Multiplex Enzyme-linked Immunosorbent Assay Research Articles

Searching for New Biomarkers of Neuroendocrine Tumors: A Comparative Analysis of Chromogranin A and Inflammatory Cytokines in Patients with Neuroendocrine Tumors.

Neuroendocrine neoplasms (NENs) present a diagnostic challenge due to their heterogeneous nature and non-specific clinical manifestations. This study aimed to explore novel biomarkers for NENs. Serum chromogranin A (CgA) levels and a panel of 48 inflammatory cytokines were analyzed in a cohort of 84 NEN patients and 40 healthy controls using enzyme-linked immunosorbent assay (ELISA) and multiplex ELISA. Significant alterations in cytokine levels were observed in the NEN patients compared to the controls, including elevated levels of pro-inflammatory cytokines, such as interleukin (IL)-6, IL-8, and tumor necrosis factor alpha (TNF-α), and reduced levels of angiogenic factors like platelet-derived growth factor-BB (PDGF-BB) and tumor necrosis factor beta (TNF-β). Notably, cytokines such as growth-regulated alpha protein (GRO-α) and TNF-β demonstrated strong potential as diagnostic markers, with receiver operating characteristic (ROC) curve analyses showing high sensitivity and specificity. Additionally, a positive correlation was found between CgA levels and several inflammatory cytokines, suggesting their synergistic role in tumor progression. These findings highlight the limited reliability of CgA alone as a diagnostic marker and underscore the importance of a multi-marker approach in diagnosing and monitoring NENs. Further research on a larger cohort is necessary to validate these biomarkers and their potential clinical applications.

Early-Stage Moderate Alcohol Feeding Dysregulates Insulin-Related Metabolic Hormone Expression in the Brain: Potential Links to Neurodegeneration Including Alzheimer's Disease.

Alzheimer's disease (AD), one of the most prevalent causes of dementia, is mainly sporadic in occurrence but driven by aging and other cofactors. Studies suggest that excessive alcohol consumption may increase AD risk. Our study examined the degree to which short-term moderate ethanol exposure leads to molecular pathological changes of AD-type neurodegeneration. Long Evans male and female rats were fed for 2 weeks with isocaloric liquid diets containing 24% or 0% caloric ethanol (n = 8/group). The frontal lobes were used to measure immunoreactivity to AD biomarkers, insulin-related endocrine metabolic molecules, and proinflammatory cytokines/chemokines by duplex or multiplex enzyme-linked immunosorbent assays (ELISAs). Ethanol significantly increased frontal lobe levels of phospho-tau, but reduced Aβ, ghrelin, glucagon, leptin, PAI, IL-2, and IFN-γ. Short-term effects of chronic ethanol feeding produced neuroendocrine molecular pathologic changes reflective of metabolic dysregulation, together with abnormalities that likely contribute to impairments in neuroplasticity. The findings suggest that chronic alcohol consumption rapidly establishes a platform for impairments in energy metabolism that occur in both the early stages of AD and alcohol-related brain degeneration.

Association of Complement Factors With Disability Progression in Primary Progressive Multiple Sclerosis.

The complement system is known to play a role in multiple sclerosis (MS) pathogenesis. However, its contribution to disease progression remains elusive. The study investigated the role of the complement system in disability progression of patients with primary progressive MS (PPMS). Sixty-eight patients with PPMS from 12 European MS centers were included in the study. Serum and CSF levels of a panel of complement components (CCs) were measured by multiplex enzyme-linked immunosorbent assay at a baseline time point (i.e., sampling). Mean (SD) follow-up time from baseline was 9.6 (4.8) years. Only one patient (1.5%) was treated during follow-up. Univariable and multivariable logistic regressions adjusted for age, sex, and albumin quotient were performed to assess the association between baseline CC levels and disability progression in short term (2 years), medium term (6 years), and long term (at the time of the last follow-up). In short term, CC played little or no role in disability progression. In medium term, an elevated serum C3a/C3 ratio was associated with a higher risk of disability progression (adjusted OR 2.30; 95% CI 1.17-6.03; p = 0.040). By contrast, increased CSF C1q levels were associated with a trend toward reduced risk of disability progression (adjusted OR 0.43; 95% CI 0.17-0.98; p = 0.054). Similarly, in long term, an elevated serum C3a/C3 ratio was associated with higher risk of disability progression (adjusted OR 1.81; 95% CI 1.09-3.40; p = 0.037), and increased CSF C1q levels predicted lower disability progression (adjusted OR 0.41; 95% CI 0.17-0.86; p = 0.025). Proteins involved in the activation of early complement cascades play a role in disability progression as risk (elevated serum C3a/C3 ratio) or protective (elevated CSF C1q) factors after 6 or more years of follow-up in patients with PPMS. The protective effects associated with C1q levels in CSF may be related to its neuroprotective and anti-inflammatory properties.

