Luminex xMAP Technology Research Articles

Prevalence and genotype distribution of human papillomavirus infection among 66000 women from 2014 to 2023 in the plateau region of Southwest China

BackgroundPersistent infection with high-risk human papillomavirus (HR-HPV) plays a key role in the onset of cervical cancer. This study was designed to examine the epidemiological trends and genotype distribution of HPV from 2014 to 2023 in the plateau region of Southwest China.MethodsThe findings could offer valuable insights for clinical screening of cervical cancer and the formulation of HPV vaccination policies. This retrospective study analyzed 66,000 women who received HPV-DNA testing at the First People’s Hospital of Qujing, Yunnan, China, between 2014 and 2023. The cohort consisted of 33,512 outpatients, 3,816 inpatients, and 28,672 individuals undergoing health examinations. Cervical cells were collected for DNA extraction, and PCR amplification along with Luminex xMAP technology were used to detect 27 HPV genotypes. The data analysis was conducted using GraphPad Prism and IBM SPSS Statistics 27 software.ResultsThe overall HPV infection rate at the First People’s Hospital of Qujing declined from 24.92% in 2014 to 16.29% in 2023, averaging 16.02%. Specific infection rates were 18.50% among outpatients, 12.97% among inpatients, and 13.53% for health examination attendees. The predominant high-risk HPV genotypes identified were HPV52 (2.61%), HPV16 (2.06%), HPV58 (1.81%), HPV53 (1.55%), and HPV39 (1.09%). Meanwhile, the most frequent low-risk HPV genotypes were HPV6 (1.30%), HPV61 (1.21%), and HPV11 (0.85%). In HPV-positive cases, the distribution of single, double, triple, and quadruple or more infections were 79.90%, 15.17%, 3.59%, and 1.33%, respectively. The proportions of pure LR-HPV, pure HR-HPV, and mixed infections were 22.16%, 67.82%, and 10.02%, respectively. Age-specific analysis revealed a bimodal distribution of HPV infection, with the infection rate rapidly decreasing from 44.02% in the ≤ 19 age group to 19.55% in the 20–29 age group and 13.84% in the 30–39 age group, followed by a gradual increase to 14.64% in the 40–49 age group, 16.65% in the 50–59 age group, and 22.98% in the ≥ 60 age group. The coverage rates of the three available vaccines are all below 50%. The results of this study indicated a declining trend in HPV prevalence in the plateau region of Southwest China over the period from 2014 to 2023, especially in the reduction of genotypes targeted by vaccines.ConclusionThere were significant variations in the genotypes prevalent among different age groups, years, and patient sources within the same region. The underwhelming vaccination rates emphasize the critical need for developing either a multivalent vaccine or a personalized vaccine that targets the HPV genotypes common in the Chinese population. Furthermore, vaccinating adolescents to curb HPV infection and ensuring regular cervical cancer screenings for postmenopausal women are crucial steps.

Molecular circadian clock disruption in the leukocytes of individuals with type 2 diabetes and overweight, and its relationship with leukocyte–endothelial interactions

Aims/hypothesisAlterations in circadian rhythms increase the likelihood of developing type 2 diabetes and CVD. Circadian rhythms are controlled by several core clock genes, which are expressed in nearly every cell, including immune cells. Immune cells are key players in the pathophysiology of type 2 diabetes, and participate in the atherosclerotic process that underlies cardiovascular risk in these patients. The role of the core clock in the leukocytes of people with type 2 diabetes and the inflammatory process associated with it are unknown. We aimed to evaluate whether the molecular clock system is impaired in the leukocytes of type 2 diabetes patients and to explore the mechanism by which this alteration leads to an increased cardiovascular risk in this population.MethodsThis is an observational cross-sectional study performed in 25 participants with type 2 diabetes and 28 healthy control participants. Clinical and biochemical parameters were obtained. Peripheral blood leukocytes were isolated using magnetic bead technology. RNA and protein lysates were obtained to assess clock-related gene transcript and protein levels using real-time PCR and western blot, respectively. Luminex XMAP technology was used to assess levels of inflammatory markers. Leukocyte–endothelial interaction assays were performed by perfusing participants’ leukocytes or THP-1 cells (with/without CLK8) over a HUVEC monolayer in a parallel flow chamber using a dynamic adhesion system.ResultsParticipants with type 2 diabetes showed increased BMAL1 and NR1D1 mRNA levels and decreased protein levels of circadian locomotor output cycles kaput (CLOCK), cryptochrome 1 (CRY1), phosphorylated basic helix-loop-helix ARNT like 1 (p-BMAL1) and period circadian protein homologue 2 (PER2). Correlation studies revealed that these alterations in clock proteins were negatively associated with glucose, HbA1c, insulin and HOMA-IR levels and leukocyte cell counts. The leukocyte rolling velocity was reduced and rolling flux and adhesion were enhanced in individuals with type 2 diabetes compared with healthy participants. Interestingly, inhibition of CLOCK/BMAL1 activity in leukocytes using the CLOCK inhibitor CLK8 mimicked the effects of type 2 diabetes on leukocyte–endothelial interactions.Conclusions/interpretationOur study demonstrates alterations in the molecular clock system in leukocytes of individuals with type 2 diabetes, manifested in increased mRNA levels and decreased protein levels of the core clock machinery. These alterations correlated with the impaired metabolic and proinflammatory profile of the participants with type 2 diabetes. Our findings support a causal role for decreased CLOCK/BMAL1 activity in the increased level of leukocyte–endothelial interactions. Overall, our data suggest that alterations in core clock proteins accelerate the inflammatory process, which may ultimately precipitate the onset of CVD in patients with type 2 diabetes.Graphical

CX3CL1 release during immunogenic apoptosis is associated with enhanced anti-tumour immunity.

