Plasma immune signatures can predict rejection-free survival in the first year after pediatric liver transplantation.

BACKGROUND & AIMS: After pediatric liver transplantation (pLT), children undergo life-long immunosuppression since reliable biomarkers for the assessment of rejection probability are scarce. In the multicenter (n = 7) prospective clinical cohort "ChilSFree" study, we aimed to characterize longitudinal dynamics of soluble and cellular immune mediators during the first year after pLT and identify early biomarkers associated with outcome. METHODS: Using a Luminex-based multiplex technique paired with flow cytometry, we characterized longitudinal dynamics of soluble immune mediators (SIMs, n = 50) and immune cells in the blood of 244 patients at eight visits over 1 year: before, and 7/14/21/28 days and 3/6/12 months after pLT. RESULTS: The unsupervised clustering of patients based on SIM profiles revealed six unique SIM signatures associated with clinical outcome. From three signatures linked to improved outcome, one was associated with 1-year-long rejection-free survival and stable graft function and was characterized by low levels of pro-inflammatory SIMs (CXCL8/9/10/12, CCL7, SCGF-ß, sICAM-1), and high levels of regenerative (SCF, TNF-ß) and pro-apoptotic (TRAIL) SIMs (all, p <0.001, fold change >100). Of note, this SIM signature appeared 2 weeks after pLT and remained stable over the entire year, pointing towards its potential as a novel early biomarker for minimizing or weaning immunosuppression. In the blood of these patients, a higher frequency of CD56bright natural killer cells (p <0.01), a known hallmark also associated with operationally tolerant pLT patients, was detected. The concordance of the model for prediction of rejection based on identified SIM signatures was 0.715, and 0.795, in combination with living-related transplantation as a covariate, respectively. CONCLUSIONS: SIM blood signatures may enable the non-invasive and early assessment of rejection risks in the first year after pLT, paving the way for improved clinical management. IMPACT AND IMPLICATIONS: ChilSFree represents the largest pediatric liver transplant (pLT) cohort with paired longitudinal data on soluble immune mediators (SIMs) and immune phenotyping in the first year after pLT. SIM signatures allow for the selection of rejection-free patients 2 weeks after pLT independently of patient diagnosis, sex, or age. The SIM signatures may enable the non-invasive and early assessment of rejection risks, paving the way for minimization or withdrawal of immunosuppression after pLT.

Effect of high-dose Spirulina supplementation on hospitalized adults with COVID-19: a randomized controlled trial.

Objective: Spirulina (arthrospira platensis) is a cyanobacterium proven to have anti-inflammatory, antiviral, and antioxidant effects. However, the effect of high-dose Spirulina supplementation on hospitalized adults with COVID-19 is currently unclear. This study aimed to evaluate the efficacy and safety of high-dose Spirulina platensis for SARS-CoV-2 infection. Study Design: We conducted a randomized, controlled, open-label trial involving 189 patients with COVID-19 who were randomly assigned in a 1:1 ratio to an experimental group that received 15.2g of Spirulina supplement plus standard treatment (44 non-intensive care unit (non-ICU) and 47 ICU), or to a control group that received standard treatment alone (46 non-ICU and 52 ICU). The study was conducted over six days. Immune mediators were monitored on days 1, 3, 5, and 7. The primary outcome of this study was mortality or hospital discharge within seven days, while the overall discharge or mortality was considered the secondary outcome. Results: Within seven days, there were no deaths in the Spirulina group, while 15 deaths (15.3%) occurred in the control group. Moreover, within seven days, there was a greater number of patients discharged in the Spirulina group (97.7%) in non-ICU compared to the control group (39.1%) (HR, 6.52; 95% CI, 3.50 to 12.17). Overall mortality was higher in the control group (8.7% non-ICU, 28.8% ICU) compared to the Spirulina group (non-ICU HR, 0.13; 95% CI, 0.02 to 0.97; ICU, HR, 0.16; 95% CI, 0.05 to 0.48). In non-ICU, patients who received Spirulina showed a significant reduction in the levels of IL-6, TNF-α, IL-10, and IP-10 as intervention time increased. Furthermore, in ICU, patients who received Spirulina showed a significant decrease in the levels of MIP-1α and IL-6. IFN-γ levels were significantly higher in the intervention group in both ICU and non-ICU subgroups as intervention time increased. No side effects related to Spirulina supplements were observed during the trial. Conclusion: High-dose Spirulina supplements coupled with the standard treatment of COVID-19 may improve recovery and remarkably reduce mortality in hospitalized patients with COVID-19. Clinical Trial Registration: https://irct.ir/trial/54375, Iranian Registry of Clinical Trials number (IRCT20210216050373N1).