Common variation in a long non-coding RNA gene modulates variation of circulating TGF-β2 levels in metastatic colorectal cancer patients (Alliance)

BackgroundHerein, we report results from a genome-wide study conducted to identify protein quantitative trait loci (pQTL) for circulating angiogenic and inflammatory protein markers in patients with metastatic colorectal cancer (mCRC). The study was conducted using genotype, protein marker, and baseline clinical and demographic data from CALGB/SWOG 80405 (Alliance), a randomized phase III study designed to assess outcomes of adding VEGF or EGFR inhibitors to systemic chemotherapy in mCRC patients. Germline DNA derived from blood was genotyped on whole-genome array platforms. The abundance of protein markers was quantified using a multiplex enzyme-linked immunosorbent assay from plasma derived from peripheral venous blood collected at baseline. A robust rank-based method was used to assess the statistical significance of each variant and protein pair against a strict genome-wide level. A given pQTL was tested for validation in two external datasets of prostate (CALGB 90401) and pancreatic cancer (CALGB 80303) patients. Bioinformatics analyses were conducted to further establish biological bases for these findings.ResultsThe final analysis was carried out based on data from 540,021 common typed genetic variants and 23 protein markers from 869 genetically estimated European patients with mCRC. Correcting for multiple testing, the analysis discovered a novel cis-pQTL in LINC02869, a long non-coding RNA gene, for circulating TGF-β2 levels (rs11118119; AAF = 0.11; P-value < 1.4e-14). This finding was validated in a cohort of 538 prostate cancer patients from CALGB 90401 (AAF = 0.10, P-value < 3.3e-25). The analysis also validated a cis-pQTL we had previously reported for VEGF-A in advanced pancreatic cancer, and additionally identified trans-pQTLs for VEGF-R3, and cis-pQTLs for CD73.ConclusionsThis study has provided evidence of a novel cis germline genetic variant that regulates circulating TGF-β2 levels in plasma of patients with advanced mCRC and prostate cancer. Moreover, the validation of previously identified pQTLs for VEGF-A, CD73, and VEGF-R3, potentiates the validity of these associations.

Granulocyte- Macrophage Colony-Stimulating Factor Reverses Immunosuppression Acutely Following a Traumatic Brain Injury and Hemorrhage Polytrauma in a Juvenile Male Rat Model.

Traumatic brain injury (TBI) is a common cause of morbidity and mortality in children. We have previously shown that TBI with a concurrent extracranial injury reliably leads to post-injury suppression of the innate and adaptive immune systems. In patients with post-injury immune suppression, if immune function could be preserved, this might represent a therapeutic opportunity. As such, we examined, in an animal injury model, whether systemic administration of granulocyte macrophage colony-stimulating factor (GM-CSF) could reverse post-injury immune suppression and whether treatment was associated with neuroinflammation or functional deficit. Prepubescent male rats were injured using a controlled cortical impact model and then subjected to removal of 25% blood volume (TBI/H). Sham animals underwent surgery without injury induction, and the treatment groups were sham and injured animals treated with either saline vehicle or 50 μg/kg GM-CSF. GM-CSF was administered following injury and then daily until sacrifice at post-injury day (PID) 7. Immune function was measured by assessing tumor necrosis factor-α (TNF-α) levels in whole blood and spleen following ex vivo stimulation with pokeweed mitogen (PWM). Brain samples were assessed by multiplex enzyme-linked immunosorbent assay (ELISA) for cytokine levels and by immunohistochemistry for microglia and astrocyte proliferation. Neuronal cell count was examined using cresyl violet staining. Motor coordination was evaluated using the Rotarod performance test. Treatment with GM-CSF was associated with a significantly increased response to PWM in both whole blood and spleen. GM-CSF in injured animals did not lead to increases in levels of pro-inflammatory cytokines in brain samples but was associated with significant increases in counted astrocytes. Finally, while injured animals treated with saline showed a significant impairment on behavioral testing, injured animals treated with GM-CSF performed similarly to uninjured animals. GM-CSF treatment in animals with combined injury led to increased systemic immune cell response in whole blood and spleen in the acute phase following injury. Improved immune response was not associated with elevated pro-inflammatory cytokine levels in the brain or functional impairment.

Clinical significance of urinary inflammatory biomarkers in patients with IgA nephropathy

