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Allergo journal international 30(5), 155-168 (2021). doi:10.1007/s40629-021-00178-2, Published by Urban & Vogel, München

In recent years, Bovine herpesvirus 4 (BoHV-4) has emerged as an attractive gene delivery viral vector, mainly for vaccination purposes in the veterinary field. In the present study, a new infectious clone of the BoHV-4 genome carrying a bacterial artificial chromosome vector (BoHV-4-BAC) was developed by homologous recombination in mammalian cell culture and bacterial systems, and exploited to express a truncated form of glycoprotein D (tgD) of Bovine herpesvirus 1 (BoHV-1) (BoHV-4-tgD∆TK) as a vaccine candidate. This construct's immunogenicity was compared to a DNA vector expressing the same antigen (pC-tgD) in a BALB/c mouse model. After the mice were immunized, total and specific antibody responses, cytokine responses, total splenocyte cells proliferation/cytotoxicity, and virus neutralization assays were conducted to analyze the immune response elicited by both constructs. Mice from both vaccine groups developed significant humoral and cellular immune responses after a booster dose regime was conducted on day 28 post-injection. In almost all immunological assays, BoHV-4-tgDΔTK induced as high an immune response as pC-tgD. In both vaccine constructs, neutralizing antibodies were a significant determining factor in protection against BoHV-1, even after the first injection. We conclude that a BoHV-4-based viral vector offers an effective immunization strategy as an alternative to DNA-based immunization platforms, at least to combat BoHV-1.

Mitochondrial DNA (mtDNA) heteroplasmy is the dynamically determined co-expression of wild type (WT) inherited polymorphisms and collective time-dependent somatic mutations within individual mtDNA genomes. The temporal expression and distribution of cell-specific and tissue-specific mtDNA heteroplasmy in healthy individuals may be functionally associated with intracellular mitochondrial signaling pathways and nuclear DNA gene expression. The maintenance of endogenously regulated tissue-specific copy numbers of heteroplasmic mtDNA may represent a sensitive biomarker of homeostasis of mitochondrial dynamics, metabolic integrity, and immune competence. Myeloid cells, monocytes, macrophages, and antigen-presenting dendritic cells undergo programmed changes in mitochondrial metabolism according to innate and adaptive immunological processes. In the central nervous system (CNS), the polarization of activated microglial cells is dependent on strategically programmed changes in mitochondrial function. Therefore, variations in heteroplasmic mtDNA copy numbers may have functional consequences in metabolically competent mitochondria in innate and adaptive immune processes involving the CNS. Recently, altered mitochondrial function has been demonstrated in the progression of coronavirus disease 2019 (COVID-19) due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Accordingly, our review is organized to present convergent lines of empirical evidence that potentially link expression of mtDNA heteroplasmy by functionally interactive CNS cell types to the extent and severity of acute and chronic post-COVID-19 neurological disorders.

The blood and immune system of coronavirus disease 2019 (COVID-19) infected patients are dysfunctional, and numerous studies have been conducted to resolve their characteristics and pathogenic mechanisms. Nevertheless, the variations of immune responses along with disease severity have not been comprehensively documented. Here, we profiled the single-cell transcriptomes of 96,313 peripheral blood mononuclear cells (PBMCs) derived from 12 COVID-19 patients (including four moderate, four severe and four critical cases) and three healthy donors. We showed that proliferative CD8 effector T cells with declined immune functions and cytotoxicity accumulated in the critical stage. By contrast, the quantity of natural killer (NK) cells was significantly reduced, while they exhibited enhanced immune activities. Notably, a gradually attenuated responseto COVID-19 along with disease severity was observed in monocytes, in terms of cellular composition, transcriptional discrepancy and transcription factor regulatory network. Furthermore, we identified immune cell-type dependent cytokine signatures distinguishing the severity of COVID-19 patients. In addition, cell interactions between CD8 effector T/NK cells and monocytes mediated by inflammatory cytokines were enhanced in moderate and severe stages, but weakened in critical cases. Collectively, our work uncovers the cellular and molecular players underlying the disordered and heterogeneous immune responses associated with COVID-19 severity, which could provide valuable insights for the treatment of critical COVID-19 patients. Electronic Supplementary Material Supplementary material is available for this article at 10.1007/s11427-020-1880-y and is accessible for authorized users.

Septic shock is one of the most significant health concerns across the world, involving hypo-perfusion and defects in tissue energy. The current study investigates the role of NLR family CARD domain containing protein 4 (NLRC4) in septic shock-induced inflammatory reactions, lung tissue injuries, and dendritic cell (DC) apoptosis. Septic shock mice models were established by modified cecal ligation and puncture and injected with retroviral vector expressing siRNA-NLRC4. DCs were then isolated and transfected with siRNA-NLRC4. The degree of lung tissue injury, cell cycle distribution, cell apoptosis and cell viability of DCs were assessed. NLRC4 was found to be expressed at high levels in mice with septic shock. NLRC4 silencing inhibited the activation of the NOD-like receptor (NLR) pathway as evidenced by the decreased levels of NOD1, NOD2, RIP2, and NF-κB. In addition, NLRC4 silencing reduced the inflammatory reaction as attributed by reduced levels of IL-1β, TNF-α and IL-6. Suppressed NLRC4 levels inhibited cell viability and promoted cell apoptosis evidenced by inhibited induction of DC surface markers (CD80, CD86, and MHC II), along with alleviated lung tissue injury. In conclusion, NLRC4 silencing ameliorates lung injury and inflammation induced by septic shock by negatively regulating the NLR pathway.

Background: Sonodynamic therapy (SDT) is a promising strategy to inhibit tumor growth and activate antitumor immune responses for immunotherapy. However, the hypoxic and immunosuppressive tumor microenvironment limits its therapeutic efficacy and suppresses immune response. Methods: In this study, mitochondria-targeted and ultrasound-responsive nanoparticles were developed to co-deliver oxygen (O2) and nitric oxide (NO) to enhance SDT and immune response. This system (PIH-NO) was constructed with a human serum albumin-based NO donor (HSA-NO) to encapsulate perfluorodecalin (FDC) and the sonosensitizer (IR780). In vitro, the burst release of O2 and NO with US treatment to generate reactive oxygen species (ROS), the mitochondria targeting properties and mitochondrial dysfunction were evaluated in tumor cells. Moreover, in vivo, tumor accumulation, therapeutic efficacy, the immunosuppressive tumor microenvironment, immunogenic cell death, and immune activation after PIH-NO treatment were also studied in 4T1 tumor bearing mice. Results: PIH-NO could accumulate in the mitochondria and relive hypoxia. After US irradiation, O2 and NO displayed burst release to enhance SDT, generated strongly oxidizing peroxynitrite anions, and led to mitochondrial dysfunction. The release of NO increased blood perfusion and enhanced the accumulation of the formed nanoparticles. Owing to O2 and NO release with US, PIH-NO enhanced SDT to inhibit tumor growth and amplify immunogenic cell death in vitro and in vivo. Additionally, PIH-NO promoted the maturation of dendritic cells and increased the number of infiltrating immune cells. More importantly, PIH-NO polarized M2 macrophages into M1 phenotype and depleted myeloid-derived suppressor cells to reverse immunosuppression and enhance immune response. Conclusion: Our findings provide a simple strategy to co-deliver O2 and NO to enhance SDT and reverse immunosuppression, leading to an increase in the immune response for cancer immunotherapy.

The immune system with its numerous and complex interactions helps to protect the host from pathogenic microorganisms, and enables cleaning of damaged tissues. It is also associated with constant “monitoring” of the appearance of malignant cells and their elimination that can occur in the human body. Such a role depends on many factors including adequate intake of nutrients, including vitamins. The effect of vitamin supplementation on the modulation of the immune response has always been the focus of numerous studies. Vitamins A and D have been shown to have the greatest immune-modulatory effect. In this review, we discuss and consider the possible roles of vitamins A and D on the immune response through innate and adaptive immune cells, with special focus on the cell population recently characterized as innate lymphoid cells. Recent literature data indicate that vitamin A and its metabolites modulate the balance between Th1 and Th2 immunity. In addition, vitamin D expresses protective effects on the innate immune system and inhibitory effects on adaptive immunity.

Effects of a large-sized cage with a low metabolizable energy and low crude protein (LME-LCP) diet on growth performance, feed cost, blood biochemistry, and antibody response of growing layers were evaluated. A total of 668 one-day-old female Gushi chicks were randomly allocated into three different cages, namely, large, medium, and small cages, referred to as Group A, Group B, and Group C, respectively, and fed LME-LCP diets. A fourth group of birds raised in small cages and fed a standard diet (STD) was designated Group D i.e. the control. Equal stocking densities were maintained among the four groups throughout the experiment, from 50–20 birds/m2. Large cages with LME-LCP diet (Group A) increased the shank length and girth as chicks grew, improved the activities of serum creatine kinase, and reduced serum triglyceride and cholesterol concentrations. The total feed intake in birds from Group A was higher than those from the other groups at every stage. The total cost (rmb/bird) of feed was 6.70% lower in Group A than that in Group D, which indicated the cost-effectiveness of large cages. In conclusion, large cages with LME-LCP diets have positive effects on body weight, shank growth, and serum biochemical indices of growing Gushi chicks, and can reduce feed costs.