Temporal patterns of cytokine and injury biomarkers in hospitalized COVID-19 patients treated with methylprednisolone.

Background: The novel coronavirus disease 2019 (COVID-19) presents with complex pathophysiological effects in various organ systems. Following the COVID-19, there are shifts in biomarker and cytokine equilibrium associated with altered physiological processes arising from viral damage or aggressive immunological response. We hypothesized that high daily dose methylprednisolone improved the injury biomarkers and serum cytokine profiles in COVID-19 patients. Methods: Injury biomarker and cytokine analysis was performed on 50 SARS-Cov-2 negative controls and 101 hospitalized severe COVID-19 patients: 49 methylprednisolone-treated (MP group) and 52 placebo-treated serum samples. Samples from the treated groups collected on days D1 (pre-treatment) all the groups, D7 (2 days after ending therapy) and D14 were analyzed. Luminex assay quantified the biomarkers HMGB1, FABP3, myoglobin, troponin I and NTproBNP. Immune mediators (CXCL8, CCL2, CXCL9, CXCL10, TNF, IFN-γ, IL-17A, IL-12p70, IL-10, IL-6, IL-4, IL-2, and IL-1ß) were quantified using cytometric bead array. Results: At pretreatment, the two treatment groups were comparable demographically. At pre-treatment (D1), injury biomarkers (HMGB1, TnI, myoglobin and FABP3) were distinctly elevated. At D7, HMGB1 was significantly higher in the MP group (p=0.0448) compared to the placebo group, while HMGB1 in the placebo group diminished significantly by D14 (p=0.0115). Compared to healthy control samples, several immune mediators (IL-17A, IL-6, IL-10, MIG, MCP-1, and IP-10) were considerably elevated at baseline (all p≤0.05). At D7, MIG and IP-10 of the MP-group were significantly lower than in the placebo-group (p=0.0431, p=0.0069, respectively). Longitudinally, IL-2 (MP-group) and IL-17A (placebo-group) had increased significantly by D14. In placebo group, IL-2 and IL-17A continuously increased, as IL-12p70, IL-10 and IP-10 steadily decreased during follow-up. The MP treated group had IL-2, IFN-γ, IL-17A and IL-12p70 progressively increase while IL-1ß and IL-10 gradually decreased towards D14. Moderate to strong positive correlations between chemokines and cytokines were observed on D7 and D14. Conclusion: These findings suggest MP treatment could ameliorate levels of myoglobin and FABP3, but appeared to have no impact on HMGB1, TnI and NTproBNP. In addition, methylprednisolone relieves the COVID-19 induced inflammatory response by diminishing MIG and IP-10 levels. Overall, corticosteroid (methylprednisolone) use in COVID-19 management influences the immunological molecule and injury biomarker profile in COVID-19 patients.

Nasal biomarkers of immune function differ based on smoking and respiratory disease status.

Respiratory biomarkers have the potential to identify airway injury by revealing inflammatory processes within the respiratory tract. Currently, there are no respiratory biomarkers suitable for clinical use to identify patients that warrant further diagnostic work-up, counseling, and treatment for toxic inhalant exposures or chronic airway disease. Using a novel, noninvasive method of sampling the nasal epithelial lining fluid, we aimed to investigate if nasal biomarker patterns could distinguish healthy nonsmoking adults from active smokers and those with chronic upper and lower airway disease in this exploratory study. We compared 28 immune mediators from healthy nonsmoking adults (n = 32), former smokers with COPD (n = 22), chronic rhinosinusitis (CRS) (n = 22), and smoking adults without airway disease (n = 13). Using ANOVA, multinomial logistic regressions, and weighted gene co-expression network analysis (WGCNA), we determined associations between immune mediators and each cohort. Six mediators (IL-7, IL-10, IL-13, IL-12p70, IL-15, and MCP-1) were lower among disease groups compared to healthy controls. Participants with lower levels of IL-10, IL-12p70, IL-13, and MCP-1 in the nasal fluid had a higher odds of being in the COPD or CRS group. The cluster analysis identified groups of mediators that correlated with disease status. Specifically, the cluster of IL-10, IL-12p70, and IL-13, was positively correlated with healthy and negatively correlated with COPD groups, and two clusters were correlated with active smoking. In this exploratory study, we preliminarily identified groups of nasal mucosal mediators that differed by airway disease and smoking status. Future prospective, age-matched studies that control for medication use are needed to validate these patterns and determine if nasosorption has diagnostic utility for upper and lower airway disease or injury.

