Luminex Assay Research Articles

Comparison of serum cytokines levels in normal-weight and overweight patients with first-episode drug-naïve major depressive disorder.

Abnormal levels of blood cytokines have been demonstrated to be associated with both excess weight and major depressive disorder (MDD). However, few studies have addressed the direct effect of body mass index (BMI) on basal serum cytokines in individuals with first-episode drug-naïve MDD. A total of 49 patients with first-episode drug-naïve MDD were categorized into normal weight (18.5 ≤ BMI < 25 kg/m2) and overweight (25 ≤ BMI < 30 kg/m2) groups according to WHO-criteria. The severity of depressive symptoms was assessed using the 24-items Hamilton Depression Scale (HAMD-24). A total of 37 cytokines were measured using Multiplex Luminex Assays. The scores of HAMD-24 and the levels of serum cytokines between normal weight group and overweight group were compared. Multiple linear regression analysis was performed to evaluate the association between abnormal serum cytokines levels and group after adjusting for HAMD-24 scores. The correlation between BMI and the scores of HAMD-24 and the levels of serum cytokines was evaluated using Pearson correlation analysis. The scores of HAMD-24 in overweight group were significantly higher than normal weight group (t = -2.930, P = 0.005). Moreover, the levels of IL-1α, IL-1RA, IL-3, CXCL10, TNF-α, and ICAM-1 in overweight patients with MDD were significantly higher than those in normal-weight patients with MDD (all P < 0.05). Furthermore, after adjustment for HAMD-24 scores, there was a significant correlation between abnormal serum cytokines levels (IL-1α, IL-1RA, IL-3, CXCL10, TNF-α, and ICAM-1) and group (all P < 0.05). Additionally, BMI was positively correlated to the serum levels of IL-1α (r = 0.428, P = 0.002), IL-3 (r = 0.529, P < 0.001), IL-6 (r = 0.285, P = 0.050), IL-10 (r = 0.423, P = 0.003), IL-12 (r = 0.367, P = 0.010), IL-15 (r = 0.300, P = 0.036), CXCL10 (r = 0.316, P = 0.030), TNF-α (r = 0.338, P = 0.021), and ICAM-1 (r = 0.440, P = 0.002) in MDD patients. These results provide direct evidence, probably for the first time, that overweight may be associated with several serum cytokines in patients with first-episode drug-naïve MDD. The underlying mechanisms are unclear and require further investigation.

Seroprevalence and systemic immune biomarkers associated with Toxoplasma gondii infection in blood donors from Southern Brazil

The high seroprevalence of Toxoplasma gondii infection in Blood Banks could be a potential risk for contamination of blood recipients. The discovery of new biomarkers may help to distinguish between seropositive and seronegative donors. This study determined the seroprevalence and profile of systemic immune biomarkers associated with Toxoplasma gondii infection among blood donors from Southern Brazil. Peripheral blood was collected from 510 blood donors (52.2 % male; mean age: 36.61), 310, and 200 from Erechim, and Chapecó municipalities, respectively. Specific Toxoplasma gondii IgG and IgM antibodies were detected by Eletrochemioluminescence. Nested PCR and qPCR were performed to detectToxoplasma gondii DNA. Twenty-seven inflammatory factors were analyzed using a high-performance Luminex assay. Among 310 blood donors from Erechim, 44.5 % (138/310) were IgM(−)/IgG(+), and 1.3 % (4/310) were IgM(+)/IgG(+), while out of 200 blood donors from Chapeco, 42.5 % (85/200) were IgM(−)/IgG(+), and 2 % (4/200) were IgM(+)/ IgG(+). We did not find Toxoplasma gondii DNA in the samples analyzed by Nested PCR and qPCR.Additionally, IgM(−)/IgG(+) donors presented higher levels ofdistinct systemic mediators, and were indicated to be high producers of several systemic mediators (CCL11, CCL2, CCL3, CCL4, CXCL10, IL-1β, IL-17, IFN-γ, IL-4, IL-9, IL-13, IL-10, IL-1Ra, vascular endothelial growth factor/VEGF, platelet-derived growth factor/PDGF, granulocyte–macrophage colony-stimulating factor/GM-CSF, and IL-7). However, IgM(+)/IgG(+) donors were found as high producers of CXCL8, CXCL10, CCL4, IL-1β, IL-1Ra, IL-9, IL-13, and PDGF, while IgM(−)/IgG(−) donors showed unaltered levels for the most soluble mediators evaluated. These distinct biomarker signatures might help identify potential factors to distinguish between IgM(−) and IgM(+) donors.

Cytokine profiles and CD8+ T cells in the occurrence of acute and chronic hepatitis B.

