. The CBA technique detected all types of BCR-ABL proteins in leukemic cells with high specificity and sensitivity. Therefore, CBAs will contribute to the fast and easy diagnosis of leukemias, the classification of leukemias and the monitoring of minimal residual disease (MRD). [ABSTRACT FROM AUTHOR]

Abstract: Pathogen detection using biosensors is commonly limited due to the need for sensitivity and specificity in detecting targets within mixed populations. These issues were addressed through development of a dual labeling method that allows for both liquid-phase fluorescence in situ hybridization (FISH) and capture antibody targeted detection (CAT-FISH). CAT-FISH was developed using Escherichia coli O157:H7 and Staphylococcus aureus as representative bacteria, and processing techniques were evaluated with regard to FISH intensities and antibody recognition. The alternative fixative solution, methacarn, proved to be superior to standard solid-phase paraformaldehyde fixation procedures, allowing both FISH labeling and antibody recognition. CAT-FISH treated cells were successfully labeled with FISH probes, captured by immunomagnetic separation using fluorescent cytometric array beads, and detected using a cytometric array biosensor. CAT-FISH treated cells were detectable with LODs comparable to the standard antibody-based technique, (~103 cells/ml in PBS), and the technique was also successfully applied to two complex matrices. Although immunomagnetic capture and detection using cytometric arrays were demonstrated, CAT-FISH is readily applicable to any antibody-based fluorescence detection platform, and further optimization for sensitivity is possible via inclusion of fluorescently tagged antibodies. Since the confidence level needed for positive identification of a detected target is often paramount, CAT-FISH was developed to allow two separate levels of specificity, namely nucleic acid and protein signatures. With proper selection of FISH probes and capture antibodies, CAT-FISH may be used to provide rapid detection of target pathogens from within complex matrices with high levels of confidence. [Copyright &y& Elsevier]

Abstract: Differential T cell trafficking through the blood compartment towards infected foci may be occurring in different stages of tuberculosis disease and infection. The aim of the present study was to identify cytokine signatures in the blood compartment in tuberculosis patients with pulmonary disease (PTB=19), recently exposed household contacts (HC=27) and nonexposed community controls (EC=37). Diluted (1:10) whole blood was cultured for 2days and cytokine secretion was assessed using Cytometric Bead Array (Th1/Th2 kit II; BD Biosciences) which included IL-2, TNF-α, IFN-γ (Type1/T1), IL-4, IL-6 and IL-10 (Type2/T2). All T1/T2 cytokines were elevated in PTB (AUROC>0.9) while HC showed selective elevation of IL-6 (AUROC>0.7) compared to EC. Principal component analysis (PCA) extracted two groupings with Eigen values >1; IL-6 separated into the second component for PTB, HC and EC. After rotation, IFN-γ was correlated with the first component for PTB and EC and the second component for HC indicating an absence of T1/T2 dichotomy. Therefore endogenous cytokine signatures may indicate differential T cell trafficking in different stages of tuberculosis infection and disease. [Copyright &y& Elsevier]

The hepatitis B virus (HBV) is frequently transmitted by sexual intercourse. Thus, HBV-guidelines recommend vaccination. However, we have identified healthy hepatitis B surface antigen and anti-HBc-negative unvaccinated sexual partners of patients with chronic hepatitis B. We investigated whether HBV-specific cellular immune responses were present that could explain the apparent protection against HBV infection. In six anti-HBc-negative HBV-exposed sexual partners, HBV-specific T-cell responses were studied by proliferation assay and cytometric bead array after stimulation with 74 overlapping peptides spanning the HBV core, pre-S and S-encoding regions. Eleven HBV-unexposed individuals served as negative controls. HBV-DNA was undetectable in serum and peripheral blood mononuclear cells in all cases. HBV-specific cytokine secretion was observed in 4/6 seronegative partners, but only in 1/11 controls. Proliferative responses were detectable in 5/6 partners and 0/11 controls. HBV-specific cytokine secretion exists in healthy seronegative virus-exposed individuals. HBV core-directed immune responses indicate past, but controlled viral replication. T-cell immunity may prevent clinical manifestation of HBV infection in the absence of humoral immunity. [ABSTRACT FROM AUTHOR]

