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Nelly Herfina Dahlan,1,* Irma Bernadette S Sitohang,1,* Wresti Indriatmi,1 Heri Wibowo,2 Liani Elisabeth Enggy3 1Department of Dermatology and Venereology, Faculty of Medicine, University of Indonesia, Dr. Cipto Mangunkusumo Hospital, Jakarta, Indonesia; 2Department of Integrated Laboratory, Faculty of Medicine, University of Indonesia, Dr. Cipto Mangunkusumo Hospital, Jakarta, Indonesia; 3Faculty of Medicine, Hasanuddin University, Makassar, Indonesia*These authors contributed equally to this workCorrespondence: Irma Bernadette S Sitohang, Department of Dermatology and Venereology, Faculty of Medicine, Universitas Indonesia, dr. Cipto Mangunkusumo Hospital, Jalan Salemba Raya No. 6, Jakarta, 10430, Indonesia, Email irma_bernadette@yahoo.comBackground: The inflammatory process in acne vulgaris (AV) is characterized by the upregulation of specific pro-inflammatory cytokines, including interleukin (IL)-1β, IL-6, and IL-8, within sebocytes and keratinocytes. Sebocytes have been identified as target cells for bioactive vitamin D. Experimental studies on animal models have demonstrated the potent comedolytic effects of topical vitamin D. However, further research is required to specifically evaluate the impact of vitamin D on inflammatory lesions in acne vulgaris (AV).Objective: To evaluate the effectiveness of topical vitamin D in treating acne vulgaris (AV) lesions by investigating its anti-inflammatory effects on pro-inflammatory cytokine modulation, specifically assessing the correlation between IL-1β levels in acne lesions and the reduction in AV severity.Materials and Methods: This study is a double-blind, randomized, placebo-controlled clinical trial with a 2-arm design over an 8-week intervention period. Participants were randomly assigned to either the topical vitamin D group (cholecalciferol 50 mcg) or the topical placebo group, with each group comprising 32 subjects. All participants received concomitant treatment with topical adapalene 0.1%. Cytokine levels within acne lesions were assessed using Luminex Polystyrene Screening Assays to detect and quantify IL-1β levels. The effectiveness of the treatment was evaluated by monitoring the reduction in the number of inflammatory lesions, while the safety of topical vitamin D was assessed by documenting and analyzing any reported side effects.Results: The study found a significant correlation between the reduction in IL-1β levels within acne lesions and the decrease in moderate and severe inflammatory lesions in acne vulgaris (p = 0.028). The topical application of vitamin D led to a significant reduction in inflammatory AV lesions (p = 0.045). No significant topical side effects were observed in either the vitamin D or placebo groups.Conclusion: This study demonstrates that the topical administration of vitamin D in acne vulgaris (AV) lesions is effective in reducing pro-inflammatory cytokine levels within acne lesions and in decreasing the severity of AV.Trial Registration: NCT05758259. September 5, 2022.Keywords: acne vulgaris, topical vitamin D, IL-1β, Luminex

High systemic doses of adeno-associated viruses (AAVs) have been associated with immune-related serious adverse events (SAEs). Although AAV was well tolerated in preclinical models, SAEs were observed in clinical trials, indicating the need for improved preclinical models to understand AAV-induced immune responses. Here, we show that mice dual-dosed with AAV9 at 4-week intervals better recapitulate aspects of human immunity to AAV. In the model, anti-AAV9 immunoglobulin G (IgGs) increased in a linear fashion between the first and second AAV administrations. Complement activation was only observed in the presence of high levels of both AAV and anti-AAV IgG. Myeloid-derived pro-inflammatory cytokines were significantly induced in the same pattern as complement activation, suggesting that myeloid cell activation to AAV may rely on the presence of both AAV and anti-AAV IgG complexes. Single-cell RNA sequencing of peripheral blood mononuclear cells confirmed that activated monocytes were a primary source of pro-inflammatory cytokines and chemokines, which were significantly increased after a second AAV9 exposure. The same activated monocyte clusters expressed both Fcγ and complement receptors, suggesting that anti-AAV-mediated activation of myeloid cells through Fcγ receptors and/or complement receptors is one mechanism by which anti-AAV antigen complexes may prime antigen-presenting cells and amplify downstream immunity.

Within the past decade, single domain antibodies (sdAbs) have been recognized as unique affinity binding reagents that can be tailored for performance in a variety of immunoassay formats. Luminex MagPlex color-coded magnetic microspheres provide a high-throughput platform that enables multiplexed immunoassays. We developed a MagPlex bead-based assay for the detection of SARS-CoV-2, using sdAbs against SARS-CoV-2 nucleocapsid (N) protein in which we engineered the sdAb capture reagents to orient them on the beads. The oriented sdAbs provided an increase in sensitivity over randomly oriented sdAbs for samples of N diluted in buffer, which also translated into better detection of SARS-CoV-2 in clinical samples. We assessed the specificity of the assay by examining seasonal coronavirus clinical samples. In summary, we provide a proof-of-concept that a bead-based assay using sdAbs to detect SARS-CoV-2 is feasible and future research combining it with other sdAbcoated beads that can detect other viruses may provide a useful diagnostic tool. [ABSTRACT FROM AUTHOR]

Chronic subdural hematoma (CSDH) development involves inflammatory, angiogenetic, and fibrinolytic mechanisms, several components of which are now unraveled through intensive research. The urokinase plasminogen activator receptor (uPAR) is part of the plasminogen activator system and possesses inflammatory, angiogenetic, and fibrinolytic capabilities. As a first, this study aims to identify uPAR in the hematoma fluid, hematoma membrane, dura mater, and systemic blood from patients with CSDH and, if present, to investigate if the uPAR level at the time of surgery may be a predictor for later developing recurrent CSDH. uPAR expression in the hematoma membrane and dura mater was analyzed using immunohistochemistry and presented as the H-score of the positive immunostaining. The uPAR levels in the hematoma fluid and systemic blood were determined using a multiplex antibody bead kit (Luminex). Samples were collected at the time of the first CSDH surgery, and in the case of recurrent CSDH within 90 days, the samples were again collected at reoperation. A comparison of uPAR expression between the hematoma membrane and dura mater, as well as uPAR levels in systemic blood and hematoma fluid, was performed using the Wilcoxon rank sum test. We included 112 patients, 26 of whom had recurrent CSDH. The median hematoma uPAR level was 22,125 (14,845–33,237) and significantly higher than the median systemic blood level of 789 pg/L (465–2,088) (p < 0.001). Similarly, the uPAR level of the hematoma membrane was 14.3 (7.54–44.8) and significantly higher than the dural uPAR level of 0.81 (0.3–1.98) (p < 0.001). For the first time, we identified uPAR in the subdural fluid, hematoma membrane, dura mater, and systemic blood from patients with CSDH. The high expression of uPAR in the subdural fluid and hematoma membrane indicates that the mechanisms of CSDH are predominantly in the subdural fluid collection and surrounding hematoma membrane. [ABSTRACT FROM AUTHOR]

Acute cellular rejection (ACR) frequently occurs following lung transplantation (LuTx) and represents a risk factor for the development of chronic lung allograft dysfunction (CLAD) as well as long-term survival. The histopathological diagnosis of ACR carries a burden of interobserver variability. The widespread utilization and cost-effectiveness of immunohistochemistry (IHC) was proven beneficial in diagnosing rejection in human kidney transplantations and LuTx rat models. However, its potential for ACR detection in patients remains unexplored. We analyzed surface markers (CD3, CD4, CD8, CD20, CD68, CD47, PD-1, PD-L1, and CD31/PECAM-1) on lung tissue cryobiopsy samples collected within 6 months post-LuTx from 60 LuTx recipients, 48 of whom were diagnosed with ACR. Additionally, serum samples from 51 patients were analyzed using a multiplex bead-based Luminex assay. The cytokines and markers included PD-L1, IL2, TNFα, IFNγ, and Granzyme B. We observed a significant increase in PD-L1 tissue expression within the rejection group, suggesting a concerted effort to suppress immune responses, especially those mediated by T-cells. Furthermore, we noted significant differences in PECAM-1 levels between ACR/non-ACR. Additionally, peripheral blood C-reactive-protein levels tended to be higher in the ACR group, while Luminex serum analyses did not reveal any significant differences between groups. In conclusion, our findings suggest the potential value of PECAM-1 and PD-L1 markers in diagnosing ACR.