We explore the expression of functional molecules on CD8+ T lymphocytes, cytokines concentration, and their correlation to occurrence of hepatitis B and hepatitis B virus (HBV) desoxyribose nucleic acid (DNA), hepatitis B surface antigen (HBsAg), hepatitis B envelope antigen (HBeAg), and alanine aminotransferase (ALT) in patients infected with HBV. This is a single center study. 32 patients with acute hepatitis B (AHB), 30 patients with immune tolerant (IT) phase chronic HBV infected, and 50 patients with chronic hepatitis B (CHB) were enrolled. The activation molecules (CD69) and the apoptosis-inducing molecules (CD178) on surface of CD8+ T lymphocytes were tested by the flow cytometry. Fms-like tyrosine kinase 3 ligand (Flt-3L), interleukin 17A (IL-17A), interferon γ (IFN-γ), and Interferon α2 (IFN-α2) were quantitated by Luminex assay. We use linear regression analysis to analyze their correlations to ALT, HBV DNA, HBsAg, and HBeAg. The frequency of CD69+CD8+ T lymphocytes in CHB and AHB groups were increased significantly compared with IT group (4.19[3.01, 6.18]% and 4.45[2.93, 6.71]% vs. 3.02[2.17, 3.44]%; H=26.207, P=0.001; H=28.585, P=0.002), and the mean fluorescence intensity (MFI) of CD69 in AHB group was significantly higher than IT and CHB groups (27.35[24.88, 32.25] vs. 20.45[19.05, 27.75] and 23.40[16.78, 28.13]; H=25.832, P=0.005 and H=22.056, P=0.008). In IT group, HBsAg levels and HBV DNA loads were negatively correlated with CD69MFI (β=-0.025, t=-2.613, P=0.014; β=-0.021, t=-2.286, P=0.030), meanwhile, HBeAg was negatively related to the frequency of CD69+CD8+ T lymphocytes (β=-61.306, t=-2.116, P=0.043). In AHB group, IFN-α2 was positively related to the frequency of CD8+ T lymphocytes (β=6.798, t=2.629, P=0.016); however, in CHB group, IFN-α2 was negatively associated with frequency of CD8+ T lymphocytes (β=-14.534, t=-2.085, P=0.043). In CHB group, HBeAg was positively associated with frequency of CD69+CD8+ T lymphocytes (β=43.912, t=2.027, P=0.048). In AHB group, ALT was positively related to CD69MFI (β=35.042, t=2.896, P=0.007), but HBsAg was negatively related to CD178MFI (β=-0.137, t=-3.273, P=0.003). The activation of CD8+ T lymphocytes was associated with the occurrence of AHB and CHB. However, due to the insufficient expression of functional molecules of CD8+ T lymphocytes and the depletion of CD8+ T lymphocytes, CHB patients were difficult to recover from HBV infection.

Systems biology analyses reveal enhanced chronic morphine distortion of gut-brain interrelationships in simian human immunodeficiency virus infected rhesus macaques.

BackgroundCommonly used opioids, such as morphine have been implicated in augmented SIV/HIV persistence within the central nervous system (CNS). However, the extent of myeloid cell polarization and viral persistence in different brain regions remains unclear. Additionally, the additive effects of morphine on SIV/HIV dysregulation of gut-brain crosstalk remain underexplored. Therefore, studies focused on understanding how drugs of abuse such as morphine affect immune dynamics, viral persistence and gut-brain interrelationships are warranted.Materials and methodsFor a total of 9 weeks, rhesus macaques were ramped-up, and twice daily injections of either morphine (n = 4) or saline (n = 4) administered. This was later followed with infection with SHIVAD8EO variants. At necropsy, mononuclear cells were isolated from diverse brain [frontal lobe, cerebellum, medulla, putamen, hippocampus (HIP) and subventricular zone (SVZ)] and gut [lamina propria (LP) and muscularis (MUSC) of ascending colon, duodenum, and ileum] regions. Multiparametric flow cytometry was used to were profile for myeloid cell polarity/activation and results corroborated with indirect immunofluorescence assays. Simian human immunodeficiency virus (SHIV) DNA levels were measured with aid of the digital droplet polymerase chain reaction (PCR) assay. Luminex assays were then used to evaluate soluble plasma/CSF biomarker levels. Finally, changes in the fecal microbiome were evaluated using 16S rRNA on the Illumina NovaSeq platform.ResultsFlow Cytometry-based semi-supervised analysis revealed that morphine exposure led to exacerbated M1 (CD14/CD16)/M2 (CD163/CD206) polarization in activated microglia that spanned across diverse brain regions. This was accompanied by elevated SHIV DNA within the sites of neurogenesis–HIP and SVZ. HIP/SVZ CD16+ activated microglia positively correlated with SHIV DNA levels in the brain (r = 0.548, p = 0.042). Simultaneously, morphine dependence depleted butyrate-producing bacteria, including Ruminococcus (p = 0.05), Lachnospira (p = 0.068) genera and Roseburia_sp_831b (p = 0.068). Finally, morphine also altered the regulation of CNS inflammation by reducing the levels of IL1 Receptor antagonist (IL1Ra).ConclusionThese findings are suggestive that morphine promotes CNS inflammation by altering receptor modulation, increasing myeloid brain activation, distorting gut-brain crosstalk, and causing selective enhancement of SHIV persistence in sites of neurogenesis.