Abstract: Lung diseases like cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) are associated with chronic airway inflammation. The aim of our study was to compare a complex biomarker profile in order to characterize specific inflammatory patterns in sputum of patients with CF and COPD. Induced sputum samples of 19 CF-, 26 COPD patients and 21 healthy controls were analyzed for concentrations of IL-1β, IL-2, IL-6, IL-8, IL-13, IP-10, MCP-1, IFN-γ and TNF-α using the new cytometric bead array (CBA) technology. Significant differences in airway biomarker profiles of CF and COPD were detected. Patients with CF showed a significant increase in IL-1β, IL-6, IL-8, IL-13, TNF-α, IFN-γ and MCP-1. COPD patients showed an increase in IL-6, IL-8, IL-13 and MCP-1 compared to healthy controls. CF and COPD compared to each other exhibited differences in IL-1β, IL-2, IL-8, TNF-α, IFN-γ and MCP-1 levels. Significant correlations between the parameters of lung function and sputum biomarker levels were found. Analyzing induced sputum allows characterization of specific airway biomarker profiles in CF and COPD and can be related to the clinical status of the patient. CBA of induced sputum seems to be a pivotal tool to characterize pulmonary inflammation. [Copyright &y& Elsevier]

Abstract: The present study was undertaken to investigate the anti-arthritic activity of hydroxychavicol (HC) a major phenolic compound isolated from the aqueous extract leaves of plant Piper betle (Piperaceae). The compound showed significant lowering of pro-inflammatory (Th1) cytokine levels in arthritic paw tissue homogenate supernatant viz. IL-2, IFN-γ, and TNF-α with maximum inhibition at higher dose levels of 2 and 4mg/kg p.o. and enhanced the production of anti-inflammatory (Th2) cytokines IL-4 and IL-5 estimated by cytometric bead array immunoassay. Cytometric bead array uses the sensitivity of amplified fluorescence detection by flowcytometer to measure soluble analytes in a particle based immune assay. This assay can accurately quantitate five cytokines in a 50-μl sample volume. The T-helper (Th1) deviated cells produce detectable level of tumor necrosis factor (TNF-α), interleukin-2 (IL-2), and interferon-gamma (IFN-γ), while the Th2 deviated cells produce significant amount of interleukin-4 (IL-4) and interleukin-5 (IL-5). HC at graded doses also significantly decreased the expression of IL-1β, PGE2, LTB4, and nitric oxide levels showing significant inhibition of these parameters. Elevated levels of CD4+ T cell specific interferon-gamma (IFN-γ) in splenocytes of arthritic animals was also inhibited in treated animals. The oral LD0 in both mice and rats was more than 1000mg/kg. [Copyright &y& Elsevier]

Abstract: Despite expanded research in stem cell biology, little is known about the mechanisms underlying migration, growth, and differentiation of adipose-derived adult mesenchymal stem cells (ASC). The simultaneous measurement of intracellular pathways opens new avenues to gain further insights in these processes. We used the Cytometric Bead Array (CBA) Flex Set technology to simultaneously analyze protein phosphorylation after stimulation of ASC and compared the results with data generated by corresponding Western blots. Signal transduction of ASC was stimulated by epidermal growth factor (EGF) and analyzed by determining phosphorylation of mitogen-activated protein kinases (MAPKs) ERK, p38, and JNK by Western blotting and CBA. After incubation with EGF, all MAPKs were significantly but differentially phosphorylated depending on time and dose. Furthermore, the ERK-response was abolished by EGF-R antagonist AG 1478 and kinase inhibitor PD98059, whereas p38 and JNK were only inhibited by AG1478. The stimulation and inhibition profiles between the two assays were highly comparable and the data were significantly correlated. In the present study we demonstrated that the CBA technology offers a reliable and convenient method for multiplexing of phospho-proteins in the evaluation of signal transduction pathways of adipose-derived mesenchymal stem cells. [Copyright &y& Elsevier]