Immunogenic cell death (ICD) has emerged as a novel option for cancer immunotherapy. The key determinants of ICD encompass antigenicity (the presence of antigens) and adjuvanticity, which involves the release of damage-associated molecular patterns (DAMPs) and various cytokines and chemokines. CX3CL1, also known as neurotactin or fractalkine, is a chemokine involved in cellular signalling and immune cell interactions. CX3CL1 has been denoted as a "find me" signal that stimulates chemotaxis of immune cells towards dying cells, facilitating efferocytosis and antigen presentation. However, in the context of ICD, it is uncertain whether CX3CL1 is an important mediator of the effects of ICD. In this study, we investigated the intricate role of CX3CL1 in immunogenic apoptosis induced by mitoxantrone (MTX) in cancer cells. The Luminex xMAP technology was used to quantify murine cytokines, chemokines and growth factors to identify pivotal regulatory cytokines released by murine fibrosarcoma MCA205 and melanoma B16-F10 cells undergoing ICD. Moreover, a murine tumour prophylactic vaccination model was employed to analyse the effect of CX3CL1 on the activation of an adaptive immune response against MCA205 cells undergoing ICD. Furthermore, thorough analysis of the TCGA-SKCM public dataset from 98 melanoma patients revealed the role of CX3CL1 and its receptor CX3CR1 in melanoma patients. Our findings demonstrate enhanced CX3CL1 release from apoptotic MCA205 and B16-F10 cells (regardless of the cell type) but not if they are undergoing ferroptosis or accidental necrosis. Moreover, the addition of recombinant CX3CL1 to non-immunogenic doses of MTX-treated, apoptotically dying cancer cells in the murine prophylactic tumour vaccination model induced a robust immunogenic response, effectively increasing the survival of the mice. Furthermore, analysis of melanoma patient data revealed enhanced survival rates in individuals exhibiting elevated levels of CD8+ T cells expressing CX3CR1. These data collectively underscore the importance of the release of CX3CL1 in eliciting an immunogenic response against dying cancer cells and suggest that CX3CL1 may serve as a key switch in conferring immunogenicity to apoptosis.

GDNF and miRNA-29a as biomarkers in the first episode of psychosis: uncovering associations with psychosocial factors.

Schizophrenia involves complex interactions between biological and environmental factors, including childhood trauma, cognitive impairments, and premorbid adjustment. Predicting its severity and progression remains challenging. Biomarkers like glial cell line-derived neurotrophic factor (GDNF) and miRNA-29a may bridge biological and environmental aspects. The goal was to explore the connections between miRNAs and neural proteins and cognitive functioning, childhood trauma, and premorbid adjustment in the first episode of psychosis (FEP). This study included 19 FEP patients who underwent clinical evaluation with: the Childhood Trauma Questionnaire (CTQ), the Premorbid Adjustment Scale (PAS), the Positive and Negative Syndrome Scale (PANSS), and the Montreal Cognitive Assessment Scale (MoCA). Multiplex assays for plasma proteins were conducted with Luminex xMAP technology. Additionally, miRNA levels were quantitatively determined through RNA extraction, cDNA synthesis, and RT-qPCR on a 7500 Fast Real-Time PCR System. Among miRNAs, only miR-29a-3p exhibited a significant correlation with PAS-C scores (r = -0.513, p = 0.025) and cognitive improvement (r = -0.505, p = 0.033). Among the analyzed proteins, only GDNF showed correlations with MoCA scores at the baseline and after 3 months (r = 0.533, p = 0.0189 and r = 0.598, p = 0.007), cognitive improvement (r = 0.511, p = 0.025), and CTQ subtests. MIF concentrations correlated with the PAS-C subscale (r = -0.5670, p = 0.011). GDNF and miR-29a-3p are promising as biomarkers for understanding and addressing cognitive deficits in psychosis. This study links miRNA and MIF to premorbid adjustment and reveals GDNF's unique role in connection with childhood trauma.

ShinyMBA: a novel R shiny application for quality control of the multiplex bead assay for serosurveillance studies

The multiplex bead assay (MBA) based on Luminex xMAP technology can be used as a tool to measure seroprevalence as part of population immunity evaluations to multiple antigens in large-scale serosurveys. However, multiplexing several antigens presents challenges for quality control (QC) assessments of the data because multiple parameters must be evaluated for each antigen. MBA QC parameters include monitoring bead counts and median fluorescence intensity (MFI) for each antigen in plate wells, and performance of assay controls included on each plate. Analyzing these large datasets to identify plates failing QC standards presents challenges for many laboratories. We developed a novel R Shiny application, shinyMBA, to expedite the MBA QC processes and reduce the risk of user error. The app allows users to rapidly merge multi-plate assay outputs to evaluate bead count, MFI, and performance of assay controls using statistical process control charts for all antigen targets simultaneously. The utility of the shinyMBA application and its various outputs are demonstrated using data from 32 synthetic xPONENT files with 3 multiplex antigens and two population serosurveillance studies that evaluated 1200 and 3871 samples, respectively, for 20 multiplexed antigens. The shinyMBA open-source code is available for download and modification at https://github.com/CDCgov/shinyMBA. Incorporation of shinyMBA into Luminex serosurveillance workflows can vastly improve the speed and accuracy of QC processes.