CSF CXCL13 and Chitinase 3-like-1 Levels Predict Disease Course in Relapsing Multiple Sclerosis

Several biomarkers from multiple sclerosis (MS) patients’ biological fluids have been considered to support diagnosis, predict disease course, and evaluate treatment response. In this study, we assessed the CSF concentration of selected molecules implicated in the MS pathological process. To investigate the diagnostic and prognostic significance of CSF concentration of target candidate biomarkers in both relapsing (RMS, n = 107) and progressive (PMS, n = 18) MS patients and in other inflammatory (OIND, n = 10) and non-inflammatory (ONIND, n = 15) neurological disorders. We measured the CSF concentration of APRIL, BAFF, CHI3L1, CCL-2, CXCL-8, CXCL-10, CXCL-12, CXCL-13 through a Luminex Assay. MS patients were prospectively evaluated, and clinical and radiological activity were recorded. CHI3L1 and CXCL13 CSF levels were significantly higher in both MS groups compared to control groups, while CCL2, BAFF, and APRIL concentrations were lower in RMS patients compared to PMS and OIND. Considering RMS patients with a single demyelinating event, higher concentrations of CHI3L1, CXCL10, CXCL12, and CXCL13 were recorded in patients who converted to clinically defined MS(CDMS). RMS patients in the CXCL13 and CHI3L1 high concentration group had a significantly higher risk of relapse (HR 12.61 and 4.57), MRI activity (HR 7.04 and 2.46), and of any evidence of disease activity (HR 12.13 and 2.90) during follow-up. CSF CXCL13 and CHI3L1 levels represent very good prognostic biomarkers in RMS patients, and therefore can be helpful in the treatment choice. Higher CSF concentrations of neuro-inflammatory biomarkers were associated with a higher risk of conversion to CDMS in patients with a first clinical demyelinating event. Differential CSF BAFF and APRIL levels between RMS and PMS suggest a different modulation of B-cells pathways in the different phases of the disease.

Investigation of type 1 collagen a1 chain in plasma as a potential novel biomarker for prediction of coronary heart disease

Abstract Background Remodeling of the extracellular matrix (ECM) plays a crucial role in development and progression of atherosclerosis. Collagens, in particular type I collagen, are the most abundant ECM proteins in an atherosclerotic plaque. Recently, type I collagen α1 chain (COL1α1) in plasma was identified as a potential predictor of coronary heart disease (CHD). Aim The aim was to further confirm the predictive value of COL1α1 and also to investigate its correlates in a population-based cohort as well as changes over time in patients with manifest CHD in Sweden. Methods In a total of 1007 well-characterized individuals (50% women), 86 CHD cases and 184 sex- and age-matched controls were identified at 13 years follow-up. CHD at follow-up was defined as first-time event of myocardial infarction (MI) or invasive coronary intervention. Plasma levels of COL1α1 was quantified by the Luminex assay while PRO-C1 and C1M, two markers of type I collagen synthesis and degradation, respectively, were quantified by ELISA. In Cox proportional hazard analysis, log2 values of biomarker levels were used. In addition, temporal change of COL1α1 levels was also examined in a cohort of 125 patients with acute MI followed for 6 months. Results COL1α1 levels were significantly associated with incident CHD, both unadjusted (HR = 0.69, 95% CI 0.56–0.87, p=0.001) and after multiple adjustment (HR = 0.55, 95% CI 0.41–0.75, p&amp;lt;0.001). PRO-C1 was similarly associated with CHD, unadjusted (HR = 0.62, 95% CI 0.47–0.82, p=0.001) as well as adjusted (HR = 0.61, 95% CI 0.43–0.86, p=0.005), while C1M was not. In patients with acute MI, COL1α1 levels remained stable over 6 months. COL1α1 was significantly correlated to PRO-C1 (r=0.73, p&amp;lt;0.001), while there were no correlations to C1M, markers of inflammation (C-reactive protein, interleukin-6, matrix metalloproteinase-9) or myocardial injury (troponin T). Conclusions Circulating COL1α1 in plasma was independently and inversely associated with incident CHD. Furthermore, COL1α1 levels appeared to be relatively stable after an acute MI. COL1α1 levels seem to reflect collagen synthesis rather than collagen degradation and inflammation. Future studies are needed to confirm whether COL1α1 is a clinically useful marker and/or predictor of CHD. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): This research was in part financed by a grant from the Region of Östergötland, Sweden, aimed towards scientists early in their career. We would like to thank the people behind this grant for contributing to this research.This research was also partly supported by Futurum - the academy for healthcare in Region Jönköping County.

Influence of gender on coronary atherosclerosis and inflammatory biomarker profile: a CT angiographic study