Background and Objectives: Kidney transplants are effective for advanced renal failure, but graft rejection can occur when the recipient’s immune system misidentifies the kidney as a foreign object. Human leukocyte antigen (HLA), a component of the host’s immunological defense system, acts as a barrier to graft rejection. Selecting potential kidney recipients and donors for transplantation requires careful consideration of histocompatibility for the HLA-A, HLA-B, and HLA-DRB1 antigens. Mismatches between donors and recipients prolong transplant therapy, leading to lower graft survival and higher mortality. The objective of the study was to analyze HLA-A, HLA-B, and HLA-DRB1 antigens frequency in live-related renal transplant recipients and donors visiting the Department of Histocompatibility and Immunopathology at the National Public Health Laboratory, Teku, Kathmandu, Nepal. Methods: A retrospective study was conducted by using data from 104 renal transplant recipients and donors whose samples had previously been collected, processed, and analyzed to identify the Class I loci (HLA-A and HLA-B) and Class II loci (DRB1) between November 2021 and February 2024. Results: The study found that the majority of donors were female, and the majority of recipients were male. In this study, 10, 17, and 12 different HLA-A, B, and DRB1 alleles were found in recipients and donors. Alleles found more frequently are A*11 (25.00%), A*24 (23.08%), sB*15 (22.60%), B*35 (12.98%), DRB1*15 (22.60%), and DRB1*12 (21.63%). Conclusions: The study suggests that both donors and recipients of renal transplants in Nepal have a diverse HLA antigen distribution, which could aid in selecting a genetically closer group as potential matched donors for transplant recipients.

Abstract Background Melioidosis is a serious bacterial infection caused by Burkholderia pseudomallei, a gram-negative bacterium commonly found in soil and water. It can affect both humans and animals, and is endemic in regions such as Southeast Asia and Northern Australia. In recent years, there have been reports of an emergence of human melioidosis in other areas, including New Caledonia. Results During standard laboratory analysis in New Caledonia in 2021, a strain of B. pseudomallei was isolated from a goat. The strain was characterized using both MLST and WGS techniques and was found to cluster with previously described local human strains from the area. In parallel, several serological tests (CFT, ELISA, Luminex (Hcp1, GroEL, BPSS1840), arrays assay and a latex agglutination test) were performed on animals from the farm where the goat originated, and/or from three other neighboring farms. Using two commercial ELISA kits, seropositive animals were found only on the farm where the infected goat originated and tests based on recombinant proteins confirmed the usefulness of the Hcp1 protein for the diagnosis of melioidosis in animals. Conclusions Despite the regular reports of human cases, this is the first confirmed case of melioidosis in an animal in New Caledonia. These results confirm the presence of the bacterium in the region and highlight the importance of vigilance for both animal and human health. It is critical that all health partners, including breeders, veterinarians, and biologists, work together to monitor and prevent the spread of the disease.

Background: Melioidosis is a serious bacterial infection caused by Burkholderia pseudomallei, a gram-negative bacterium commonly found in soil and water. It can affect both humans and animals, and is endemic in regions such as Southeast Asia and Northern Australia. In recent years, there have been reports of an emergence of human melioidosis in other areas, including New Caledonia. Results: During standard laboratory analysis in New Caledonia in 2021, a strain of B. pseudomallei was isolated from a goat. The strain was characterized using both MLST and WGS techniques and was found to cluster with previously described local human strains from the area. In parallel, several serological tests (CFT, ELISA, Luminex (Hcp1, GroEL, BPSS1840), arrays assay and a latex agglutination test) were performed on animals from the farm where the goat originated, and/or from three other neighboring farms. Using two commercial ELISA kits, seropositive animals were found only on the farm where the infected goat originated and tests based on recombinant proteins confirmed the usefulness of the Hcp1 protein for the diagnosis of melioidosis in animals. Conclusions: Despite the regular reports of human cases, this is the first confirmed case of melioidosis in an animal in New Caledonia. These results confirm the presence of the bacterium in the region and highlight the importance of vigilance for both animal and human health. It is critical that all health partners, including breeders, veterinarians, and biologists, work together to monitor and prevent the spread of the disease. [ABSTRACT FROM AUTHOR]

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

Background and Objectives: The aim of this study was to evaluate the levels of selected cytokines and their possible influence on the development of cardiovascular and pulmonary complications in patients hospitalized at the Silesian Centre for Heart Disease in Zabrze after having undergone COVID-19. Materials and methods: The study included 76 randomly selected patients from the SILCOVID-19 database. The median time from symptom onset to the study visit was 102 (86–118) days. The median age of the study group was 53 (44–60) years. Assays of a panel of 30 cytokines were carried out in the serum of patients on a Luminex100 platform using the Milliplex MAP kit from Merck KGaA Germany. Results: There were no statistically significant differences in most of the cytokines analyzed between patients with confirmed or excluded lung lesions or cardiac abnormalities. Additionally, no statistically significant differences in cytokine concentrations according to gender, age, comorbidity of diabetes, renal disease, hypertension, increased risk of thrombotic disease, or psychological disorders were demonstrated. There were high concentrations of cytokines such as platelet-derived growth actor-AA (PDGF-AA), monocyte chemoattractant protein-1 (MCP-1), monokine-induced gamma interferon (MIG), and vascular endothelial growth factor-A (VEGF-A). Conclusions: No direct impact of the dependencies between a panel of cytokines and the incidence of cardiovascular and pulmonary complications in patients hospitalized at the Silesian Centre for Heart Disease in Zabrze after having undergone COVID-19 was demonstrated. The demonstration of high levels of certain cytokines (PDGF-AA, VEGF, MIG, and IP10) that are of significance in the development of many lung diseases, as well as cytokines (MCP-1) that influence the aetiopathogenesis of cardiovascular diseases seems to be highly concerning in COVID-19 survivors. This group of patients should receive further monitoring of these cytokine levels and diagnostic imaging in order to detect more severe abnormalities as early as possible and administer appropriate therapy. [ABSTRACT FROM AUTHOR]

Within the past decade, single domain antibodies (sdAbs) have been recognized as unique affinity binding reagents that can be tailored for performance in a variety of immunoassay formats. Luminex MagPlex color-coded magnetic microspheres provide a high-throughput platform that enables multiplexed immunoassays. We developed a MagPlex bead-based assay for the detection of SARS-CoV-2, using sdAbs against SARS-CoV-2 nucleocapsid (N) protein in which we engineered the sdAb capture reagents to orient them on the beads. The oriented sdAbs provided an increase in sensitivity over randomly oriented sdAbs for samples of N diluted in buffer, which also translated into better detection of SARS-CoV-2 in clinical samples. We assessed the specificity of the assay by examining seasonal coronavirus clinical samples. In summary, we provide a proof-of-concept that a bead-based assay using sdAbs to detect SARS-CoV-2 is feasible and future research combining it with other sdAb-coated beads that can detect other viruses may provide a useful diagnostic tool.

Metastatic colorectal cancer (mCRC) currently lacks reliable biomarkers for precision medicine, particularly for chemotherapy-based treatments. This study examines the behavior of 11 CXC chemokines in the blood of 104 mCRC patients undergoing first-line oxaliplatin-based treatment to pinpoint predictive and prognostic markers. Serum samples were collected before treatment, at response evaluation (EVAR), and at disease progression or last follow-up. Chemokines were assessed in all samples using a Luminex® custom panel. CXCL13 levels increased at EVAR in responders, while in non-responders it decreased. Increasing levels of CXCL13 at EVAR, independently correlated with improved progression-free survival (PFS) and overall survival (OS). Nanostring® analysis in primary tumor samples showed CXCL13 gene expression's positive correlation not only with gene profiles related to an immunogenic tumor microenvironment, increased B cells and T cells (mainly CD8+) but also with extended OS. In silico analysis using RNAseq data from liver metastases treated or not with neoadjuvant oxaliplatin-based combinations, and deconvolution analysis using the MCP-counter algorithm, confirmed CXCL13 gene expression's association with increased immune infiltration, improved OS, and Tertiary Lymphoid Structures (TLSs) gene signatures, especially in neoadjuvant-treated patients. CXCL13 analysis in serum from 36 oxaliplatin-treated patients from the METIMMOX study control arm, reported similar findings. In conclusion, the increase of CXCL13 levels in peripheral blood and its association with the formation of TLSs within the metastatic lesions, emerges as a potential biomarker indicative of the therapeutic efficacy in mCRC patients undergoing oxaliplatin-based treatment

