A Microsphere Immunoassay for the Quantitative Detection of Antigens in Cell Culture Supernatant.

Quantitative immunoassays, such as the traditional enzyme-linked immunosorbent assay (ELISA), are used to determine concentrations of an antigen in a matrix of unknown antigen concentration. Magnetic immunoassays, such as the Luminex xMAP technology, allow for the simultaneous detection of multiple analytes and offer heightened sensitivity, specificity, low sample volume requirements, and high-throughput capabilities. Here, we describe a quantitative immunoassay using the Luminex MAGPIX® System to determine the antigen concentration from liquid samples with unknown concentrations. In detail, we describe a newly developed assay for determining production yields of Drosophila S2-produced Marburg virus (MARV) glycoprotein in insect-cell-culture-derived supernatant. The potential applications of this assay could extend to the quantification of viral antigens in fluids derived from both in vitro and in vivo models infected with live MARV, thereby providing additional applications for virological research.

Sulfonamide resistance evaluation in five animal species and first report of sul4 in companion animals.

Sulfonamides are one of the oldest groups of antibacterial agents with a broad-spectrum, used as first line treatment in bacterial infections. Their widespread use produced a selective pressure on bacteria, as observed by the high incidence of sulfonamides resistance mainly in Gram negative bacteria isolated from animals. In this research, the presence of sulfonamide resistance genes (sul1, sul2, sul3, and sul4) in phenotypically resistant Escherichia coli isolates has been studied. These genes were amplified in isolates recovered from five animal species, with different interactions to humans: cattle, swine, poultry as livestock, and dogs and cats as companion animals. Isolates were collected according to their phenotypic resistance, and the magnetic bead-based Luminex technology was applied to simultaneously detect sul target genes. The frequency of sul genes was highest in swine, among livestock isolates. The sul1 and sul2 were the most frequently sulfonamide resistance genes detected in all phenotypically resistant isolates. Notably, in companion animals, with a closest interaction with human, sul4 gene was detected. To our knowledge, this is the first report of the presence of sul4 gene in E. coli collected from animals, whereas previously the presence of this gene was reported in environmental, municipal wastewater and human clinical isolates. These results highlighted the importance of continuous antimicrobial resistant genes monitoring in animal species, with a special care to companion animals.

Age-Dependent Signature of Serum Inflammatory Cytokines in Healthy Children and Young Adults.

The study of sensitive and specific biomarkers, such as blood inflammatory cytokines, could provide an answer to the challenges faced in the differential diagnosis of patients with systemic inflammation. Limited data exist on the impact of age on serum levels of inflammatory cytokines. We collected serum samples of 42 healthy children and young adults (1 month to 21 years). Serum levels of interleukin 1 receptor antagonist (IL-1Ra), IL-1ß, IL-6, IL-18, tumor necrosis factor-alpha (TNF-α), CXCL9, and CXCL10 were measured. Data were analyzed for three different age groups (<6, 6-17, and 18-21 years). IL-18, TNF-α, and CXCL9 values varied significantly according to age group. Median values of IL-18 and TNF-α decline with age, whereas CXCL9 and CXCL10 are lowest at 6-17 years. IL-1Ra is stable among age groups. In the majority of cases, IL-1ß and IL-6 are not measurable above the lower limit of quantification. A scoping literature review revealed highly variable data on IL-1Ra, IL-18, TNF-α, and CXCL10. For CXCL9, pediatric reference data are scarce. In conclusion, we report an age-dependent signature of multiple inflammatory cytokines measured in the serum of healthy children and young adults, suggesting the need to use age-specific reference values in future pediatric studies.

Changes In Serum CXCL13 Levels Are Associated With Outcomes of Colorectal Cancer Patients Undergoing First-Line Oxaliplatin-Based Treatment.

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.

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

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

Sources of variability in Luminex bead-based cytokine assays: Evidence from twelve years of multi-site proficiency testing.