Abstract 6642: Exploring the x-ray psoralen activated cancer therapy mechanism of action in a genetically engineered mouse model of soft tissue sarcoma

Abstract BACKGROUND: Psoralen is a natural ultraviolet activated compound that has been used for over 70 years to treat skin cancer, psoriasis, and vitiligo. Recently, to expand its use beyond external treatment, a novel therapeutic called X-PACT (X-ray psoralen activated cancer therapy) was created to treat internal tumors. A combination of 8-methoxypsoralen and phosphors that convert low doses of x-ray radiation to ultraviolet (UV) light are injected into solid tumors and subsequently activated by x-ray photons with considerable antineoplastic effect. Notably, canine clinical trials have observed significant abscopal effects of X-PACT therapy on metastatic lesions throughout patients, but the immunomodulatory mechanisms of action are unclear (Nolan et al, in submission). Here, we present preliminary data investigating the immune response to X-PACT therapy in a murine soft tissue sarcoma (STS) model. METHODS: Six mice of the inducible KrasG12D/+; p53flox/flox (KP) murine STS model with palpable, gastrocnemius tumors were used in this initial study. Four mice underwent treatment with X-PACT. In this group, psoralen was mixed with energy converters and injected into the tumor. The mice then received low-level radiation to activate the drug. Treatment was performed five times over a 15-day period and the animals were sacrificed within 48 hours of final treatment. The other two mice with induced tumors received no treatment prior to sacrifice. For both groups, a cardiac blood draw was performed at the time of sacrifice to obtain plasma serum for analysis. Luminex xMAP technology was used for multiplexed quantification of 45 mouse cytokines, chemokines and growth factors. RESULTS: There were five markers that showed significantly different levels between the treated and control groups. Results indicated significantly higher (p = 0.006) plasma concentrations of macrophage derived chemokine (MDC) following five rounds of treatment with X-PACT therapy. Additionally, the treated mice displayed significantly lower concentrations of granulocyte colony-stimulating factor (G-CSF, p = 0.003), eotaxin (p = 0.016), tissue inhibitor of matrix metalloproteinase 1 (TIMP1, p = 0.024), and interleukin-1β (IL-1β, p = 0.033) compared to untreated mice with STS. CONCLUSION: In humans, MDC levels are associated with longer survival and lower cancer severity. G-CSF, eotaxin, TIMP1, and IL-1β are upregulated in cancer patients and correlate with disease severity. Together, the lower concentrations of G-CSF, Eotaxin, TIMP1, and IL-1β suggest a softening of cancer severity following X-PACT treatment. Further studies and larger sample sizes are necessary to confirm and expand upon these preliminary findings. Citation Format: Beatrice Schleupner, Alexandra Hunter Aitchison, Elle MacAlpine, Nicole Cantor, Jason Somarelli, Brian Brigman, Julia Visgauss, Michael Monument, William Eward. Exploring the x-ray psoralen activated cancer therapy mechanism of action in a genetically engineered mouse model of soft tissue sarcoma [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 6642.

Cecropin A: investigation of a host defense peptide with multifaceted immunomodulatory activity in a chicken hepatic cell culture.

Host defense peptides (HDPs) are increasingly referred to as promising candidates for the reduction of the use of conventional antibiotics, thereby combating antibiotic resistance. As HDPs have been described to exert various immunomodulatory effects, cecropin A (CecA) appears to be a potent agent to influence the host inflammatory response. In the present study, a chicken primary hepatocyte-non-parenchymal cell co-culture was used to investigate the putative immunomodulatory effects of CecA alone and in inflammatory conditions evoked by polyinosinic-polycytidylic acid (Poly I:C). To examine the viability of the cells, the extracellular lactate dehydrogenase (LDH) activity was determined by colorimetric assay. Inflammatory markers interleukin (IL)-8 and transforming growth factor-ß1 (TGF-ß1) were investigated using the ELISA method, whereas concentrations of IL-6, IL-10, and interferon-γ (IFN-γ) were assayed by Luminex xMAP technology. Extracellular H2O2 and malondialdehyde levels were measured by fluorometric and colorimetric methods, respectively. Results of the lower concentrations suggested the safe application of CecA; however, it might contribute to hepatic cell membrane damage at its higher concentrations. We also found that the peptide alleviated the inflammatory response, reflected by the decreased production of the pro-inflammatory IL-6, IL-8, and IFN-γ. In addition, CecA diminished the levels of anti-inflammatory IL-10 and TGF-ß1. The oxidative markers measured remained unchanged in most cases of CecA exposure. CecA displayed a multifaceted immunomodulatory but not purely anti-inflammatory activity on the hepatic cells, and might be suggested to maintain the hepatic inflammatory homeostasis in Poly I:C-triggered immune response. To conclude, our study suggests that CecA might be a promising molecule for the development of new immunomodulatory antibiotic-substitutive agents in poultry medicine; however, there is still a lot to clarify regarding its cellular effects.