Background Muscle-invasive bladder cancer (MIBC) patients are subject to unfavorable treatment options and a high recurrence rate. The status of TP53 mutations played an essential role in the progression and the prognosis of MIBC. The present study proposed to investigate the association between TP53 mutations and immunophenotype in MIBC. Results We established an immune prognostic model (IPM) ground on the immune-associated genes derived from variation analysis between wild-type TP53 and mutated TP53 TCGA-MIBC patients, and validated in another cohort from GEO database. Based on IPM, we divided MIBC patients into low and high risk subgroups. The high risk MIBC patients had higher proportions of macrophages M1, and lower proportions of T cells regulatory (Tregs) and activated dendritic cells than the low risk MIBC patients. Moreover, the expression of immune checkpoints genes (PD1, CTLA4, LAG3, HAVCR2 and TIGIT) was higher in the high risk patients than the low risk patients. In clinical application, IPM exhibited better survival prediction than conventional clinical characteristics. Conclusions Our investigation presented practical prognostic significance for MIBC patients and displayed the overarching landscape of the immune response in the MIBC microenvironment. Methods Data were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). Differentially expressed genes (DEGs) analysis between the TP53 mutated and wild-type MIBC patients was conducted. The CIBERSORT algorithm was performed to evaluate the proportion of immune cell types. Gene expression profiles from the TCGA and GEO were used as training and testing cohorts to build and validate an immune-related prognostic model (IPM). Genes in the IPM model were first screened by univariate Cox analysis, then filtered by the least absolute shrinkage and selection operator (LASSO) Cox regression. A nomogram was finally established and evaluated by combining both the IPM and other clinical factors.

Physical frailty’s impact on hemagglutination inhibition antibody titers (HAI) and peripheral blood mononuclear cell (PBMC) transcriptional responses after influenza vaccination is unclear. Physical frailty was assessed using the 5-item Fried frailty phenotype in 168 community- and assisted-living adults ≥55 years of age during an observational study. Blood was drawn before, 3, 7, and 28 days post-vaccination with the 2017-2018 inactivated influenza vaccine. HAI response to the A/H1N1 strain was measured at Days 0 and 28 using seropositivity, seroconversion, log2 HAI titers, and fold-rise in log2 HAI titers. RNA sequencing of PBMCs from Days 0, 3 and 7 was measured in 28 participants and compared using pathway analyses. Frailty was not significantly associated with any HAI outcome in multivariable models. Compared with non-frail participants, frail participants expressed decreased cell proliferation, metabolism, antibody production, and interferon signaling genes. Conversely, frail participants showed elevated gene expression in IL-8 signaling, T-cell exhaustion, and oxidative stress pathways compared with non-frail participants. These results suggest that reduced effectiveness of influenza vaccine among older, frail individuals may be attributed to immunosenescence-related changes in PBMCs that are not reflected in antibody levels.

Chemotherapy resistance eventually develops in patients with gastric cancer (GC). Intra-tumoral heterogeneity (ITH) refers to the intercellular genetic variations and phenotypic diversity that affect responses to drug therapy. We measured ITH using mutant-allele tumor heterogeneity (MATH) derived from whole-exome sequencing data of patients with GC in The Cancer Genome Atlas (TCGA) database. The study included 385 patients from the TCGA database with available data regarding gastrectomy, survival, and whole-exome sequencing. Further analysis was performed in 171 GC patients with available data regarding adjuvant chemotherapy. Multiple factor analysis showed that MATH was an independent predictor of OS (hazard ratio [HR], 1.432; 95% confidence interval [CI], 1.073–1.913; P = 0.015) in patients with GC. Moreover, MATH was also an independent predictor of OS among the 171 GC patients who received adjuvant chemotherapy (HR, 2.016; 95% CI, 1.236–3.289; P = 0.005). Pathway enrichment and immune cell analyses revealed significantly higher infiltration by 20 types of immune cells in the low/intermediate group, compared to the group with high MATH scores. In conclusion, low/intermediate MATH scores predicted longer OS, when compared to those with high MATH scores. The immune response was obviously upregulated in patients with GC and low/intermediate MATH scores.

Legionnaires’ Disease (LD) is a severe pneumonia mainly caused in Europe by Legionella pneumophila serogroup 1 (Lp1). Sequence-based typing methods reveal that some sequence types (ST) are overrepresented in clinical samples such as ST1 and ST47, suggesting that some strains are more fit for infection than others. In the present study, a collection of 108 Lp1 clinical isolates were used to evaluate the strain-dependent immune responses from human macrophages. Clinical Lp1 isolates induced differential TNFα secretion from macrophages. ST1 isolates induced a significantly higher TNF-α secretion than non-ST1, whereas ST47 isolates induced a significantly lower TNF-α secretion than non-ST47 isolates. ST1 isolates induced a significantly higher cell death than ST47 isolates evaluated by lactate dehydrogenase activity (cytotoxicity) and caspase-3 activity (apoptosis). Treatment of macrophages with anti-TNF-α antibodies significantly reduced the cell death in macrophages infected with ST1 or ST47 strains. The TNF-α secretion was neither explained by a differential bacterial replication nor by the number or type (bystander or infected) of TNF-α producing cells following infection but by a differential response from macrophages. The Paris ST1 reference strain elicited a significantly higher TNF-α gene transcription and a higher induction of NF-κB signaling pathway than the Lorraine ST47 reference strain. Clinical Lp1 isolates induce a diverse immune response and cell death, which could be related to the genotype. The two predominant sequence-types ST1 and ST47 trigger opposite inflammatory response that could be related to the host susceptibility.

The complement systems play an important role in innate and adaptive immunity. In this study, the complement C3 gene, designated CagC3, was cloned and sequenced from Gibel carp (Carassius auratus gibelio). The expression pattern of CagC3 in different tissues of healthy Gibel carp and after challenge with Cyprinid herpesvirus 2 (CyHV-2) were evaluated using quantitative real-time PCR. The full-length CagC3 cDNA was 5131 bp with an ORF of 4950 bp, encoding a predicted protein of 1649 amino acids. The deduced amino acid sequence showed that CagC3 has conserved domains and residues known to be critical for C3 function. Phylogenetic analysis demonstrated that CagC3 clustered with homologs from common carp and grass carp (Ctenopharyngodon idella). CagC3 is expressed in all examined tissues of healthy Gibel carp, with the highest expression in liver. In vivo, after CyHV-2 challenge, CagC3 transcription was significantly upregulated in liver, spleen and kidney with the peaks at 24 hr, 2 d, and 2 d, respectively. In vitro, CagC3 expression in the Gibel carp brain cell line showed the same pattern as that in vivo after stimulation with CyHV-2 or poly(I:C). However, CagC3 expression was downregulated at 24 hr after induction with lipopolysaccharide (LPS), and then reached the peak at 2 d. These results suggest that CagC3 is involved in the innate immune response of Gibel carp to viral infection.

Background: The clinical benefit of immune response is largely unknown. We systematically explored the correlation of immune response with patient outcome in human cancers. Results: The global immune gene signature was primarily located on the plasma membrane with a high gene density at 6p21 and 1q23-1q24. Immune responses varied with a wide range in human cancers. A total of 11 cancer types exhibited significant correlation of immune response with overall survival. Higher immune response was significantly associated with longer overall survival in 7 types and with shorter overall survival in 4 types. In addition, 11 cancer types exhibited significant correlation of immune response with progression-free interval. Higher immune response was significantly associated with longer progression-free interval in 7 types and with shorter progression-free interval in 4 types. Methods: The Ingenuity Knowledge Base and human genome assembly GRCh38 were used to annotate the immune gene signature by cellular components and genomic coordinates, respectively. We devised an mRNA-based metric of pre-existing immune conditions by using the gene signature, and calculated the metric for 10,062 The Cancer Genome Atlas tumor samples across 32 different cancer types. The Kaplan-Meier method was used to evaluate the overall survival and progression-free interval differences between dichotomic groups stratified by the median metric for each cancer type. Conclusions: Immune responses have different impacts on patient outcome in different human cancers. Prospective verification is needed before the findings can be applied for clinical trial development.

This report describes efforts to understand the immune mechanism of action that led to a complete response in a patient with progressive, refractory, metastatic melanoma after treatment with a therapeutic vaccine consisting of autologous dendritic cells (DC) loaded with autologous tumor antigens (ATA) derived from cells that were self-renewing in cell culture. Her histocompatibility type proved to be HLA B27 with extensive mutations in the HLA-A locus. Exomic analysis of proliferating tumor cells revealed more than 2800 non-synonymous mutations compared to her leukocytes. Histology of resected tumor lesions showed no evidence of an existing or suppressed immune response. In in vitro mixed cell cultures, DC loaded with ATA induced increased IL-22 expression, and a four-fold increase in CD8 + T lymphocytes. Cryopreserved blood samples obtained at week-0, 1 week before the first of three-weekly vaccine injections, and at week-4, 1 week after the third dose, were analyzed by protein array and compared for 110 different serum markers. At baseline, she had marked elevations of amyloid A, IL-12p40, IL21, IL-22, IL-10, IL-16, GROa, TNF-alpha, IL-3, and IL-2, and a lesser elevation of IL-15. One week after 3 weekly vaccinations she had a further 80% increase in amyloid A, a further 66% increase in IL-22, a 92% decrease in IL12p40, a 45% decrease in TGF-β and 26% decrease in IL-10. The data suggested that by 3 weeks after the first DCV injection, vaccine-induced changes in this particular patient were most consistent with enhanced innate and Th1 immune responses rather than Th2 or Th17.