BackgroundIgA nephropathy (IgAN) is the most common type of primary glomerulonephritis, although the definitive markers are unknown. We aimed to investigate the clinical significance of urinary cytokines in patients with IgAN.MethodsFrom 2009 to 2018, the patients were divided into three groups: IgAN (n = 191), disease control (n = 53), and normal control (n = 76). We used a multiplex enzyme-linked immunosorbent assay to measure 16 selected urinary inflammatory cytokines, evaluated the correlation between clinical and pathological features following regression analysis on progression.ResultsThe IgAN group exhibited significantly different levels of urinary cytokines compared to the normal control and disease control groups. Urinary levels of B-cell-activating factor, vascular endothelial growth factor receptor-2, monocyte chemoattractant protein-1, C–X–C motif chemokine 10, C–X–C motif ligand 16, epidermal growth factor (EGF), endocan, endostatin, growth/differentiation factor-15 (GDF-15), interleukin-6 (IL-6), mannose-binding lectin, transferrin receptor, and kidney injury molecule-1 were significantly correlated with both the estimated glomerular filtration rate and urine protein–creatinine ratio. In a multivariate Cox regression analysis, urinary EGF (hazard ratio [HR] 0.40, 95% confidence interval [CI] 0.17–0.95, P = 0.04), GDF-15 (HR 2.45, 95% CI 1.01–5.94, P = 0.048), and IL-6 (HR 3.02, 95% CI 1.05–8.64, P = 0.04) were associated with progression in IgAN.ConclusionsUrinary inflammatory biomarkers may serve as alternative predictive biomarkers in patients with IgAN. Further studies are needed to elucidate the physiological mechanisms and confirm the results.

Single-cell RNA sequencing reveals recruitment of the M2-like CCL8high macrophages in Lewis lung carcinoma-bearing mice following hypofractionated radiotherapy

BackgroundTumor-associated macrophages (TAMs) play a pivotal role in reshaping the tumor microenvironment following radiotherapy. The mechanisms underlying this reprogramming process remain to be elucidated.MethodsSubcutaneous Lewis lung carcinoma (LLC) murine model was treated with hypofrationated radiotherapy (8 Gy × 3F). Single-cell RNA sequencing was utilized to identify subclusters and functions of TAMs. Multiplex assay and enzyme-linked immunosorbent assay (ELISA) were employed to measure serum chemokine levels. Bindarit was used to inhibit CCL8, CCL7, and CCL2. The infiltration of TAMs after combination treatment with hypofractionated radiotherapy and Bindarit was quantified with flow cytometry, while the influx of CD206 and CCL8 was assessed by immunostaining.ResultsTranscriptome analysis identified a distinct subset of M2-like macrophages characterized by elevated Ccl8 expression level following hypofractionated radiotherapy in LLC-bearing mice. Remarkbly, hypofractionated radiotherapy not only promoted CCL8high macrophages infiltration but also reprogrammed them by upregulating immunosuppressive genes, thereby fostering an immunosuppressive tumor microenvironment. Additioinally, hypofractionated radiotherapy enhanced the CCL signaling pathway, augmenting the pro-tumorigenic functions of CCL8high macrophages and boosting TAMs recruitment. The adjunctive treatment combining hypofractionated radiotherapy with Bindarit effectively reduced M2 macrophages infiltration and prolonged the duration of local tumor control.ConclusionsHypofractionated radiotherapy enhances the infiltration of CCL8high macrophages and amplifies their roles in macrophage recruitment through the CCL signaling pathway, leading to an immunosuppressive tumor microenvironment. These findings highlight the potential of targeting TAMs and introduces a novel combination to improve the efficacy of hypofractionated radiotherapy.

A distinct immune cytokine profile is associated with morning cortisol and repeated stress.

The objective of this study was to investigate possible immune cytokine trends throughout a week-long surgical simulation mass-casualty training session in order to determine the effects of stress inoculation on the immune system. Thirty-seven military medical students participated in a hyper-realistic surgical simulation training event conducted at Strategic Operations site in San Diego, California. Salivary samples were collected every morning of the stress training exercise for 4 consecutive days. Cortisol, along with a panel of 42 immune cytokines, was measured using multiplex enzyme-linked immunosorbent assays from Eve Technologies. The determined concentrations were averaged and plotted on a scatter plot, and then points were fit to a second-order polynomial trendline of best fit to measure. The cytokines epidermal growth factor, growth-related oncogene-α, interleukin (IL)-1α, and platelet-derived growth factor-AA followed a noted pattern of cortisol decrease throughout the week. In addition, cytokines IL-27, granulocyte colony stimulating factor, IL-10, and IL-13 demonstrated a late peak, followed by a return to baseline at the conclusion of training. Finally, the cytokine monocyte chemoattractant protein-1 displayed a decline throughout the week followed by an increase on the last day of stress training. Altogether, these results help to identify important biomarkers that may help to improve long-term stress adaptation and prevent post-traumatic stress disorder following exposure to repeated stress.

Automatic microdispenser-integrated multiplex enzyme-linked immunosorbent assay device with autonomously driven centrifugal microfluidic system.

In this study, we established the control and design theory of an autonomously driven dispenser at a steady rotation speed and proposed a dispenser-integrated multiplex enzyme-linked immunosorbent assay (ELISA) device. In establishing the theory of the dispenser, we estimated the flow rate in the dispenser and the applied pressure onto the passive valves, so that the suitable burst pressure of the valves and flow rate could be designed. The dispenser-integrated multiplex ELISA device has the potential to perform flow control for executing an ELISA of 6 samples/standards per chip or 18 samples/standards per compact disk by just steadily rotating a chip. In the immunoassay evaluation of the device using mouse IgG detection, it was confirmed that the device could assay 5 μL of several standards in just 30 min without nonspecific reactions, and although this system has a high limit of detection (LOD, 63.4-164 pg mL-1) it is equal to that of manual assay with a titer plate. The device can be fabricated by transferring the microchannel pattern from a mold without complex assembly or alignment, and it can control the liquid operation by just steadily rotating. Thus, the device system developed will contribute to reducing the cost of fabricating chips and control equipment for ELISA systems. Consequently, a compact, portable, and low-cost ELISA system for point-of-care testing is expected to be realized.