Powassan virus is an emerging tick-borne pathogen capable of causing severe neuroinvasive disease. As the incidence of human Powassan virus grows both in magnitude and geographical range, the development of sensitive detection methods for diagnostics and surveillance is critical. In this study, a Taqman-based triplex real-time PCR assay was developed for the simultaneous and quantitative detection of Powassan virus and Powassan virus lineage II (deer tick virus) in Ixodes scapularis ticks. An exon–exon junction internal control was built-in to allow for accurate detection of RNA quality and the failure of RNA extraction. The newly developed assay was also applied to survey deer tick virus in tick populations at 13 sites on Cape Cod and Martha's Vineyard Island in Massachusetts. The assay's performance was compared with the Luminex xMAP MultiFLEX Vector-borne Panel 2. The results suggested that the real-time PCR method was more sensitive. Powassan virus infection rates among ticks collected from these highly endemic tick areas ranged from 0.0 to 10.4%, highlighting the fine-scale geographic variations in deer tick virus presence in this region. Looking forward, our PCR assay could be adopted in other Powassan virus surveillance systems. [ABSTRACT FROM AUTHOR]

Atherosclerotic plaques are sites of chronic inflammation with diverse cell contents and complex immune signaling. Plaque progression and destabilization are driven by the infiltration of immune cells and the cytokines that mediate their interactions. Here, we attempted to compare the systemic cytokine profiles in the blood plasma of patients with atherosclerosis and the local cytokine production, using ex vivo plaque explants from the same patients. The developed method of 41-plex xMAP data normalization allowed us to differentiate twenty-two cytokines produced by the plaque that were not readily detectable in free circulation and six cytokines elevated in blood plasma that may have other sources than atherosclerotic plaque. To verify the xMAP data on the putative atherogenesis-driving chemokines MCP-1 (CCL2), MIP-1α (CCL3), MIP-1β (CCL4), RANTES (CCL5), and fractalkine (CX3CL1), qPCR was performed. The MIP1A (CCL3), MIP1B (CCL4), FKN (CX3CL1), and MCP1 (CCL2) genes were expressed at high levels in the plaques, whereas RANTES (CCL5) was almost absent. The expression patterns of the chemokines were restricted to the plaque cell types: the MCP1 (CCL2) gene was predominantly expressed in endothelial cells and monocytes/macrophages, MIP1A (CCL3) in monocytes/macrophages, and MIP1B (CCL4) in monocytes/macrophages and T cells. RANTES (CCL5) was restricted to T cells, while FKN (CX3CL1) was not differentially expressed. Taken together, our data indicate a plaque-specific cytokine production profile that may be a useful tool in atherosclerosis studies. [ABSTRACT FROM AUTHOR]

In an ineffective transfusion context, solid‐phase immunoassays using the Luminex platform for the detection and characterization of HLA antibodies are currently used to select HLA‐compatible platelet products. A new HLA antibody identification method, the HISTO SPOT® HLA AB test (BAG Health care GmbH, Lich, Germany), based on the detection of antibodies directed against a recombinant single antigen (SA) by colored spots detected by HISTO MATCH HLA AB module software, runs fully automated on the MR.SPOT®. The aim of this study was to compare the ability of the HISTO SPOT HLA AB and C1qScreen™ (C1q SAB) assays with that of the Labscreen single antigen class I (OL SAB) assay to detect anti‐HLA class I antibodies in 56 serum samples from 54 platelet refractory acute myeloid leukemia patients who received HLA mismatch platelet concentrates at a single oncohematology center. In total, 1414 class I specificities, 433 HLA‐A and 981 HLA‐B, were detected by the OL SAB test. The mean fluorescence intensity (MFI) was >5000 for 874 antigens and <5000 for 655 antigens. The HISTO SPOT® HLA AB and C1q SAB tests identified 85% and 79% of OL SA‐detected antigens with an MFI >5000, respectively, but did not identify 34% and 44% of OL SAB‐detected antigens, highlighting the lower sensitivity of these techniques. Interestingly, the donor‐specific antibodies (DSAs) identified by the HISTO SPOT® HLA AB and C1q SAB assays reacted against HLA mismatch platelet concentrates with the same specificity (86%) and positive predictive (77%) value as in the OL SAB test when the MFI threshold was >2000 for DSA detection. Although the HISTO SPOT® HLA AB test is less sensitive than the OL SAB test, this test could be used for the selection of HLA‐compatible platelet products. [ABSTRACT FROM AUTHOR]

Background and Objectives: Kidney transplants are effective for advanced renal failure, but graft rejection can occur when the recipient's immune system misidentifies the kidney as a foreign object. Human leukocyte antigen (HLA), a component of the host's immunological defense system, acts as a barrier to graft rejection. Selecting potential kidney recipients and donors for transplantation requires careful consideration of histocompatibility for the HLA-A, HLA-B, and HLA-DRB1 antigens. Mismatches between donors and recipients prolong transplant therapy, leading to lower graft survival and higher mortality. The objective of the study was to analyze HLA-A, HLA-B, and HLA-DRB1 antigens frequency in live-related renal transplant recipients and donors visiting the Department of Histocompatibility and Immunopathology at the National Public Health Laboratory, Teku, Kathmandu, Nepal. Methods: A retrospective study was conducted by using data from 104 renal transplant recipients and donors whose samples had previously been collected, processed, and analyzed to identify the Class I loci (HLA-A and HLA-B) and Class II loci (DRB1) between November 2021 and February 2024. Results: The study found that the majority of donors were female, and the majority of recipients were male. In this study, 10, 17, and 12 different HLA-A, B, and DRB1 alleles were found in recipients and donors. Alleles found more frequently are A*11 (25.00%), A*24 (23.08%), sB*15 (22.60%), B*35 (12.98%), DRB1*15 (22.60%), and DRB1*12 (21.63%). Conclusions: The study suggests that both donors and recipients of renal transplants in Nepal have a diverse HLA antigen distribution, which could aid in selecting a genetically closer group as potential matched donors for transplant recipients. [ABSTRACT FROM AUTHOR]

We investigated the characteristics of the microbial community of the nasal sinuses in patients with chronic rhinosinusitis with nasal polyps (CRSwNP) and identified the correlations of the nasal microbiome with the inflammatory microenvironment of the nasal cavity. We collected matched nasal secretion and polyp tissue samples from 77 CRSwNP patients. Then, we extracted microbial DNA from cotton swabs, used high-throughput sequencing technology based on 16S ribosomal RNA (rRNA) to detect the bacterial community composition, and detected cytokines such as interleukin (IL)-5, IL-8, IL-17a, IL-17e, IL-18, IL-27 and interferon (INF)-gamma in the polyp tissue samples using Luminex. Eosinophils and neutrophils in the peripheral blood and polyp tissue were counted, and the relationships between inflammatory factors or inflammatory cell counts and nasal microbial diversity were analyzed. Among the inflammatory factors evaluated, IL-5 had a positive rate of 32.47%, IFN-γ had a positive rate of 84.42%, IL-17A and IL-17E had positive rates of 75.32%, IL-18 had a positive rate of 94.81%, IL-27 had a positive rate of 68.83%, and IL-8 had a positive rate of 100%. IL-17a and IL-27 were negatively correlated with both Enterobacter and Anaerococcus , IL-8 was negatively correlated with both Enterobacter and Staphylococcus , IL-18 was positively correlated with Candidatus Arthromitus and negatively correlated with Haemophilus , and IL-27 was positively correlated with Faecalibaculum. Lactobacillus and Enterococcus were positively correlated with the degree of neutrophil infiltration in nasal polyp tissue. In Southwest China, inflammation of the nasal polyps exhibits a variety of patterns. Enterobacteria and anaerobic bacteria may be correlated with the inflammatory pattern of nasal polyps. The neutrophil-mediated inflammatory response plays an important role in patients with CRSwNP in Southwest China. [ABSTRACT FROM AUTHOR]