// Yuexin Liu 1 1 Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA Correspondence to: Yuexin Liu, email: yliu8@mdanderson.org Keywords: clinicopathological characteristics; immune response; immunogenicity; cancer Received: August 01, 2019 Accepted: September 24, 2019 Published: October 08, 2019 ABSTRACT Background: The clinicopathologic association of tumor immune response is largely unknown. We systematically investigated this matter in human cancers. Results: Different cancer types exhibited distinct immune gene profiling. Four cancer types exhibited a significant and positive correlation of immune response with patient age. Significant but inconsistent correlation of immune response was observed with gender, surgical stage, and TNM stage in a small number of cancer types. In contrast, the histological grade appears to have much stronger and more consistent association with immune response as compared to the other clinicopathologic factors. Specifically, patients with high grade had significantly higher immune responses than those with low grade in 5 out of 12 analyzed cancer types. In addition, both histological and molecular classifications had a significant and strong association with tumor immune response. Methods: t-distributed stochastic neighbor embedding was used to assess similarity of immune gene profiling in human cancers. The Mann-Whitney or Kruskal-Wallis test was, respectively, used to compare the tumor immune response in two or more groups that were stratified by patient clinicopathological characteristics, such as gender, grade, stage (including surgical and TNM stage), histology, and molecular subtypes. Spearman correlation with student’s t -test was used to examine the association of patient age with immune response. Multiple tests with the Benjamini-Hochberg correction also were performed. Conclusions: Tumor grade should be taken into account in selection of patient candidates for immunotherapy. Prospective verification is needed before use of the findings for clinical practice.

It focused on the antiviral immune regulation of biofilm-localized protein kinase Dbf2p-related kinase 1 (NDR1) in viral pneumonia. Mouse alveolar monocyte RAW264.7 was used as blank control, and viral pneumonia cell model was prepared by infecting cells with respiratory syncytial virus (RSV). NDR1 overexpression vector and siRNA interference sequences were synthesized, and overexpression/silence NDR1 cell model was fabricated. About 50 ng/mL interleukin 17 (IL-17) was given to stimulate. Enzyme-linked immunosorbent assay (ELISA), quantitative reverse transcription PCR (RT-qRCR), and Western blot were performed to detect cytokines and chemokines, mRNA of inflammatory factors, and signal molecule protein expression. Notably, RSV infection increased RSV-F mRNA in RAW264.7 cells and reduced NDR1 mRNA and protein. Secretion levels of IL-6, interferon β (IFN-β), chemokine (C-X-C motif) ligand 2 (CXCL2), and chemokine (C-C motif) ligand 2 (CCL20) increased in the model group versus blank control (P 0.05). In short, NDR1 regulated innate immune response to viral pneumonia induced by IL-17, which can be used as a new target for the treatment of IL-17-induced inflammatory response and autoimmune diseases.

Rationale: Around 10%-20% patients with glioblastoma (GBM) are diagnosed with more than one tumor lesions or multifocal GBM (mGBM). However, the understanding on genetic, DNA methylomic, and transcriptomic characteristics of mGBM is still limited. Methods: In this study, we collected nine tumor foci from three mGBM patients followed by whole genome sequencing, whole genome bisulfite sequencing, RNA sequencing, and immunohistochemistry. The data were further examined using public GBM databases and GBM cell line. Results: Analysis on genetic data confirmed common features of GBM, including gain of chr.7 and loss of chr.10, loss of critical tumor suppressors, high frequency of PDGFA and EGFR amplification. Through profiling DNA methylome of individual tumor foci, we found that promoter methylation status of genes involved in detection of chemical stimulus, immune response, and Hippo/YAP1 pathway was significantly changed in mGBM. Although both CNV and promoter methylation alteration were involved in heterogeneity of different tumor foci from same patients, more CNV events than promoter hypomethylation events were shared by different tumor foci, implying CNV were relatively earlier than promoter methylation alteration during evolution of different tumor foci from same mGBM. Moreover, different tumor foci from same mGBM assumed different molecular subtypes and mesenchymal subtype was prevalent in mGBM, which might explain the worse prognosis of mGBM than single GBM. Interestingly, we noticed that LIF and CCL2 was tightly correlated with mesenchymal subtype tumor focus in mGBM and predicted poor survival of GBM patients. Treatment with LIF and CCL2 produced mesenchymal-like transcriptome in GBM cells. Conclusions: Together, our work herein comprehensively profiled multi-omics features of mGBM and emphasized that components of extracellular microenvironment, such as LIF and CCL2, contributed to the evolution and prognosis of tumor foci in mGBM patients.

Recent studies have gained a better appreciation of the potential impacts of enteric infections beyond symptomatic diarrhea. It is recognized that infections by several enteropathogens could be associated with growth deficits in children and intestinal and systemic inflammation may play an important underlying role. With enterotoxigenic E. coli (ETEC) being one of the leading causes of diarrhea among children in the developing world and important contributor to stunting, a better understanding of the impact of ETEC infection beyond diarrhea is timely and greatly needed. To address this, we evaluated if ETEC infection induces intestinal and systemic inflammation and its impact on colonization and immune responses to ETEC vaccine-specific antigens in a dose descending experimental human challenge model using ETEC strain H10407. This study demonstrates that the concentrations of myeloperoxidase (MPO) in stool and intestinal fatty acid-binding protein (an indicator of compromised intestinal epithelial integrity) in serum, significantly increased following ETEC infection in both diarrhea and asymptomatic cases and the magnitudes and kinetics of MPO are dose and clinical outcome dependent. Cytokines IL-17A and IFN-γ were significantly increased in serum post-ETEC challenge. In addition, higher pre-challenge concentrations of cytokines IL-10 and GM-CSF were associated with protection from ETEC diarrhea. Interestingly, higher MPO concentrations were associated with higher intestinal colonization of ETEC and lower seroconversions of colonization factor I antigen, but the reverse was noted for seroconversions to heat-labile toxin B-subunit. Together this study has important implications for understanding the acute and long-term negative health outcomes associated with ETEC infection.

Recent studies demonstrated that CD155 plays an important role in anti-tumor immune responses. However, its role in glioma remains unclear. Here, we identify CD155 as a promising immune target in glioma. CD155 expression was significantly highly expressed in glioblastoma but not in normal brain tissue. Subsequent analysis based on genetic and clinical data from 1173 glioma patients in Rembrandt and TCGA dataset suggested that CD155 related genes of immune response were mainly positively correlated with CD155 expression. CD155 expression was positively correlated with immune-related metagenes STAT1, HCK, LCK, and MHC I but negatively associated with IgG. CD155 expression was positively correlated with biomarker gene expression of infiltrating immune cells, suggested that high CD155 expression in gliomas tend to have more infiltrating immune cells compared with gliomas with low CD155 expression. Pearson correlation analysis showed that CD155 is associated with CD96, CD226, Nectin4, PD-L1, B7-H2, NR2F6 and GITR, implying the potential synergistic effects of these checkpoint proteins. These findings implied that CD155 is a promising immunotherapy target, combined with existing immune checkpoint blockade therapies for glioma.

Phytochemicals which exist in various plants and fungi are non-nutritive compounds that exert numerous beneficial bioactive actions for animals. In recent years following the restriction of antibiotics, phytochemicals have been regarded as a primal selection when dealing with the challenges during the producing process in the poultry industry. The selected fast-growing broiler breed was more fragile when confronting the stressors in their growing environments. The disruption of oxidative balance that impairs the production performance in birds may somehow be linked to the immune system since oxidative stress and inflammatory damage are multi-stage processes. This review firstly discusses the individual influence of oxidative stress and inflammation on the poultry industry. Next, studies related to the application of phytochemicals or botanical compounds with the significance of their antioxidant and immunomodulatory abilities are reviewed. Furthermore, we bring up nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and nuclear factor kappa B (NF-κB) for they are respectively the key transcription factors involved in oxidative stress and inflammation for elucidating the underlying signal transduction pathways. Finally, by the discussion about several reports using phytochemicals to regulate these transcription factors leading to the improvement of oxidative status, heme oxygenase-1 gene is found crucial for Nrf2-mediated NF-κB inhibition.

Background The comprehensive characterization and prognostic relevance of immune activation in endometrial cancer remain largely unknown. Results We systematically reported a subset of endometrioid-type endometrial cancer characterized by multifaceted immune features such as low tumor purity, high leukocyte percentage, and striking CD8 lymphocytic infiltration with anti-tumor efficacy along with marked upregulation of immunosuppressive gene markers. We also showed that genes whose expression was significantly correlated with better survival were significantly enriched in the immune-related signaling pathways, suggesting that tumor-infiltrating lymphocytes give rise to a favorable prognosis in endometrial cancer. Furthermore, we showed that immune cell recruitment in this subset of tumors is likely due to the transcriptional activation of the STAT1 signaling network. Methods We obtained the multi-dimensional genomic data from publicly available databases and correlated them with the four gene expression-based subtypes we recently identified in endometrial cancer. Upstream regulator analysis was used to identify the most significantly enriched transcription regulators and Ingenuity pathway analysis was applied to determine enrichment of signaling pathways in survival-associated genes. Gene set enrichment analysis was performed on the 200-gene T-cell tumor infiltration gene signature comparing Cluster IV with the other three clusters combined. All statistical tests were two-sided, and a P value of less than 0.05 is considered significant across all analyses performed. Conclusion This study helps to identify patients with immune activation who are likely to benefit from emerging immune checkpoint inhibitors.