This research aims to quantify pro-inflammatory cytokines from the exudates of dental pulp tissues are helpful in the diagnosis of pulpitis, thus, laying down the foundation for further vital pulp therapy on irreversible pulpitis. The authors selected patients admitted to Beijing Stomatological Hospital from October 2016 to March 2018. Exudates were collected from pulp exposure and were divided into four groups, which encompass normal pulps (Group 1), early stage of chronic pulpitis (Group 2), late stage of chronic pulpitis (Group 3) and acute attack of chronic pulpitis (Group 4). The cytokines IL-1β, IL-6, IL-8 and TNF from the exudates of dental pulp tissues were quantified by cytometric bead array using enhanced sensitivity flex sets. All statistical analyses were performed using SPSS 22.0 and the 2-sided significance level was set at p < 0.05. The Kruskal-Wallis test, Mann-Whitney U test and Spearman correlation analysis were employed to process data. There were 32, 37, 14, 29 samples in Groups 1, 2, 3 and 4, respectively. Only small amount of IL-1β, IL-8 was expressed in normal pulps and almost no TNF, IL-6 could be detected in Group 1. No difference was observed in the concentration of TNF between Group 2, 3, 4. [ABSTRACT FROM AUTHOR]

Populations in high infectious exposure countries are at low risk of some immune-mediated diseases such as Crohn’s disease and allergy. This low risk is maintained upon immigration to an industrialized country, but the offspring of such immigrants have a higher immune-mediated disease risk than the indigenous population. We hypothesize that early life exposures in a developing country shape the maternal immune system, which could have implications for the offspring born in a developed country with a low infectious load. The aim of this study was to investigate if exposures in childhood (indicated by country of origin) and subsequent exposures influence immunologic characteristics relevant to stimulation of offspring. Breast milk components among 64 mothers resident in Sweden, 32 of whom immigrated from a developing country, were examined using the ELISA and Cytometric Bead Array methods. Immigrants from a developing country had statistically significantly higher levels of breast milk interleukin-6 (IL-6), IL-8 and transforming growth factor-β1. A larger number of previous pregnancies were associated with down-regulation of several substances, statistically significant for soluble CD14 and IL-8. The results suggest that maternal country of birth may influence adult immune characteristics, potentially relevant to disease risk in offspring. Such a mechanism may explain the higher immune-mediated disease risk among children of migrants from a developing to developed country. Older siblings may influence disease risk through the action of previous pregnancies on maternal immune characteristics. [ABSTRACT FROM AUTHOR]

We compared two methods to determine IL-6 at rest and following exercise. On two occasions, plasma IL-6 was determined in 10 men and 13 women before and immediately following 90 min of cycling at 65% V(O)(2max) using a cytometric bead array (CBA) and an ELISA. The CBA detected IL-6 in 63% of pre-exercise samples and 84% of post-exercise samples; the ELISA detected IL-6 in all samples. In samples with detectable IL-6, resting values were comparable between the CBA and ELISA (2.3 +/- 1.0 and 2.4 +/- 1.2 pg/mL, respectively; p = 0.99); the IL-6 response to exercise was underestimated (p = 0.0003) by the CBA by ~45%, compared with the ELISA. CBA- and ELISA-determined IL-6 values were moderately correlated (r = 0.66, p < 0.0001), but exhibited poor agreement (mean bias +/- 1SD = -1.4 +/- 2.1 pg/mL). The CBA provided concordant IL-6 concentrations at rest, when detected, but underestimated exercise-induced increases in IL-6 versus the ELISA method. [ABSTRACT FROM AUTHOR]

Over six million people in nine districts of West Bengal, India are exposed to very high levels of arsenic primarily through their drinking water. More than 300,000 people showed arsenic-induced skin lesions in these districts. This is regarded as the greatest arsenic calamity in the world. Chronic arsenicosis causes varied dermatological signs ranging from pigmentation changes, hyperkeratosis to non-melanocytic cancer of skin, and also malignancies in different internal organs. Higher incidences of opportunistic infections are found in the arsenic-exposed individuals, indicating that their immune systems may be impaired somehow. We have thus investigated the effect of arsenic on T-cell proliferation and cytokine secretion in 20 individuals with arsenic-induced skin lesions and compared the results with 18 arsenic-unexposed individuals. A marked dose-dependent suppression of Concanavalin A (Con A) induced T-cell proliferation was observed in the arsenic-exposed individuals compared with the unexposed (P < 0.001) individuals. This correlated with a significant decrease in the levels of secreted cytokines by the T cells (TNF-α, IFN-γ, 1L2, IL10, 1L5, and 1L4) in the exposed individuals (P < 0.001). Thus it can be inferred that arsenic exposure can cause immunosuppression in humans. [ABSTRACT FROM AUTHOR]