Plasma proteomics reveals early, broad release of chemokine, cytokine, TNF, and interferon mediators following trauma with delayed increases in a subset of chemokines and cytokines in patients that remain critically ill.

Severe injury is known to cause a systemic cytokine storm that is associated with adverse outcomes. However, a comprehensive assessment of the time-dependent changes in circulating levels of a broad spectrum of protein immune mediators and soluble immune mediator receptors in severely injured trauma patients remains uncharacterized. To address this knowledge gap, we defined the temporal and outcome-based patterns of 184 known immune mediators and soluble cytokine receptors in the circulation of severely injured patients. Proteomics (aptamer-based assay, SomaLogic, Inc) was performed on plasma samples drawn at 0, 24, and 72 hours (h) from time of admission from 150 trauma patients, a representative subset from the Prehospital Plasma during Air Medical Transport in Trauma Patients at Risk for Hemorrhagic Shock (PAMPer) trial. Patients were categorized into outcome groups including Early Non-Survivors (died within 72 h; ENS; n=38), Non-Resolvers (died after 72 h or required ≥7 days of intensive care; NR; n=78), and Resolvers (survivors that required < 7 days of intensive care; R; n=34), with low Injury Severity Score (ISS) patients from the Tranexamic Acid During Prehospital Transport in Patients at Risk for Hemorrhage After Injury (STAAMP) trial as controls. The major findings include an extensive release of immune mediators and cytokine receptors at time 0h that is more pronounced in ENS and NR patients. There was a selective subset of mediators elevated at 24 and 72 h to a greater degree in NR patients, including multiple cytokines and chemokines not previously described in trauma patients. These findings were validated in a quantitative fashion using mesoscale discovery immunoassays (MSD) from an external validation cohort (VC) of samples from 58 trauma patients matched for R and NR status. This comprehensive longitudinal description of immune mediator patterns associated with trauma outcomes provides a new level of characterization of the immune response that follows severe injury.

An inflammatory Signature of Glucose Impairment in Cystic Fibrosis.

Objective and Design: Cystic fibrosis-related diabetes (CFRD) is a severe complication associated with increased morbidity and mortality in cystic fibrosis (CF) patients. Extensive inflammatory state in CF leads to pancreas damage and insulin resistance with consequent altered glucose tolerance and CFRD development. The aim of the present study was to identify circulating levels of inflammatory markers specifically associated with impaired glucose tolerance (IGT) and overt CFRD in a sample of young adults with CF. Materials and Methods: Sixty-four CF outpatients, without evident active pulmonary exacerbation, infectious and autoimmune diseases, were enrolled in the study and the levels of 45 inflammatory serum mediators were measured through x magnetic bead panel multiplex technology. Results: Serum levels of PDGF-AA, CCL20/MIP3α, IFNα, CCL11/eotaxin, CXCL1/GROα, GMCSF, B7H1/PDL1, IL13, IL7, VEGF, and TGFα were all significantly (p<0.05) elevated in patients according to glycemic status and directly correlated with glycated hemoglobin and C-reactive protein levels. Conclusion: Our findings suggest that increased levels of specific circulating inflammatory mediators are directly associated with impaired glucose tolerance in CF patients, thus, potentially implicating them in CFRD pathogenesis and warranting larger longitudinal studies to validate their monitoring as predictor of CFRD onset.

Atorvastatin attenuates NS1 (Non-structural protein-1) of dengue type-2 serotype-induced expressions of matrix metalloproteinases in HL-60 cells, differentiated to neutrophils: Implications for the immunopathogenesis of dengue viral disease.

