Multiplex Bead-based Immunoassay Research Articles

Assessment of Malaria Rapid Diagnostic Tests and Histidine-Rich Protein 2 Deletions among Asymptomatic Children and Adults in Bagamoyo, Tanzania.

Malaria rapid diagnostic tests (mRDTs) that detect histidine-rich protein 2 (HRP2) remain the mainstay of falciparum malaria diagnosis in Sub-Saharan Africa. Understanding their test characteristics when used for surveillance in asymptomatic populations is important. We explored the rate of false-positive and false-negative mRDT results among asymptomatic persons >5 years old screened for malaria at schools and clinics in the rural Bagamoyo District using 18S ribosomal RNA real-time polymerase chain reaction (qPCR) as the reference test. Among 5,966 persons screened using mRDTs, microscopy, and qPCR tests from 2018 to 2021, 14% (832) were mRDT-positive. Twelve percent of these (98/832) were negative by both microscopy and qPCR, with children overrepresented among those with false-positive mRDTs. Among those who were mRDT-negative, 22% (1,136/5,134) tested qPCR-positive, predominantly because of low-density parasitemia (92% had <100 p/µL by qPCR). Among mRDT-negative samples with >100 p/µL, we looked for evidence of hrp2 or histidine-rich protein 3 (hrp3) deletion using two methods, multiplexed qPCR and multiplex bead-based immunoassay. When sufficient parasite material existed for a reliable deletion assessment, 12/34 (35%) had evidence of hrp2/3 deletion by qPCR (nine hrp2-/3+ and three hrp2-/3-), and 20/52 (38%) had evidence of deletion by immunoassay. Only three isolates showed evidence of hrp2 deletion by both assays. In an area of low to moderate transmission in Tanzania, false-positive mRDTs are relatively common (12% of positive tests), and false-negative mRDTs are even more common (22% of negative tests), but hrp2/3 deletion causing false-negative mRDTs remains rare (<1% of negative tests).

Effects of Different Voided Urine Sample Storage Time, Temperature, and Preservatives on Analysis with Multiplex Bead-Based Oncuria Bladder Cancer Immunoassay.

Background/Objectives: Urinalysis accuracy requires reliable sample stability that is dependent on the chosen collection and storage conditions. The multiplex Oncuria bladder cancer immunoassay currently needs urine samples stored at 4 °C until analysis, which requires more effort, equipment, and workflow than storing samples at room temperature. Thus, successful sample storage at room temperature (20 °C) may reduce laboratory handling time and expenses. This study evaluated whether different voided urine sample collection and storage parameters affected subsequent biomarker analysis with Oncuria. The Oncuria simultaneously quantifies 10 protein analytes in urine to generate a bladder cancer diagnostic signature. Methods: Samples were stored at varied temperatures (20 °C, 4 °C, -20 °C) for up to 1 month. The effects of adding two commercial urine sample stabilizers and antibiotics (trimethoprim) were also assessed. Subsequently, multiple potential biospecimen stabilizers were tested in urine samples and evaluated with Oncuria in hopes of allowing the urine sample to remain at room temperature for extended periods of time. Results: First, it was demonstrated that voided urine samples stored at room temperate without such stabilizers had different levels of the 10 analytes associated with the Oncuria test compared to voided urine samples stored at 4 °C. Next, we evaluated the effects of commercially available biospecimen stabilizers. Despite the addition of these stabilizers, the levels of the 10 analytes were altered when the samples were stored at room temperature for prolonged periods of time. Therefore, we could not identify a suitable biospecimen stabilizer that would not require sample refrigeration. Conclusions: To minimize sample degradation/alteration after collection, voided urine samples should be refrigerated until analyzed with Oncuria as the refrigeration is advantageous for the storage and the transport of these urine samples.

Impact of Long-term Fasting on Breath Volatile Sulphur Compounds, Inflammatory Markers and Saliva Microbiota Composition.