A novel XNA-based Luminex assay to detect UBA1 somatic mutations associated with VEXAS syndrome

ObjectivesPatients with VEXAS syndrome carry mutations of UBA1 gene coding for the E1 enzyme. The three most frequent mutations are p.M41T(122T > C), p.M41V (c.121A > G), and p.M41L (c.121A > C) in codon 41 of exon 3. Currently, sanger sequencing was mainly used to detect these mutations, which has low sensitivity and low throughput. There is a need of high sensitivity, simple and high throughput method to characterize patients with VEXAS syndrome. MethodsBased on our proprietary XNA technology, we have developed a QClamp® Plex platform to detect eight mutations in a single reaction using the Luminex xMap technology. The assay sensitivity, specificity and precision were subsequently evaluated. Furthermore, the reference interval and clinical sensitivity/specificity were estimated using clinical healthy/positive DNA samples and the sanger sequencing method was used for comparison. ResultsWith spiking synthetic mutant DNA in wildtype GM24385 cell line DNA, this assay can detect UBA1 mutations with a detection limit of variant allele frequency (VAF) at 0.1–5%. Our assay shows 100% concordance with Sanger sequencing results when used for analyzing 15 positive and 19 negative clinical samples. ConclusionsThe QClamps® Plex UBA1 Mutation Detection Assay is a quicker, simpler, and more sensitive assay that can accurately detect the UBA1 mutations even at early stages with low mutation frequency.

Characteristic cytokine profile of the aqueous humor in eyes with congenital cataract and pre-existing posterior capsule dysfunction.

To investigate the characteristic cytokine profile of the aqueous humor in eyes with congenital cataract and pre-existing posterior capsule dysfunction (PCD). In this cross-sectional study, the enrolled eyes with congenital cataract and PCD were included in the PCD group, while those with an intact posterior capsule were included in the control group. Demographic data and biometric parameters were recorded. The levels of 17 inflammatory factors in the aqueous humor collected from the enrolled eyes were detected using Luminex xMAP technology, and intergroup differences in the collected data were analyzed. The PCD group comprised 41 eyes from 31 patients with congenital cataract and PCD, whereas the control group comprised 42 eyes from 27 patients with congenital cataract and an intact posterior capsule. Lens thickness was significantly thinner in the PCD group than in the control group. However, the levels of monocyte chemoattractant protein-1 (MCP-1), transforming growth factor-β2 (TGF-β2), and vascular endothelial growth factor (VEGF) were significantly higher in the PCD group than in the control group. Multivariate logistic regression confirmed that lens thickness and TGF-β2 level were independent risk factors for PCD. A thinner lens thickness in eyes with congenital cataract and PCD could serve as a biometric feature of these eyes. The higher levels of MCP-1, TGF-β2, and VEGF in eyes with PCD indicated a change in their intraocular inflammatory microenvironment, which possibly led to cataract progression. Lens thickness and TGF-β2 level are independent risk factors for PCD.

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.

Novel Gene Mutations Associated with Disease-Free Survival in Elderly Patients with Acute Myeloid Leukemia Receiving First-Line Induction-Consolidation with or without Azacitidine Post-Remission Treatment

Background: The achievement of complete remission (CR) is an important milestone for patients with acute myeloid leukemia (AML) undergoing curative-intent therapy. However, patients who achieve CR with induction therapy for AML will relapse within months. Therefore, research is focused on prolonging leukemia-free survival (DFS). We have recently reported on the randomized phase III study to compare the efficacy and safety of azacitidine (AZA) versus best supportive care (BSC) for treatment of AML in elderly patients who achieved first CR after a homogeneous intensive induction and consolidation phase (QoLESS AZA-AMLE, Oliva et al. Cancers 2023). At 2 and 5 years, DFS was 6.0 (95% CI:0.2-11.7) months in the BSC arm vs. 10.8 (95% CI:1.9-19.6 p=0.23) months in the AZA arm. We report on the ancillary translational study (QOL-ONE Trans-2) to evaluate biological changes in bone marrow samples through Next Generation Sequencing (NGS). Objective: In patients enrolled in the trial who reached CR and were randomized to receive AZA or BSC, the endpoint of the present study was to evaluate the effect modification by gene mutations at AML diagnosis on the relationship between treatment allocation and relapse (disease-free survival, DFS). Methods: The study was performed on available biological samples collected at baseline, randomization, and 6-month post remission. DNA was subjected to high throughput NGS using genes commonly mutated in myeloid malignancies and prepared with a home-set of 350 genes for Illumina (University of Kyoto, Japan) for all subjects with available vials. Only variants with high-quality reads, were considered. A Variant Allele Frequency (VAF) ≥ 4% was considered an appropriate threshold for minimal burden of clonality to be reported. Results: Samples were available for 24 patients, 12 males, of median age 71 (65-74) years, 11 allocated in AZA arm and 13 in BSC arm. At diagnosis, mutations were detected in all patients with a range of 5 to 17 (median 10) simultaneous mutations, the most frequent being DNMT3A (42%), NPM1 (33%) and TET2 (33%). The most frequently mutated genes at diagnosis are represented in the figure. Only FANCA gene, mutated in 4 patients was associated with a hazard risk (HR) 4.96 (95% CI 1.34 - 18.35; p=0.02) of relapse and, interestingly, HLA-A, which was mutated in 7 patients, was associated with a HR 0.277 (95% CI 0.08 - 0.95; p=0.049). Patients with HLA-A mutation had a significantly longer survival (Figure 2). Three HLA-A mutated patients were allocated in 5-Aza arm and 4 in BSC arm. In a multivariate COX model, the effect of baseline HLA-A mutation on DFS is independent of the allocation arm (P=0.041). HLA Class I and Class II alleles were assessed based on the Luminex xMAP technology: for all cases, the HLA-SNPs are located on chromosome 6 in exon 2, 3 and 4 within protein-codifying sequences. Conclusions: HLA gene mutations are not usually investigated for diagnostic or prognostic purposes in AML. The extensive 350-gene panel used in this study included HLA genes and has revealed a possible favorable role of HLA-A polimorphisms for the prognosis of elderly patients with AML undergoing intensive chemotherapy. Further specific HLA NGS analyses are undergoing to determine whether this concerns genuine somatic mutations. The present finding is worthy of further investigation to confirm its value in prognosis and its therapeutic implications.