BACKGROUND: The dengue is a vector borne viral infection in humans. Bite of mosquito infected with a dengue virus transmits the disease. The neutrophils support more to the innate immune response by switching to infected tissues and triggering immunomodulatory mechanisms including the release of proteases and host defence peptides. METHODS: Cell viability by MTT and trypan blue dye exclusion assay, bright field microscopy for assessment of cell morphology, cytokines measurements by ELISA, estimation of protein by Bradford assay were done. Assessments of matrix metalloproteinase genes mRNA expressions were done using real-time PCR. RESULTS: In the present study, we have for the first time unveiled that, NS1 antigen of dengue type-2 serotype, induce and stimulate the neutrophils cells to express high levels of matrix metalloproteases. NS1 exposure of HL-60 cells differentiated to neutrophils affected cell morphology and in 24 h of exposure. We have demonstrated that, the NS1 antigen has induced MMP-2, MMP-14 and MMP-9 expressions in neutrophils in a 24hrs exposure time. NS1 exposure has also further upregulated MMP-1, MMP-13, and MMP-8 expressions in neutrophils in a 24hrs exposure time. Notably, treatment with atorvastatin concentrations downregulated the expression profile of the all matrix metalloprotease significantly. Importantly, NS1 antigen has significantly increased the IL-6, IL-13 release by the HL,60 cells which was reversed by atorvastatin. On the other hand, NS1 exposure enhanced the mRNA expressions of VEGF-A and VEGF-D which was reversed by atorvastatin. However, we found that, NS1 exposure reduced the mRNA expressions profile of VEGF-C, which was reversed by atorvastatin. CONCLUSION: In conclusion, we report that, neutrophils associated matrix metalloprotease are involved in the pathogenesis of dengue viral disease. VEGF growth factors may also be released by the neutrophils which may subsequently participate in the endothelial dysfunctions leading to dengue shock syndrome.

Acute kidney injury associated to COVID-19 leads to a strong unbalance of circulant immune mediators.

BACKGROUND: Severe cases of coronavirus disease 2019 (COVID-19) have increased risk for acute kidney injury (AKI). The exacerbation of the immune response seems to contribute to AKI development, but the immunopathological process is not completely understood. OBJECTIVES: To analyze levels of circulant immune mediators in COVID-19 patients evolving with or without AKI. We have also investigated possible associations of these mediators with viral load and clinical outcomes. METHODS: This is a longitudinal study performed with hospitalized patients with moderate to severe COVID-19. Serum levels of 27 immune mediators were measured by a multiplex immunoassay. Data were analyzed at two timepoints during the follow-up: within the first 13 days of the disease onset (early sample) and from the 14th day to death or hospital discharge (follow-up sample). RESULTS: We studied 82 COVID-19 patients (59.5 ± 17.5 years, 54.9% male). Of these, 34 (41.5%) developed AKI. These patients presented higher SARS-CoV-2 viral load (P = 0.03), higher frequency of diabetes (P = 0.01) and death (P = 0.0004). Overall, AKI patients presented significantly higher and sustained levels (P < 0.05) of CCL-2, CCL-3, CCL-4, CXCL-8, CXCL-10, IFN-γ, IL-2, IL-6, TNF-α, IL-1Ra, IL-10 and VEGF. Importantly, higher levels of CCL-2, CXCL-10, IL-2, TNF-α, IL-10, FGFb, and VEGF were observed in AKI patients independently of death. ROC curves demonstrated that early alterations in CCL-2, CXCL-8, CXCL-10, IFN-γ, IL-6, IL-1Ra and IL-10 show a good predictive value regarding AKI development. Lastly, immune mediators were significantly associated with each other and with SARS-CoV-2 viral load in AKI patients. CONCLUSIONS: COVID-19 associated AKI is accompanied by substantial alterations in circulant levels of immune mediators, which could significantly contribute to the establishment of kidney injury.

Timeline Kinetics of Systemic and Airway Immune Mediator Storm for Comprehensive Analysis of Disease Outcome in Critically Ill COVID-19 Patients.

In the present study, the levels of serum and airway soluble chemokines, pro-inflammatory/regulatory cytokines, and growth factors were quantified in critically ill COVID-19 patients (total n=286) at distinct time points (D0, D2-6, D7, D8-13 and D>14-36) upon Intensive Care Unit (ICU) admission. Augmented levels of soluble mediators were observed in serum from COVID-19 patients who progress to death. An opposite profile was observed in tracheal aspirate samples, indicating that systemic and airway microenvironment diverge in their inflammatory milieu. While a bimodal distribution was observed in the serum samples, a unimodal peak around D7 was found for most soluble mediators in tracheal aspirate samples. Systems biology tools further demonstrated that COVID-19 display distinct eccentric soluble mediator networks as compared to controls, with opposite profiles in serum and tracheal aspirates. Regardless the systemic-compartmentalized microenvironment, networks from patients progressing to death were linked to a pro-inflammatory/growth factor-rich, highly integrated center. Conversely, patients evolving to discharge exhibited networks of weak central architecture, with lower number of neighborhood connections and clusters of pro-inflammatory and regulatory cytokines. All in all, this investigation with robust sample size landed a comprehensive snapshot of the systemic and local divergencies composed of distinct immune responses driven by SARS-CoV-2 early on severe COVID-19.