Despite substantial evidence supporting the role of resident bacterial communities in therapeutic fasting outcomes, research has primarily focused on gut microbiota, leaving changes in oral microbiota largely unexplored. The clinical significance of oral health changes during fasting is nonetheless underscored by the documented development of halitosis in fasting individuals. However, no scientific studies have comprehensively examined the interplay between salivary microbiota alterations, inflammatory changes in the gingival crevice, and the production of malodorous volatile compounds. We examined volatile sulphur compounds (VSC) in breath during fasting, cytokine levels in the gingival crevice, and oral microbiota composition of the saliva in a single-arm interventional study involving 36 subjects who fasted for 10 ± 3 days. Participants fasted according to Buchinger fasting guidelines. VSC were evaluated every morning before any food or drink intake using the OralChroma gas chromatography device. Saliva and gingival crevicular fluid (GCF) samples were collected at the clinical site before fasting, at the end of fasting, and at the end of food reintroduction. Follow-up saliva samples were sent to the patients after 1 and 3 months. Saliva samples were processed and analysed by targeted sequencing of 16S rRNA gene amplicons, whereas the expression of 6 inflammatory markers in the GCF were analysed using a multiplex fluorescent bead-based immunoassay. The quantification of volatile compounds in the breath demonstrated a statistically significant increase in dimethylsulfide levels during fasting, which corroborates the occurrence of bad breath as a common side effect of fasting. Salivary microbiota profiling showed a shift in microbial composition, including reduction in the levels of Neisseria, Gemella and Porphyromonas spp., concomitant with an increase in the levels of Megasphaera, Dialister, Prevotella, Veillonella, Bifidobacteria, Leptotrichia, Selenomonas, Alloprevotella, and Atopobium. We further demonstrated a reduction in the levels of the pro-inflammatory cytokine interleukin-8 in the GCF. Dimethylsulfide concentrations in the breath increased during fasting, and this was correlated to changes in the oral microbiota. Future studies are needed to illuminate the possible impact of these changes on oral and general health status.

Anti-Inflammatory Cytokine Profiles in Thrombotic Thrombocytopenic Purpura-Differences Compared to COVID-19.

Thromboinflammation/immunothrombosis plays a role in several diseases including thrombotic thrombocytopenic purpura (TTP) and COVID-19. Unlike the extensive research that has been conducted on COVID-19 cytokine storms, the baseline and acute phase cytokine profiles of TTP are poorly characterized. Moreover, we compared the cytokine profiles of TTP and COVID-19 to identify the disease-specific/general characteristics of thromboinflammation/immunothrombosis. Plasma concentrations of 33 soluble mediators (SMs: cytokines, chemokines, soluble receptors, and growth factors) were measured by multiplex bead-based LEGENDplex™ immunoassay from 32 COVID-19 patients (32 non-vaccinated patients in three severity groups), 32 TTP patients (remission/acute phase pairs of 16 patients), and 15 control samples. Mainly, the levels of innate immunity-related SMs changed in both diseases. In TTP, ten SMs decreased in both remission and acute phases compared to the control, one decreased, and two increased only in the acute phase compared to remission, indicating mostly anti-inflammatory changes. In COVID-19, ten pro-inflammatory SMs increased, whereas one decreased with increasing severity compared to the control. In severe COVID-19, sixteen SMs exceeded acute TTP levels, with only one higher in TTP. PCA identified CXCL10, IL-1RA, and VEGF as the main discriminators among their cytokine profiles. The innate immune response is altered in both diseases. The cytokine profile of TTP suggests a distinct pathomechanism from COVID-19 and supports referring to TTP as thromboinflammatory rather than immunothrombotic, emphasizing thrombosis over inflammation as the driving force of the acute phase.

Association between calcium-channel blockers and gingival enlargement: A case-control study

ObjectivesAs reported by the existing literature, calcium-channel blockers (CCB) can lead to gingival enlargement. The aims of this study were to investigate the factors associated with gingival enlargement in patients on CCB and to assess the saliva and gingival crevicular fluid (GCF) profile of patients on CCB with gingival enlargement. MethodsA total of 131 participants were included. Data were collected from 91 patients taking CCB for treatment of systemic hypertension. The presence of drug-induced gingival enlargement (DIGE) was assessed clinically and associated with patient factors. Patients with DIGE were group-matched for gender and ethnicity with an equal number of consecutive CCB non-DIGE patients (control 1), no-CCB no-DIGE (control 2) and periodontally healthy with no DIGE (control 3) for the saliva and GCF analysis. A bead-based multiplex immunoassay was used to assess a panel of biomarkers. ResultsTwenty-two percent of patients on CCB were diagnosed with DIGE. Lack of daily interdental cleaning and self-reported diagnosis of type II diabetes were associated with the diagnosis of DIGE. When analysing patients only on CCB, those with DIGE had higher GCF levels of vascular endolthelial growth factor (VEGF) (p = 0.032), epidermal growth factor (EGF) (p = 0.030) and matrix metalloproteinase-8 (MMP-8) (p = 0.008). Among the salivary markers, only MMP-8 showed a statistically significant difference across groups (p < 0.001). ConclusionsThis is the first study investigating saliva and GCF biomarkers in patients with DIGE and different control groups, suggesting that causes of the overgrowth might involve inflammatory processes, tissue damage pathways, and potentially an impact on growth factors like VEGF. Future research should verify these results in independent populations and explore the underlying pathogenic mechanisms in-depth. Clinical significanceCalcium-channel blockers (CCB) can lead to gingival enlargement. This study confirms lack of interdental cleaning and type II diabetes as risk factors. Elevated levels of VEGF, EGF, and MMP-8 in gingival crevicular fluid and MMP-8 in saliva suggest inflammatory processes and growth factors might play roles in this condition.