Considering the complexity of the immunological events triggered during active visceral Leishmaniasis (VL), the relevance of the segregation of the immune response during human VL into type 1 and type 2 still remains unclear. For this purpose, in individuals living in risk areas for VL, we have evaluated especially asymptomatic individuals and patients with active VL, the plasmatic levels of cytokines and reactive nitrogen species under ex vivo conditions. In addition, we have also performed an analysis of intracellular cytokine patterns of circulating leucocytes after short-term culture, particularly in the absence of antigenic-specific stimulation, in order to reflect dynamic events of immune response in vivo during Leishmania chagasi infection. Although asymptomatic individuals and non-infected subjects presented a similar immunological profile, an outstanding inflammatory/regulatory profile, based on higher plasmatic levels of cytokines such as interleukin (IL)-8, interferon (IFN)-γ, tumour necrosis factor (TNF)-α, IL-6 and IL-10, was associated with clinical status observed in active VL. In this context, we hypothesize that IL-10, through its ability to inhibit anti-leishmanial macrophage activation, associated with the lower frequency of TNF-α+ monocytes and ordinary levels of nitrite and nitrate are the major mechanisms associated with disease onset. [ABSTRACT FROM AUTHOR]

In order to detect T cells against several chronic myeloid leukaemia (CML)-associated antigens we used: (i) a novel T-cell assay [cytometric bead array (CBA)]; (ii) γ-interferon enzyme-linked immunoSPOT ( γ-IFN-ELISpot); and (iii) tetramer staining in peripheral blood from CML patients. Peptide-specific cytokine release was detected by CBA in some patients, whereas standard γ-IFN-ELISpot and tetramer staining were negative in the vast majority of cases. In CBA, peptide-specific cytokine release was predominantly tumour necrosis factor- α, raising questions about the responding cells and their functional status. CBA appears to be a new useful tool for the detection of leukaemia-reactive T cells. [ABSTRACT FROM AUTHOR]

Abstract: Renal rejection is associated with an active immune response regulated by cytokines and in which immunocompetent cells are involved. Previous studies have measured high levels of cytokines in the urine and plasma in various renal dysfunction states. However, some methods used to measured cytokines hinder their use as a diagnostic tool in renal rejection. In this report, cytokine levels were determined in the plasma and urine of kidney transplant patients, with renal rejection and without it, using a cytometric bead array (CBA) technique. Concentrations of six human cytokines (IL-2, IL-4, IL-5, IL-10, TNF-α and INF-γ) were established. Results show that patients who develop renal rejection presented high levels of IL-10 and IFN-γ cytokines in plasma and urine compared to patients without renal rejection. The CBA technique displayed greater sensitivity in the determination of cytokines in urine than the conventional ELISA technique. Finally, when standard cytokines in plasma and in urine were compared, it was observed that, in plasma, levels of IL-4, IL-5, IL-10, TNF-α and IFN-γ were detected, whereas in urine the levels detected were of IL-4, IL-5, IL-10 and IFN-γ. These results indicate that the CBA assay is a sensitive method to measure cytokines in urine. In kidney transplant patients undergoing acute renal rejection, the presence of cytokines in urine reflects renal damage and could be a useful method in the diagnosis of renal rejection. [Copyright &y& Elsevier]

Paajanen L, Vaarala O, Karttunen R, Tuure T, Korpela R, Kokkonen J. Increased IFN-γ secretion from duodenal biopsy samples in delayed-type cow's milk allergy. Pediatr Allergy Immunol 2005: 16: 439–443. © 2005 Blackwell Munksgaard A delayed and local gastrointestinal hypersensitivity to cow's milk (CM) protein is difficult to diagnose and there are limited data on this disorder. The aim of this study was to investigate local intestinal cytokine secretion in the upper small intestine in children with delayed-type cow's milk allergy (CMA). Duodenal biopsy samples from 31 children with delayed CMA, 14 with celiac disease (CD), and 14 healthy controls were studied for the spontaneous release of IFN- γ, TNF- α, IL-2, IL-4, IL-5, and IL-10, measured by cytometric bead array, and of TGF- β and IL-6 measured by ELISA. The children with delayed CMA secreted more IFN- γ than the controls (p = 0.006) and the children with CD (p = 0.006). The children with CD secreted more IL-6 compared to the controls (p = 0.008) and the children with delayed CMA (p = 0.002). The children with delayed CMA who had continuously been exposed to CM secreted less TGF- β than the children with delayed CMA who avoided CM (p = 0.050), and showed a tendency towards lower secretion compared to the controls (p = 0.078). Secretions of TNF- α, IL-2, IL-4, IL-5, and IL-10 were low in general; however, the children with delayed CMA who did not avoid CM secreted more IL-4 and IL-10 than the controls (p = 0.016, 0.059). In conclusion, the children with delayed CMA showed up-regulation of IFN- γ. Interestingly, TGF- β secretion was up-regulated in those children with delayed CMA who avoided CM suggesting recovery of regulation mechanisms. [ABSTRACT FROM AUTHOR]