Different effectiveness of fungal pathogen-associated molecular patterns (PAMPs) in activating rat peritoneal mast cells.

Mast cells (MCs) are the first immune cell type that can contact with the external environment, where they may rapidly sense the presence of pathogens. These cells are directly involved in innate defense through their ability to pathogen destruction by several mechanisms and the pattern recognition receptors (PRRs) they express. Several studies have focused on the aspects of MC responses to bacterial and viral pathogens or their specific components and the role of those cells in antibacterial or antiviral defense mechanisms. However, to date, the knowledge of the influence of various fungi-derived molecules on MC activity is primarily based on limited data. Thus, this study aims to compare the effect of the major fungi cell wall-associated antigens, i.e., two ß-(1,3)-glucans: zymosan - ß-(1,3)-glucan containing mannan and chitin, and curdlan - purified linear model ß-(1,3)-glucan as well as mannan on peritoneal MC activity. In particular, the potency of various fungal cell wall components to induce MC migration, degranulation, and generation and/or release of de novo-synthesized mediators/cytokines/chemokines was analyzed. The most striking result to emerge from the data is that MC activation differs depending on the fungal stimuli. Our study outlines that components of the inner layer of the fungi cell wall - ß-glucans, i.e., zymosan and curdlan, are more potent stimulators of MC activity compared to mannan. On this note, the data described here may provide a foundation for further studying the role of MC in antifungal immunity and be helpful for a better understanding of host-pathogenic fungi interactions.

Comparison of laboratory and immune characteristics of the initial and second phase of tick-borne encephalitis.

Tick-borne encephalitis (TBE) usually has a biphasic course which begins with unspecific febrile illness, followed by central nervous system involvement. Because TBE is not yet suspected during the initial phase, knowledge of early TBE pathogenesis is incomplete. Herein we evaluated laboratory and immune findings in the initial and second (meningoencephalitic) phase of TBE in 88 well-defined adult patients. Comparison of nine laboratory blood parameters in both phases of TBE revealed that laboratory abnormalities, consisting of low leukocyte and platelet counts and increased liver enzymes levels, were predominately associated with the initial phase of TBE and resolved thereafter. Assessment of 29 immune mediators in serum during the initial phase, and in serum and cerebrospinal fluid (CSF) during the second phase of TBE revealed highly distinct clustering patterns among the three groups. In the initial phase of TBE, the primary finding in serum was a rather heterogeneous immune response involving innate (CXCL11), B cell (CXCL13, BAFF), and T cell mediators (IL-27 and IL-4). During the second phase of TBE, growth factors associated with angiogenesis (GRO-α and VEGF-A) were the predominant characteristic in serum, whereas innate and Th1 mediators were the defining feature of immune responses in CSF. These findings imply that distinct immune processes play a role in the pathophysiology of different phases of TBE and in different compartments.

Identifying Rare Genetic Variants of Immune Mediators as Risk Factors for Autism Spectrum Disorder.

Autism spectrum disorder (ASD) affects more than 1% of children, and there is no viable pharmacotherapeutic agent to treat the core symptoms of ASD. Studies have shown that children with ASD show changes in their levels of immune response molecules. Our previous studies have shown that ASD is more common in children with folate receptor autoantibodies. We also found that children with ASD have abnormal gut immune function, which was characterized by a significant increase in the content of immunoglobulin A and an increase in gut-microbiota-associated epitope diversity. These studies suggest that the immune mechanism plays an important role in the occurrence of ASD. The present study aims to systematically assess gene mutations in immune mediators in patients with ASD. We collected genetic samples from 72 children with ASD (2−12 years old) and 107 healthy controls without ASD (20−78 years old). We used our previously-designed immune gene panel, which can capture cytokine and receptor genes, the coding regions of MHC genes, and genes of innate immunity. Target region sequencing (500×) and bioinformatics analytical methods were used to identify variants in immune response genes associated with patients with ASD. A total of 4 rare variants were found to be associated with ASD, including HLA-B: p.A93G, HLA-DQB1: p.S229N, LILRB2: p.R322H, and LILRB2: c.956-4C>T. These variants were present in 44.44% (32/72) of the ASD patients and were detected in 3.74% (4/107) of the healthy controls. We expect these genetic variants will serve as new targets for the clinical genetic assessment of ASD, and our findings suggest that immune abnormalities in children with ASD may have a genetic basis.