Abstract Background and aims The burden of coronary atherosclerosis differs between men and women. Beyond traditional cardiovascular risk factors, inflammatory biomarkers can influence plaque formation and progression. We analyzed the influence of gender difference on coronary atherosclerosis and inflammatory cytokines. Methods Coronary CT-Angiography was performed in 301 patients and plaque morphology was quantitatively and qualitatively assessed using semi-automated software (Autoplaque version 2.5, Cedars-Sinai Medical Center, Los Angeles, CA). We analyzed total (TPV), non-calcified (NCPV), calcified (CPV) and low-density plaque volume in mm3 (LDPV, defined as plaque attenuation &amp;lt;30HU). Serum was analyzed for various cytokines including Interleukin (IL)-1a, IL-1b, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-13, Il-15, IL-17, TNF-a, IFNg, CRP, MCP-1, MIP-1a, Eotaxin, G_CSF and GM-CSF using Luminex assay. Results Out of 301 patients, 94 (31%) were female and 207 (69%) were male. As compared to women, men were significantly younger (62 vs. 57 years, p&amp;lt;0.001) and showed a higher BMI (28 vs. 26 kg/m2, p=0.005). No differences were seen for hypertension, hyperlipidemia, diabetes, smoking habits and family history of CAD. All plaque characteristics showed significant higher values in men as compared to women (all p&amp;lt;0.05). In men, cytokine profile showed significantly lower serum levels for IL-2 (3.20 [3.20; 7.01] vs. 4.31 [3.20; 9.68]; p=0.01) and Interferon-gamma (3.20 [3.20; 14.72] vs. 8.76 [3.20; 26.26]; p&amp;lt;0.001) and significantly higher levels for MCP-1 (224 [117; 327] vs. 155 [49; 260]; p&amp;lt;0.001). In multivariable logistic regression analysis, Interferon-gamma showed significant inverse association to male gender (OR 0.35; 95% CI: 0.17–0.72; p=0.004), but IL-2 and MCP-1 did not. The association of Interferon-gamma to gender was independent of age (OR 0.57; 95% CI: 0.43–0.76; p&amp;lt;0.001) and BMI (OR 2.12; 95% CI 1.25–3.62; p=0.006) Conclusion Coronary plaque volume and morphology significantly differs between men and women and so does the inflammatory cytokine profile. Albeit significantly lower coronary plaque burden in women, serum levels of interferon-gamma - a known pro-inflammatory cytokine - was significantly higher in women with independent association to gender. Whether interferon-gamma plays contradicting roles in the process of coronary atherosclerosis in men and women needs to be explored in future studies to identify potential gender-specific targets for therapeutic interventions. Funding Acknowledgement Type of funding sources: None.

Anti-Glutathione S Transferase T1 Antibodies After Renal Transplant and Their Impact on Graft Outcome.

Anti-glutathione S transferase T1 (GSTT1) antibodies, a type of non-HLA antibody, have been associated with chronic hepatic graft rejection. Despite the presence of this enzyme in the kidney, there are not enough studies on the development of anti-GSTT1 antibodies and their impact on renal grafts. Our objective was to evaluate the presence of anti-GSTT1 antibodies after renal transplant and their impact on graft outcomes. We conducted an ambispective cohort study. We performed real-time polymerase chain reaction to screen for GSTT1 alleles in 293 recipients and their donors. In null GSTT1 (GSTT1*0) genotype recipients of GSTT1-positive donors, the presence of anti-GSTT1 antibodies was evaluated using indirect immunofluorescence and Luminex assays, and their effects on graft function were evaluated. The median follow-up period was 54.3 months. Of the 293 patients studied, 42 recipients (14.4%) with GSTT1-positive donors did not have the GSTT1 allele (GSTT1-positive donor/GSTT1*0 recipient). Using Luminex assay, we detected antibodies in 16 patients (38.1%), 12 of which were already present at the time of transplant. Of these cases, 37.5% with antibodies had undergone a previous renal transplant. Using indirect immunofluorescence, we found that only 12 patients tested positive, 4 at the time of transplant. Antibody presence did not effect graft glomerular filtration rates or graft loss at 1 year, at 2 years, or end of follow-up. The presence of anti-GSTT1 antibodies is frequent in renal transplant GSTT1*0 recipients of GSTT1-positive donors but has no effects on graft outcome.

Decreased frequency of a novel T-lymphocyte subset, CD3+ CD4- CD7+ CD57- T cells, in hepatitis B virus-related end-stage liver disease might contribute to disease progression.

CD7 and CD57 are related to the differentiation and functional stages of CD8+ T cells. However, the role of their combined presence in CD8+ T cells in patients with chronic hepatitis B virus (HBV) infection, especially those with end-stage liver disease, remains unclear. Blood samples from healthy volunteers and patients with chronic hepatitis B were analyzed via Luminex assay and ELISA to measure plasma cytokine levels. Further, recombinant IL-22 was used to stimulate peripheral blood mononuclear cells from healthy volunteers, and the frequency of CD3+ CD4- CD7+ CD57- T cells and apoptosis rates were investigated via flow cytometry. Patients with end-stage liver disease, particularly those with acute to chronic liver failure, showed decreased CD3+ CD4- CD7+ CD57- T cell frequency. Furthermore, the prevalence of CD3+ CD4- CD7+ CD57- T cells was negatively correlated with disease severity, prognosis, and complications (ascites). We also observed that IL-22 promoted apoptosis and brought about a decrease in the number of CD3+ CD4- CD7+ CD57- T cells in a dose-dependent manner. CD3+ CD4- CD7+ CD57- T cells displayed a B and T lymphocyte attenuator(BTLA)high CD25high CD127high immunosuppressive phenotype and showed low interferon-γ, tumor necrosis factor-α, granzyme A, and perforin expression levels. The present findings will elucidate the pathogenesis of HBV-related end-stage liver disease and aid the identification of novel drug targets.