Bead array assays, such as those sold by Luminex, BD Biosciences, Sartorius, Abcam and other companies, are a well-established platform for multiplexed quantification of cytokines and other biomarkers in both clinical and discovery research environments. In 2011, the National Institute of Allergy and Infectious Diseases (NIAID)-funded External Quality Assurance Program Oversight Laboratory (EQAPOL) established a proficiency assessment program to monitor participating laboratories performing multiplex cytokine measurements using Luminex bead array technology. During every assessment cycle, each site was sent an assay kit, a protocol, and blinded samples of human sera spiked with recombinant cytokines. Site results were then evaluated for performance relative to peer laboratories. After over a decade of biannual assessments, the cumulative dataset contained over 15,500 bead array observations collected at more than forty laboratories in twelve countries. These data were evaluated alongside post-assessment survey results to empirically test factors that may contribute to variability and accuracy in Luminex bead-based cytokine assays. Bead material, individual technical ability, analyte, analyte concentration, and assay kit vendor were identified as significant contributors to assay performance. In contrast, the bead reader instrument model and the use of automated plate washers were found not to contribute to variability or accuracy, and sample results were found to be highly-consistent between assay kit-manufacturing lots and over time. In addition to these statistical analyses, subjective evaluations identified technical ability, instrument failure, protocol adherence, and data transcription errors as the most common causes of poor performance in the proficiency program. The findings from the EQAPOL multiplex program were then used to develop recommended best practices for bead array monitoring of human cytokines. These included collecting samples to assay as a single batch, centralizing analysis, participating in a quality assurance program, and testing samples using paramagnetic-bead kits from a single manufacturer using a standardized protocol.

Designing a Multiplex PCR-xMAP Assay for the Detection and Differentiation of African Horse Sickness Virus, Serotypes 1-9.

African horse sickness is a severe and often fatal disease affecting all species of equids. The aetiological agent, African horse sickness virus (AHSV), can be differentiated into nine serotypes. The identification of AHSV serotypes is vital for disease management, as this can influence vaccine selection and help trace disease incursion routes. In this study, we report the development and optimisation of a novel, molecular-based assay that utilises multiplex PCR and microsphere-based technology to expedite detection and differentiation of multiple AHSV serotypes in one assay. We demonstrated the ability of this assay to identify all nine AHSV serotypes, with detection limits ranging from 1 to 277 genome copies/µL depending on the AHSV serotype. An evaluation of diagnostic sensitivity and specificity revealed a sensitivity of 88% and specificity of 100%. This method can serotype up to 42 samples per run and can be completed in approximately 4-6 h. It provides a powerful tool to enhance the rapidity and efficiency of AHSV serotype detection, thereby facilitating the generation of epidemiological data that can help understand and control the incidence of AHSV worldwide.

Development of a Bead-Based Multiplex Fluorescent Immunoassay to Detect Antibodies against Maedi-Visna Virus in Sheep.

The Maedi-visna virus (MVV) causes a persistent infection in small ruminants, and its high genetic heterogeneity affects the performance of diagnostic tests when used in different populations. Therefore, the aim of this study was to develop a bead-based multiplex immunoassay tailored to detect antibodies against a Norwegian MVV strain. We used tissue samples from 14 PCR-positive sheep from a recent MVV outbreak in Norway to sequence the viral strain and produced recombinant antigens based on sequences from one animal. The assay included commercial TM-A and recombinant Norwegian p25, p16-25 and SU5 antigens. Cut-off values for each antigen were determined using receiver operating characteristic curves on 40 ELISA-negative and 67 ELISA-positive samples from the outbreak. The intraplate and interplate repeatability were investigated by testing a quadruplicate of five samples over three days, while the analytical sensitivity (aSe) and specificity (aSp) were measured in comparison to a commercial ELISA. The repeatability showed a coefficient of variation below 15% for most positive samples. The aSe was equal or higher for the multiplex assay than the ELISA, and the aSp of each antigen was 91.7, 93.3, 95.0 and 93.3% for p25, p16-25, SU5 and TM-A, respectively. The assay shows promising results; however, further evaluations of diagnostic characteristics are necessary before implementation in the Norwegian surveillance programme.