Lifetime sexual violence exposure in women compromises systemic innate immune mediators associated with HIV pathogenesis: A cross-sectional analysis.

OBJECTIVES: Violence and HIV/AIDS syndemic highly prevalent among women impairs HIV prevention efforts. Prolonged exposure to violence results in physical trauma and psychological distress. Building on previous findings regarding genital immune dysregulation following sexual abuse exposure, we investigate here whether systemic changes occur as well. METHODS: Using the Women's Interagency HIV Study repository, 77 women were stratified by HIV serostatus and categorized into four subgroups: (1) no sexual abuse history and lower depression score (Control); (2) no sexual abuse history but higher depression score (Depression); (3) high sexual abuse exposure and lower depression score (Abuse); (4) high sexual abuse exposure and higher depression score (Abuse + Depression). Inflammation-associated immune biomarkers (TNF-α, IL-6, IL-1α, IL-1ß, TGF-ß, MIP-3α, IP-10, MCP-1, and Cathepsin-B) and anti-inflammatory/anti-HIV biomarkers (Secretory leukocyte protease inhibitor, Elafin, human beta-defensin-2 (HBD-2), alpha-defensins 1-3, Thrombospondin, Serpin-A1, and Cystatin-C) were measured in plasma using enzyme-linked immunosorbent assay. Within each HIV serostatus, differences in biomarker levels between subgroups were evaluated with Kruskal-Wallis and Dunn's test with Bonferroni correction. Spearman correlations between biomarkers were assessed for each subgroup. RESULTS: Compared to the Control and Depression groups, Abuse + Depression was associated with significantly higher levels of chemokines MIP-3α and IP-10 (p < 0.01) and lower levels of inflammatory cytokine IL-1ß (p < 0.01) in the HIV-uninfected population. Human beta-defensin-2 was lowest in the Abuse + Depression group (p < 0.05 versus Depression). By contrast, among HIV-infected, Abuse and Abuse + Depression were associated with lower levels of MIP-3α (p < 0.05 versus Control) and IP-10 (p < 0.05, Abuse versus Control). Inflammatory cytokine IL-6 was higher in both Abuse groups (p < 0.05 versus Control), while Elafin was lowest in the Abuse + Depression group (p < 0.01 versus Depression). CONCLUSION: We report compromised plasma immune responses that parallel previous findings in the genital mucosa, based on sexual abuse and HIV status. Systemic biomarkers may indicate trauma exposure and impact risk of HIV acquisition/transmission.

Immune-enhancing effect of water-soluble beta-glucan derived from enzymatic hydrolysis of yeast glucan.

Immunostimulants play an important role in the treatment of immunodeficiency. Macrophages are the first line in our immune defense system and play a critical role in the immune response. Therefore, finding new and better substances to induce an immune response by activating macrophages is an attractive research topic, especially in the fields of immunopharmacology and cancer prevention. Keratinocytes actively crosstalk with immune cells during wound repair, so enhancing the function of keratinocytes is also an important part of improving immunity. Beta-glucans are naturally occurring polysaccharides, consisting of d-glucose monomers linked by beta-glycosidic bonds. Several studies have investigated the immunomodulatory effects of beta-glucan, such as its anti-inflammatory and antibacterial properties. However, the use of yeast cell wall glucan has been limited because it is not soluble in water. In this study, we produced low-molecular-weight water-soluble yeast glucan (WSY glucan) and confirmed various aspects of its immune-enhancing effect. The structure of the beta-(1→3) and (1→6) bonds of WSY glucan were confirmed by nuclear magnetic resonance spectroscopy (1H-NMR) analysis. Our results showed that treatment with WSY glucan significantly and dose-dependently induced the production of inflammatory mediators (prostaglandin E2 (PGE2) and nitric oxide (NO)) and pro-inflammatory cytokines (tumor necrosis factor (TNF)-α and interleukin (IL)-6) in macrophages. In addition, WSY glucan treatment showed changes in the morphological structure of the macrophages and promoted phagocytic activity of the macrophages and wound healing in keratinocytes. Based on these results, WSY glucan is considered as a potential candidate for the treatment of diseases related to the weakening of the immune system without the limitation of insolubility.