Syndecan-1 as the Effect or Effector of the Endothelial Inflammatory Response?

IntroductionSyndecan-1 is a heparan sulfate proteoglycan found in the glycocalyx of vascular endothelial cells. Serum levels of syndecan-1 have repeatedly been demonstrated to increase following traumatic injury and shock, but it is unclear whether syndecan-1 plays an active role in the inflammatory response or is simply a biomarker of a state of hypoperfusion. The aim of this study was to identify the role of syndecan-1 role in the inflammatory process in the absence of trauma. MethodsMale mice were randomized into five groups (n = 3). Four groups received increasing concentrations of syndecan-1 (1, 10, 100, and 1000pg/mL per blood volume) and a fifth group was given normal saline as a control via intravenous injection. These concentrations were selected based on previous syndecan-1 enzyme-linked immunosorbent assay data acquired following induced hemorrhagic shock in mice resulting in serum levels of 10-6000 pg/mL. Mice from each group were sacrificed at 1-, 4-, and 24-h time points for serum biomarker evaluation. A multiplex enzyme-linked immunosorbent assay was performed to analyze proinflammatory cytokines and chemokines including interleukin (IL)-1a, IL-1b, IL-2, IL-3, IL-4, IL-6, IL-10, IL-12, IL-17, monocyte chemoattractant protein-1, TNF-α, macrophage inflammatory protein-1α, granulocyte-macrophage colony-stimulating factor, and normal T cell expressed and presumably secreted levels. Whole blood was analyzed via rotational thromboelastometry in a separate group of mice dosed with syndecan-1 at 1000 pg/mL and compared to sham mice at 1 h. ResultsTumor necrosis factor-α was significantly elevated in the 1000 pg/mL group compared to sham animals. There were no significant changes in IL-1a, IL-1b, IL-2, IL-3, IL-4, IL-6, IL-10, IL-12, monocyte chemoattractant protein--1, macrophage inflammatory protein-1α, granulocyte-macrophage colony-stimulating factor, or normal T cell expressed and presumably secretedat 1, 4, and 24 h for any group when compared to mice receiving saline alone. No significant differences were noted in coagulability between the 1000 pg/mL syndecan-1 group and shams at 1 h ConclusionsInflammatory cytokine concentrations did not change with increasing dosage of syndecan-1 within mice at any timepoint, except for an acute change in tumor necrosis factor-α which was transient. Based on our results, syndecan-1 appears to be a biomarker for inflammation rather than an active participant in eliciting an inflammatory response. Further research will focus on the role of syndecan-1 following hemorrhagic shock.

Roles of Inflammatory Biomarkers in Exhaled Breath Condensates in Respiratory Clinical Fields.

Exhaled condensates contain inflammatory biomarkers; however, their roles in the clinical field have been under-investigated. We prospectively enrolled subjects admitted to pulmonology clinics. We collected exhaled breath condensates (EBC) and analysed the levels of six and 12 biomarkers using conventional and multiplex enzyme-linked immunosorbent assay, respectively. Among the 123 subjects, healthy controls constituted the largest group (81 participants; 65.9%), followed by the preserved ratio impaired spirometry group (21 patients; 17.1%) and the chronic obstructive pulmonary disease (COPD) group (21 patients; 17.1%). In COPD patients, platelet derived growth factor-AA exhibited strong positive correlations with COPD assessment test (ρ=0.5926, p=0.0423) and COPD-specific version of St. George's Respiratory Questionnaire (SGRQ-C) score (total, ρ=0.6725, p=0.0166; activity, ρ=0.7176, p=0.0086; and impacts, ρ=0.6151, p=0.0333). Granzyme B showed strong positive correlations with SGRQ-C score (symptoms, ρ=0.6078, p=0.0360; and impacts, ρ=0.6007, p=0.0389). Interleukin 6 exhibited a strong positive correlation with SGRQ-C score (activity, ρ=0.4671, p=0.0378). The absolute serum eosinophil and basophil counts showed positive correlations with pro-collagen I alpha 1 (ρ=0.6735, p=0.0164 and ρ=0.6295, p=0.0283, respectively). In healthy subjects, forced expiratory volume in 1 second (FEV1)/forced vital capacity demonstrated significant correlation with CC chemokine ligand 3 (CCL3)/macrophage inflammatory protein 1 alpha (ρ=0.3897 and p=0.0068). FEV1 exhibited significant correlation with CCL11/eotaxin (ρ=0.4445 and p=0.0017). Inflammatory biomarkers in EBC might be useful to predict quality of life concerning respiratory symptoms and serologic markers. Further studies are needed.