Association of Inflammatory Cytokine Levels with Extra Glandular Manifestations, Fatigue, and Disease Activity in Primary Sjögren's Syndrome in Saudi Patients: A Cross-Sectional Study.

Primary Sjögren's syndrome (pSS) is an autoimmune disease that can cause fatigue and extraglandular manifestations (EGMs). pSS is associated with cytokine network dysregulation, which may be related to the immune-mediated destruction of exocrine glands. We determined cytokine levels and their relationship to EGMs, the European League Against Rheumatism (EULAR) Sjögren's syndrome disease activity index (ESSDAI), and fatigue in Saudi patients with pSS. This study was a cross-sectional, single-center study. We included forty-one patients and 71 controls. Serum samples were collected from random healthy people and pSS patients who were followed in the rheumatology and pulmonary clinics of King Saud University Medical City in Riyadh, Saudi Arabia. Levels of the frequently studied cytokines were measured using Luminex xMAP technology. Each ESSDAI score and EGM were recorded, and the Arabic version of the fatigue severity scale (FSS) was applied to assess fatigue. The main outcome measures were cytokine levels in pSS Saudi patients using/not using immune-suppressive medications (ISMs). Thirty-six (87.8%) patients had one or more EGMs, and the mean ESSDAI score was 9.95 ± 7.73. There was a significant decrease in TNFα and IL-21 levels in the pSS group compared to those in the control group (p = 0.034 and p < 0.001, respectively), whereas IL-12 levels were significantly elevated in the pSS group (p = 0.002). Cytokine levels in patients who used ISMs were the same as those in patients who did not use medications. Decreased IL-1β (p = 0.014), IL-2 (p = 0.035), IL-6 (p = 0.014), and IL-35 (p = 0.010) levels were observed in patients who had EGMs. Patients who had low disease activity exhibited low IL-10 (p = 0.018) and high IFN-α (p = 0.049), IFN-β (p = 0.049), IL-1β (p = 0.006), and IL-35 (p = 0.032) levels compared to patients with high disease activity. A negative association between a positive fatigue score and IL-1β (p = 0.010), IL-2 (p = 0.037), IFN-α (p = 0.025), TNFα (p = 0.030), IL-17 (p = 0.029), IL-12 (p = 0.046), and IL-21 (p = 0.005) levels was found. Cytokine profiles correlate with EGMs, ESSDAI, and fatigue. Patients with controlled disease activity have a normal cytokine profile that is similar to that of controls.

Amyloid-β Pathology-Specific Cytokine Secretion Suppresses Neuronal Mitochondrial Metabolism

IntroductionNeuroinflammation and metabolic dysfunction are early alterations in Alzheimer’s disease (AD) brain that are thought to contribute to disease onset and progression. Glial activation due to protein deposition results in cytokine secretion and shifts in brain metabolism, which have been observed in AD patients. However, the mechanism by which this immunometabolic feedback loop can injure neurons and cause neurodegeneration remains unclear.MethodsWe used Luminex XMAP technology to quantify hippocampal cytokine concentrations in the 5xFAD mouse model of AD at milestone timepoints in disease development. We used partial least squares regression to build cytokine signatures predictive of disease progression, as compared to healthy aging in wild-type littermates. We applied the disease-defining cytokine signature to wild-type primary neuron cultures and measured downstream changes in gene expression using the NanoString nCounter system and mitochondrial function using the Seahorse Extracellular Flux live-cell analyzer.ResultsWe identified a pattern of up-regulated IFNγ, IP-10/CXCL10, and IL-9 as predictive of advanced disease. When healthy neurons were exposed to these cytokines in proportions found in diseased brain, gene expression of mitochondrial electron transport chain complexes, including ATP synthase, was suppressed. In live cells, basal and maximal mitochondrial respiration were impaired following cytokine stimulation.ConclusionsWe identify a pattern of cytokine secretion predictive of progressing amyloid-β pathology in the 5xFAD mouse model of AD that reduces expression of mitochondrial electron transport complexes and impairs mitochondrial respiration in healthy neurons. We establish a mechanistic link between disease-specific immune cues and impaired neuronal metabolism, potentially causing neuronal vulnerability and susceptibility to degeneration in AD.

Natural Acetogenins, Chatenaytrienins-1, -2, -3 and -4, Mitochondrial Potential Uncouplers and Autophagy Inducers-Promising Anticancer Agents.