Characterization of Early Peripheral Immune Responses in Patients with Sepsis and Septic Shock.

(1) Background: Sepsis is a life-threatening condition caused by an abnormal host response to infection that produces altered physiological responses causing tissue damage and can result in organ dysfunction and, in some cases, death. Although sepsis is characterized by a malfunction of the immune system leading to an altered immune response and immunosuppression, the high complexity of the pathophysiology of sepsis requires further investigation to characterize the immune response in sepsis and septic shock. (2) Methods: This study analyzes the immune-related responses occurring during the early stages of sepsis by comparing the amounts of cytokines, immune modulators and other endothelial mediators of a control group and three types of severe patients: critically ill non-septic patients, septic and septic shock patients. (3) Results: We showed that in the early stages of sepsis the innate immune system attempts to counteract infection, probably via neutrophils. Conversely, the adaptive immune system is not yet fully activated, either in septic or in septic shock patients. In addition, immunosuppressive responses and pro-coagulation signals are active in patients with septic shock. (4) Conclusions: The highest levels of IL-6 and pyroptosis-related cytokines (IL-18 and IL-1α) were found in septic shock patients, which correlated with D-dimer. Moreover, endothelial function may be affected as shown by the overexpression of adhesion molecules such as s-ICAM1 and E-Selectin during septic shock.

Elevated Levels of Interleukins, Leukocyte Protein and Cathelicidin Antimicrobial Peptide are Strongly Associated with Early to Mid-Stage of Pythium insidiosum Infection in Rabbit Corneas.

PURPOSE: Corneal infection in humans caused by Pythium insidiosum can lead to blindness and the host ocular immune response to it is less studied. Herein, we investigate the expression of mediators of innate and adaptive immune responses in a rabbit model. METHODS: P. insidiosum zoospores were injected intracorneally in right eye of the nine New Zealand White rabbits while left eye was injected with 1XPBS. RT-qPCR and multiplex ELISA (mELISA) were used to study the expression of antimicrobial peptides (AMPs) and immune mediators in infected cornea on 3rd, 7th and 9th day of post-infection(PI). STRING-11.0 analysis was used to predict the interactions of immune mediators. mRNA expressions of pathogen recognition receptors (PRRs) were determined in human corneal epithelial cells (HCECs) stimulated with P. insidiosum zoospores. Data was analyzed using one-way ANOVA with Post-Hoc Tukey HSD test and p-value <0.05 was considered significant. RESULTS: mRNA expression assay for IL-1ß, IL-6, IL-8 and Cathelicidin antimicrobial peptide (CAP)-18 showed significant upregulation (p-value < 0.05) on 7th day post-infection (PI) compared to 3rd and 9th day while Leukocyte protein (LeukoP) was elevated significantly on 3rd day followed by 7th and 9th day PI . Only IL-17A among other adaptive immune cytokines showed significant upregulation on 7th day compared to 9th day PI. Expressed in pg/mL, mELISA showed significant higher levels (p-value < 0.05) of IL-1ß, IL-8 in infected tissue in each of the time points compared to control. STRING analysis revealed co-expression of IL-1ß, IL-8 and IL-6. Among PRRs, Dectin 1 and TLR4 showed significant upregulation in HCECs at 12 hrs compared to 6 hrs. CONCLUSION: In the rabbit P. insidiosum keratitis model, innate immune mediators: IL-1ß, IL-8, IL-6, AMPs: LeukoP and CAP-18 are strongly associated in early to mid-stage of corneal infection. Dectin 1 and TLR4 were observed to be associated with recognition of P. insidiosum.

Opposing Immune-Metabolic Signature in Visceral Versus Subcutaneous Adipose Tissue in Patients with Adenocarcinoma of the Oesophagus and the Oesophagogastric Junction.