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

Background and Objectives: Detection of anti‐platelet antibodies is required for the diagnosis of foetal/neonatal alloimmune thrombocytopaenia. The most commonly used methods for anti‐platelet antibody detection are the monoclonal antibody‐specific immobilization of platelet antigens (MAIPA) and the Luminex bead assay (PakLx). However, for economic reasons, the use of the PakLx assay is limited. Materials and Methods: In the present study, we evaluated the performance of an optimized protocol based on a half‐volume of PakLx reagents. We compared two alternative procedures: one with a half‐volume of all components including patient samples, and another based on a half‐volume of reagents but a standard volume of patient sample. Results: Our results obtained with a panel of 67 samples demonstrate improved sensitivity when using a standard sample volume. Conclusion: In the event of an inconclusive result with this optimized protocol (e.g., incomplete panel of positive Luminex beads), we recommend testing the sample with an alternative protocol (e.g., MAIPA or the original PakLx protocol). [ABSTRACT FROM AUTHOR]

Abstract Background Although the role of viral agents, such as human papillomavirus (e.g. HPV16, HPV18) in colorectal cancer (CRC) has been previously investigated, results remain inconclusive. Methods To further evaluate the involvement of oncogenic HPV types in CRC, 40 frozen neoplastic and 40 adjacent colonic tissues collected from Italian patients were analyzed by Luminex-based assays that detect a broad spectrum of HPV types, i.e. Alpha (n = 21), Beta (n = 46) and Gamma HPVs (n = 52). In addition, 125 frozen CRC samples and 70 surrounding mucosal tissues were collected from Czech patients and analyzed by broad spectrum PCR protocols: (i) FAP59/64, (ii) FAPM1 and (iii) CUT combined with Next Generation Sequencing (NGS). Results Using Luminex-basedassays, DNA from HPV16 was detected in 5% (2/40) CRC tissues from Italian patients. One HPV16 DNA-positive CRC case was subsequently confirmed positive for E6*I mRNA. Cutaneous beta HPV types were detected in 10% (4/40) adjacent tissues only, namely HPV111 (n = 3) and HPV120 (n = 1), while gamma HPV168 (n = 1) and HPV199 (n = 1) types were detected in adjacent and in tumor tissues, respectively. The NGS analysis of the CRC Czech samples identified HPV sequences from mucosal alpha-3 (HPV89), alpha-7 (HPV18, 39, 68 and 70) and alpha-10 species (HPV11), as well as cutaneous beta-1 (HPV20, 24, 93, 98, 105,124) beta-2 (HPV23), beta-3 (HPV49) and gamma-1 species (HPV205). Conclusions Our findings indicate that HPV types belonging to the mucosal alpha, and the ‘cutaneous’ beta and gamma genera can be detected in the colonic mucosal samples with a low prevalence rate and a low number of HPV reads by Luminex and NGS, respectively. However, additional studies are required to corroborate these findings.

High systemic doses of adeno-associated viruses (AAVs) have been associated with immune-related serious adverse events (SAEs). Although AAV was well tolerated in preclinical models, SAEs were observed in clinical trials, indicating the need for improved preclinical models to understand AAV-induced immune responses. Here, we show that mice dual-dosed with AAV9 at 4-week intervals better recapitulate aspects of human immunity to AAV. In the model, anti-AAV9 immunoglobulin G (IgGs) increased in a linear fashion between the first and second AAV administrations. Complement activation was only observed in the presence of high levels of both AAV and anti-AAV IgG. Myeloid-derived pro-inflammatory cytokines were significantly induced in the same pattern as complement activation, suggesting that myeloid cell activation to AAV may rely on the presence of both AAV and anti-AAV IgG complexes. Single-cell RNA sequencing of peripheral blood mononuclear cells confirmed that activated monocytes were a primary source of pro-inflammatory cytokines and chemokines, which were significantly increased after a second AAV9 exposure. The same activated monocyte clusters expressed both Fcγ and complement receptors, suggesting that anti-AAV-mediated activation of myeloid cells through Fcγ receptors and/or complement receptors is one mechanism by which anti-AAV antigen complexes may prime antigen-presenting cells and amplify downstream immunity.

Automation of diagnostic assays generally aims to increase reproducibility and throughput while decreasing human errors and hands-on time. Here, we introduce a protocol for the automated chemical conjugation of glycans to color-coded magnetic beads using the KingFisher Flex magnetic particle processor. The resulting glycan-coupled magnetic beads allow the detection of anti-glycan antibodies of different isotypes from various species. By generating anti-glycan antibody profiles, monoclonal antibodies can be screened for their specificity and cross-reactivity, while anti-glycan antibody profiles from different human body fluids can aid in predicting response to treatment or outcome of disease. This efficient, scalable protocol can also be adapted to attach proteins and other biomolecules to beads, making it useful for a wider range of applications that require bead-based laboratory methods.

Background: We aimed to evaluate the performance of a novel multiplex serological assay, able to simultaneously detect IgG of six infections, as a screening tool for imported diseases in migrants. Methods: Six panels of 40 (n = 240) anonymized serum samples with confirmed infections were used as positive controls to assess the multiplex assay's sensitivity. One panel of 40 sera from non-infected subjects was used to estimate the seropositivity cutoffs, and 32 non-infected sera were used as negative controls to estimate each serology's sensitivity and specificity. The multi-infection screening test was validated in a prospective cohort of 48 migrants from endemic areas.The sensitivity of the Luminex assay was calculated as the proportion of positive results over all positive samples identified by reference tests. The specificity was calculated using 32 negative samples. Uncertainty was quantified with 95 % confidence intervals using receiver operating characteristic analyses. Results: The sensitivity/specificity were 100 %/100 % for HIV (gp41 antigen), 97.5 %/100 % for Hepatitis B virus (HBV-core antigen), 100 %/100 % for Hepatitis C virus (HCV-core antigen), 92.5 %/90.6 % for strongyloidiasis [31-kDa recombinant antigen (NIE)], 97.5 %/100 % for schistosomiasis (combined serpin Schistosoma mansoni and S.haematobium antigens) and 95 %/90.6 % for Chagas disease [combined Trypanosoma cruzi kinetoplastid membrane protein-11 (KMP11) and paraflagellar rod proteins 2 (PFR2) antigens].In the migrant cohort, antibody response to the combination of the T.cruzi antigens correctly identified 100 % individuals, whereas HBV-core antigen correctly identified 91.7 % and Strongyloides-NIE antigen 86.4 %. Conclusions: We developed a new, robust and accurate 8-plex Luminex assay that could facilitate the implementation of screening programmes targeting migrant populations.

In the field of immunology, a systems biology approach is crucial to understanding the immune response to infection and vaccination considering the complex interplay between genetic, epigenetic, and environmental factors. Significant progress has been made in understanding the innate immune response, including cell players and critical signaling pathways, but many questions remain unanswered, including how the innate immune response dictates host/pathogen responses and responses to vaccines. To complicate things further, it is becoming increasingly clear that the innate immune response is not a linear pathway but is formed from complex networks and interactions. To further our understanding of the crosstalk and complexities, systems-level analyses and expanded experimental technologies are now needed. In this review, we discuss the most recent immunoprofiling techniques and discuss systems approaches to studying the global innate immune landscape which will inform on the development of personalized medicine and innovative vaccine strategies.

Combination and polyvalent vaccines not only provide protection against several different pathogens at the same time but can also increase vaccine protection against pathogens that have closely related pathogenic strains or serotypes. Multiplexed serological testing is a preferred method for determining the efficacy of combination and polyvalent vaccines, as it reduces the need for conducting multiple individual assays to confirm immune responses and cross-reactivity, uses less sample, and can be faster, more reliable, and more cost-effective. Bead-based suspension array technologies, such as the Luminex® xMAP® Technology, are often used for development of multiplexed serological assays for various vaccine trials and for routine testing in clinical laboratories to determine immune status of vaccinated individuals. This article reviews publications describing the development and implementation of bead-based multiplexed serological assays for detection of immune responses to polyvalent polysaccharide and conjugate vaccines against Streptococcus pneumoniae. Many of these serological assays on the bead array platform have been further optimized and expanded over time and are still widely used today. [ABSTRACT FROM AUTHOR]