Immediate outcome prognostic value of plasma factors in patients with acute ischemic stroke after intravenous thrombolytic treatment

In the present study, we explored multiple plasma factors to predict the outcomes of patients with AIS after IVT. Fifty AIS patients who received IVT with alteplase were recruited and divided into two groups according to their NIHSS scores. Serum from all subjects was collected to quantitatively analyze the levels of different plasma factors, IL-6, MMP-9, ADAMTS13, TNC, GSN and TRX, using Luminex assays or ELISA measurements. Compared with the levels assessed at the onset of AIS, the levels of MMP-9 (P < 0.001), ADAMTS13 (P < 0.001), and TRX (P < 0.001) significantly decreased after IVT. The level of IL-6 was significantly increased in the NIHSS > 5 group at admission (P < 0.001) compared to the NIHSS ≤ 5 group. AIS patients with a poor prognosis had lower levels of ADAMTS13 at 72 h post-IVT compared with patients with a good prognosis (P = 0.021). IL-6 also was notably higher in the poor outcome group (P = 0.012). After adjusting for confounders, ADAMTS13 at 72 h post-IVT was an independent protective factor for prognosis in AIS patients with an adjusted OR of 0.07 (P = 0.049), whereas IL-6 was an independent predictor of risk for AIS patients with an adjusted OR of 1.152 (P = 0.028). IVT decreased MMP-9, ADAMTS13, and TRX levels in the plasma of AIS patients. Patients with a NIHSS score of less than 5 exhibited lower IL-6 levels, indicating that increased levels of IL-6 correlated with AIS severity after IVT. Therefore, IL-6 and ADAMTS13 might be useful plasma markers to predict the prognosis in AIS patients at 90-days after IVT.

Reduction of natural killer cells is associated with poor outcomes in patients with hepatitis B virus-related acute-on-chronic liver failure.

Natural killer (NK) cells are critical innate effectors that respond to viral infections and contribute to immunopathology. Here, we aimed to investigate the role of NK cells in hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) and elucidate the underlying mechanism by examining their phenotypic and functional profiles. We included patients with HBV-ACLF (n = 37) and chronic hepatitis B (n = 19), and healthy controls (n = 13) in our study. We examined the phenotype and function of different subsets of peripheral NK cells using flow cytometry and RNA-sequencing analysis, and screened liver NK cells using immunohistochemistry. We detected inflammatory cytokines using a Luminex assay. In addition, we analyzed the relationships between these parameters and disease severity. Peripheral NK cells were decreased and characterized by high expression of caspase-3, Ki67, CXCR3, NKG2D, NKp46, CD107a, and GM-CSF, and typified by higher cell migration and immune response by RNA-sequencing analysis in patients with HBV-ACLF than in those with chronic hepatitis B. Accumulations of CXCL-10 and NK cells were found in the liver, and excessive production of CXCL-10 in the peripheral blood contributed to the apoptosis of NK cells in vitro. The decrease in NK cells was associated with the level of HBV DNA and disease severity and had good prognostic performance in predicting the outcome of patients with HBV-ACLF through AUROC analysis. NK cells were significantly decreased and showed dysfunction of phenotypic and functional profiles across distinct subsets in the peripheral blood of patients with ACLF. Crosstalk between CXCL-10 and NK cells may mediate the unbalanced distribution of NK cells. Understanding the dysfunction and decrease in NK cells may provide new insights into ACLF pathogenesis.

SARS-CoV-2 Seroprevalence Study in Pediatric Patients and Health Care Workers Using Multiplex Antibody Immunoassays.

SARS-CoV-2 infection has become a global health problem specially exacerbated with the continuous appearance of new variants. Healthcare workers (HCW) have been one of the most affected sectors. Children have also been affected, and although infection generally presents as a mild disease, some have developed the Pediatric Inflammatory Multisystem Syndrome Temporally Associated with SARS-CoV-2 (PIMS-TS). We recruited 190 adults (HCW and cohabitants, April to June 2020) and 57 children (April 2020 to September 2021), of whom 12 developed PIMS-TS, in a hospital-based study in Spain. Using an in-house Luminex assay previously validated, antibody levels were measured against different spike and nucleocapsid SARS-CoV-2 proteins, including the receptor-binding domain (RBD) of the Alpha, Beta, Gamma, and Delta variants of concern (VoC). Seropositivity rates obtained from children and adults, respectively, were: 49.1% and 11% for IgG, 45.6% and 5.8% for IgA, and 35.1% and 7.3% for IgM. Higher antibody levels were detected in children who developed PIMS-TS compared to those who did not. Using the COVID-19 IgM/IgA ELISA (Vircell, S.L.) kit, widely implemented in Spanish hospitals, a high number of false positives and lower seroprevalences compared with the Luminex estimates were found, indicating a significantly lower specificity and sensitivity. Comparison of antibody levels against RBD-Wuhan versus RBD-VoCs indicated that the strongest positive correlations for all three isotypes were with RBD-Alpha, while the lowest correlations were with RBD-Delta for IgG, RBD-Gamma for IgM, and RBD-Beta for IgA. This study highlights the differences in antibody levels between groups with different demographic and clinical characteristics, as well as reporting the IgG, IgM, and IgA response to RBD VoC circulating at the study period.