Evaluating the modulation of peripheral immune profile in people living with HIV and (Neuro)cysticercosis.

The parasitic infection caused by Taenia solium represents a significant public health concern in developing countries. Larval invasion of body tissues leads to cysticercosis (CC), while central nervous system (CNS) involvement results in neurocysticercosis (NCC). Both conditions exhibit diverse clinical manifestations, and the potential impact of concomitant HIV infection especially prevalent in sub-Saharan Africa on peripheral and CNS immune responses remains poorly understood. This study aimed to identify the potential impact of HIV coinfection in CC and NCC patients. A nested study within a cross-sectional analysis in two Tanzanian regions was performed and 234 participants (110 HIV+ and 124 HIV-) were tested for cysticercosis antibodies, antigens, CD4 counts and serum Th1 and Th2 cytokines via multiplex bead-based immunoassay. 127 cysticercosis seropositive individuals underwent cranial computed tomography (CCT) and clinical symptoms were assessed. Multiple regression analyses were performed to identify factors associated with cytokine modulation due to HIV in CC and NCC patients. Serologically, 18.8% tested positive for cysticercosis antibodies, with no significant difference HIV+ and HIV+. A significantly higher rate of cysticercosis antigen positivity was found in HIV+ individuals (43.6%) compared to HIV- (28.2%) (p = 0.016). CCT scans revealed that overall 10.3% had active brain cysts (NCC+). Our study found no significant changes in the overall cytokine profiles between HIV+ and HIV- participants coinfected CC and NCC, except for IL-5 which was elevated in HIV+ individuals with cysticercosis. Furthermore, HIV infection in general was associated with increased levels of pro-and some anti-inflammatory cytokines e.g. TNF-α, IL-8, and IFN-γ. However, based on the interaction analyses, no cytokine changes were observed due to HIV in CC or NCC patients. In conclusion, while HIV infection itself significantly modulates levels of key cytokines such as TNF-α, IL-8, and IFN-γ, it does not modulate any cytokine changes due to CC or NCC. This underscores the dominant influence of HIV on the immune system and highlights the importance of effective antiretroviral therapy in managing immune responses in individuals coinfected with HIV and CC/NCC.

Immunogenicity, reactogenicity, and IgE-mediated immune responses of a mixed whole-cell and acellular pertussis vaccine schedule in Australian infants: A randomised, double-blind, noninferiority trial.