Follow-up of humoral immune response after HPV vaccination using first-void urine: A longitudinal cohort study.

Assessment of humoral immune responses following human papillomavirus (HPV) vaccination currently relies on invasive blood sampling. This longitudinal cohort study explores the usability of first-void urine as a noninvasive alternative sample for antibody detection. In this study, 58 women receiving three doses of the 9vHPV vaccine within a Gardasil9 (9vHPV) Phase III randomized controlled trial were included. Participants provided paired first-void urine and blood samples before vaccination (M0), 1 month after the third dose (M7), and ~3 years after the third dose (M43). Type-specific antibody responses to the 9vHPV types were analyzed in 174 first-void urine and 172 serum samples using a virus-like particle-based IgG multiplex enzyme-linked immunosorbent assay. Additionally, total human IgG concentrations were determined using the BioPlex assay. At M7, 1 month after complete 9vHPV vaccination, 95%-100% of first-void urine and 100% of serum samples had detectable concentrations, varying by HPV type. At M43, 84%-100% of first-void urine and 98%-100% of serum samples had HPV-specific antibody concentrations. Results show significant Spearman rank correlations between type-specific HPV-antibody concentrations for paired first-void urine and serum at all time points. This study confirms the potential feasibility of utilizing first-void urine as a noninvasive immunological sample within HPV vaccine trials.

A Prospective Study of Bevacizumab and Neoadjuvant Concurrent Chemoradiation in Locally Advanced Esophageal Squamous Cell Carcinoma: Paradoxical Increase in Circulating Vascular Endothelial Growth Factor-A and Effect on Outcome

In the prior prospective biomarker study, high serum vascular endothelial growth factor-A (VEGF-A) was associated with a poor prognosis. We conducted a prospective phase II trial of adding Bevacizumab, an anti-VEGF-A monoclonal antibody, to neoadjuvant concurrent chemoradiation (neoCCRT) for patients with locally advanced esophageal squamous cell carcinoma (LA-ESCC). This prospective biomarker study aims to evaluate the expressions of angiogenesis-associated circulating biomarkers before and after neoCCRT and compare clinical outcomes for patients receiving platinum/5-fluorouracil (PF) with or without Bevacizumab. Patients with biopsy-proven resectable non-T4 LA-ESCC were enrolled for the prospective phase II trial investigating PF-neoCCRT plus Bevacizumab (BPF group). A parallel patient cohort enrolled in a prospective biomarker study receiving PF-neoCCRT was included in the present analysis as the control group (PF group). Radiotherapy was delivered with 40 Gy in 20 fractions. All patients had restaging workups after enoCCRT and underwent radical esophagectomy if the disease remained resectable. Serums were collected before and after neoCCRT. The serum concentrations of angiogenesis-associated biomarkers were determined by the multiplex enzyme-linked immunosorbent assay. Survival analyses were performed by the Kaplan-Meier method. The t-test and log-rank test were used to compare differences in biomarker expression and survival between groups. From 2016 to 2019, 43 patients (BPF/PF group: 21/22) were enrolled in the study. Twenty patients in each group had serum samples available for biomarker analysis. 15 out of 21 patients in the BPF group and 20 out of 22 patients in the PF group underwent radical esophagectomy. Six patients in the BPF group and nine patients in the PF group achieved pathological complete responses. The median overall survival for the BPF and PF group was 20.8 months and not-reached, respectively (hazard ratio = 1.33, long rank p = 0.58). In the BPF group, the serum VEGF-A level was significantly increased from an average value of 446 pg/mL to 723 pg/mL after neoCCRT (p = 0.037), while its level was decreased from 815 ng/mL to 380 pg/mL in the PF group (p = 0.104). In addition, the expression value of circulating Angiopoietin-1 was not changed in the BPF group (before neoCCRT, mean value = 828 pg/mL; after neoCCRT, mean value 762 pg/mL, p = 0.67). In contrast, serum Angiopoietin-1 level was reduced from an average value of 659 pg/mL before neoCCRT to 271 pg/mL after neoCCRT (p = 0.002) in the PF group. The addition of Bevacizumab to PF-neoCCRT did not improve pathological or survival outcomes in patients with resectable LA-ESCC. Adding a single dose of Bevacizumab paradoxically increases circulating VEGF-A while maintaining the Angiopoietin-1 serum level after neoCCRT. Further investigation by using additional VEGF-A inhibition may be required to achieve sustained angiogenesis blocked for tumor control.