The present paper details the complete stereoselective synthesis of four natural acetogenins, chatenaytrienins-1, -2, -3 and -4, previously isolated from the roots of fruit trees of the family Annonaceae (A. nutans and A. muricata), as an inseparable mixture. The novel organometallic reactions, developed by the authors, of Ti-catalyzed cross-cyclomagnesiation of O-containing and aliphatic allenes using available Grignard reagents were applied at the key stage of synthesis. We have studied the biological activity of the synthesized individual chatenaytrienins-1, -2, -3 and -4 in vitro, including their cytotoxicity in a panel of tumor lines and their ability to induce apoptosis, affect the cell cycle and mitochondria, and activate the main apoptotic signaling pathways in the cell, applying modern approaches of flow cytometry and multiplex analysis with Luminex xMAP technology. It has been shown that chatenaytrienins affect mitochondria by uncoupling the processes of mitochondrial respiration, causing the accumulation of ROS ions, followed by the initiation of apoptosis. The most likely mechanism for the death of cortical neurons from the consumption of tea from the seeds of Annona fruit is long-term chronic hypoxia, which leads to the development of an atypical form of Parkinson's disease that is characteristic of the indigenous inhabitants of Guam and New Caledonia.

Visfatin and Subclinical Atherosclerosis in Type 2 Diabetes: Impact of Cardiovascular Drugs.

Background and Objectives: The role of adipokines in the development of atherosclerosis in type 2 diabetes (T2DM) has not yet been fully elucidated. The effects of drugs on adipokine concentrations have only been evaluated in very few studies, although they may be of clinical importance. This study aimed to assess whether the concentrations of circulating adipokines could predict subclinical atherosclerosis in patients with T2DM, as well as their interactions with commonly used cardiovascular drugs. Materials and Methods: Our population-based cross-sectional multicentric study included 216 participants with T2DM but without previously diagnosed atherosclerosis. The carotid artery intima-media thickness (IMT), plaque and ankle-brachial index (ABI) metrics were measured. Resistin, visfatin, retinol-binding protein 4, high molecular weight adiponectin and leptin levels were evaluated using Luminex's xMAP technology. Results: Visfatin and resistin concentrations correlated positively with IMT (p = 0.002 and p = 0.009, respectively). The correlation of visfatin to IMT ≥ 1.0 mm was significant in males (p < 0.001). Visfatin had a positive correlation with IMT ≥ 1.0 mm or plaque (p = 0.008) but resistin only correlated with plaque (p = 0.049). Visfatin predicted IMT ≥ 1.0 mm or plaque in patients on β-blocker monotherapy (p = 0.031). Visfatin lost its ability to predict subclinical atherosclerosis in patients taking angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, calcium channel blockers or statins. After adjustments for risk factors for atherosclerosis and cardiovascular drugs, visfatin maintained an independent association with mean IMT (p = 0.003), IMT ≥ 1.0 mm or plaque (p = 0.005) and ABI ≤ 0.9 (p = 0.029). Conclusions: Visfatin could be used as a marker of subclinical atherosclerosis in patients with T2DM, especially in males. The assessment of visfatin concentration could aid in identifying individuals who could benefit from implementing preventive measures against atherosclerosis.

Post-Traumatic Stress Response and Appendicitis in Children-Clinical Usefulness of Selected Biomarkers.

Acute appendicitis is an inflammatory process which is one of the most frequent global causes of surgical interventions in children. The goal of the study was to determine whether acute phase proteins, that is, C-reactive protein (CRP), procalcitonin (PCT) and neutrophil gelatinase-associated lipocalin (NGAL), interleukin 6 (IL-6), transforming growth factor-beta1 (TGF-β1) and cortisol (HC) play a role in the pathomechanism of post-trauma stress response of the organism and to establish the impact of the applied surgical procedure and/or of inflammation on their concentrations. An additional purpose was to establish the clinical usefulness of the studied biomarkers in the diagnostics of appendicitis. CRP concentrations were quantified via the immunoturbidimetric method, while the levels of IL-6 and PCT were assessed using a bead-based multiplexed immunoassay system in a microplate format (Luminex xMAP technology); NGAL, TGF-β1 and cortisol concentrations were determined via the enzyme-linked immunosorbent assay (ELISA) technique. All the investigated biomarkers were assayed twice, i.e., immediately before the surgery and 12-24 h after its completion. Significant increases in CRP, IL-6 and PCT concentrations were found in all children subjected to laparoscopic surgeries (p = 0.001, p = 0.006, and p = 0.009, respectively) and open (classic) surgeries (p = 0.001, p = 0.016, and p = 0.044, respectively) compared to the initial concentrations. The patients undergoing classical surgery moreover presented with significant (p = 0.002, and p = 0.022, respectively) increases in NGAL and TGF-β1 levels after the procedures. In a group of children undergoing laparoscopic surgery, the appendicitis induced an increase in cortisol concentration, whereas in patients undergoing classical surgery the increase in the levels of this biomarker was caused by the type of performed surgical procedure. Simultaneously assaying the levels of CRP, NGAL and IL-6 (p = 0.008, p = 0.022, and p = 0.000, respectively) may prove useful in clinical practice, enabling the diagnosis of appendicitis in paediatric patients reporting to a hospital with abdominal pains, in addition to data from anamnesis and from clinical or ultrasound examination. The performed study confirms the participation of examined biomarkers in the pathomechanism of post-injury stress reaction of the organism to surgical trauma.