Neuroinflammatory markers at school age in preterm born children with neurodevelopmental impairments.

Background: Immune system activation in the neonatal period is associated with white matter injury in preterm infants. In animal studies, neonatal priming of the immune system leads to chronic activation of i.e. microglia cells and altered neuroinflammatory responses potentially years after preterm birth. This may contribute further to brain injury and neurodevelopmental impairment. It is unknown to what extend this also occurs in human. Aim: To identify neuro-inflammatory markers at school age that relate to motor, cognitive and behavioral impairments in preterm born children in a pilot case-control study. Methods: We included n = 20 preterm born children (GA < 28 weeks) in this study, of which n = 10 with motor, cognitive and behavorial impairments and n = 10 preterm born controls next to n = 30 healthy adult controls. In the preterm children, at 8-9 years, 39 inflammatory markers were assessed by Luminex assay in blood serum samples. Firstly, the preterm concentrations of these markers were compared to n = 30 adult controls. Then a univariate analysis was performed to determine differences in values between preterm children with and without impairment at school age. Finally, a principal component analysis and hierarchical clustering was performed to identify protein profiles in preterm born children that relate to impairment at school age. Results: Inflammatory proteins in preterm children at school age differed from values of adult controls. Within the group of preterm children, we found significantly higher levels of GM-CSF in preterms with impairment (p < 0.01) and a trend towards significance for Gal1 and TRAIL (p = 0.06 and p = 0.06 respectively) when compared to preterms without impairment. In addition, differences in clustering of proteins between preterm children was observed, however this variance was not explained by presence of neurodevelopmental impairments. Conclusion: The inflammatory profile at school age in preterm children is different from that of adult controls. The immune modulating cytokines GM-CSF, Gal1 and TRAIL were higher in preterm children with impairment than control preterm children, suggesting that immune responses are altered in these children. No specific cluster of inflammatory markers could be identified. Results indicate that even at school age, neuroinflammatory pathways are activated in preterm born children with neurodevelopmental impairments.

Development and validation of a 6-plex Luminex-based assay for measuring human serum antibodies to group B streptococcus capsular polysaccharides.

Six serotypes (Ia, Ib, II, III, IV, and V) cause nearly all group B streptococcal (GBS) disease globally. Capsular polysaccharide (CPS) conjugate vaccines aim to prevent GBS disease, however, licensure of a vaccine would depend on a standardized serological assay for measuring anti-CPS IgG responses. A multiplex direct Luminex-based immunoassay (dLIA) has been developed to simultaneously measure the concentration of serum IgG specific for the six prevalent GBS CPS serotypes. Assay validation was performed using serum samples obtained from human subjects vaccinated with an investigational 6-valent GBS CPS conjugate vaccine. Results for the assay are expressed as IgG concentrations (µg/mL) using a human serum reference standard composed of pooled sera from vaccinated subjects. The lower limits of quantitation (LLOQ) for all serotypes covered in the 6-plex GBS IgG dLIA fell within the range of 0.002-0.022 µg/mL IgG. Taken together, the 6-plex GBS IgG dLIA platform is specific for the six GBS serotypes included in Pfizer's investigational vaccine, has a wide dilution adjusted assay range, and is precise (<18.5% relative standard deviation) for all serotypes, and, therefore, is suitable for quantitatively measuring vaccine-induced or naturally acquired serotype-specific anti-CPS IgG responses against GBS.

Comprehensive domain-specific analysis and immunoglobulin G profiling of anti-factor VIII antibodies using a bead-based multiplex immunoassay.