Amaryllidaceae Alkaloids Decrease the Proliferation, Invasion, and Secretion of Clinically Relevant Cytokines by Cultured Human Colon Cancer Cells.

Alkaloids isolated from members of the Amaryllidaceae plant family are promising anticancer agents. The purpose of the current study was to determine if the isocarbostyrils narciclasine, pancratistatin, lycorane, lycorine, crinane, and haemanthamine inhibit phenomena related to cancer progression in vitro. To achieve this, we examined the proliferation, adhesion, and invasion of cultured human colon cancer cells via MTT assay and Matrigel-coated Boyden chambers. In addition, Luminex assays were used to quantify the secretion of matrix metalloproteinases (MMP) and cytokines associated with poor clinical outcomes. We found that all alkaloids decreased cell proliferation regardless of TP53 status, with narciclasine exhibiting the greatest potency. The effects on cell proliferation also appear to be specific to cancer cells. Narciclasine, lycorine, and haemanthamine decrease both adhesion and invasion but with various potencies depending on the cell line. In addition, narciclasine, lycorine, and haemanthamine decreased the secretion of MMP-1, -2, and -7, as well as the secretion of the cytokines pentraxin 3 and vascular endothelial growth factor. In conclusion, the present study shows that Amaryllidaceae alkaloids decrease phenomena and cytokines associated with colorectal cancer progression, supporting future investigations regarding their potential as multifaceted drug candidates.

Evaluation of the immunomodulatory effects of interleukin-10 on peripheral blood immune cells of COVID-19 patients: Implication for COVID-19 therapy.

ObjectiveSeveral therapies with immune-modulatory functions have been proposed to reduce the overwhelmed inflammation associated with COVID-19. Here we investigated the impact of IL-10 in COVID-19, through the ex-vivo assessment of the effects of exogenous IL-10 on SARS-CoV-2-specific-response using a whole-blood platform.MethodsTwo cohorts were evaluated: in “study population A”, plasma levels of 27 immune factors were measured by a multiplex (Luminex) assay in 39 hospitalized “COVID-19 patients” and 29 “NO COVID-19 controls” all unvaccinated. In “study population B”, 29 COVID-19 patients and 30 NO COVID-19-Vaccinated Controls (NO COVID-19-VCs) were prospectively enrolled for the IL-10 study. Whole-blood was stimulated overnight with SARS-COV-2 antigens and then treated with IL-10. Plasma was collected and used for ELISA and multiplex assay. In parallel, whole-blood was stimulated and used for flow cytometry analysis.ResultsBaseline levels of several immune factors, including IL-10, were significantly elevated in COVID-19 patients compared with NO COVID-19 subjects in “study population A”. Among them, IL-2, FGF, IFN-γ, and MCP-1 reached their highest levels within the second week of infection and then decreased. To note that, MCP-1 levels remained significantly elevated compared with controls. IL-10, GM-CSF, and IL-6 increased later and showed an increasing trend over time. Moreover, exogenous addition of IL-10 significantly downregulated IFN-γ response and several other immune factors in both COVID-19 patients and NO COVID-19-VCs evaluated by ELISA and a multiplex analysis (Luminex) in “study population B”. Importantly, IL-10 did not affect cell survival, but decreased the frequencies of T-cells producing IFN-γ, TNF-α, and IL-2 (p<0.05) and down-modulated HLA-DR expression on CD8+ and NK cells.ConclusionThis study provides important insights into immune modulating effects of IL-10 in COVID-19 and may provide valuable information regarding the further in vivo investigations.

LBA41 Phase II study SECOMBIT (sequential combo immuno and target therapy study): 4-years OS data and preliminary biomarkers evaluation

To investigate the best sequential strategy in BRAFV600-mutated melanoma, the SECOMBIT study was carried out, a three parallel arms phase 2 randomized trial (NCT02631447). We used the combination of encorafenib/binimetinib (E+B) as targeted therapy (T-T) and the combination of ipilimumab/nivolumab (I+N) as immunotherapy (I-O). We explored T-T (Arm A) or I-O (Arm B) as first line with switch at progression, as well as the “sandwich” strategy with T-T first preplanned switched to combo I-O after 8 weeks, returning back to T-T after progression (Arm C). In our previous report overall survival (OS) and total progression free survival (tPFS) rates at 2 and 3 years showed a better trend in Arm B and C. From Nov 2016 to May 2019, 251 patients (pts) from 37 centers in 9 countries with untreated, metastatic BRAFV600 melanoma were enrolled. Pts were randomized to Arm A, Arm B or C. OS is the primary endpoint of the study. Secondary endpoints include tPFS, 4-year survival rate and a preliminary biomarkers evaluation. NGS analysis and 21 inflammatory cytokines through Luminex assay or HS-ELISA were evaluated at baseline from 83 pts (29,24,30 for Arm A,B,C) and 93 pts (28,29, 36 for Arm A,B,C) respectively. The OS at 4 years, with median follow up of 43 months (IQR: 37-51), was calculated in the three arms for all pts, and in the subgroups normal or elevate LDH level and < 3 or ≥ 3 metastatic sites. OS and tPFS rates at 4 years with subgroup analysis are shown in table 1. In the biomarkers analysis we observed a correlation of JAK mutation with survival, IFN-gamma and CXCL5 cytokines with ORR and PFS.Table: LBA41Arm A (T-T)Arm B (I-O)Arm C (sandwich)4-year tPFS29%55%54%4-year OS46%64%59%4-year tPFS <3 ms33%59%59%4-year OS <3 ms55%65%62%4-year tPFS ≥3 ms23%51%46%4-year OS ≥3 ms32%63%54%4-year tPFS nLDH31%58%51%4-year OS nLDH53%70%56%3-year tPFS eLDH23%48%60%3-year OS eLDH42%53%65%4-year tPFS eLDH18%48%60%4-year OS eLDH42%53%65%Biomarkers Analysis at 4 yearsJAK WT -OS57%53%71%JAK WT -PFS38%53%53%JAK MUT - OS75%100%65%JAK MUT - PFS75%100%61%IFN-g high - ORR67%0%56%IFN-g low - ORR67%33%74%CXCL5 high - PFS>2y27%45%40%CXCL5 low - PFS>2y56%71%63% Open table in a new tab The OS and tPFS rates at 4 years revealed positive trend in pts on Arm B and Arm C indicating planned switch could be beneficial treatment strategy. In Arm B, IFN Gamma low seems to be related to ICI response. JAK mutations are associated with long term OS. Further biomarkers evaluations are ongoing.