The introduction of flow cytometric bead-based technology has added a new approach for investigators to simultaneously measure multiple analytes in biological and environmental samples. This new technology allows for (1) evaluation of multiple analytes in a single sample; (2) utilization of minimal sample volumes to glean data; (3) reproducibility and results comparative with previous experiments; (4) direct comparison with existing assays; and (5) a more rapid evaluation of multiple samples in a single platform. The cytometric bead array (CBA) system enables simultaneous measurement of multiple analytes in sample volumes too small for traditional immunoassays. Results have been presented for the analysis of a variety of human cytokines. In addition, the technology allows for the design and creation of assays to measure a variety of analytes including inflammatory mediators, chemokines, immunoglobulin isotypes, intracellular signaling molecules, apoptotic mediators, adhesion molecules, and antibodies. New initiatives put forward by the Human Genome Project and the FDA require the development and use of assays for the rapid simultaneous quantitation of multiple analytes. The CBA technology provides the ability to quantify multiple proteins within a given sample, with precision and consistency. [Copyright &y& Elsevier]

Background: Psoriasis is a chronic inflammatory skin disease characterized by hyperproliferation and incomplete differentiation of epidermis, and accumulation of neutrophils and proinflammatory T cells in epidermis and dermis. Chemokines are believed to be the main players mediating the chemotaxis of leucocytes to the lesional site. Previous studies have established the role of various chemokine ligands and receptors at the lesional site in psoriasis.Aims: In this study, we have compared the serum levels of various chemokines, namely, inducible protein-10 (IP-10) (CXCL10), MCP-1 (CCL-2), monokine induced by gamma interferon (MIG) (CXCL-9), RANTES (CCL5), interleukin (IL)-8, and eotaxin in patients with chronic plaque psoriasis with that of healthy controls. We also studied whether the chemokine levels varied within different patient groups based on various clinical and demographic parameters, and if any of these chemokines correlated with disease activity.Methods: We studied 40 patients with chronic plaque psoriasis from a single center. Their clinical and demographic details were recorded in predesigned prforma. Patients with unstable forms of psoriasis like guttate, erythrodermic, or pustular psoriasis were excluded. The serum chemokine levels were measured by flow cytometry-based bead array set system. The serum levels of the patients were compared with that of 25 healthy controls. A subgroup analysis was also done to study the correlation of chemokine levels with age, sex, duration, and severity of disease.Results: We observed a significant decrease in serum level of all these chemokines in patients, when compared with that of healthy controls. We also found that MIG levels showed a positive correlation with disease severity based on Psoriasis Area and Severity Index.Limitations: The major limitation of the study is lack of data on the lesional chemokine levels compared to serum chemokines.Conclusion: The inflammatory process in psoriasis is orchestrated through chemokines. MIG is a potential serum biomarker for assessing disease severity. [ABSTRACT FROM AUTHOR]

We herein describe a case of Sweet's syndrome (SS) in a patient being treated for pulmonary toxoplasmosis complicated with myelodysplastic syndrome (MDS). The patient's SS developed after the pulmonary toxoplasmosis improved following treatment. We searched his cytokine profiles comprehensively using a bead-based immunoassay. The results showed no elevation of interleukin (IL)-2, interferon (IFN)-γ or IL-17A, and IL-6 was only observed to have increased at the onset of SS, suggesting that the pulmonary toxoplasmosis had been well controlled and that chronic inflammation may have been the cause of SS. Pulmonary toxoplasmosis is an extremely rare occurrence. The cytokine profile can help to clarify the pathological condition of SS and MDS complicated with severely invasive infectious diseases.