In many countries, infant vaccination with acellular pertussis (aP) vaccines has replaced use of more reactogenic whole-cell pertussis (wP) vaccines. Based on immunological and epidemiological evidence, we hypothesised that substituting the first aP dose in the routine vaccination schedule with wP vaccine might protect against IgE-mediated food allergy. We aimed to compare reactogenicity, immunogenicity, and IgE-mediated responses of a mixed wP/aP primary schedule versus the standard aP-only schedule. OPTIMUM is a Bayesian, 2-stage, double-blind, randomised trial. In stage one, infants were assigned (1:1) to either a first dose of a pentavalent wP combination vaccine (DTwP-Hib-HepB, Pentabio PT Bio Farma, Indonesia) or a hexavalent aP vaccine (DTaP-Hib-HepB-IPV, Infanrix hexa, GlaxoSmithKline, Australia) at approximately 6 weeks old. Subsequently, all infants received the hexavalent aP vaccine at 4 and 6 months old as well as an aP vaccine at 18 months old (DTaP-IPV, Infanrix-IPV, GlaxoSmithKline, Australia). Stage two is ongoing and follows the above randomisation strategy and vaccination schedule. Ahead of ascertainment of the primary clinical outcome of allergist-confirmed IgE-mediated food allergy by 12 months old, here we present the results of secondary immunogenicity, reactogenicity, tetanus toxoid IgE-mediated immune responses, and parental acceptability endpoints. Serum IgG responses to diphtheria, tetanus, and pertussis antigens were measured using a multiplex fluorescent bead-based immunoassay; total and specific IgE were measured in plasma by means of the ImmunoCAP assay (Thermo Fisher Scientific). The immunogenicity of the mixed schedule was defined as being noninferior to that of the aP-only schedule using a noninferiority margin of 2/3 on the ratio of the geometric mean concentrations (GMR) of pertussis toxin (PT)-IgG 1 month after the 6-month aP. Solicited adverse reactions were summarised by study arm and included all children who received the first dose of either wP or aP. Parental acceptance was assessed using a 5-point Likert scale. The primary analyses were based on intention-to-treat (ITT); secondary per-protocol (PP) analyses were also performed. The trial is registered with ANZCTR (ACTRN12617000065392p). Between March 7, 2018 and January 13, 2020, 150 infants were randomised (75 per arm). PT-IgG responses of the mixed schedule were noninferior to the aP-only schedule at approximately 1 month after the 6-month aP dose [GMR = 0·98, 95% credible interval (0·77 to 1·26); probability (GMR > 2/3) > 0·99; ITT analysis]. At 7 months old, the posterior median probability of quantitation for tetanus toxoid IgE was 0·22 (95% credible interval 0·12 to 0·34) in both the mixed schedule group and in the aP-only group. Despite exclusions, the results were consistent in the PP analysis. At 6 weeks old, irritability was the most common systemic solicited reaction reported in wP (65 [88%] of 74) versus aP (59 [82%] of 72) vaccinees. At the same age, severe systemic reactions were reported among 14 (19%) of 74 infants after wP and 8 (11%) of 72 infants after aP. There were 7 SAEs among 5 participants within the first 6 months of follow-up; on blinded assessment, none were deemed to be related to the study vaccines. Parental acceptance of mixed and aP-only schedules was high (71 [97%] of 73 versus 69 [96%] of 72 would agree to have the same schedule again). Compared to the aP-only schedule, the mixed schedule evoked noninferior PT-IgG responses, was associated with more severe reactions, but was well accepted by parents. Tetanus toxoid IgE responses did not differ across the study groups. Trial registered at the Australian and New Zealand Clinical 207 Trial Registry (ACTRN12617000065392p).

Development and validation of a multiplex bead-based immunoassay for the simultaneous detection of fifteen pathogenic biological agents

In this study, a multiplex bead-based capture sandwich immunoassay (MIA) for the simultaneous detection of fifteen plant and bacterial Gram-positive and Gram-negative pathogenic biological agents (PBAs) was developed. This study included the selection of “sandwich” pairs of monoclonal antibodies, the development of an analytical protocol, and an evaluation of the specificity and sensitivity of detection. The working range, linearity, precision, and limits of detection of the developed assay were evaluated. The LLOQ of the multiplex assay was around 104 – 105 CFU/ml for bacteria and 0,4 ng/ml – 6,6 ng/ml for antigens. The assay can be used for the simultaneous detection of 15 PBAs, including Francisella tularensis, Brucella abortus, Bacillus anthracis, Legionella pneumophila, Staphylococcus aureus, Staphylococcus haemolyticus, Pseudomonas aeruginosa, and Salmonella typhimurium, as well as cholera toxin beta-subunit, viscumin, ricin, staphylococcal enterotoxins A and B, and botulinum toxins A and B.

Gut microbiome and inflammation among athletes in wheelchair in a crossover randomized pilot trial of probiotic and prebiotic interventions

Disorders related to gut health are a significant cause of morbidity among athletes in wheelchair. This pilot feasibility trial aims to investigate whether probiotics compared to prebiotics can improve inflammatory status and gut microbiome composition in elite athletes in wheelchair. We conducted a 12-week, randomized, cross-over controlled trial involving 14 elite Swiss athletes in wheelchair. Participants were given a multispecies-multistrain probiotic or prebiotic (oat bran) daily for 4 weeks (Clinical trials.gov NCT04659408 09/12/2020). This was followed by a 4-week washout and then crossed over. Thirty inflammatory markers were assessed using bead-based multiplex immunoassays (LegendPlex) from serum samples. The gut microbiome was characterized via 16S rRNA sequencing of stool DNA samples. Statistical analyses were conducted using linear mixed-effect models (LMM). At baseline, most athletes (10/14) exhibited low levels of inflammation which associated with higher gut microbiome alpha diversity indices compared to those with high inflammation levels. The use of probiotic had higher decrease in 25 (83%) inflammatory markers measured compared to prebiotic use. Probiotic has the potential in lowering inflammation status and improving the gut microbiome diversity. The future trial should focus on having sufficient sample sizes, population with higher inflammation status, longer intervention exposure and use of differential abundance analysis.