Abstract P082: Exposure To Excess Albumin Causes Inflammatory Activation Of The Proximal Tubule.

In previous studies, we used chronic servo control experiments to show that renal inflammation is a secondary event in salt-sensitive hypertension, with pressure amplifying renal immune cell infiltration in Dahl salt-sensitive (SS) rats. In contrast, albuminuria occurs early in the development of hypertension and facilitates its progression. In this study, we tested the hypothesis that albuminuria causes release of pro-inflammatory cytokines in the proximal tubule, which in turn may initiate renal inflammation.We exposed the apical membrane of human proximal tubule cell monolayers (RPTEC-TERT1) to human serum albumin (HSA, 10 mg/ml, n=8) for 24 and 48hrs. Control cells were kept in standard media without HSA (n=8). The concentrations of 47 cytokines were measured in the basolateral (BM) and apical (AM) media using Luminex assays. After 24hrs we found that 18 cytokines were significantly higher in the BM and 14 in the AM relative to controls. At 48hrs, 17 cytokines were significantly higher in the BM and 15 in the AM. Notably, we found significant increases in basolateral release of several chemokines, including CCL2 (24hrs: 6.1±0.9 vs. 40.6±8.9, 48hrs: 20.1±4.5 vs. 85.8±15.6 pg/mg), CXCL1 (24hrs: 1.3±0.3 vs. 39.5±12.2, 48hrs: 3.4±1.1 vs. 94.2±41.4 pg/mg), and IL-8 (24hrs: 1.3±0.4 vs. 11.6±3.7, 48hrs: 2.1±0.5 vs. 10.1±3 pg/mg). We propose that HSA-induced chemokine secretion facilitates immune cell recruitment and the initiation of renal inflammation. To track the progression of albuminuria and proximal tubule injury in SS rats we performed daily urine collections in rats fed either a high-salt (HS, 4.0% NaCl, n=6) or a control-salt (CS, 0.4% NaCl, n=5) diet for 7-days. In SS rats fed HS, albuminuria preceded proximal tubule injury. Urinary albumin was elevated after 2-days of HS (22.6±6.5 vs. 61.1±18.7 mg/day) whereas Kidney Injury Molecule-1 (KIM-1) was increased after 5-days of HS (15.5±1.5 vs. 31.3±5.6 ng/day).To conclude, in vitro exposure to excess albumin causes significant increases in the levels of multiple cytokines involved in the initiation of inflammation. In SS rats fed HS, albuminuria precedes proximal tubule injury. Together these data suggest that albuminuria may facilitate hypertension through the initiation of renal inflammation.

Quantitative Plasma Proteomics to Identify Candidate Biomarkers of Relapse in Pediatric/Adolescent Hodgkin Lymphoma.

Classical pediatric Hodgkin Lymphoma (HL) is a rare malignancy. Therapeutic regimens for its management may be optimized by establishing treatment response early on. The aim of this study was to identify plasma protein biomarkers enabling the prediction of relapse in pediatric/adolescent HL patients treated under the pediatric EuroNet-PHL-C2 trial. We used untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics at the time of diagnosis—before any therapy—as semiquantitative method to profile plasma proteins specifically associated with relapse in 42 children with nodular sclerosing HL. In both the exploratory and the validation cohorts, six proteins (apolipoprotein E, C4b-binding protein α chain, clusterin, fibrinogen γ chain, prothrombin, and vitronectin) were more abundant in the plasma of patients whose HL relapsed (|fold change| ≥ 1.2, p < 0.05, Student’s t-test). Predicting protein function with the Gene Ontology classification model, the proteins were included in four biological processes (p < 0.01). Using immunoblotting and Luminex assays, we validated two of these candidate biomarkers—C4b-binding protein α chain and clusterin—linked to innate immune response function (GO:0045087). This study identified C4b-binding protein α chain and clusterin as candidate early plasma biomarkers of HL relapse, and important for the purpose of shedding light on the molecular scenario associated with immune response in patients treated under the EuroNet-PHL-C2 trial.