BACKGROUND: Antibodies against factor (F)VIII are a major complication in the treatment of patients with severe hemophilia A. The Nijmegen-Bethesda assay (NBA) is the gold standard for detection of neutralizing antibodies (inhibitors), whereas both inhibitors and nonneutralizing antibodies can be detected by immunoassays such as enzyme-linked immunosorbent assay (ELISA) and multiplex bead-based assays. OBJECTIVES: Evaluation of an in-house Luminex bead-based assay (LumiTope) compared with a commercially available ELISA and NBA. METHODS: The LumiTope method comprised full-length and B-domain-deleted FVIII as well as 9 purified FVIII single or multidomains. The respective proteins were coupled to magnetic beads to detect domain-specific immunoglobulin (IgG; IgG1-4) anti-FVIII antibodies in a large cohort of patients with hemophilia A with and without inhibitors. RESULTS: Overall, LumiTope assay had a high sensitivity (94.9%) and specificity (91.2%), particularly in patients with low-titer inhibitors compared with ELISA (sensitivity of 72.2% vs 27.7%). IgG4 was the most abundant IgG subclass in NBA-positive patients. NBA-positive and -negative patients showed different domain profiles. Patients with genetic variants in the heavy chain predominantly exhibited antibodies specific to this chain, while those with a light-chain variant showed a more diverse distribution of antibody specificities. Patients with an intron 22 inversion resembled those with a light-chain defect, with a majority of antibodies targeting the light chain. CONCLUSION: LumiTope assay provides a sensitive and specific method for not only detection but also domain specification of anti-FVIII-antibodies. Implementation of bead-based assays could improve antibody detection, profiling, and comparability of results and complement NBA.

A novel immunocomplex capture fluorescence assay (ICFA) using fluorescent beads and transfected cells for specific identification of human neutrophil antigen (HNA)-1a and -1b antibodies.

BACKGROUND: To identify specific human neutrophil antigen (HNA) antibodies, assays using neutrophils such as monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA) are recommended. However, these assays are limited by labor-intensive neutrophil preparation and varying antigen expression levels. METHODS: We evaluated a newly developed immunocomplex capture fluorescence assay (ICFA) for identifying HNA-1 antibodies and compared it to MAIGA and LABScreen Multi (LABM), which utilizes recombinant HNA-coated Luminex beads. For ICFA, HNA-1a or HNA-1b transfected cells replaced neutrophils. Cells incubated with serum were lysed, and immune complexes were captured using five CD16 monoclonal antibody-conjugated Luminex beads. Nine antisera with known specificity and 26 samples suspected of containing HNA antibodies were analyzed by ICFA and MAIGA using neutrophils or transfected cells (ICFA-N or ICFA-T, and MAIGA-N or MAIGA-T, respectively). RESULTS: ICFA-T and MAIGA-N accurately determined the specificity of all antibodies in the nine antiserum samples. The ICFA-T detection limit was 2048-fold for anti-HNA-1a and 256-fold for anti-HNA-1b; the limits of MAIGA-T, MAIGA-N, and LABM were 32-, 4 ~ 64-, and 128-fold for anti-HNA-1a and 64-, 16 ~ 64-, and 32-fold for anti-HNA-1b, respectively. Twelve and 7 of the remaining 26 samples tested negative and positive, respectively, in both ICFA-T and MAIGA-N. Antibody specificity against HNA-1a or HNA-1b determined using ICFA-T agreed with that determined using MAIGA-N and LABM. Another seven samples tested positive in ICFA-T but negative in MAIGA-N. CONCLUSION: The novel ICFA is highly sensitive and exhibits specificity similar to MAIGA and LABM for detecting HNA-1 antibodies.

Development of a multiplexed Luminex assay for simultaneous detection of enteric viruses in cattle.