With a broad range of hosts, Salmonella enterica serovar Typhimurium (S. Typhimurium) is the major cause of gastroenteritis in human beings and systemic disease in susceptible mice strains. However, different S. Typhimurium strains differ in regard to virulence and host adaptation. Here, C57BL/6 mice were infected, respectively, with different S. Typhimurium strains SL1344 (calf), CVCC541 (chicken) and CMCC50115 (mutton) to determine their virulence and host immune responses. It was found that mice were less susceptible to infection by S. Typhimurium CVCC541 and CMCC50115 strains, with lower lethality and decreased bacterial burden in liver and spleen. Besides, S. Typhimurium strains CVCC541 and CMCC50115 enhanced host innate immune responses by increased frequencies of macrophages and neutrophils 3 days after infection. But SL1344 strain evaded immune response by inducing apoptosis of macrophages. Moreover, CVCC541 could elicit adaptive immune responses of host 11 days after infection upon examination of the proliferation and activation of CD4+ T cells. In addition, 125 and 138 unique mutant coding genes, respectively, in S. Typhimurium strains CVCC541 and CMCC50115 and 78 shared mutant coding genes were annotated by genomic alignment to SL1344 genome and the signal pathways involving these genes were further analyzed. The acquired results indicate that different original S. Typhimurium strains show differential virulence and may induce diverse immune responses in the same host infected.

Streptococcus suis is a major porcine bacterial pathogen and emerging zoonotic agent. S. suis 5ʹ-nucleotidase is able to convert adenosine monophosphate to adenosine, resulting in inhibiting neutrophil functions in vitro and it is an important virulence factor. Here, we show that S. suis 5ʹ-nucleotidase not only enables producing 2ʹ-deoxyadenosine from 2ʹ-deoxyadenosine monophosphate by the enzymatic assay and reversed-phase high performance liquid chromatography (RP-HPLC) analysis in vitro, but also synthesizes both 2ʹ-deoxyadenosine and adenosine in mouse blood in vivo by RP-HPLC and liquid chromatography with tandem mass spectrometry analyses. Cellular cytotoxicity assay and Western blot analysis indicated that the production of 2ʹ-deoxyadenosine by 5ʹ-nucleotidase triggered the death of mouse macrophages RAW 264.7 in a caspase-3-dependent way. The in vivo infection experiment showed that 2ʹ-deoxyadenosine synthesized by 5ʹ-nucleotidase caused monocytopenia in mouse blood. The in vivo transcriptome analysis in mouse blood showed the inhibitory effect of 5ʹ-nucleotidase on neutrophil functions and immune responses probably mediated through the generation of adenosine. Taken together, these findings indicate that S. suis synthesizes 2ʹ-deoxyadenosine and adenosine by 5ʹ-nucleotidase to dampen host immune responses, which represents a new mechanism of S. suis pathogenesis.

Superoxide dismutase 1 (SOD1) binds copper and zinc ions and is one of three superoxide dismutases responsible for destroying free superoxide radicals in the body. Reactive oxygen species (ROS), including free superoxide radicals, play important roles in colitis. However, the role of SOD1 in oxidative stress under colitis remains unclear. Here, we examined the role of SOD1 in the DSS-induced mouse model of colitis. SOD1 deficiency resulted in severe oxidative stress with body weight loss, epithelial barrier disruption and decreased antioxidant enzyme activities. The levels of neutrophils, monocytes, pro-inflammatory CD11c+ macrophages and CD11b+CD103- dendritic cells (DCs) were increased, while anti-inflammatory CD206+ macrophages and CD11b−CD103+ DCs were decreased, in DSS-treated SOD1-knockout (KO) mice compared to DSS-treated wild-type mice. Furthermore, rescue of SOD activity in SOD1-KO mice by oral gavage of B. amyloliquefaciens SOD (BA SOD) significantly ameliorated enhanced DSS-induced colitis in these mice by suppressing p38-MAPK/NF-κB signaling, which can induce inflammation and apoptosis. Taken together, our results suggest that SOD1-mediated inhibitory responses play a crucial role in limiting the development of DSS-induced colitis, and that BA SOD is a promising candidate for treating colitis., Graphical abstract Image 1, Highlights • Superoxide dismutase 1(SOD1) deficiency exacerbates DSS-induced colitis. • SOD1 deficiency increases ROS production and reduces antioxidant enzyme activities. • SOD1 deficiency increases the infiltration of pro-inflammatory immune cells. • Rescue of SOD1 by BA SOD administration ameliorates acute colitis. • BA SOD administration suppresses p38-MAPK/NF-κB signaling.

Temperature influences fish’s susceptibility to infectious disease through an immune response. However, the mechanism underlying this regulation is yet to be elucidated. In this study, we compared the susceptibility of crucian carp that were grown at 18°C and 33°C, respectively, to Aeromonas sobrial infection and found that crucian carp was more susceptible when grown at 33°C. These distinct susceptibilities of fish at different temperatures to infection may partially be explained by their differences in the metabolism as revealed by comparative metabolomics profiling: crucian carp demonstrated enhanced TCA cycle but reduced fatty acid biosynthesis; Our study also found that maltose was the most suppressed metabolite in fish grown at 33°C. Importantly, exogenous injection of maltose enhances crucian carp survival grown at 33°C by 30%. Further study showed that exogenous maltose downregulated the production of several cytokines but enhanced the lysozyme (lyz) and complement component c3, which involves the humoral innate immunity. Our results suggest that maltose promotes the survival of crucian carp likely through fine tuning the immune gene expression, and this finding provides a novel approach to manage bacterial infection.

As one of the 10 most common cancers in men, the incidence of renal cell carcinoma (RCC) has been increasing in recent years. Clear cell renal cell carcinoma (ccRCC) is the most common pathological type of RCC, counting for 80%-90% of cases. Immunotherapy is becoming increasingly important in the treatment of advanced RCC. Tumor mutation burden (TMB) is a potent marker for predicting the response to immune checkpoint blockade (ICB) treatment. Here, we analyzed somatic mutation data for ccRCC from The Cancer Genome Atlas datasets. We found that the frequently mutated gene SYNE1 is associated with higher TMBs and with a poor clinical prognosis. To further investigate the relationship between SYNE1 mutation and the immune system, we used Gene Set Enrichment Analysis and the CIBERSORT algorithm. They showed that SYNE1 mutations correlate with immune system pathways and immune cell tumor infiltration. We also found that SYNE1 mutation correlated with a better response to ICB therapy. Thus, mutation of SYNE1 correlates with a higher TMB and a poorer outcome in ccRCC, but may mediate better responses to ICB therapy.

In December 2019, cases of severe coronavirus 2019 (COVID-19) infection rapidly progressed to acute respiratory failure. This study aims to assess the association between the neutrophil-to-lymphocyte ratio (NLR) and the incidence of severe COVID-19 infection. A retrospective cohort study was conducted on 210 patients with COVID-19 infection who were admitted to the Central Hospital of Wuhan from 27 January 2020 to 9 March 2020. Peripheral blood samples were collected and examined for lymphocyte subsets by flow cytometry. Associations between tertiles of NLR and the incidence of severe illness were analysed by logistic regression. Of the 210 patients with COVID-19, 87 were diagnosed as severe cases. The mean NLR of the severe group was higher than that of the mild group (6.6 vs. 3.3, P < 0.001). The highest tertile of NLR (5.1–19.7) exhibited a 5.9-fold (95% CI 1.3–28.5) increased incidence of severity relative to that of the lowest tertile (0.6–2.5) after adjustments for age, diabetes, hypertension and other confounders. The number of T cells significantly decreased in the severe group (0.5 vs. 0.9, P < 0.001). COVID-19 might mainly act on lymphocytes, particularly T lymphocytes. NLR was identified as an early risk factor for severe COVID-19 illness. Patients with increased NLR should be admitted to an isolation ward with respiratory monitoring and supportive care.

Tumor-associated tertiary lymphoid structures (TLS) play a critical role in the progression of various tumors. However, the dynamics of lymphocyte recruitment during hepatocellular carcinoma (HCC) clinical progression have not been fully elucidated. In the present study, tissue microarrays and hematoxylin-eosin staining were used to evaluate the existence and degree of TLS in HCC patients. Nine immune biomarkers in intratumoral tissues were examined by immunohistochemical staining. A total of 462 patients were recruited for the study. Kaplan-Meier analysis showed that TLS was inversely correlated with the risk of early tumor recurrence (P=0.014), whereas no association was found between TLS and overall survival. Cox regression analysis identified TLS as an independent prognostic factor for early HCC recurrence (P=0.005). In addition, TLS was associated with increased intratumoral CD3+, CD8+, CD20+, and decreased infiltration of Foxp3+ and CD68+ cells. A lower density of PD1+, TIM3+, and LAG3+ were found in TLS+ cases. Sub-analysis revealed the prognostic value of TLS on early-stage HCC (BCLC 0-A, TNM stage I-II) and HCC with solitary nodule. The validation cohort verified the prognostic value of TLS in early-stage HCC patients. These results suggest that TLS-targeted immune-modulating therapies may be a potential strategy for effective immune-mediated tumor suppression.