Abstract PO-068: Harmony between transdifferentiated neuron and immunosuppressive tumor microenvironment

Abstract Solid tumors sculpt their microenvironments to maximize their growth and metastatic potential. Previously, we showed that the loss of p53 in head and neck squamous cell carcinoma (HNSCC) cells reprograms tumor-associated neurons (TANs) towards an adrenergic phenotype and promotes tumor progression. The impact of this neuronal reprogramming on the tumor immune microenvironment remains undefined. To understand how neuronal transdifferentiation regulates the antitumor immune response, we tested our hypothesis that shuttling cancer-derived microRNAs to TANs via exosomes induces an immunosuppressive neuronal phenotype in the tumor microenvironment (TME) that undermines the anti-tumor immune response. We first checked the stability of HNSCC-derived exosomes and their cargo (i.e., microRNA [miR]). Then, we assessed the miR transfer to TANs both in vitro and in vivo using HNSCC syngeneic models, and we measured miR enrichment in the trigeminal ganglia neurons after exosome injection. We used RNA sequencing to assess the trigeminal ganglia’s transcriptome and a single-cell multiplex enzyme-linked immunosorbent assay to evaluate the functional status of CD8+ T cells cocultured with cancer cells with or without TANs. Cancer cell–derived exosomes and their cargo were stable for up to 14 days in the TME and were taken up by TANs both in vitro and in vivo. The injection of exosomes into the peripheral nerves led to miR324a and miR21 upregulation in the associated trigeminal ganglionic soma, indicating that cancer-derived exosomes mediated crosstalk between cancer and neuronal cells. The RNA sequencing of human and mouse neuronal cells transfected with miR21 and miR324a revealed many differentially regulated immune pathways, including the neuroinflammation, T-cell receptor signaling, toll-like receptor, macrophage differentiation and growth inhibition, antigen-processing, interleukin (IL)-6, programmed cell death protein 1/programmed death-ligand 1, acute-phase response signaling, IL-8, wound-healing, IL-9, IL-17A, IL-23, IL-2, IL-10, IL-7, IL-4, IL-33, IL-36, CD28, CD40, signal transducer and activator of transcription 3, and nuclear factor kappa B pathways. Furthermore, the single-cell proteomic assay indicated that the CD8+ T cells cultured with neuronal cells were functionally distinct from the activated T cells co-cultured with neuronal and cancer cells. The co-culture with cancer cells and neuronal cells significantly downregulated the effector phenotype markers (e.g., granzyme B, interferon gamma, tumor necrosis factor alpha) in activated T cells, indicating that the interaction of cancer cells with neurons regulates the immunosuppressive TME. Our study showed the ability of cancer-derived exosomes to reprogram neurons and downstream to that to regulate the tumor immune environment in HNSCC. Our data suggest that transdifferentiating of TANs induce immunosuppression by direct inhibitory signal and immune checkpoint expression in the TME of HNSCC. Future studies are needed to investigate TANs as a potential target in immune oncology. Citation Format: Shamima Akhter, Tongxin Xie, Simone Anfossi, Adewale Adebayo, Frederico O. Gleber-Netto, Melanie Winkle, Masayoshi Shimitzu, Weilu Zhao, Megan L. Uhelski, Yan Li, George A. Calin, Moran Amit. Harmony between transdifferentiated neuron and immunosuppressive tumor microenvironment [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-068.

Predictive Value of Early Inflammatory Markers in Trauma Patients Based on Transfusion Status

IntroductionSeven key inflammatory biomarkers were recently found to be associated with the risk of mortality in a multicenter study of massively transfused patients. The aim of this prospective single-center study was to determine which of these early inflammatory markers could predict 30-d mortality among all critically injured trauma patients. MethodsSerum samples were collected at 6, 24, and 72 h from 238 consecutive patients admitted to the intensive care unit following traumatic injury. Inflammatory markers syndecan-1, eotaxin, IL-1ra, IL-6, IL-8, IL-10, IP-10, and MCP-1 were analyzed via multiplex enzyme-linked immunosorbent assay. Subgroup analysis was performed for patients undergoing massive transfusion (≥5 red blood cells), submassive transfusion (1-4 red blood cells), or no transfusion during the first 4 h postinjury. The primary outcome of 30-d survival was modeled as a function of each biomarker and confounders using repeat measures logistic regression. ResultsPatients had a median age of 51.3 y [33.7, 70.2], 70.6% were male, 17.4% experienced penetrating trauma, and had a median injury severity score of 22 [14, 33]. IL-1ra, IL-8, IL-10, and MCP-1 were significantly increased during the first 72 h in nonsurvivors (n = 31). Elevated IL-1ra, IL-8, IL-10, and MCP-1 at 6 h postinjury were associated with 30-d mortality. By contrast, serum syndecan-1 and eotaxin levels were not associated with mortality at any time point. IL-8 and lactate were increased at 6 h in 30-d nonsurvivors for patients receiving submassive transfusion (n = 78). ConclusionsEarly evaluations of IL-1ra, IL-8, IL-10, and IP-10 within 6 h of injury are useful predictors of 30-d mortality. Subgroup analysis suggests that transfusion status does not significantly affect early inflammatory markers. Level of EvidenceLevel III, prognostic/epidemiological.