Oesophageal adenocarcinoma (OAC) is an exemplar model of obesity-associated cancer. Previous work in our group has demonstrated that overweight/obese OAC patients have better responses to neoadjuvant therapy, but the underlying mechanisms are unknown. Unravelling the immune-metabolic signatures of adipose tissue may provide insight for this observation. We hypothesised that different metabolic pathways predominate in visceral (VAT) and subcutaneous adipose tissue (SAT) and inflammatory secretions will differ between the fat depots. Real-time ex vivo metabolic profiles of VAT and SAT from 12 OAC patients were analysed. These samples were screened for the secretion of 54 inflammatory mediators, and data were correlated with patient body composition. Oxidative phosphorylation (OXPHOS) was significantly higher in VAT when compared to SAT. OXPHOS was significantly higher in the SAT of patients receiving neoadjuvant treatment. VEGF-A, VEGF-C, P1GF, Flt-1, bFGF, IL-15, IL-16, IL-17A, CRP, SAA, ICAM-1, VCAM-1, IL-2, IL-13, IFN-γ, and MIP-1ß secretions were significantly higher from VAT than SAT. Higher levels of bFGF, Eotaxin-3, and TNF-α were secreted from the VAT of obese patients, while higher levels of IL-23 and TARC were secreted from the SAT of obese patients. The angiogenic factors, bFGF and VEGF-C, correlated with visceral fat area. Levels of OXPHOS are higher in VAT than SAT. Angiogenic, vascular injury and inflammatory cytokines are elevated in VAT versus SAT, indicating that VAT may promote inflammation, linked to regulating treatment response.

Plasma-derived extracellular vesicles discriminate type-1 allergy subjects from non-allergic controls.

BACKGROUND: Allergies are on the rise globally, with an enormous impact on affected individuals' quality of life as well as health care resources. They cause a wide range of symptoms, from slightly inconvenient to potentially fatal immune reactions. While allergies have been described and classified phenomenologically, there is an unmet need for easily accessible biomarkers to stratify the severity of clinical symptoms. Furthermore, biomarkers marking the success of specific immunotherapy are urgently needed. OBJECTIVES: Plasma extracellular vesicles (pEV) play a role in coordinating the immune response and may be useful future biomarkers. A pilot study on differences in pEV content was carried out between patients with type I allergy, suffering from rhinoconjunctivitis with or without asthma, and voluntary non-allergic donors. METHODS: We examined pEV from 38 individuals (22 patients with allergies and 16 controls) for 38 chemokines, cytokines, and soluble factors using high-throughput data mining approaches. RESULTS: Patients with allergies had a distinct biomarker pattern, with 7 upregulated (TNF-alpha, IL-4, IL-5, IL-6, IL-17F, CCL2, and CCL17) and 3 downregulated immune mediators (IL-11, IL-27, and CCL20) in pEV compared to controls. This reduced set of 10 factors was able to discriminate controls and allergic patients better than the total array. CONCLUSIONS: The content of pEV showed potential as a target for biomarker research in allergies. Plasma EV, which are readily measurable via blood test, may come to play an important role in allergy diagnosis. In this proof-of-principle study, it could be shown that pEV's discriminate patients with allergies from controls. Further studies investigating whether the content of pEVs may predict the severity of allergic symptoms or even the induction of tolerance to allergens are needed.

Psychological Stresses in Children Trigger Cytokine- and Kynurenine Metabolite-Mediated Abdominal Pain and Proinflammatory Changes.

Recurrent abdominal pain (RAP) is a common medically unexplained symptom among children worldwide. However, the biological mechanisms behind the development of functional and behavioral symptoms and changes in blood markers have not been well explored. This study aimed to assess changes in the concentrations of inflammatory markers, including cytokines and tryptophan catabolites, in the serum of children with RAP compared to those with subclinical infections. Children with RAP but without organic diseases were included, and those with asymptomatic intestinal parasitic infections were used as a subclinical infection cohort. Blood samples were collected and used to measure the cytokine profile using Multiplex Immunoassay and tryptophan catabolites using high performance liquid chromatography. Children with RAP showed significantly higher concentrations of serum tumor necrotic factor-α, p<0.05, but lower concentrations of IL-10, p<0.001, IL-6, p<0.001 and brain-derived neurotrophic factors (BDNF) p<0.01. In addition, a significant increase in the metabolite of the kynurenine pathway, 3-hydroxyanthranilic acid (3-HAA) p<0.01, a significant decrease in the concentrations of anthranilic acid (AA) p<0.001, together with an increased ratio of serum 3-HAA to AA (3-HAA/AA) p<0.001, was found in this cohort. These findings indicate the significant activation of the immune system and presence of inflammation in children with RAP than those with subclinical parasitic infections. Moreover, children with RAP tested with the Strengths and Difficulties Questionnaire (SDQ), displayed high psychological problems though these SDQ scores were not statistically associated with measured cytokines and kynurenine metabolites. We however could hypothesize that the pro-inflammatory state together with concomitant low concentrations of BDNF in those children with RAP could play a role in psychological stress and experiencing medically unexplained symptoms.