Background: Although the role of viral agents, such as human papillomavirus (e.g. HPV16, HPV18) in colorectal cancer (CRC) has been previously investigated, results remain inconclusive. Methods: To further evaluate the involvement of oncogenic HPV types in CRC, 40 frozen neoplastic and 40 adjacent colonic tissues collected from Italian patients were analyzed by Luminex-based assays that detect a broad spectrum of HPV types, i.e. Alpha (n = 21), Beta (n = 46) and Gamma HPVs (n = 52). In addition, 125 frozen CRC samples and 70 surrounding mucosal tissues were collected from Czech patients and analyzed by broad spectrum PCR protocols: (i) FAP59/64, (ii) FAPM1 and (iii) CUT combined with Next Generation Sequencing (NGS). Results: Using Luminex-basedassays, DNA from HPV16 was detected in 5% (2/40) CRC tissues from Italian patients. One HPV16 DNA-positive CRC case was subsequently confirmed positive for E6*I mRNA. Cutaneous beta HPV types were detected in 10% (4/40) adjacent tissues only, namely HPV111 (n = 3) and HPV120 (n = 1), while gamma HPV168 (n = 1) and HPV199 (n = 1) types were detected in adjacent and in tumor tissues, respectively. The NGS analysis of the CRC Czech samples identified HPV sequences from mucosal alpha-3 (HPV89), alpha-7 (HPV18, 39, 68 and 70) and alpha-10 species (HPV11), as well as cutaneous beta-1 (HPV20, 24, 93, 98, 105,124) beta-2 (HPV23), beta-3 (HPV49) and gamma-1 species (HPV205). Conclusions: Our findings indicate that HPV types belonging to the mucosal alpha, and the 'cutaneous' beta and gamma genera can be detected in the colonic mucosal samples with a low prevalence rate and a low number of HPV reads by Luminex and NGS, respectively. However, additional studies are required to corroborate these findings. [ABSTRACT FROM AUTHOR]

Luminex single antigen bead (SAB) kits from One Lambda (OL) and Lifecodes (LC) are widely used for HLA antibody detection but have substantial differences in design and assay protocol resulting in different mean fluorescence intensity (MFI) values. Here, we present a non‐linear modeling approach to accurately convert MFI values between two vendors and to establish user‐independent MFI cutoffs when analyzing big datasets. HLA antibody data from a total of 47 EDTA‐treated sera tested using both OL and LC SAB kits were analyzed. MFI comparisons were made for the common 84 HLA class I and 63 class II beads. In the exploration set (n = 24), a non‐linear hyperbola model on raw MFI corrected by locus‐specific highest self MFI subtraction yielded the highest correlation (class I r2: 0.946, class II r2: 0.898). Performance of the model was verified in an independent validation set (n = 12) (class I r2: 0.952, class II r2: 0.911). Furthermore, in an independent cohort of post‐transplant serum samples (n = 11) using the vendor‐specific MFI cutoffs dictated by the current model, we found 94% accuracy in bead‐specific reactivity assignments by the two vendors. We recommend using the non‐linear hyperbola modeling approach with self HLA correction and locus‐specific analyzes to harmonize MFI values between two vendors in particular research datasets. As there are considerable variations between the two assays, using MFI conversion for individual patient samples is not recommended. [ABSTRACT FROM AUTHOR]

• Multiplexing technologies, such as bead-based suspension microarrays, have advanced the ability to perform high-throughput nucleic acid measurements. • These analysis systems require the nucleic acids to be extracted and purified from the specimens and sample types of interest. • The method used must be capable of providing purified nucleic acids sufficient for use with the up-front assay chemistry (e.g., PCR, primer extension, branched DNA, etc.). • For bead-based microarrays, the nucleic acid sample must be sufficiently pure to undergo hybridization, often with a heat denaturation step, without leading to agglutination of the beads. Multiplexing in biological assays allows the simultaneous detection of multiple analytes in a single reaction, which reduces time, labor, and cost as compared to single reaction-based detection methods. Microsphere- or bead-based suspension array technologies, such as the Luminex® xMAP® system, offer high-throughput detection of nucleic acids through a variety of different assay chemistries. Common with most nucleic acid chemistries, for bead-based or other microarray technologies, is the need for efficient extraction and purification of the nucleic acids from the specimen of interest. Often, the optimal method will be dictated by the requirements of the up-front enzymatic chemistry, such as PCR, primer extension, branched DNA (bDNA), etc. For bead-based microarray platforms, the user must also be cognizant of proteins and other contaminants present in reactions that require heat denaturation, as that can lead to bead aggregation or agglutination, preventing the reading of assay results. This review describes and highlights some of the nucleic acid extraction and purification methods that have been used successfully for bead-based nucleic acid analysis, for both prokaryotic and eucaryotic nucleic acids, from a variety of sample types. [ABSTRACT FROM AUTHOR]

BackgroundMultiplex analyses allow for detection of dozens of cytokines/chemokines in small sample volumes. Although several commercially available assay kits are available, there are no comparative data in plasma measurements among pediatric or epilepsy cohorts.New methodCohort study of 38 children with epilepsy. We evaluated plasma levels of cytokines/chemokines using three different assays: Luminex® xMAP high-sensitivity (HS) and standard-sensitivity (SS) assays, and Meso-Scale Discovery (MSD). We calculated recovery rates of each analyte, correlation coefficients between assays, and level of agreement between measurements. We repeated analyses in a subset of samples after a single freeze-thaw cycle.ResultsAmong ten analytes common to all assays, HS had high recovery (

Shao-Long Chen,1 Hong Xiao,2 Guo-Jun Li,3 Yao-Jie Shen4 1Shulan International Medical College, Zhejiang Shuren University, Hangzhou, 310015, People’s Republic of China; 2Department of Infectious Diseases, Shanghai Public Health Clinical Center, Fudan University, Shanghai, 201508, People’s Republic of China; 3Department of Hepatology, The Second Hospital of Yinzhou of Ningbo, Ningbo, 315100, People’s Republic of China; 4Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, 200040, People’s Republic of ChinaCorrespondence: Shao-Long Chen, Shulan International Medical College, Zhejiang Shuren University, Hangzhou, 310015, People’s Republic of China, Email chenshaolong@zjsru.edu.cnPurpose: Chronic hepatitis B virus (CHB) infection is a worldwide health problem. Polyethylene glycol (PEG)ylated interferon (PEG-IFN) is an available therapy for CHB that has antiviral and immunomodulatory effects. However, PEG-IFN therapy is limited by the fact that only a subset of patients show a sustained response, its severe side effects, and high cost. The aim of this study was to explore novel biomarkers for the early prediction of PEG-IFN treatment response and to uncover its underlying mechanism.Patients and Methods: We enrolled 10 paired patients with Hepatitis B e antigen (HBeAg)-positive CHB who received PEG-IFN-α 2a monotherapy. Patient serum samples were collected at 0, 4, 12, 24, and 48 weeks and serum samples were collected from eight healthy people as healthy controls. For confirmation, we enrolled 27 patients with HBeAg-positive CHB receiving PEG-IFN therapy and serum samples at 0 and 12 weeks were obtained. Serum samples were analyzed using Luminex technology.Results: Among 27 assessed cytokines, 10 cytokines were identified to have high expression levels. Among them, six cytokines had significant differences in their levels between the patients with HBeAg-positive CHB and the healthy controls (P < 0.05). Potentially, treatment response could be predicted using the early time points of 4, 12, and 24 weeks. Moreover, after 12 weeks of PEG-IFN treatment, increased levels of pro-inflammatory cytokines and decreased levels of anti-inflammatory cytokines were observed. The fold change of IP-10 between 12 weeks and 0 weeks correlated with the decrease in ALT levels from 0 to 12 weeks (r = 0.2675, P = 0.0024).Conclusion: In patients with CHB, we observed a certain pattern in the levels of cytokines during treatment with PEG-IFN, and the cytokine IP-10 might be a potential biomarker for treatment response.Keywords: chronic hepatitis B, PEGylated interferon, cytokine, chemokine, Luminex

Combination and polyvalent vaccines not only provide protection against several different pathogens at the same time but can also increase vaccine protection against pathogens that have closely related pathogenic strains or serotypes. Multiplexed serological testing is a preferred method for determining the efficacy of combination and polyvalent vaccines, as it reduces the need for conducting multiple individual assays to confirm immune responses and cross-reactivity, uses less sample, and can be faster, more reliable, and more cost-effective. Bead-based suspension array technologies, such as the Luminex® xMAP® Technology, are often used for development of multiplexed serological assays for various vaccine trials and for routine testing in clinical laboratories to determine immune status of vaccinated individuals. This article reviews publications describing the development and implementation of bead-based multiplexed serological assays for detection of immune responses to polyvalent polysaccharide and conjugate vaccines against Streptococcus pneumoniae. Many of these serological assays on the bead array platform have been further optimized and expanded over time and are still widely used today.