P2. Effects of smoking status on metabolic response of cervical and lumbar degenerative intervertebral discs

<h3>BACKGROUND CONTEXT</h3> Many studies have validated the clinical signficance of smoking on disc degeneration and adverse outcome in spine health. A paucity of literature exists on the relationship between the biomarkers and the disc degeneration. <h3>PURPOSE</h3> Smoking is associated with IVDD (intervertebral disc degeneration). This study was designed to characterize metabolic responses in cervical and lumbar IVDs based on smoking status. <h3>STUDY DESIGN/SETTING</h3> All patients undergoing elective anterior cervical fusion, lumbar discectomy and lumbar interbody fusion in an ongoing disc biomarker study were included after the IRB approval and obtaiing informed consent. <h3>OUTCOME MEASURES</h3> Significance between smoking status with region and Pfirrmann grade were determined using a One-Way ANOVA with Tukey post-hoc analysis with significance set at p<0.05. Smoking was recorded as nonsmoking (NS), former smoker (FS) or current smoker (CS). <h3>METHODS</h3> IVD tissues from surgery were recovered, and two explants/IVD were created. They were cultured for 3 days and tested for MMPs (1, 2, 3, 7, 8, 9, 13), TIMPs (1, 2, 3, 4), GRO-α, MCP-1, MCP-3, TNF-α, IL-1RA, IL-6, IL-8, MIP-1α, MIP-1ß, RANTES, PGE2, PDGF-AA, PDGF-AB/BB, and VEGF using Luminex assays. <h3>RESULTS</h3> Lumbar IVDs from FS produced significantly lower IL-RA and MMP-13 compared to NS, and MMP-7 compared to CS. Cervical IVDs from CS produced significantly higher levels of MMP-2, MCP-1, RANTES, IL-1RA, and PDGF-ABBB, and lower levels of MCP-3, compared to NS and/or FS. FS produced significantly lower levels of MMP-8 compared to NS and CS. For NS and CS, inflammatory and degrative biomarkers increased as Pfirrmann grade increased. FS produced significantly lower levels of MMP-8 compared to NS and CS. At Pfirrmann grade 4, NS produced lower levels of TIMP-3 and TIMP-4 than FS, and FS produced lower levels of MMP-2 than CS. <h3>CONCLUSIONS</h3> CS produced significantly higher levels of inflammatory and degradative biomarkers at each Pfirrmann grade. FS produced significantly lower levels of MMP-8. NS produced significantly lower levels of MMP-2 and RANTES at Pfirrmann grade 3 and MMP-7 at Pfirrmann grade 5 than CS. NS and FS produced lower levels of PDGF-AB/BB than CS. Inflammatory and degradative biomarkers increased as Pfirrmann grade increased in NS more than CS. Smoking has a greater effect on cervical IVD responses than lumbar IVD. <h3>FDA DEVICE/DRUG STATUS</h3> This abstract does not discuss or include any applicable devices or drugs.

High-throughput DNA methylation analysis in ITP confirms NOTCH1 hypermethylation through the Th1 and Th2 cell differentiation pathways

BackgroundImmune thrombocytopenia (ITP) is a prevalent autoimmune disease with a complex aetiology where DNA methylation changes are becoming triggers. MethodTo investigate novel abnormally methylated genes in the pathogenesis of ITP, we performed a high-throughput methylation analysis on 21 ITP patients and 9 normal control samples. We analysed the extent of key methylated genes and their downstream cytokines through Luminex assay or qRT–PCR. Then, bone marrow mononuclear cells were extracted from ITP patients, and decitabine (demethylation drug) was added to the culture medium of cultured cells. qRT–PCR and ELISA were used to detect whether decitabine could effectively affect target genes and related cytokines. ResultsThrough the STRING and Metascape databases, hypermethylated NOTCH1 can be identified and can influence ITP by regulating many downstream cytokines through Th1 and Th2 cell differentiation pathways. Compared with those in the normal control group, the expression levels of NOTCH1 and its downstream Th2 cytokines (IL-4, IL-10, and GATA3) were significantly decreased and those of Th1 cytokines (IFN-γ, IL-12, and TNF-α) were significantly increased in the ITP group. Decitabine exerts its demethylation effect, so the expression of NOTCH1 and its related cytokines in the ITP group treated with 100 nM decitabine were significantly reversed. ConclusionsOur results suggest that the pathogenesis of ITP may exert its influence on epigenetics through alteration of DNA methylation at regulatory regions of the target NOTCH1 gene in the Th1 and Th2 cell differentiation pathways. At the same time, decitabine may achieve a therapeutic effect on ITP by demethylation.

Intermittent fasting was superior to a low-fat diet in impacting tumor growth in a pre-clinical model of obesity-driven endometrial cancer (125)

Intermittent fasting was superior to a low-fat diet in impacting tumor growth in a pre-clinical model of obesity-driven endometrial cancer (125)