Development of a human papillomavirus (HPV) multiplex immunoassay to profile HPV antibodies.

Neutralizing antibodies (NAbs) are considered the primary mechanism of vaccine-mediated protection against human papillomaviruses (HPV), the causative agent of cervical cancer. However, the minimum level of NAb needed for protection is currently unknown. The HPV pseudovirion-based neutralization assay (PBNA) is the gold standard method for assessing HPV antibody responses but is time-consuming and labor-intensive. With the development of higher valency HPV vaccines, alternative serological assays with the capacity for multiplexing would improve efficiency and output. Here we describe a multiplex bead-based immunoassay to characterize the antibody responses to the seven oncogenic HPV types (HPV16/18/31/33/45/52/58) contained in the current licensed nonavalent HPV vaccine. This assay can measure antibody isotypes and subclasses (total IgG, IgM, IgA1-2, IgG1-4), and can be adapted to measure other antibody features (e.g., Fc receptors) that contribute to vaccine immunity. When tested with serum samples from unvaccinated and vaccinated individuals, we found high concordance between HPV-specific IgG using this multiplex assay and NAbs measured with PBNA. Overall, this assay is high-throughput, sample-sparing, and time-saving, providing an alternative to existing assays for the measurement and characterization of HPV antibody responses.

Abstract A006: Bladder cancer risk stratification with the Oncuria 10 plex bead-based urinalysis assay using three different Luminex xMAP instrumentation platforms

Abstract Introduction and Objectives Oncuria® is a bead-based multiplex fluorescence immunoassay that coordinately measures 10 protein biomarkers in urine samples. The current study compared assay performance and output when urine samples were evaluated with the Oncuria assay using three different fluorescence-analyzing instruments commonly used in diagnostic laboratories worldwide. Methods We compared the performance of the clinically validated Oncuria bladder cancer (BC) multiplex immunoassay when data output was generated on three different analyzer systems. Voided urine samples from 36 subjects (18 with BC and 18 Controls) were reacted with Oncuria test reagents in three 96 well microtiter plates, and consecutively evaluated on the LED/image based MagPix, and laser/flow based Luminex 200 and FlexMap 3D (all xMAP instruments from Luminex Corp., Austin, TX). The BC assay uses magnetic bead based fluorescence technology (xMAP, Multi-analyte profiling; Luminex) to simultaneously quantify 10 protein analytes in urine specimens [i.e., angiogenin (ANG), apolipoprotein E (ApoE), carbonic anhydrase IX (CA9), CXCL8/interleukin-8 (IL-8), matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-10 (MMP-10), serpin A1/alpha-1 anti-trypsin (A1AT), serpin E1/plasminogen activator inhibitor-1 (PAI-1), CD138/syndecan-1 (SDC1), and vascular endothelial growth factor-A (VEGF-A)]. Results All three platforms categorized all 10 analytes in identical samples at nearly identical concentrations, with variance across systems typically &amp;lt;5%. While the most contemporary instrument, the FlexMap 3D, output higher raw fluorescence values than the two comparator systems, standard curve slopes and analyte concentrations determined in urine samples were concordant across all three units. Forty-four percent of BC samples registered ≥1 analyte above the highest standard concentration, i.e., A1AT (n=7/18), IL-8 (n=5), and/or ANG (n=2). In Controls, A1AT was higher in one sample. Conclusions In conclusion, the Oncuria BC assay performed similarly well across three different flow analysis platforms for all 10 analytes simultaneously evaluated in urine samples. This agreement across instruments indicates that the test is amenable to standardized performance in laboratories using existing xMAP, without requiring costly outlays for new equipment. Citation Format: Toru Sakatani, Sunao Tanaka, Kaoru Murakami, Richard Waldron, Wayne Hogrefe, Charles Rosser, Hideki Furuya. Bladder cancer risk stratification with the Oncuria 10 plex bead-based urinalysis assay using three different Luminex xMAP instrumentation platforms [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A006.

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.

Longitudinal study of immunity to SARS-CoV2 in ocrelizumab-treated MS patients up to 2 years after COVID-19 vaccination.