Dietary soy prevents fetal demise, intrauterine growth restriction, craniofacial dysmorphic features, and impairments in placentation linked to gestational alcohol exposure: Pivotal role of insulin and insulin-like growth factor signaling networks.

Prenatal alcohol exposure can impair placentation and cause intrauterine growth restriction (IUGR), fetal demise, and fetal alcohol spectrum disorder (FASD). Previous studies showed that ethanol's inhibition of placental insulin and insulin-like growth factor, type 1 (IGF-1) signaling compromises trophoblastic cell motility and maternal vascular transformation at the implantation site. Since soy isolate supports insulin responsiveness, we hypothesized that dietary soy could be used to normalize placentation and fetal growth in an experimental model of FASD. Pregnant Long-Evans rat dams were fed with isocaloric liquid diets containing 0% or 8.2% ethanol (v/v) from gestation day (GD) 6. Dietary protein sources were either 100% soy isolate or 100% casein (standard). Gestational sacs were harvested on GD19 to evaluate fetal resorption, fetal growth parameters, and placental morphology. Placental insulin/IGF-1 signaling through Akt pathways was assessed using commercial bead-based multiplex enzyme-linked immunosorbent assays. Dietary soy markedly reduced or prevented the ethanol-associated fetal loss, IUGR, FASD dysmorphic features, and impairments in placentation/maturation. Furthermore, ethanol's inhibitory effects on the placental glycogen cell population at the junctional zone, invasive trophoblast populations at the implantation site, maternal vascular transformation, and signaling through the insulin and IGF1 receptors, Akt and PRAS40 were largely abrogated by co-administration of soy. Dietary soy may provide an economically feasible and accessible means of reducing adverse pregnancy outcomes linked to gestational ethanol exposure.

MultiSero: An Open-Source Multiplex-ELISA Platform for Measuring Antibody Responses to Infection.

A multiplexed enzyme-linked immunosorbent assay (ELISA) that simultaneously measures antibody binding to multiple antigens can extend the impact of serosurveillance studies, particularly if the assay approaches the simplicity, robustness, and accuracy of a conventional single-antigen ELISA. Here, we report on the development of multiSero, an open-source multiplex ELISA platform for measuring antibody responses to viral infection. Our assay consists of three parts: (1) an ELISA against an array of proteins in a 96-well format; (2) automated imaging of each well of the ELISA array using an open-source plate reader; and (3) automated measurement of optical densities for each protein within the array using an open-source analysis pipeline. We validated the platform by comparing antibody binding to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) antigens in 217 human sera samples, showing high sensitivity (0.978), specificity (0.977), positive predictive value (0.978), and negative predictive value (0.977) for classifying seropositivity, a high correlation of multiSero determined antibody titers with commercially available SARS-CoV-2 antibody tests, and antigen-specific changes in antibody titer dynamics upon vaccination. The open-source format and accessibility of our multiSero platform can contribute to the adoption of multiplexed ELISA arrays for serosurveillance studies, for SARS-CoV-2 and other pathogens of significance.

High levels of pro-inflammatory SARS-CoV-2-specific biomarkers revealed by in vitro whole blood cytokine release assay (CRA) in recovered and long-COVID-19 patients.

Cytokines induced by SARS-CoV-2 infection play a crucial role in the pathophysiology of COVID-19 and hyperinflammatory responses have been associated with poor clinical outcomes, with progression to severe conditions or long-term subacute complications named as long-COVID-19. In this cross-sectional study, we aimed to evaluate a set of antigen-specific inflammatory cytokines in blood from recovered COVID-19 individuals or who suffered a post-acute phase of SARS-CoV-2 infection compared to healthy individuals with no history of COVID-19 exposition or infection. Interferon-gamma (IFN-γ), IFN-γ-induced protein 10 (IP-10), tumor necrosis factor (TNF), IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, and IL-17A were quantified by multiplex cytometric bead assay and enzyme-linked immunosorbent assay after stimulation of whole blood with recombinant Spike protein from SARS-CoV-2. Additionally, all participants have evaluated for anti-(S) protein-specific IgG antibodies. Clinical specimens were collected within two months of COVID-19 diagnosis. A total of 47 individuals were enrolled in the study, a median age of 43 years (IQR = 14.5), grouped into healthy individuals with no history of infection or exposure to SARS-CoV-2 (unexposed group; N = 21); and patients from the Health Complex of the Rio de Janeiro State University (UERJ), Brazil, who were SARS-CoV-2 positive by RT-PCR (COVID-19 group)-categorized as recovered COVID-19 (N = 11) or long-COVID-19 (N = 15). All COVID-19 patients presented at least one signal or symptom during the first two weeks of infection. Six patients were hospitalized and required invasive mechanical ventilation. Our results showed that COVID-19 patients had significantly higher levels of IFN-γ, TNF, IL-1β, IL-2, IL-6, IL-8, and IP-10 than the unexposed group. The long-COVID-19 group has presented significantly high levels of IL-1β and IL-6 compared to unexposed individuals, but not from recovered COVID-19. A principal-component analysis demonstrated 84.3% of the total variance of inflammatory-SARS-CoV-2 response in the first two components, and it was possible to stratify IL-6, TNF, IL-1β, IL-10, and IL-2 as the top-five cytokines which are candidates to discriminate COVID-19 group (including long-COVID-19 subgroup) and healthy unexposed individuals. We revealed important S protein-specific differential biomarkers in individuals affected by COVID-19, bringing new insights into the inflammatory status or SARS-CoV-2 exposition determination.