Purpose We investigated serum cytokine and T-cell responses directed against tumour-associated antigens (TAAs) in association with survival of patients with glioblastoma multiforme (GBM). Patients and Methods Peripheral blood from 205 treatment-naïve patients with glioma (GBM = 145; non-GBM = 60) was obtained on the day of surgery to measure (i) circulating T-cells reacting to viral antigens and TAAs, in the presence or absence of cytokine conditioning with IL-2/IL-15/IL-21 or IL-2/IL-7, and (ii) serum cytokine levels (IL-4, IL-5, IL-6, TNF-α, IFN-γ and IL-17A). Patients were followed-up for at least 1000 days post-surgery. Survivin protein and gene expression in resected GBM tumour tissue were confirmed by immunohistochemistry and real-time polymerase chain reaction, respectively. Antigen-specific T-cell responses were gauged by ICS (intracellular cytokine production). Associations between patient survival and immunological reactivity patterns were analysed using univariate and multivariate statistics. Results Approximately 2% of patients with GBM and 18% of patients with non-GBM glioma, were alive beyond 1000 days of surgery. Univariate analysis indicated that the combination of three cytokines (IL-4/IL-5/IL-6, p = .0022; IFN-γ/TNF-α/IL-17A, p = .0083) but not a ‘partial’ combination of these cytokines, the IFN-γ immune response to EBV-EBNA-1 (p, Highlights • Cytokine combinations of IL-4/5/6 or IFN-γ/TNF-α/IL-17A may predict survival in patients with GBM. • IL-2/15/21-amplified survivin peptide-specific T-cell responses may independently predict survival among patients with GBM. • Large-scale clinical studies to validate these readouts as biomarkers of survival in GBM are warranted.

The TP53 family consists of three sets of transcription factor genes, TP53, TP63 and TP73, each of which expresses multiple RNA variants and protein isoforms. Of these, TP53 is mutated in 25-30% of breast cancers. How TP53 mutations affect the interaction of TP53 family members and their isoforms in breast cancer is unknown. To investigate this, 3 independent breast cancer cohorts were stratified into 4 groups based on oestrogen receptor (ER) and TP53 mutation status. Using bioinformatic methodologies, principal signalling pathways associated with the expression of TP53 family members were identified. Results show an enrichment of IFN-γ signalling associated with TP63 RNA in wild type TP53 (wtTP53), ER negative (ER-) tumours and with Δ133TP53 RNA in mutant TP53 (mTP53) ER positive (ER+) tumours. Moreover, tumours with low IFN-γ signalling were associated with significantly poorer patient outcome. The predicted changes in expression of a subset of RNAs involved in IFN-γ signalling were confirmed in vitro. Our data show that different members of the TP53 family can drive transcription of genes involved in IFN-γ signalling in different breast cancer subgroups.

Cryoablation is moderately effective against prostate cancer. Of note, the off-target or enlarged therapeutic effect after cryoablation is reported in routine clinical practice. To uncover it, we constructed a bilateral inguinal transplantation model of prostate cancer. All the mice were randomly subdivided into three groups: Group A (Control group), Group B (Surgery group), and Group C (Cryoablation group). All the procedures in three groups were conducted only for tumors in the target region (right groin). The tumors in untargeted region (left groin) received no treatment. We measured the growth of untargeted tumors and lung metastasis rate, and then explored the changes in a series of immune cells and danger signals. First, our results revealed the protective effect of cryoablation treatment against the abscopal tumor. The possible mechanism was mediated by an increase in the number of CD4+ T cells, CD8+ T cells, ratio T helper 1 (Th1)/Th2, the killing activity of cytotoxic T lymphocytes and NK cells. Hsp70 may be involved in the modulation of the immune response. The combination of weakened Ki67 activity and activated immune response delayed spectator tumor growth, decreased the pulmonary metastasis rate, and prolonged animal survival, with an inducible abscopal effect.

CD147, encoded by BSG, is a highly glycosylated transmembrane protein that belongs to the immunological superfamily and expressed on the surface of many types of cancer cells. While CD147 is best known as a potent inducer of extracellular matrix metalloproteinases, it can also function as a key mediator of inflammatory and immune responses. To systematically elucidate the function of CD147 in cancer cells, we performed an analysis of genome-wide profiling across the Cancer Cell Line Encyclopedia (CCLE). We showed that CD147 mRNA expression was much higher than that of most other genes in cancer cell lines. CD147 varied widely across these cell lines, with the highest levels in the ovary (COLO704) and stomach (SNU668), intermediate levels in the lung (RERFLCKJ, NCIH596 and NCIH1651) and lowest levels in hematopoietic and lymphoid tissue (UT7, HEL9217, HEL and MHHCALL3) and the kidney (A704 and SLR20). Genome-wide analyses showed that CD147 expression was significantly negatively correlated with immune-related genes. Our findings implicated CD147 as a novel regulator of immune-related genes and suggest its important role as a master regulator of immune-related responses in cancer cell lines. We also found a high correlation between the expression of CD147 and FOXC1, and proved that CD147 was a direct transcriptional target of FOXC1. Our findings demonstrate that FOXC1 is a novel regulator of CD147 and confirms its role as a master regulator of the immune response.

Although there is ample evidence that the chemotherapeutic drugs trigger an immune response, the efficient tumor rejection or regression is not guaranteed probably due to the massive immunosuppression within the tumor microenvironment. Thus, a rational delivery platform that overcomes immunosuppression is needed to maximally achieve both cytotoxic and immune-modulatory functions of chemotherapeutics. Accumulating evidence suggests that platinum-based drugs might be suitable for this application. Methods: The dendrigraft polylysine (DGL) with its uniform size and multifunctional groups was employed as the polymeric core and conjugated with platinum-based compounds as therapeutics and WKYMVm peptide (Wpep) as a targeting ligand to construct the novel delivery platform Wpep-DGL/Pt. A series of in vitro and in vivo analyses, including physical and chemical characterizations, targeting property, biosafety, and antitumor efficacy of Wpep-DGL/Pt were systematically carried out. Results: Wpep-DGL/Pt showed potent antitumor efficacy in MDA-MB-231 cells tumor-bearing nude mice with a deficient immune system, demonstrating targeted delivery of chemotherapeutics and the resultant cytotoxicity. Furthermore, in immunocompetent mice bearing 4T1 cells tumors, Wpep-DGL/Pt activated immune cells and induced cell death proving their dual function of chemotherapeutic and immunomodulatory efficacy. Conclusion: This work represents a novel approach for cancer immunotherapy by integrating nanotechnology and platinum-based therapeutics which not only efficiently exerts the chemotherapeutic cytotoxic effect on tumor cell but also restores immune response of immunological cells within the tumor microenvironment.

Background We and others have previously demonstrated that the endothelium is a primary target of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and L-arginine has been shown to improve endothelial dysfunction. However, the effects of L-arginine have never been evaluated in coronavirus disease 2019 (COVID-19). Methods This is a parallel-group, double-blind, randomized, placebo-controlled trial conducted on patients hospitalized for severe COVID-19. Patients received 1.66 g L-arginine twice a day or placebo, administered orally. The primary efficacy endpoint was a reduction in respiratory support assessed 10 and 20 days after randomization. Secondary outcomes were the length of in-hospital stay, the time to normalization of lymphocyte number, and the time to obtain a negative real-time reverse transcription polymerase chain reaction (RT-PCR) for SARS-CoV-2 on nasopharyngeal swab. This clinical trial had been registered at ClinicalTrials.gov, identifier: NCT04637906. Findings We present here the results of the initial interim analysis on the first 101 patients. No treatment-emergent serious adverse events were attributable to L-arginine. At 10-day evaluation, 71.1% of patients in the L-arginine arm and 44.4% in the placebo arm (p

In this study, experimental pathology, flow cytometry (FCM), quantitative real-time polymerase chain reaction (qRT-PCR), and western blot (WB) were used to evaluate the effects of sodium fluoride (NaF) on hepatocellular cell cycle progression in mice. A total of 240 ICR mice were divided equally into four groups; the experimental groups received 12, 24, or 48 mg/kg NaF intragastrically for 42 days, while the control group received distilled water. Doses of NaF above 12 mg/kg increased the percentage of cells in S phase (S-phase arrest), reduced percentages of cells in G0/G1 or G2/M phase, and activated the ATM-p53-p21 and ATR-Chk1-Cdc25A pathways. Activation of these pathways was characterized by up-regulation of ATM, p53, p21, ATR, and Chk1 mRNA and protein expression, and down-regulation of Cdc25A, cyclin E, cyclin A, CDK2, CDK4, and proliferating cell nuclear antigen (PCNA) mRNA and protein expression. These results indicate that NaF caused S-phase arrest by activating the ATM-p53-p21 and ATR-Chk1-Cdc25A pathways.