P-506 Administration of low molecular weight heparin alleviate immune physiology in recurrent implantation failure modulating CCL2-CCR2 axis during frozen embryo transfer: effort from clinic to bench

Abstract Study question Does low molecular-weight heparin (LMWH) administration modulate CCL2-CCR2 axis in recurrent implantation failure (RIF) patients undergoing frozen embryo transfer (FET) to improve pregnancy salvage? Summary answer LMWH treatment attests beneficial immunological tolerance in vivo and in vitro through modulation of CCL2-CCR2 axis suggesting possible restoration of weak pro-inflammatory response in RIF. What is known already Immune imbalance in proportion of activated macrophages (M1) and alternatively activated macrophages (M2) impede endometrial receptivity and contribute to RIF. CCL2 (M2-asscociated) - CCR2 (t-helper-2 associated) axis orders macrophage polarization by influencing expression of functionally relevant and polarization associated genes and down-modulate pro-inflammatory cytokine production. Immunological effects of LMWH regulate pregnancy-promoting processes at foetal-maternal interface to provide an appropriate implantation process; outcomes being controversial. We aim to investigate the potential immunoregulatory properties of LMWH in patients with RIF undergoing FET, with particular regard to effects on macrophages and T-helper (Th) cells; two key players in promoting immune tolerance during pregnancy. Study design, size, duration This prospective cohort study constituted 121 women with RIF (≥3 failed embryo transfer) undergoing FET at Institute of Reproductive Medicine between January to October 2022. An incremental dose (4-12mg/day) of estradiol with (Group A; n = 62) or without (Group B; n = 59) LMWH (40 mg/day) from d2 to d12 with sequential ultrasound following intra-muscular progesterone (100mg/day) for 4 days was administered in all participants. Blood samples were collected to isolate plasma and peripheral-blood-mononuclear-cells (PBMCs). Participants/materials, setting, methods Monocytes were purified from PBMCs and cultured with GM-CSF or M-CSF to analyze factor/s which control CCL2-CCR2 expression along macrophage polarization by flow cytometry. Influence of CCL2 on GM-CSF-cultured macrophages (GM-MØ) and M-CSF-cultured macrophages (M-MØ) specific gene markers was analysed using quantitative RT-PCR (qRT-PCR). To assess immunological effects of LMWH in-vivo, Th-specific cytokine profile/s and respective chemokines were measured from plasma by Luminex xMAP technology. Statistical significance was set at p &amp;lt; 0.05. Main results and the role of chance The clinical pregnancy rate was higher in group A. (A vs B: 37.09% vs. 27.11%, p = 0.24). LMWH exposure significantly decreased the secretion of CCL2 (p &amp;lt; 0.01) and CCL22 (p &amp;lt; 0.001) in GM-CSF-cultured macrophages, while it increased CCL2, CCL20 production (p &amp;lt; 0.001) in both GM-MØ and M-MØ. However, expression of CCL7 was significantly lower (p &amp;lt; 0.01) in M-MØ and GM-MØ generated from CD14+ CD16+ monocytes and CD14 ++ CD16- monocytes respectively under LMWH exposure. qPCR analysis revealed a significant increase (p &amp;lt; 0.01) in the expression of various GM-MØ–specific genes including EGLN3, and SERPINE1 in macrophage supernatant post LMWH treatment. Expression of various M-MØ-specific marker/s such as IGF1, FOLR2, and HTR2B, is significantly up-regulated (p &amp;lt; 0.01) in Group B. Group A, although documented a decreased concentration/s (pg/ml) of Th17-type cytokine/s (IL-6 (1.88 ± 0.65 vs. 0.91 ± 0.43; p &amp;lt; 0.04 and IL-23 (17.94 ± 9.76 vs. 7.78 ± 3.43; p &amp;lt; 0.01)), at end of treatment, concentration of TGF-β was significantly higher (3909.05 ± 1248.35 vs. 2469.83 ± 1058.71; p &amp;lt; 0.01) which cue to probable maintenance in balance of inflammatory response at the time of implantation. No differences were observed in Th1-type cytokine level/s (IFN-γ and TNF-α). CXCL10, CXCL11, CXCL1, CXCL8, CCL17, CCL22 do not differ in vivo. Limitations, reasons for caution Beneficial effects of LMWH administration with a good sample size are clearly needed to confirm this hypothesis; hence result/s should be dealt with caution. Further to this, inclusion of other arm/s (corticosteroids, intralipid and/or intravenous immunoglobulin) will make the study much robust and conclusive. Wider implications of the findings The identification of atypical Th1-associated pro-inflammatory effects of LMWH drives to evaluate modulation of CCL2 expression to limit or potentiate macrophage activation in RIF; especially where anticoagulant effect is not the primary goal. Further, modification of cytokine expression/s might potentiate novel strategy derived from deregulated macrophage polarization. Trial registration number Not Applicable

1201-P: Circulating C1q/TNF-Related Protein 6 (CTRP6) and CTRP9 as Potential Biomarkers for Early Gestational Diabetes Mellitus