(1) To plot the trajectory of humoral and cellular immune responses to the primary (two-dose) COVID-19 mRNA series and the third/booster dose in B-cell-depleted multiple sclerosis (MS) patients up to 2 years post-vaccination; (2) to identify predictors of immune responses to vaccination; and (3) to assess the impact of intercurrent COVID-19 infections on SARS CoV-2-specific immunity. Sixty ocrelizumab-treated MS patients were enrolled from NYU (New York) and University of Colorado (Anschutz) MS Centers. Samples were collected pre-vaccination, and then 4, 12, 24, and 48 weeks post-primary series, and 4, 12, 24, and 48 weeks post-booster. Binding anti-Spike antibody responses were assessed with multiplex bead-based immunoassay (MBI) and electrochemiluminescence (Elecsys®, Roche Diagnostics), and neutralizing antibody responses with live-virus immunofluorescence-based microneutralization assay. Spike-specific cellular responses were assessed with IFNγ/IL-2 ELISpot (Invitrogen) and, in a subset, by sequencing complementarity determining regions (CDR)-3 within T-cell receptors (Adaptive Biotechnologies). A linear mixed-effect model was used to compare antibody and cytokine levels across time points. Multivariate analyses identified predictors of immune responses. The primary vaccination induced an 11- to 208-fold increase in binding and neutralizing antibody levels and a 3- to 4-fold increase in IFNγ/IL-2 responses, followed by a modest decline in antibody but not cytokine responses. Booster dose induced a further 3- to 5-fold increase in binding antibodies and 4- to 5-fold increase in IFNγ/IL-2, which were maintained for up to 1 year. Infections had a variable impact on immunity. Humoral and cellular benefits of COVID-19 vaccination in B-cell-depleted MS patients were sustained for up to 2 years when booster doses were administered.

Inflammation and tissue remodeling mediator expression during gingivitis: A comparison between experimental, naturally occurring gingivitis, and periodontal health.

The aim of this study is to evaluate the immune regulation and tissue remodeling responses during experimental gingivitis (EG) and naturally occurring gingivitis (NG) to provide a comprehensive analysis of host responses. Gingival crevicular fluid (GCF) was obtained from 2 human studies conducted in university settings. The EG study enrolling 26 volunteers provided controls for the baseline (Day 0) from healthy disease-free participants, while Day 21 (the end of EG induction of the same group) was used to represent EG. Twenty-six NG participants age-matched with those of the EG group were recruited. GCF samples were analyzed for 39 mediators of inflammatory/immune responses and tissue remodeling using commercially available bead-based multiplex immunoassays. The differences in GI and mediator expression among groups were determined at a 95% confidence level (p ≤ 0.05) by a 2-way analysis of variance (ANOVA) with a post-hoc Tukey's test. Our findings showed that EG had a greater gingival index than NG and was healthy (p<0.01 of all comparisons). Furthermore, EG showed significantly higher levels of MPO (p<0.001), CCL3 (p<0.05), and IL-1B (p<0.001) than NG. In contrast, NG had increased levels of MIF (p<0.05), Fractalkine (p<0.001), angiogenin (p<0.05), C3a (p<0.001), BMP-2 (p<0.001), OPN (p<0.05), RANKL (p<0.001), and MMP-13 (p<0.001) than EG. Consistent with the findings from chronic (NG) versus acute (EG) inflammatory lesions, these data reveal that NG displays greater immune regulation, angiogenesis, and bone remodeling compared to EG.

Intravital Imaging of the Human Cornea Reveals the Differential Effects of Season on Innate and Adaptive Immune Cell Morphodynamics