// Yu-Jie Wang 1,2,* , Lin Li 1,4,* , Wei-Juan Song 3,* , Yan-Jun Zhou 1 , Meng-Da Cao 1 , Xiang-Rong Zuo 1,2 and Ji-Fu Wei 1 1 Research Division of Clinical Pharmacology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, China 2 Department of Critical Care Medicine, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, China 3 Department of Clinical Laboratory, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, China 4 Department of Emergency Medicine, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, China * These authors have contributed equally to this article Correspondence to: Xiang-Rong Zuo, email: // Ji-Fu Wei, email: // Keywords : Can f 6, dog allergy, epitopes, allergenicity, Chinese children, Immunology and Microbiology Section, Immune response, Immunity Received : July 07, 2017 Accepted : September 20, 2017 Published : October 13, 2017 Abstract Dog allergy is common worldwide. However, the allergenicity of dog allergy is still unclear in China as well as in special group, such as children. In this study, we chose Can f 6, a major dog allergen which belongs to the lipocalin to study its allergenicity in Chinese dog allergic children. Can f 6 gene was subcloned into pET-28a vector and transformed into E. coli BL21 (DE3) cells for expression. The recombinant Can f 6 was purified by nickel affinity chromatography, identified by SDS-PAGE, and tested for its allergenicity by Western blot with sera and basophil activation test. Secondary structures, B cell epitopes and homology modeling of Can f 6 were predicted by using a series of bioinformatical approaches. And the verification of B cell epitopes was detected by ELISA. The recombinant allergen showed an explicit band with the molecular weight of 20 kDa by SDS-PAGE. Sera from 56.3 % (18/32) of dog-allergic children patients reacted with Can f 6. The induction of the expression of CD63 and CCR3 of dog allergic children in passively sensitized basophils was up to approximately 5.0 times higher than healthy subjects. The secondary structure of Can f 6 contains 3 α-helices, 9 β-sheets and random coils. Five B cell epitopes of Can f 6 were predicted and were confirmed successfully by ELISA. The results showed Can f 6 is a major allergen in Chinese children, which provides a basis for further study of Can f 6 in diagnosis and treatment of symptoms in children in China. The structural information of Can f 6 will help to form a foundation for the future design of vaccines and therapies for Can f 6 related allergies.

In osteoarthritis (OA), activated synoviocytes and articular chondrocytes produce pro-inflammatory cytokines, such as IL-1β, that promote chondrocyte apoptosis and activate the NF-κB signaling pathway to induce catabolic factors. In this study, we examined the anti-inflammatory and anti-apoptotic effect of baicalein on IL-1β signaling and NF-κB-regulated gene products in rat chondrocytes. Rat chondrocytes were pretreated with 10 ng/ml IL-1β for 24 h and then co-treated with 10 ng/ml IL-1β and 50 μM baicalein for 0, 12, 24, 36 and 48h. The expression levels of poly(ADP-ribose) polymerase (PARP), Bcl-2, caspase-3, matrix metalloproteinase (MMP)-9, MMP-3, cyclooxygenase (COX)-2 and SOX-9 were detected by Western blot and quantitative reverse transcription-PCR (qPCR). The effects of baicalein on the translocation and phosphorylation of the NF-κB system were studied by Western blotting and immunofluorescence. Baicalein stimulated the expression of anti-apoptotic genes and reduced the pro-apoptotic and pro-inflammatory gene products in chondrocytes. Baicalein promoted SOX-9 expression in a time-dependent manner in chondrocytes. Baicalein inhibited the NF-κB activation that was induced by IL-1β in a time-dependent manner in chondrocytes. Our results suggest that the anti-inflammatory and anti-apoptotic effects of baicalein are mediated through the inhibition of the translocation of phosphorylated p65 to the nucleus.

// Zhaoduan Liang 1 , Ye Tian 1 , Wenxuan Cai 1 , Zhiming Weng 3 , Yanyan Li 1 , Huanling Zhang 1,2 , Yifeng Bao 1 and Yi Li 1,3 1 State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China 2 School of Life Sciences, University of Science and Technology of China, Hefei, China 3 XiangXue Life Sciences Research Center, XiangXue Pharmaceutical Co. Ltd., Guangzhou, China Correspondence to: Yi Li, email: // Keywords : high affinity, programmed cell death protein 1 (PD-1), PD-ligand 1 (PD-L1), soluble PD-L1, T cell, Immunology and Microbiology Section, Immune response, Immunity Received : August 15, 2017 Accepted : September 03, 2017 Published : October 10, 2017 Abstract The activated T cells can be suppressed by programed death-1 (PD-1) axis through low affinity interaction between PD-1 and PD-ligand 1 (PD-L1) in solution or on antigen presenting cells. In clinic, the concentration of soluble PD-L1 in peripheral blood negatively correlates with cancer prognosis. However, there is little information about the relation between the affinity of PD-1/PD-L1 interaction and the suppressive capacity of PD-1 axis. In this study, we analyzed inhibitory roles of high affinity soluble human PD-L1 (hPD-L1) variants, which were generated with directed molecular evolution. Resultant two clones L3C7-hPD-L1 and L3B3-hPD-L1 showed over 20 folds greater affinity than that of native hPD-L1. We found that L3B3-hPD-L1 and L3C7-hPD-L1 could compete with an anti-PD-1 antibody (EH12.1) for binding to hPD-1. More importantly, although native soluble hPD-L1 can induce suppressive effects on activated T cells, we found L3B3-hPD-L1 and L3C7-hPD-L1 attenuated the strength of PD-1 axis for suppressing the proliferation and interferon γ (IFN-γ) secretion of PBMC. In conclusion, our data provide direct evidence in which immune checkpoint receptor-ligand interactive strength can alter the the suppressive function, in particular, the suppressive capacity of PD-1 axis could be decreased with enhanced affinity of soluble PD-L1 and PD-1 interaction. Our study might provide a new direction for manipulating immune checkpoints.

// Kyung-Ah Cho 1 , Minhwa Park 1 , Yu-Hee Kim 1 , Kyung-Ha Ryu 2 and So-Youn Woo 1 1 Department of Microbiology, College of Medicine, Ewha Womans University, Seoul, Republic of Korea 2 Department of Pediatrics, College of Medicine, Ewha Womans University, Seoul, Republic of Korea Correspondence to: So-Youn Woo, email: // Keywords : mesenchymal stem cells, osteoprotegerin, Th17 cells, osteoclasts, psoriasis, Immunology and Microbiology Section, Immune response, Immunity Received : February 20, 2017 Accepted : September 05, 2017 Published : September 28, 2017 Abstract Th17 cells play a critical role in several autoimmune diseases, including psoriasis and psoriatic arthritis (PsA). Psoriasis is a chronic inflammatory skin disease associated with systemic inflammation and comorbidities, such as PsA. PsA develops in nearly 70% of patients with psoriasis, and osteoclasts associated bone erosion is a hallmark of the disease. Thus far, the effect of Th17 cells on osteoclastogenesis via direct cell-to-cell interactions is less understood. In this study, we observed that Th17 cells directly promote osteoclast differentiation and maturation via expression of receptor activator of nuclear factor-κ β ligand (RANKL) in vitro . We investigated the impact of conditioned medium obtained from human palatine tonsil-derived mesenchymal stem cells (T-CM) on the interactions between osteoclasts and Th17 cells. T-CM effectively blunted the RANK-RANKL interaction between the osteoclast precursor cell line RAW 264.7 and Th17 cells via osteoprotegerin (OPG) activity. The frequency of tartrate-resistant acid phosphatase (TRAP)-positive cells in the bone marrow of an imiquimod (IMQ)-induced psoriasis mouse model was decreased following T-CM injection. Therefore, our data provide novel insight into the therapeutic potential of tonsil-derived mesenchymal stem cell-mediated therapy ( via OPG production) for the treatment of pathophysiologic processes induced by osteoclasts under chronic inflammatory conditions such as psoriasis.

// Danlin Yao 1,2,* , Ling Xu 1,2,* , Jiaxiong Tan 1,2 , Yikai Zhang 1,2 , Shuai Lu 1 , Mingde Li 1,2 , Sichun Lu 1,2 , Lijian Yang 1 , Shaohua Chen 1 , Jie Chen 2 , Jing Lai 2 , Yuhong Lu 2 , Xiuli Wu 1,2 , Xianfeng Zha 3 and Yangqiu Li 1,2 1 Key Laboratory for Regenerative Medicine of Ministry of Education, Institute of Hematology, Jinan University, School of Medicine, Jinan University, Guangzhou, China 2 Department of Hematology, First Affiliated Hospital, Jinan University, Guangzhou, China 3 Department of Clinical Laboratory, First Affiliated Hospital, Jinan University, Guangzhou, China * These authors have contributed equally to this work Correspondence to: Yangqiu Li, email: // Keywords : memory T cells, TSCM, CML, TKI, Immunology and Microbiology Section, Immune response, Immunity Received : June 28, 2017 Accepted : August 27, 2017 Published : September 16, 2017 Abstract T cell immune surveillance is considered an important host protection process for inhibiting carcinogenesis. The full capacity of T cell immune surveillance is dependent on T cell homeostasis, particularly for central memory T (T CM ) cells and stem cell memory T (T SCM ) cells. In this study, distribution of T cell subsets in peripheral blood from 12 patients with chronic myeloid leukemia (CML) and 12 cases with CML in complete remission (CR) was analyzed using a multicolor flow cytometer, and 16 samples from healthy individuals (HIs) served as control. The proportion of CD8 + T SCM and CD4 + and CD8 + T CM cells were lower, while CD4 + effector memory T (T EM ) cells and CD4 + and CD8 + terminal effector T (T EF ) cells were higher in CML patients compared with HIs. Moreover, the proportion of CD8 + CD28 - T cells, which were found to have the immune suppressive function, increased in the naive T (T N ) cell and T CM subsets in CML patients compared with HIs. Our study reveals that elimination of leukemia cells by treating with tyrosine kinase inhibitors (TKIs) restores the memory T cell distribution from a skewed pattern in CML patients who are under leukemia burden, indicating that leukemia-specific immune responses mediated by T cells might be induced and maintained in CML patients, however, these responsive T cells might gradually become exhausted due to the continued existence of leukemia cells and their environment; therefore, T cell activation using a different approach remains a key point for enhancing global T cell immunity in CML patients, even for those with CR status.