Proteomics in respiratory allergic diseases has such a battery of techniques and programs that one would almost think there is nothing impossible to find, invent or mold. All the resources that we document here are involved in solving problems in allergic diseases, both diagnostic and prognostic treatment, and immunotherapy development. The main perspectives, according to this version, are in three strands and/or a lockout immunological system: (1) Blocking the diapedesis of the cells involved, (2) Modifications and blocking of paratopes and epitopes being understood by modifications to antibodies, antagonisms, or blocking them, and (3) Blocking FcεRI high-affinity receptors to prevent specific IgEs from sticking to mast cells and basophils. These tools and targets in the allergic landscape are, in our view, the prospects in the field. However, there are still many allergens to identify, including some homologies between allergens and cross-reactions, through the identification of structures and epitopes. The current vision of using proteomics for this purpose remains a constant; this is also true for the basis of diagnostic and controlled systems for immunotherapy. Ours is an open proposal to use this vision for treatment. [ABSTRACT FROM AUTHOR]

Introduction: Bovine mastitis is caused by over 150 different microorganisms. Specific identification and quantification of multiple bacteria in a single milk sample becomes essential for rapid intervention. Methods: In the present study a Luminex beads based multiplex assay emphasizing on the precise identification of six major bacterial pathogens of mastitis was developed. Assay was developed in two triplex sets, triplex 1 comprised of Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis while triplex 2 consisted of Staphylococcus aureus, E. coli and Klebsiella pneumoniae. Results: The analytical sensitivity was 10 6 copies per reaction mixture for all the six bacteria. A 100% analytical specificity was observed for simultaneous detection of these bacteria. Clinical milk samples from 100 bovine quarters were tested for validation. Discussion: The analytical sensitivity was similar to the findings reported earlier in real time PCR multiplex assay targeting the DNA of the 11 most common bacterial species or groups in mastitis. The analytical specificity of the optimized assay was 100% similar to reported earlier for simultaneous detection of Mycoplasma spp. and for seven entric viruses of humans.The developed assay indicates a concept proof of a rapid, cost effective high throughput diagnostic tool for identification of major bacteria causing mastitis. [ABSTRACT FROM AUTHOR]

Bat-borne zoonotic pathogens belonging to the family Paramxyoviridae, including Nipah and Hendra viruses, and the family Filoviridae, including Ebola and Marburg viruses, can cause severe disease and high mortality rates on spillover into human populations. Surveillance efforts for henipaviruses and filoviruses have been largely restricted to the Old World; however, recent studies suggest a potentially broader distribution for henipaviruses and filoviruses than previously recognized. In the current study, we screened for henipaviruses and filoviruses in New World bats collected across 4 locations in Trinidad near the coast of Venezuela. Bat tissue samples were screened using previously established reverse-transcription polymerase chain reaction assays. Serum were screened using a multiplex immunoassay to detect antibodies reactive with the envelope glycoprotein of viruses in the genus Henipavirus and the family Filoviridae. Serum samples were also screened by means of enzyme-linked immunosorbent assay for antibodies reactive with Nipah G and F glycoproteins. Of 84 serum samples, 28 were reactive with ≥1 henipavirus glycoprotein by ≥1 serological method, and 6 serum samples were reactive against ≥1 filovirus glycoproteins. These data provide evidence of potential circulation of viruses related to the henipaviruses and filoviruses in New World bats.

Zika virus (ZIKV) is an emerging, mosquito-borne flavivirus responsible for recent epidemics across the Americas, and it is closely related to dengue virus (DENV). Here, we study samples from 46 DENV-naive and 43 DENV-immune patients with RT-PCR-confirmed ZIKV infection at early-acute, late-acute, and convalescent time points from our pediatric cohort study in Nicaragua. We analyze the samples via RNA sequencing (RNA-seq), CyTOF, and multiplex cytokine/chemokine Luminex to generate a comprehensive, innate immune profile during ZIKV infection. Immunophenotyping and analysis of cytokines/chemokines reveal that CD14+ monocytes play a key role during ZIKV infection. Further, we identify CD169 (Siglec-1) on CD14+ monocytes as a potential biomarker of acute ZIKV infection. Strikingly distinct transcriptomic and immunophenotypic signatures are observed at all three time points. Interestingly, pre-existing dengue immunity has minimal impact on the innate immune response to Zika. Finally, this comprehensive immune profiling and network analysis of ZIKV infection in children serves as a valuable resource.

Chemokines are involved in the humoral regulation of body homeostasis. Changes in the blood level of chemokines were found in cancer, atherosclerosis, diabetes, and other systemic diseases. It is essential to distinguish the effects of co-morbid pathologies and cancer on the level of chemokines in the blood. We aimed to analyze, by multiplex cytometry, the levels of chemokines in the blood of healthy young volunteers as well as of intact mice and mice with CT26 colon and Pan02 pancreatic tumors. Two types of chemokines were identified both in human and murine plasmas: homeostatic ones, which were found in high concentrations (>100 pg/mL), and inducible ones, which can be undetectable or determined at very low levels (0–100 pg/mL). There was a high variability in the chemokine levels, both in healthy humans and mice. To analyze chemokine levels during tumor growth, C57BL/6 and BALB/c were inoculated with Pan02 or CT26 tumor cells, accordingly. The tumors significantly differed in the growth and the mortality of mice. However, the blood chemokine levels did not change in tumor-bearing mice until the very late stages. Taken collectively, blood chemokine level is highly variable and reflects in situ homeostasis. Care should be taken when considering chemokines as prognostic parameters or therapeutic targets in cancer.

Background: Hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) has significant morbidity and mortality and is associated with the induction of cytokines/chemokines, which might contribute to the pathogenesis of liver injury. This study aimed to explore the cytokine/chemokine profiles of patients with HBV-ACLF and develop a composite clinical prognostic model. Methods: We prospectively collected blood samples and the clinical data of 107 patients with HBV-ACLF admitted to the Beijing Ditan Hospital. The concentrations of 40-plex cytokines/chemokines were measured in 86 survivors and 21 non-survivors using the Luminex assay. Discrimination between the cytokine/chemokine profiles in different prognosis groups was analyzed using the multivariate statistical techniques of principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). An immune-clinical prognostic model was obtained using multivariate logistic regression analysis. Results: The PCA and PLS-DA indicated that cytokine/chemokine profiling could clearly distinguish patients with different prognoses. A total of 14 cytokines, namely, IL-1b, IL-6, IL-8, IL-10, TNF-a, IFN-g, CXCL1, CXCL2, CXCL9, CXCL13, CX3CL1, GM-SCF, CCL21, and CCL23, were significantly correlated with disease prognosis. Multivariate analysis identified CXCL2, IL-8, total bilirubin, and age as independent risk factors that constituted the immune-clinical prognostic model, which showed the strongest predictive value of 0.938 compared with those of the Chronic Liver Failure Consortium (CLIF-C) ACLF (0.785), Model for End-Stage Liver Disease (MELD) (0.669), and MELD-Na (0.723) scores (p < 0.05 for all). Conclusion: The serum cytokine/chemokine profiles correlated with the 90-day prognosis of patients with HBV-ACLF. The proposed composite immune-clinical prognostic model resulted in more accurate prognostic estimates than those of the CLIF-C ACLF, MELD, and MELD-Na scores. [ABSTRACT FROM AUTHOR]

Introduction/Aim: The Luminex assay, where beads are coated with a single HLA antigen, has been shown to detect HLA antibodies with more sensitivity and specificity as compared to microlymphocytotoxicity (CDC) assay and flow cross match (FCXM). We report the impact of low Mean Flourescence intensity (MFI) pre‐transplant DSA by Luminex with negative CDC and FCXM on acute rejection, graft function, and survival. Methods: In this retrospective study between January 2015 to December 2021, 45 recipients had pre‐transplant anti‐HLA donor‐specific antibodies (DSAs) detected by Luminex. Two control groups of 45 patients each matched for age and gender, first with non‐DSA HLA antibodies and second with no antibodies by Luminex were selected to compare outcomes with DSA group. Results: In the DSA group of 45, 22 (48.8%) had class I (MFI mean 4043 ± 1909, range: 1096–7111), 20 (44.4%) class II (MFI mean 3601 ± 2310, range: 1031–9259), and 3 (6.6%) both class I (MFI mean 4746 ± 1922) and class II (MFI mean 3940 ± 2312) antibodies. Acute rejection episodes were reported in 15.6%, DSA group, 17.8% in non‐DSA, and 24.4% in no antibody group (p =.538). Death censored graft survival at 1 and 5 years was 98% and 93% in DSA group, 100% and 95% in non‐DSA and 93% and 85% in the no antibody group (p =.254). Conclusions: Patients with low MFI DSA pre‐transplant, with a negative CDC and FCXM under ATG induction, have similar graft outcomes at 1 and 5 years when compared to non‐DSA and no antibody groups. [ABSTRACT FROM AUTHOR]