Defining how the invivo immune status of peripheral tissues is shaped by the external environment has remained a technical challenge. We recently developed Functional invivo confocal microscopy (Fun-IVCM) for dynamic, longitudinal imaging of corneal immune cells in living humans. This study investigated the effect of seasonal-driven environmental factors on the morphodynamic features of human corneal immune cell subsets. Longitudinal, observational clinical study. Sixteen healthy participants (aged 18-40 years) attended 2 visits in distinct seasons in Melbourne, Australia (Visit 1, November-December 2021 [spring-summer]; Visit 2, April-June 2022 [autumn-winter]). Environmental data were collected over each period. Participants underwent ocular surface examinations and corneal Fun-IVCM (Heidelberg Engineering). Corneal scans were acquired at 5.5 ± 1.5-minute intervals for up to 5 time points. Time-lapse Fun-IVCM videos were created to analyze corneal immune cells, comprising epithelial T cells and dendritic cells (DCs), and stromal macrophages. Tear cytokines were analyzed using a multiplex bead-based immunoassay. Difference in the density, morphology, and dynamic parameters of corneal immune cell subsets over the study periods. Visit 1 was characterized by higher temperature, lower humidity, and higher air particulate and pollen levels compared with Visit 2. Clinical ocular surface parameters and the density of immune cell subsets were similar across visits. At Visit 1 , corneal epithelial DCs were larger, with a lower dendrite probing speed (0.38 ± 0.21 vs. 0.68 ± 0.33 μm/min; P < 0.001) relative to Visit 2; stromal macrophages were more circular and had less dynamic activity (Visit 1, 7.2 ± 1.9 vs. Visit 2, 10.3 ± 3.7 dancing index; P < 0.001). Corneal T cell morphodynamics were unchanged across periods. Basal tear levels of interleukin 2 and CXCL10 were relatively lower during spring-summer. This study identifies that the invivo morphodynamics of innate corneal immune cells (DCs, macrophages) are modified by environmental factors, but such effects are not evident for adaptive immune cells (T cells). The cornea is a potential invivo window to investigate season-dependent environmental influences on the human immune system. Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

A Multiplex Bead-Based Immunoassay to Quantify Multiple Cytokine Targets

A Multiplex Bead-Based Immunoassay to Quantify Multiple Cytokine Targets

A Multiplex Bead-Based Immunoassay to Quantify Multiple Cytokine Targets

A Multiplex Bead-Based Immunoassay to Quantify Multiple Cytokine Targets

Effects of aflibercept and bevacizumab on cell viability, cell metabolism and inflammation in hypoxic human Müller cells.

Anti-VEGF (vascular endothelial growth factor) drugs such as aflibercept (AFL) and bevacizumab (BVZ) inhibit pathological neo-angiogenesis and vascular permeability in retinal vascular diseases. As cytokines and growth factors are produced by Müller glial cells under stressful and pathological conditions, we evaluated the in vitro effect of AFL (Eylea®, 0.5 mg/mL) and BVZ (Avastin®, 0.5 mg/mL) on cell viability/metabolism, and cytokine/growth factor production by Müller cells (MIO-M1) under cobalt chloride (CoCl2)-induced hypoxia after 24h, 48h and 72h. Cell viability/metabolism were analyzed by Trypan Blue and MTT assays and cytokine/growth factors in supernatants by Luminex xMAP-based multiplex bead-based immunoassay. Cell viability increased with AFL at 48h and 72h and decreased with BVZ or hypoxia at 24h. BVZ-treated cells showed lower cell viability than AFL at all exposure times. Cell metabolism increased with AFL but decreased with BVZ (72h) and hypoxia (48h and72h). As expected, AFL and BVZ decreased VEGF levels. AFL increased PDGF-BB, IL-6 and TNF-α (24h) and BVZ increased PDGF-BB (72h). Hypoxia reduced IL-1β, -6, -8, TNF-α and PDGF-BB at 24h, and its suppressive effect was more prominent than AFL (EGF, PDGF-BB, IL-1β, IL-6, IL-8, and TNF-α) and BVZ (PDGF-BB and IL-6) effects. Hypoxia increased bFGF levels at 48h and 72h, even when combined with anti-VEGFs. However, the stimulatory effect of BVZ predominated over hypoxia for IL-8 and TNF-α (24h), as well as for IL-1β (72h). Thus, AFL and BVZ exhibit distinct exposure times effects on MIO-M1 cells viability, metabolism, and cytokines/growth factors. Hypoxia and BVZ decreased MIO-M1 cell viability/metabolism, whereas AFL likely induced gliosis. Hypoxia resulted in immunosuppression, and BVZ stimulated inflammation in hypoxic MIO-M1 cells. These findings highlight the complexity of the cellular response as well as the interplay between anti-VEGF treatments and the hypoxic microenvironment.