// Parvin Forghani 1,* , Christopher T. Petersen 1,* and Edmund K. Waller 1 1 Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, Georgia, USA * These authors have contributed equally to this work Correspondence to: Parvin Forghani, email: // Edmund K. Waller, email: // Keywords : CMV, myeloid-derived suppressor cells (MDSC), VIP, mice, Immunology and Microbiology Section, Immune response, Immunity Received : June 01, 2017 Accepted : August 14, 2017 Published : September 07, 2017 Abstract Vasoactive intestinal peptide (VIP) is recognized as a potent anti-inflammatory factor which affects both the innate and adaptive arms of the immune system. These effects include, but are not limited to, inhibition of T cell proliferation and disruption of immune homeostasis. Myeloid-derived suppressor cells (MDSC) are an immune regulatory cell type that has been described in settings of cancer and infectious disease.Here we demonstrate a reduced circulating monocytic MDSCs in the VIP -/- vs. wild type MCMV. VIP-/- MDSCs secretes less NO upon stimulation with LPS and interferon that relatively lose the ability to suppress T cells activation in vitro compared to wild type MDSCs.Considering the importance of VIP in immunomodulation, the possible effect of VIP in the suppressive function of MDSC populations following CMV infection remains unknown. We describe the possible role of VIP in the regulation of anti-CMV activity of T cells through the activation of MDSCs.

// Yun-Jung Choi 1 , EunSu Kim 1 , Abu Musa Md Talimur Reza 1 , Kwonho Hong 1 , Hyuk Song 1 , Chankyu Park 1 , Seong-Keun Cho 2 , Kiho Lee 3 , Randall S. Prather 4 and Jin-Hoi Kim 1 1 Department of Stem Cell and Regenerative Biotechnology, Humanized Pig Research Center (SRC), Konkuk University, Seoul, Republic of Korea 2 Department of Animal Science, Pusan National University, Miryang, Gyeongnam, Republic of Korea 3 Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, VA, USA 4 Division of Animal Science, University of Missouri-Columbia, Columbia, MO, USA Correspondence to: Jin-Hoi Kim, email: // Keywords : severe combined immunodeficient, SCID, recombination activating gene-2, RAG-2, Talen, Immunology and Microbiology Section, Immune response, Immunity Received : June 22, 2017 Accepted : August 23, 2017 Published : September 02, 2017 Abstract This study comparatively investigated the transcriptional, physiological, and phenotypic differences of the immune disorder between severe combined immunodeficient (SCID) mouse and pig models. We discovered that the recombination activating gene-2 ( Rag-2 ) SCID mice, but not RAG-2 SCID pigs, showed intense, infrequent, and mild cluster of CD3 + -, CD4 + -, and CD8 + signals respectively, suggesting that distinct species-specific effects exist. Furthermore, the expression of six relevant genes ( NFATC1 , CD79B , CD2 , BLNK , FOXO1 , and CD40) was more downregulated than that in the Rag-2 SCID mice, which provides a partial rationale for the death of T/B cells in the lymphoid organs of RAG-2 SCID pigs but not in Rag-2 SCID mice. Further, NK cell maturation-related gene expression was significantly lower in RAG-2 SCID pigs than in Rag-2 SCID mice. Consistently, the RAG-2 SCID pigs, but not Rag-2 SCID mice, developed human induced pluripotent stem cell-derived teratomas that were the same as those of perforin / Rag-2 SCID mice. Therefore, these unexpected findings indicate the superiority of RAG-2 SCID pigs over Rag-2 SCID mice as a suitable model for investigating human diseases.

We sought to define the host immune response, a.k.a, the “cytokine storm” that has been implicated in fatal COVID-19 using an AI-based approach. Over 45,000 transcriptomic datasets of viral pandemics were analyzed to extract a 166-gene signature using ACE2 as a ‘seed’ gene; ACE2 was rationalized because it encodes the receptor that facilitates the entry of SARS-CoV-2 (the virus that causes COVID-19) into host cells. Surprisingly, this 166-gene signature was conserved in all viral pandemics, including COVID-19, and a subset of 20-genes classified disease severity, inspiring the nomenclatures ViP and severe-ViP signatures, respectively. The ViP signatures pinpointed a paradoxical phenomenon wherein lung epithelial and myeloid cells mount an IL15 cytokine storm, and epithelial and NK cell senescence and apoptosis determines severity/fatality. Precise therapeutic goals were formulated and subsequently validated in high-dose SARS-CoV-2-challenged hamsters using neutralizing antibodies that abrogate SARS-CoV-2•ACE2 engagement or a directly acting antiviral agent, EIDD-2801. IL15/IL15RA were elevated in the lungs of patients with fatal disease, and plasma levels of the cytokine tracked with disease severity. Thus, the ViP signatures provide a quantitative and qualitative framework for titrating the immune response in viral pandemics and may serve as a powerful unbiased tool to rapidly assess disease severity and vet candidate drugs.One Sentence SummaryThe host immune response in COVID-19.PANEL: RESEARCH IN CONTEXTEvidence before this studyThe SARS-CoV-2 pandemic has inspired many groups to find innovative methodologies that can help us understand the host immune response to the virus; unchecked proportions of such immune response have been implicated in fatality. We searched GEO and ArrayExpress that provided many publicly available gene expression data that objectively measure the host immune response in diverse conditions. However, challenges remain in identifying a set of host response events that are common to every condition. There are no studies that provide a reproducible assessment of prognosticators of disease severity, the host response, and therapeutic goals. Consequently, therapeutic trials for COVID-19 have seen many more ‘misses’ than ‘hits’. This work used multiple (> 45,000) gene expression datasets from GEO and ArrayExpress and analyzed them using an unbiased computational approach that relies upon fundamentals of gene expression patterns and mathematical precision when assessing them.Added value of this studyThis work identifies a signature that is surprisingly conserved in all viral pandemics, including Covid-19, inspiring the nomenclature ViP-signature. A subset of 20-genes classified disease severity in respiratory pandemics. The ViP signatures pinpointed the nature and source of the ‘cytokine storm’ mounted by the host. They also helped formulate precise therapeutic goals and rationalized the repurposing of FDA-approved drugs.Implications of all the available evidenceThe ViP signatures provide a quantitative and qualitative framework for assessing the immune response in viral pandemics when creating pre-clinical models; they serve as a powerful unbiased tool to rapidly assess disease severity and vet candidate drugs.

// Ming-Jie Wang 1,* , Xiao-Liang Xu 2,* , Guo-Liang Yao 3,* , Qiang Yu 4 , Chun-Fu Zhu 4 , Zhi-Jun Kong 4 , Hui Zhao 5 , Li-Ming Tang 4 and Xi-Hu Qin 4 1 Department of Orthopedics, Affiliated Hospital of Nanjing Medical University, Changzhou Second People’s Hospital, Changzhou, China 2 Liver Surgery of Jiangsu Province People’s Hospital, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China 3 Department of Thoracic Surgery, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China 4 Department of General Surgery, Affiliated Hospital of Nanjing Medical University, Changzhou Second People’s Hospital, Changzhou, China 5 Department of General Surgery, Wuxi Third People’s Hospital, Wuxi, China * These authors have contributed equally to this study Correspondence to: Zhi-Jun Kong, email: // Hui Zhao, email: // Xi-Hu Qin, email: // Keywords : inflammatory bowel disease; Crohn’s disease; ulcerative colitis; MYO9B; polymorphism; Immunology and Microbiology Section; Immune response; Immunity Received : March 25, 2016 Accepted : July 19, 2016 Published : August 19, 2016 Abstract Myosin IXB ( MYO9B ) gene polymorphisms have been extensively investigated in terms of their associations with inflammatory bowel disease (IBD), with contradictory results. The aim of this meta-analysis was to evaluate associations between MY09B gene polymorphisms and the risk of IBD, Crohn’s disease (CD) and ulcerative colitis (UC). Eligible studies from PubMed, Embase, and CNKI databases were identified. Pooled odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated. Ten studies published in eight papers reporting 8,975 cases and 9,482 controls were included in this meta-analysis. Five MY09B gene polymorphisms were evaluated: rs1545620, rs962917, rs1457092, rs2305764, and rs2305767. Our data suggested that the rs1545620 polymorphism was associated with a decreased risk of IBD. A similar result was found for rs2305767 and UC. The rs962917 single nucleotide polymorphism (SNP) increased the risk of IBD, CD and UC. Moreover, rs1457092 increased the risk of IBD and UC. Rs2305764 was also associated with an increased risk of IBD. Furthermore, stratification analyses indicated that rs1545620 decreased the risk of IBD, while rs962917 increased the risk of IBD, CD and UC in Caucasian populations. To sum up, our data indicate that these five SNPs in MY09B are significantly associated with the risk of IBD.