Viral and bacterial gastroenteritis and diarrhea have long been a problem in livestock with devastating effects on animal health and production causing a heavy financial burden on producers. Therefore, the bead-based multiplex detection assay was created for simultaneous detection of three livestock viral diarrheic agents viz. bovine rotavirus (BRV), bovine coronavirus (BCoV) and bluetongue virus (BTV). The primers and probes for triplex MAGPIX assay for simultaneous detection of three enteric viruses were designed and the assay was optimized for hybridization temperature, primer-probe and bead concentrations. The newly developed MAGPIX assay was used to determine the prevalence of these diarrhea-associated viruses by testing 200 fecal samples collected from Haryana state of India during 2018-2019. The limit of detection of the developed triplex assay was 1 × 105, 1 × 104, and 1 × 105 RNA copies for BRV, BCoV, and BTV, respectively, being lower than the reverse transcription-quantitative polymerase chain reaction (RT-qPCR). However, it was higher than the conventional RT-PCR, showing it to be more sensitive. The newly developed MAGPIX assay was a rapid, cost-effective and high throughput diagnostic tool for identification of three major entero-pathogenic diarrhea associated viruses, either alone or in tandem, with the aim to prevent and control viral diarrhea in animals.

Isolation of Burkholderia pseudomallei from a goat in New Caledonia: implications for animal and human health monitoring and serological tool comparison.

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.

Evaluation of the Levels of Selected Cytokines and Their Possible Influence on the Development of Cardiovascular and Pulmonary Complications in Patients after COVID-19.

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.

Active Surveillance of Powassan Virus in Massachusetts Ixodes scapularis Ticks, Comparing Detection Using a New Triplex Real-Time PCR Assay with a Luminex Vector-Borne Panel.

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.

Diversity in the HLA-I Recognition of HLA-F Monoclonal Antibodies: HLA-F or HLA-Ib Monospecific, HLA-E or HLA-G Bispecific Antibodies with or without HLA-Ia Reactivity.

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.

Discovery of a novel cytokine signature for the diagnosis of autism spectrum disorder in young Arab children in Qatar.

Background: Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental disorder characterized by impaired social interaction and communication and the occurrence of stereotyped and repetitive behaviors. Several studies have reported altered cytokine profiles in ASD and hence may serve as potential diagnostic biomarkers of the disorder. This study aims to identify diagnostic biomarkers for ASD in a well-defined study cohort in Qatar. Methods: We measured the protein levels of 45 cytokines in the plasma samples of age- and gender-matched children (2-4 years) with ASD (n = 100) and controls (n = 60) using a Luminex multiplex assay. We compared the differences in the levels of these cytokines between the two study groups and then fitted the significantly altered cytokines into a logistic regression model to examine their diagnostic potential for ASD. Results: We found elevated levels of IFN-γ, FGF-2, IL-1RA, and IL-13 and reduced levels of eotaxin, HGF, IL-1 alpha, IL-22, IL-9, MCP-1, SCF, SDF-1 alpha, VEGFA, and IP-10 in the plasma of children with ASD compared to controls. Furthermore, we observed that elevated levels of IFN-γ (odds ratio (OR) = 1.823; 95% (confidence interval) CI = 1.206, 2.755; p = 0.004) and FGF-2 (OR = 2.528; 95% CI = 1.457, 4.385; p < 0.001) were significantly associated with increased odds of ASD, whereas reduced levels of eotaxin (OR = 0.350; 95% CI = 0.160, 0.765; p = 0.008) and HGF (OR = 0.220; 95% CI = 0.070, 0.696; p = 0.010) were significantly associated with lower odds of ASD relative to controls. The combination of these four cytokines revealed an area under the curve (ROC-AUC) of 0.829 (95% CI = 0.767, 0.891; p < 0.001), which demonstrates the diagnostic accuracy of the four-cytokine signature. Conclusions: Our results identified a panel of cytokines that could discriminate between children with ASD and controls in Qatar. In addition, our findings support the predominance of a Th1 immune phenotype in ASD children and emphasize the need to validate these results in larger populations.

Cytokine Profiling of Plasma and Atherosclerotic Plaques in Patients Undergoing Carotid Endarterectomy.

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-1ß (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.

Urokinase Plasminogen Activator Receptor: An Important Focal Player in Chronic Subdural Hematoma?

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.