Introduction: The role of tumour secretory cytokines and peripheral circulatory cytokines in tumour progression has received increasing attention; however, the role of tumour-related inflammatory cytokines in colorectal cancer (CRC) remains unclear. In this study, the concentrations of various cytokines in the peripheral blood of healthy controls and patients with CRC at different stages were compared. Methods: Peripheral blood samples from 4 healthy participants and 22 colorectal cancer patients were examined. Luminex beads were used to evaluate concentration levels of 40 inflammatory cytokines in peripheral blood samples. Results: In peripheral blood, compared with healthy controls and early stage (I + II) CRC patients, advanced CRC (III + IV) patients had increased concentrations of mononuclear/macrophage chemotactic-related proteins (CCL7, CCL8, CCL15, CCL2, and MIF), M2 polarization-related factors (IL-1β, IL-4), neutrophil chemotactic and N2 polarization-related cytokines (CXCL2, CXCL5, CXCL6, IL-8), dendritic cells (DCs) chemotactic-related proteins (CCL19, CCL20, and CCL21), Natural killer (NK) cell related cytokines (CXCL9, CXCL10), Th2 cellrelated cytokines (CCL1, CCL11, CCL26), CXCL12, IL-2, CCL25, and CCL27, and decreased IFN-γ and CX3CL1 concentrations. The differential upregulation of cytokines in peripheral blood was mainly concentrated in CRC patients with distant metastasis and was related to the size of the primary tumour; however, there was no significant correlation between cytokine levels in peripheral blood and the propensity and mechanism of lymph node metastasis. Discussion: Different types of immune cells may share the same chemokine receptors and can co-localise in response to the same chemokines and exert synergistic pro-tumour or anti-tumour functions in the tumour microenvironment. Chemokines and cytokines affect tumour metastasis and prognosis and may be potential targets for treatment. [ABSTRACT FROM AUTHOR]

IntroductionBovine mastitis is caused by over 150 different microorganisms. Specific identification and quantification of multiple bacteria in a single milk sample becomes essential for rapid intervention.MethodsIn the present study a Luminex beads based multiplex assay emphasizing on the precise identification of six major bacterial pathogens of mastitis was developed. Assay was developed in two triplex sets, triplex 1 comprised of Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis while triplex 2 consisted of Staphylococcus aureus, E. coli and Klebsiella pneumoniae.ResultsThe analytical sensitivity was 10 6 copies per reaction mixture for all the six bacteria. A 100% analytical specificity was observed for simultaneous detection of these bacteria. Clinical milk samples from 100 bovine quarters were tested for validation.DiscussionThe analytical sensitivity was similar to the findings reported earlier in real time PCR multiplex assay targeting the DNA of the 11 most common bacterial species or groups in mastitis. The analytical specificity of the optimized assay was 100% similar to reported earlier for simultaneous detection of Mycoplasma spp. and for seven entric viruses of humans.The developed assay indicates a concept proof of a rapid, cost effective high throughput diagnostic tool for identification of major bacteria causing mastitis.

Background and Objectives: The aim of this study was to evaluate the levels of selected cytokines and their possible influence on the development of cardiovascular and pulmonary complications in patients hospitalized at the Silesian Centre for Heart Disease in Zabrze after having undergone COVID-19. Materials and methods: The study included 76 randomly selected patients from the SILCOVID-19 database. The median time from symptom onset to the study visit was 102 (86–118) days. The median age of the study group was 53 (44–60) years. Assays of a panel of 30 cytokines were carried out in the serum of patients on a Luminex100 platform using the Milliplex MAP kit from Merck KGaA Germany. Results: There were no statistically significant differences in most of the cytokines analyzed between patients with confirmed or excluded lung lesions or cardiac abnormalities. Additionally, no statistically significant differences in cytokine concentrations according to gender, age, comorbidity of diabetes, renal disease, hypertension, increased risk of thrombotic disease, or psychological disorders were demonstrated. There were high concentrations of cytokines such as platelet-derived growth actor-AA (PDGF-AA), monocyte chemoattractant protein-1 (MCP-1), monokine-induced gamma interferon (MIG), and vascular endothelial growth factor-A (VEGF-A). Conclusions: No direct impact of the dependencies between a panel of cytokines and the incidence of cardiovascular and pulmonary complications in patients hospitalized at the Silesian Centre for Heart Disease in Zabrze after having undergone COVID-19 was demonstrated. The demonstration of high levels of certain cytokines (PDGF-AA, VEGF, MIG, and IP10) that are of significance in the development of many lung diseases, as well as cytokines (MCP-1) that influence the aetiopathogenesis of cardiovascular diseases seems to be highly concerning in COVID-19 survivors. This group of patients should receive further monitoring of these cytokine levels and diagnostic imaging in order to detect more severe abnormalities as early as possible and administer appropriate therapy.

Powassan virus is an emerging tick-borne pathogen capable of causing severe neuroinvasive disease. As the incidence of human Powassan virus grows both in magnitude and geographical range, the development of sensitive detection methods for diagnostics and surveillance is critical. In this study, a Taqman-based triplex real-time PCR assay was developed for the simultaneous and quantitative detection of Powassan virus and Powassan virus lineage II (deer tick virus) in Ixodes scapularis ticks. An exon–exon junction internal control was built-in to allow for accurate detection of RNA quality and the failure of RNA extraction. The newly developed assay was also applied to survey deer tick virus in tick populations at 13 sites on Cape Cod and Martha’s Vineyard Island in Massachusetts. The assay’s performance was compared with the Luminex xMAP MultiFLEX Vector-borne Panel 2. The results suggested that the real-time PCR method was more sensitive. Powassan virus infection rates among ticks collected from these highly endemic tick areas ranged from 0.0 to 10.4%, highlighting the fine-scale geographic variations in deer tick virus presence in this region. Looking forward, our PCR assay could be adopted in other Powassan virus surveillance systems.

Abstract Background Since 1995, a surveillance program for Salmonella has been applied in the Danish pig industry in order to reduce cases of human salmonellosis. The objective of this study was to develop a bead-based Multiplexed Fluorometric ImmunoAssay (MFIA) as an improved serological surveillance method compared to the Salmonella mix ELISA, which has been the national reference immunoassay in the Danish Salmonella surveillance program for about 20 years. Results An MFIA for detection of antibodies to Salmonella serogroup B and C1 was developed and optimized with regard to coupling of beads with Salmonella lipopolysaccharide antigens and establishing suitable assay conditions. The Salmonella MFIA was validated by testing sera from experimentally infected pigs as well as field sera from non-infected and infected pig herds, and by comparing to results from the Salmonella mix ELISA, which was run in parallel. Sensitivity and specificity was evaluated using receiver operating curve analysis showing an area under curve for the serogroup B and C1 MFIA of 0.984 and 0.998, respectively. The Salmonella MFIA was shown to detect more antibody-positive samples in seropositive herds compared to the Salmonella mix ELISA, and Bayesian statistics confirmed that the MFIA had a considerably higher sensitivity (94.5%) compared to the mix ELISA (75.1%). The assay specificity was slightly lower for the Salmonella MFIA (96.8%) compared to Salmonella mix ELISA (99.5%). Coupled beads were stable for at least 1 year at 4˚C, and MFIA reproducibility and repeatability of the Salmonella MFIA were acceptable. Results from proficiency tests also indicated that the Salmonella MFIA was more sensitive than the Salmonella mix ELISA and that they had similar specificity. Conclusions A bead-based MFIA for simultaneous detection of porcine serum antibodies to Salmonella enterica serogroup B and C1 was developed and implemented in the Danish porcine serological Salmonella surveillance program in 2018. The Salmonella MFIA can distinguish, as opposed to the Salmonella mix ELISA, between antibodies to serogroup B and C1 and the MFIA shows considerably better sensitivity.

BackgroundHepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) has significant morbidity and mortality and is associated with the induction of cytokines/chemokines, which might contribute to the pathogenesis of liver injury. This study aimed to explore the cytokine/chemokine profiles of patients with HBV-ACLF and develop a composite clinical prognostic model.MethodsWe prospectively collected blood samples and the clinical data of 107 patients with HBV-ACLF admitted to the Beijing Ditan Hospital. The concentrations of 40-plex cytokines/chemokines were measured in 86 survivors and 21 non-survivors using the Luminex assay. Discrimination between the cytokine/chemokine profiles in different prognosis groups was analyzed using the multivariate statistical techniques of principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). An immune-clinical prognostic model was obtained using multivariate logistic regression analysis.ResultsThe PCA and PLS-DA indicated that cytokine/chemokine profiling could clearly distinguish patients with different prognoses. A total of 14 cytokines, namely, IL-1β, IL-6, IL-8, IL-10, TNF-α, IFN-γ, CXCL1, CXCL2, CXCL9, CXCL13, CX3CL1, GM-SCF, CCL21, and CCL23, were significantly correlated with disease prognosis. Multivariate analysis identified CXCL2, IL-8, total bilirubin, and age as independent risk factors that constituted the immune-clinical prognostic model, which showed the strongest predictive value of 0.938 compared with those of the Chronic Liver Failure Consortium (CLIF-C) ACLF (0.785), Model for End-Stage Liver Disease (MELD) (0.669), and MELD-Na (0.723) scores (p

Objectives: To examine the chemokine profile in the cerebrospinal fluid (CSF) of patients with glial fibrillary acidic protein astrocytopathy (GFAP‐A), central nervous system immune‐related adverse event (CNS‐irAE), neurosarcoidosis (NS), neuromyelitis optica spectrum disorders (NMOSD), multiple sclerosis (MS), and human T‐cell leukemia virus‐1 (HTLV‐1)‐associated myelopathy (HAM). Methods: The study included 38 patients presenting to St. Marianna University Hospital between May 2013 and November 2021 with GFAP‐A, CNS‐irAE, NMOSD, MS, NS, HAM and noninflammatory neurological diseases (NIND). We recorded the age, sex, duration of disease, brain/spinal lesions on magnetic resonance imaging (MRI), blood data, and measured chemokines (CXCL9, −10, −13, CCL3, −4, −17, −20, −22) in CSF. In patients with GFAP‐A, clinical symptoms, and CSF CXCL10 levels were compared before and after steroid treatment. Results: Patients with GFAP‐A had higher CSF levels of CXCL10, CXCL13, and CCL22 (10736.1 [8786.7–149079.0] pg/ml (p <.05), 378.4 [239.9–412.2] pg/ml (p <.01) and 159.9 [130.5–413.9] pg/ml (p <.01), respectively). The CSF levels of CXCL10 improved from 10736.1 [8786.7–149079.0] pg/ml to 1879.0 [783.9–4360.0] pg/ml in patients with GFAP‐A by steroid therapy. Conclusion: CSF CXCL10 levels were particularly high in GFAP‐A, and changes in levels after treatment correlated with clinical improvements, suggesting CXCL10 involvement in GFAP‐A pathogenesis. [ABSTRACT FROM AUTHOR]

Advances over the last decades have made renal transplantation an important therapy for patients with end-stage renal disease, as the incidences of acute rejection and short-term transplant loss have been significantly reduced. However, long-term transplant survival remains a challenge in the renal transplantation community. The main causes of long-term graft loss are acute and chronic rejection, as well as the complications related to immunosuppression therapy. In spite of the breakthroughs achieved in recent years, histology is the gold standard technique to confirm the activation of the immune system against the graft with all the ensuing problems that taking biopsies brings to immunosuppressed patients. For this reason, several assays have been developed to try to monitor the immune function, but they show serious constraints owing to the fact that they require substantial laboratory work, they are not clinically available and they provide controversial results, so the combination of multiple assays is often needed to obtain a reliable diagnosis. Thus, the aim of this review is to perform a retrospective study of the immune system in renal transplantation, with special emphasis on the cutting-edge technological developments for monitoring, classification and early detection of rejection episodes in order to contribute to a better adjustment of immunosuppressive therapies and, hence, to a more personalized medicine that improves the quality of life of patients. [ABSTRACT FROM AUTHOR]

Background and Objectives: Testing for anti-human leukocyte antigen (HLA) antibodies both before and after transplantation is of fundamental significance for the success of lung transplantation. The aim of this study was the evaluation of anti-HLA immunization of patients before and after lung transplant who were subjected to qualification and transplantation. Materials and Methods: Prior to the transplantation, patients were examined for the presence of IgG class anti-HLA antibodies (anti-human leukocyte antigen), the so-called panel-reactive antibodies (PRA), using the flow cytometry method. After the transplantation, the class and specificity of anti-HLA antibodies (also IgG) were determined using Luminex. Results: In the group examined, the PRA results ranged from 0.1% to 66.4%. Low (30%) and average (30–80%) immunization was found in only 9.7% of the group examined. Presence of class I anti-HLA antibodies with MFI (mean fluorescence intensity) greater than 1000 was found in 42.7% of the patients examined, while class II anti-HLA antibodies were found in 38.4%. Immunization levels before and after the transplantation were compared. In 10.87% of patients, DSA antibodies (donor-specific antibodies) with MFI of over 1000 were found. Conclusions: It seems that it is possible to confirm the correlation between pre- and post-transplantation immunization with the use of the two presented methods of determining IgG class anti-HLA antibodies by increasing the size of the group studied and conducting a long-term observation thereof. [ABSTRACT FROM AUTHOR]

Objective To compare three kinds of platelet antibody detection methods used to identify alloantibodies in patients with platelet transfusion refractory(PTR). Methods The 83 samples from PTR patients were analyzed base on three different methods, including solid phase ELISA, Luminex, and capture. The sensitivity, reproducibility, and consistency of different kits were evaluated. Results A total of 71 (62 positive and 9 negative) out of 83 samples showed consistent results by three methods. The consistency between Luminex and solid phase ELISA was 95.2% (Kapp value=0.829, P

Abstract Background A thorough understanding of a patient’s inflammatory response to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is crucial to discerning the associated, underlying immunological processes and to the selection and implementation of treatment strategies. Defining peripheral blood biomarkers relevant to SARS-CoV-2 infection is fundamental to detecting and monitoring this systemic disease. This safety-focused study aims to monitor and characterize the immune response to SARS-CoV-2 infection via analysis of peripheral blood and nasopharyngeal swab samples obtained from patients hospitalized with Coronavirus disease 2019 (COVID-19), in the presence or absence of bamlanivimab treatment. Methods 23 patients hospitalized with COVID-19 were randomized to receive a single dose of the neutralizing monoclonal antibody, bamlanivimab (700 mg, 2800 mg or 7000 mg) or placebo, at study initiation (Clinical Trial; NCT04411628). Serum samples and nasopharyngeal swabs were collected at multiple time points over 1 month. A Proximity Extension Array was used to detect inflammatory profiles from protein biomarkers in the serum of hospitalized COVID-19 patients relative to age/sex-matched healthy controls. RNA sequencing was performed on nasopharyngeal swabs. A Luminex serology assay and Elecsys® Anti-SARS-CoV-2 immunoassay were used to detect endogenous antibody formation and to monitor seroconversion in each cohort over time. A mixed model for repeated measures approach was used to analyze changes in serology and serum proteins over time. Results Levels of IL-6, CXCL10, CXCL11, IFNγ and MCP-3 were > fourfold higher in the serum of patients with COVID-19 versus healthy controls and linked with observations of inflammatory and viral-induced interferon response genes detected in nasopharyngeal swab samples from the same patients. While IgA and IgM titers peaked around 7 days post-dose, IgG titers remained high, even after 28 days. Changes in biomarkers over time were not significantly different between the bamlanivimab and placebo groups. Conclusions Similarities observed between nasopharyngeal gene expression patterns and peripheral blood biomarker profiles reveal a connection between the circulation and processes in the nasopharyngeal cavity, reinforcing the potential utility of systemic blood biomarker profiling for therapeutic monitoring of patient response. Serological antibody responses in patients correlated closely with reductions in the COVID-19 inflammatory protein biomarker signature. Bamlanivimab did not affect the biomarker dynamics in this hospitalized patient population.