Colony-Stimulating Factor-1 Receptor Inhibition Transiently Attenuated the Peripheral Immune Response to Experimental Traumatic Brain Injury.

To investigate microglial mechanisms in central and peripheral inflammation after experimental traumatic brain injury (TBI), we inhibited the colony-stimulating factor-1 receptor (CSF-1R) with PLX5622 (PLX). We hypothesized that microglia depletion would attenuate central inflammation acutely with no effect on peripheral inflammation. After randomization, male mice (n = 105) were fed PLX or control diets (21 days) and then received midline fluid percussion injury or sham injury. Brain and blood were collected at 1, 3, or 7 days post-injury (DPI). Immune cell populations were quantified in the brain and blood by flow cytometry. Cytokines (interleukin [IL]-6, IL-1β, tumor necrosis factor-α, interferon-γ, IL-17A, and IL-10) were quantified in the blood using a multi-plex enzyme-linked immunosorbent assay. Data were analyzed using Bayesian multi-variate, multi-level models. PLX depleted microglia at all time points and reduced neutrophils in the brain at 7 DPI. PLX also depleted CD115+ monocytes, reduced myeloid cells, neutrophils, and Ly6Clow monocytes in blood, and elevated IL-6. TBI induced a central and peripheral immune response. TBI elevated leukocytes, microglia, and macrophages in the brain and elevated peripheral myeloid cells, neutrophils, Ly6Cint monocytes, and IL-1β in the blood. TBI lowered peripheral CD115+ and Ly6Clow monocytes in the blood. TBI PLX mice had fewer leukocytes and microglia in the brain at 1 DPI, with elevated neutrophils at 7 DPI compared to TBI mice on a control diet. TBI PLX mice also had fewer peripheral myeloid cells, CD115+, and Ly6Clow monocytes in the blood at 3 DPI, but elevated Ly6Chigh, Ly6Cint, and CD115+ monocyte populations at 7 DPI, compared to TBI mice on a control diet. TBI PLX mice had elevated proinflammatory cytokines and lower anti-inflammatory cytokines in the blood at 7 DPI compared to TBI mice on a control diet. CSF-1R inhibition reduced the immune response to TBI at 1 and 3 DPI, but elevated peripheral inflammation at 7 DPI.

Impact of soluble tumor necrosis factor-related apoptosis-inducing ligand released by engineered adipose mesenchymal stromal cells on white blood cells

Background aimsThe proapoptotic protein tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is physiologically expressed by immune cells and performs regulatory functions in infections, autoimmune diseases and cancer, where it acts as a tumor suppressor. Adipose-derived mesenchymal stromal cells (AD-MSCs) also may play immunomodulatory roles in both primary and acquired immune responses. We have previously demonstrated the efficacy of an anticancer gene therapy based on AD-MSC engineered to secrete a soluble TRAIL variant (sTRAIL) against pancreatic cancer. However, the impact of AD-MSC sTRAIL on leukocyte subsets has been not yet considered also to predict a possible immunotoxicity profile in the clinical translation of this cell-based anticancer strategy. MethodsMonocytes, polymorphonuclear cells and T lymphocytes were freshly isolated from the peripheral blood of healthy donors. Immunophenotype and functional (DR4 and DR5) and decoy (DcR1 and DcR2) TRAIL receptors were tested by flow cytometry. The viability of white blood cells treated with sTRAIL released by gene-modified AD-MSC or co-cultured with AD-MSC sTRAIL was then evaluated by both metabolic assays and flow cytometry. In addition, cytokine profile in co-cultures was analyzed by multiplex enzyme-linked immunosorbent assay. ResultsMonocytes and polymorphonuclear cells showed high positivity for DR5 and DcR2, respectively, whereas T cells revealed negligible expression of all TRAIL receptors. Irrespective of TRAIL receptors’ presence on the cell membrane, white blood cells were refractory to the proapoptotic effect displayed by sTRAIL secreted by gene-modified AD-MSC, and direct cell-to-cell contact with AD-MSC sTRAIL had negligible impact on T-cell and monocyte viability. Cytokine crosstalk involving interleukin 10, tumor necrosis factor alpha, and interferon gamma secreted by T lymphocytes and vascular endothelial growth factor A and interleukin 6 released by AD-MSC was highlighted in T-cell and AD-MSC sTRAIL co-cultures. ConclusionsIn summary, this study demonstrates the immunological safety and thus the clinical feasibility of an anticancer approach based on AD-MSC expressing the proapoptotic molecule sTRAIL.