Abstract 3773: Bladder cancer risk stratification with the Oncuria 10 plex bead-based urinalysis assay using three different Luminex xMAP instrumentation platforms

Abstract Introduction and Objectives: Oncuria® is a bead-based multiplex fluorescence immunoassay that coordinately measures 10 protein biomarkers in urine samples. The current study compared assay performance and output when urine samples were evaluated with the Oncuria assay using three different fluorescence-analyzing instruments commonly used in diagnostic laboratories worldwide. Methods: We compared the performance of the clinically validated Oncuria bladder cancer (BC) multiplex immunoassay when data output was generated on three different analyzer systems. Voided urine samples from 36 subjects (18 with BC and 18 Controls) were reacted with Oncuria test reagents in three 96 well microtiter plates, and consecutively evaluated on the LED/image based MagPix, and laser/flow based Luminex 200 and FlexMap 3D (all xMAP instruments from Luminex Corp., Austin, TX). The BC assay uses magnetic bead based fluorescence technology (xMAP, Multi-analyte profiling; Luminex) to simultaneously quantify 10 protein analytes in urine specimens [i.e., angiogenin (ANG), apolipoprotein E (ApoE), carbonic anhydrase IX (CA9), CXCL8/interleukin-8 (IL-8), matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-10 (MMP-10), serpin A1/alpha-1 anti-trypsin (A1AT), serpin E1/plasminogen activator inhibitor-1 (PAI-1), CD138/syndecan-1 (SDC1), and vascular endothelial growth factor-A (VEGF-A)]. Results: All three platforms categorized all 10 analytes in identical samples at nearly identical concentrations, with variance across systems typically &amp;lt;5%. While the most contemporary instrument, the FlexMap 3D, output higher raw fluorescence values than the two comparator systems, standard curve slopes and analyte concentrations determined in urine samples were concordant across all three units. Forty-four percent of BC samples registered ≥1 analyte above the highest standard concentration, i.e., A1AT (n=7/18), IL-8 (n=5), and/or ANG (n=2). In Controls, A1AT was higher in one sample. Conclusions: In conclusion, the Oncuria BC assay performed similarly well across three different flow analysis platforms for all 10 analytes simultaneously evaluated in urine samples. This agreement across instruments indicates that the test is amenable to standardized performance in laboratories using existing xMAP, without requiring costly outlays for new equipment. Citation Format: Sunao Tanaka, Toru Sakatani, Kaoru Murakami, Richard T. Waldron, Walter Morales, Wayne Hogrefe, Charles J. Rosser, Hideki Furuya. Bladder cancer risk stratification with the Oncuria 10 plex bead-based urinalysis assay using three different Luminex xMAP instrumentation platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3773.

Anti-histone and anti-nucleosome rather than anti-dsDNA antibodies associate with IFN-induced biomarkers in Sudanese and Swedish Systemic Lupus Erythematosus patients.

In SLE, anti-dsDNA can co-occur with autoantibodies against other chromatin components, like histones and nucleosomes. These antibodies induce type-1 interferon production, a hallmark of SLE. We measured antinuclear antibody (ANA) sub-specificities and investigated their associations to inflammatory biomarkers including interferon-regulated chemokines. We included 93 Sudanese and 480 Swedish SLE patients and matched controls (N = 104 + 192). Autoantibodies targeting ANA-subspecificites: dsDNA, Sm, Sm/U1RNPcomplex, U1RNP, SSA/Ro52, SSA/Ro60, SSB/La, ribosomal P, PCNA and histones were quantified in all subjects, anti-nucleosome only in the Swedish patients, with a bead-based multiplex immunoassay. Levels of 72 plasma biomarkers were determined with Proximity Extension Assay technique or ELISA. Among Sudanese patients, the investigated antibodies significantly associated with 9/72 biomarkers. Anti-histone antibodies showed the strongest positive correlations with MCP-3 and S100A12 as well as with interferon I-inducible factors MCP-1 and CXCL10. Anti-dsDNA antibodies associated with CXCL10 and S100A12, but in multivariate analyses, unlike anti-histone, associations lost significance.Among Swedish patients, MCP-1, CXCL10, SA100A12 also demonstrated stronger associations to anti-histone and anti-nucleosome antibodies, compared with anti-dsDNA and other ANA sub-specificities. In multiple regression models, anti-histone/nucleosome retained the strongest associations. When excluding anti-histone or anti-nucleosome positive patients, the associations between MCP-1/CXCL10 and anti-dsDNA were lost. In contrast, when excluding anti-dsDNA positive patients, associations with anti-histone and anti-nucleosome remained significant. In two cohorts of different ethnical origin, autoantibodies targeting chromatin correlate stronger with IFN-induced inflammatory biomarkers than anti-dsDNA or other ANA sub-specificities. Our results suggest that anti-histone/nucleosome autoantibodies may be main drivers of type-1 interferon activity in SLE.