// Ming Zhou 1,* , Lei Wang 1,* , Songqin Zhou 1,* , Zhao Wang 1 , Juncheng Ruan 1 , Lijun Tang 3 , Ziming Jia 3 , Min Cui 1 , Ling Zhao 1 and Zhen F. Fu 1,2 1 State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China 2 Department of Pathology, University of Georgia, Athens, GA, USA 3 Hubei Provincial Key Laboratory for Applied Toxicology, Hubei Provincial Academy of Preventive Medicine, Wuhan, China * These authors have contributed equally to this paper Correspondence to: Ling Zhao, email: // Zhen F. Fu, email: // Keywords : rabies, recombinant rabies virus, GM-CSF, oral vaccine, dog, Immunology and Microbiology Section, Immune response, Immunity Received : July 12, 2015 Accepted : August 26, 2015 Published : September 30, 2015 Abstract Developing efficacious oral rabies vaccines is an important step to increase immunization coverage for stray dogs, which are not accessible for parenteral vaccination. Our previous studies have demonstrated that recombinant rabies virus (RABV) expressing cytokines/chemokines induces robust protective immune responses after oral immunization in mice by recruiting and activating dendritic cells (DCs) and B cells. To develop an effective oral rabies vaccine for dogs, a recombinant attenuated RABV expressing dog GM-CSF, designated as LBNSE-dGM-CSF was constructed and used for oral vaccination in a dog model. Significantly more DCs or B cells were activated in the peripheral blood of dogs vaccinated orally with LBNSE-dGM-CSF than those vaccinated with the parent virus LBNSE, particularly at 3 days post immunization (dpi). As a result, significantly higher levels of virus neutralizing antibodies (VNAs) were detected in dogs immunized with LBNSE-dGM-CSF than with the parent virus. All the immunized dogs were protected against a lethal challenge with 4500 MICLD 50 of wild-type RABV SXTYD01. LBNSE-dGM-CSF was found to replicate mainly in the tonsils after oral vaccination as detected by nested RT-PCR and immunohistochemistry. Taken together, our results indicate that LBNSE-dGM-CSF could be a promising oral rabies vaccine candidate for dogs.

IFITM3 is involved in cell adhesion, apoptosis, immune, and antivirus activity. Furthermore, IFITM3 gene has been considered as a preferential marker for inflammatory diseases, and positive correlation to pathological grades. Therefore, we assumed that IFITM3 was regulated by different signal pathways. To better understand IFITM3 function in inflammatory response, we cloned swine IFITM3 gene, and detected IFITM3 distribution in tissues, as well as characterized this gene. Results indicated that the length of swine IFITM3 gene was 438 bp, encoding 145 amino acids. IFITM3 gene expression abundance was higher in spleen and lungs. Moreover, we next constructed the eukaryotic expression vector PBIFM3 and transfected into PK15 cells, finally obtained swine IFITM3 gene stable expression cell line. Meanwhile, we explored the effects of LPS on swine IFITM3 expression. Results showed that LPS increased IFITM3 mRNA abundance and exhibited time-dependent effect for LPS treatment. To further demonstrate the mechanism that IFITM3 regulated type I IFNs production, we also detected the important molecules expression of TLR4 signaling pathway. In transfected and non-transfected IFITM3 PK15 cells, LPS exacerbated the relative expression of TLR4-NFκB signaling molecules. However, the IFITM3 overexpression suppressed the inflammatory development of PK15 cells. In conclusion, these data indicated that the overexpression of swine IFITM3 could decrease the inflammatory response through TLR4 signaling pathway, and participate in type I interferon production. These findings may lead to an improved understanding of the biological function of IFITM3 in inflammation.

// Liu Zhenjiang 1 , Martin Rao 1 , Thomas Poiret 1,2 , Silvia Nava 1 , Qingda Meng 1 , Anna von Landenberg 1 , Jiri Bartek Jr. 3,4,5 , Shanshan Xie 6 , Georges Sinclair 3 , Inti Peredo 3 , Ernest Dodoo 3,* and Markus Maeurer 1,2,* 1 Department of Laboratory Medicine, Division of Therapeutic Immunology, Karolinska Institutet, Stockholm, Sweden 2 Centre for Allogeneic Stem Cell Transplantation, Karolinska University Hospital Huddinge, Stockholm, Sweden 3 Department of Neurosurgery, Karolinska University Hospital, Stockholm, Sweden 4 Department of Clinical Neuroscience and Department of Medicine, Karolinska Institutet, Stockholm, Sweden 5 Department of Neurosurgery, Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark 6 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden * These authors have contributed equally to this work Correspondence to: Markus Maeurer, email: // Keywords : glioblastoma, mesothelin, T-cell response, interferon gamma, immunotherapy, Immunology and Microbiology Section, Immune response, Immunity Received : February 25, 2017 Accepted : June 11, 2017 Published : August 16, 2017 Abstract Glioblastoma multiforme (GBM) presents the most malignant form of glioma, with a 5-year survival rate below 3% despite standard therapy. Novel immune-based therapies in improving treatment outcomes in GBM are therefore warranted. Several molecularly defined targets have been identified mediating anti-GBM cellular immune responses. Mesothelin is a tumor-associated antigen (TAA) which is expressed in several solid tumors with different histology. Here, we report the immunological significance of mesothelin in human malignant glioma. Expression of mature, surface-bound mesothelin protein was found to bein human GBM defined by immunofluorescence microscopy, and on freshly isolated, single cell suspension of GBM tumor cells and GBM tumor cell lines, determined by based on flow cytometric analysis. Peripheral blood (PB) from patients with GBM, stimulated with mesothelin peptides and IL-2, IL-15 and IL-21, exhibited increased antigen-specific IFN-γ and TNF-α production. Anti-mesothelin directed T-cell responses could also be detected in tumor - infiltrating lymphocytes (TIL) isolated from GBM speciments. Furthermore, T cells cultured in the presence of IL-2, IL-15 and IL-21 displayed enhanced mesothelin-specific CD4+ and CD8+ subset proliferation, based on ELISA and flow cytometric readouts. Mesothelin-specific IgG antibodies as well as (shed) mature mesothelin protein were detected in plasma samples from patients with GBM by indirect ELISA. Finally yet importantly, we identified distinct immune recognition hotspots within the mature mesothelin component, defined by peptide-specific IFN-γ responses from peripheral T-cells from patients with GBM. Mesothelin may therefore qualify as a viable target for immunotherapeutic approaches for patients with GBM.

// Yingyun Fu 1,3,* , Yazhen Li 1,* , Lan Xu 1 , Shengguo Liu 1 , Minlian Wang 3 , Lu Xiao 1 , Song Liu 2 and Yong Dai 2 1 Department of Respiratory and Critical Care Medicine, The Second Medical College of Jinan University, Shenzhen People’s Hospital, Luohu District, Shenzhen, China 2 Clinical Medical Research Center, The Second Clinical Medical College of Jinan University, Shenzhen People’s Hospital, Luohu District, Shenzhen, China 3 Shenzhen Key Laboratory of Respiratoty Disease, Shenzhen, China * These authors have contributed equally to this work Correspondence to: Yingyun Fu, email: // Keywords : sarcoidosis, immune system, CDR3, TCR BV CDR3, PMBCs, Immunology and Microbiology Section, Immune response, Immunity Received : April 08, 2017 Accepted : July 30, 2017 Published : August 09, 2017 Abstract Sarcoidosis is a systemic granulomatous disorder highly related with immune response. The diversity and stability of the immune system could be measured by hypervariable complementarity-determining region 3 (CDR3) segments of the T cell receptor (TCR). Here we used a combination of multiplex PCR and next-generation sequencing to conduct a good quality analysis of the T-cell receptor BV complementarity-determining region 3 (TCR BV CDR3) gene in peripheral blood mononuclear cells (PBMCs) from 7 sarcoidosis patients and lung sarcoidosis tissue from 6 patients. The length distribution of CDR3 sequences identified a significant difference among CD4+, CD8+ and tissue samples. The analysis of Gini coefficient, Shannon entropy and HEC number showed that they all presents in sarcoidosis tissue group clones in a more skewed manner than that of in PMBCs groups. 2 nucleotide sequences and 2 amino acid sequences were shared by all samples. The comparison of TRBV, TRBJ usage and VJ combination frequency identified 2 TRBV genes, 2 TRBJ genes differentially expressed among different groups and different higher usage and lower usage of V-J combinations between each group.

Since urine samples more directly reflect kidney alterations and damage than blood samples, we investigated whether urine anti-PLA2R antibody (uPLA2R-Ab) could be utilized similarly to serum anti-PLA2R antibody (sPLA2R-Ab) as a noninvasive biomarker of idiopathic membranous nephropathy (IMN). In this study, we performed a qualitative analysis using an indirect immunofluorescence test (IIFT) and measured uPLA2R-Ab and sPLA2R-Ab concentrations using an enzyme-linked immunosorbent assay (ELISA) in 28 patients with biopsy-proven IMN and 12 patients with secondary membranous nephropathy (SMN). Overall, 64.3% (n=18) of patients with IMN had IIFT-positive sPLA2R-Ab, 67.9% (n=19) of patients with IMN had IIFT-positive uPLA2R-Ab, and none of the SMN patients had IIFT-positive sPLA2R-Ab or uPLA2R-Ab. The titers of the anti-PLA2R antibody from the IMN patients in the urine (10.72±22.24 RU/μmol, presented as uPLA2R-Ab/urine creatinine) and serum (107.36±140.93 RU/ml) were higher than those from the SMN patients (0.51±0.46 RU/μmol, 0.008±0.029 RU/ml, respectively, p