Background: The growing evidence has been verified that C1q/TNF-related protein 6 (CTRP6) and CTRP9, secreted by adipose tissue, can regulate glucose and lipid metabolism. However, the effect of CTRP6 and CTRP9 in gestational diabetes mellitus (GDM) is scarcely known. This study aimed to evaluate the potential clinical value of CTRP6 and CTRP9 in GDM. Methods: A total of 35 GDM subjects and 37 healthy controls, between 24 and 28 weeks of gestation, were included in the study. Diagnosis of GDM was made according to American Diabetes Association criteria. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of CTRP6 and CTRP9. Fasting insulin (FINS), IL-6, and TNF-α were detected by Luminex-xMAP technology. The main clinical characteristics, such as Anthropological data and metabolic parameters, were also obtained. Results: Circulating CTRP6, CTRP9, IL-6 and TNF-α were significantly elevated in GDM subjects compared with controls. Pearson correlation analysis showed that CTRP6 and CTRP9 were positively correlated with FPG (r = 0.280, P = 0.017 and r = 0.559, P &amp;lt; 0.001, respectively) and HOMA-IR (r = 0.250, P = 0.035 and r = 0.382, P = 0.001, respectively). Moreover, CTRP9 was also positively correlated with FINS (r = 0.253, P = 0.032), IL-6 (r = 0.283, P = 0.016) and TNF-α (r = 0.266, P = 0.024). The area under the receiver operating characteristic curve (AUC-ROC) indicated that the combination of CTRP6 and CTRP9 was more effective in predicting GDM than single indicators. Conclusion: Elevated CTRP6 and CTRP9 in GDM patients may be related to insulin resistance. And inflammatory factors may mediate the role of CTRP9 in insulin resistance. In addition, CTRP6 and CTRP9 may serve as candidate predictors in patients with early GDM. Disclosure Y.Liu: None. Y.Chen: None. W.Gu: None. L.Yin: None. H.Cao: None. M.Shi: None. Funding National Natural Science Foundation of China (81700723)

A surge in serum mucosal cytokines associated with seroconversion in children at risk for type 1 diabetes.

Autoantibodies to pancreatic islet antigens identify young children at high risk of type 1 diabetes. On a background of genetic susceptibility, islet autoimmunity is thought to be driven by environmental factors, of which enteric viruses are prime candidates. We sought evidence for enteric pathology in children genetically at-risk for type 1 diabetes followed from birth who had developed islet autoantibodies ("seroconverted"), by measuring mucosa-associated cytokines in their sera. Sera were collected 3 monthly from birth from children with a first-degree type 1 diabetes relative, in the Environmental Determinants of Islet Autoimmunity (ENDIA) study. Children who seroconverted were matched for sex, age, and sample availability with seronegative children. Luminex xMap technology was used to measure serum cytokines. Of eight children who seroconverted, for whom serum samples were available at least 6 months before and after seroconversion, the serum concentrations of mucosa-associated cytokines IL-21, IL-22, IL-25, and IL-10, the Th17-related cytokines IL-17F and IL-23, as well as IL-33, IFN-γ, and IL-4, peaked from a low baseline in seven around the time of seroconversion and in one preceding seroconversion. These changes were not detected in eight sex- and age-matched seronegative controls, or in a separate cohort of 11 unmatched seronegative children. In a cohort of children at risk for type 1 diabetes followed from birth, a transient, systemic increase in mucosa-associated cytokines around the time of seroconversion lends support to the view that mucosal infection, e.g., by an enteric virus, may drive the development of islet autoimmunity.

Abstract 5298: Multiplex immunoassay characterization of 48 cytokines, chemokines, and growth factors in colorectal cancer

Abstract Cytokines, chemokines, and growth factors are critical mediators of immune system function capable of signaling through autocrine, paracrine, and endocrine mechanisms. Their pleiotropic immunomodulatory properties allow these biomolecules to react to diverse stimuli and regulate the immune response. We developed a novel immunoassay, using Luminex xMAP technology, for the simultaneous measurement of 48 cytokines, chemokines, and growth factors in serum, plasma, and cell culture supernatant. Many of these proteins have been proposed as biomarkers of colorectal cancer (CRC) and as key regulators of the tumor microenvironment (TME). While this complex milieu can support antitumor immunity, CRC is frequently characterized by chronic inflammation driving disease progression, with the TME serving as a potent reservoir from which the cytokines, chemokines, and growth factors exert their influence. The MILLIPLEX Human Cytokine/Chemokine/Growth Factor Panel B (HCYTB-60K) assays were performed in 96-well plates and run on the Luminex LX200™ instrument. Data was acquired via xPONENT v. 4.3 software. Data analysis was performed for all immunoassays using the Belysa Immunoassay Curve Fitting Software. Commercially available serum samples obtained from colorectal cancer patients and healthy controls were evaluated for cytokine, chemokine, and growth factor expression. Of the 48 analytes tested, statistically significant increases in BAFF, sFas, SCF, and MPIF-1 were observed in colorectal cancer serum. In contrast, IL-20 and ENA-78 decreased relative to healthy controls. While the immunoassay was verified for measurement of serum and plasma samples, additional sample types may be of interest to cancer researchers. Therefore, we evaluated the ability of the 48-plex immunoassay to measure these biomarkers in CSF, urine, milk, tumor samples and exosomal isolates, establishing a profile for these proteins in each sample type. Altogether, this study uses a novel multiplex immunoassay to characterize the immunological profile of colorectal cancer serum, focusing on 48 cytokines, chemokines, and growth factors. Additionally, alternative sample types were evaluated to establish the utility of this technology for applications beyond serum, plasma, and cell culture supernatant. Citation Format: Wen-Rong Lie, Harold Steiner, Sasha Williams, Munmun Banerjee, Anthony Saporita, Brooke Gilliam, Qiang Xiao. Multiplex immunoassay characterization of 48 cytokines, chemokines, and growth factors in colorectal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5298.