Glutathione peroxidase 4-regulated neutrophil ferroptosis induces systemic autoimmunity.

The linkage between neutrophil death and the development of autoimmunity has not been thoroughly explored. Here, we show that neutrophils from either lupus-prone mice or patients with systemic lupus erythematosus (SLE) undergo ferroptosis. Mechanistically, autoantibodies and interferon-α present in the serum induce neutrophil ferroptosis through enhanced binding of the transcriptional repressor CREMα to the glutathione peroxidase 4 (Gpx4, the key ferroptosis regulator) promoter, which leads to suppressed expression of Gpx4 and subsequent elevation of lipid-reactive oxygen species. Moreover, the findings that mice with neutrophil-specific Gpx4 haploinsufficiency recapitulate key clinical features of human SLE, including autoantibodies, neutropenia, skin lesions and proteinuria, and that the treatment with a specific ferroptosis inhibitor significantly ameliorates disease severity in lupus-prone mice reveal the role of neutrophil ferroptosis in lupus pathogenesis. Together, our data demonstrate that neutrophil ferroptosis is an important driver of neutropenia in SLE and heavily contributes to disease manifestations.

Molecular characterization of PANoptosis-related genes with features of immune dysregulation in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease. PANoptosis is a novel form of programmed cell death involved in various inflammatory diseases. This study aimed to identify the differentially-expressed PANoptosis-related genes (PRGs) involved in immune dysregulation in SLE. Five key PRGs, including ZBP1, MEFV, LCN2, IFI27, and HSP90AB1, were identified. The prediction model with these 5 key PRGs showed a good diagnostic performance in distinguishing SLE patients from controls. These key PRGs were associated with memory B cells, neutrophils and CD8 + T cells. Besides, these key PRGs were significantly enriched in pathways involving the type I interferon responses and IL-6-JAK-STAT3 signaling. The expression levels of the key PRGs were validated in peripheral blood mononuclear cells (PBMCs) of patients with SLE. Our findings suggest that PANoptosis may be implicated in the immune dysregulation in SLE by regulating the interferons and JAK-STAT signaling pathways in memory B cells, neutrophils and CD8 + T cells.

Monoclonal gammopathy in autoimmune diseases: Analysis and follow-up of 160 cases in a tertiary center in China.

Monoclonal gammopathy (MG) is common in autoimmune diseases (AID), but its progression to hematological neoplasm (HN) and the predictors for the progression are unclear. Patients diagnosed with AID and MG in our hospital from January 2010 to June 2017 were reviewed and followed. Cox proportional hazard regression analysis was applied. Of 160 patients with AID and MG, the most common AID was primary SjÓ§gren's syndrome (37, 23.1%). Thirty-nine (24.4%) patients developed HN during follow-up (median: 3.7 years, IQR: 0.3-5.5 years). The cumulative probability of HN progression was 21.8% at one year and 29.3% at six years after the finding of MG. High levels of monoclonal protein (> 14.35% of total serum protein) (HR 11.71, 95%CI: 5.37-25.54), significant weight loss (HR 6.24, 95%CI: 2.87-13.59), and reduction of other types of immunoglobulins (HR 3.02, 95%CI: 1.40-6.48) are independent risk indicators for HN whose presence warrants vigorous follow-up and monitoring.

Pathological mechanisms and crosstalk among different forms of cell death in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disorder characterized by a profound immune dysregulation and the presence of a variety of autoantibodies. Aberrant activation of programmed cell death (PCD) signaling and accelerated cell death is critical in the immunopathogenesis of SLE. Accumulating cellular components from the dead cells and ineffective clearance of the dead cell debris, in particular the nucleic acids and nucleic acids-protein complexes, provide a stable source of self-antigens, which potently activate auto-reactive B cells and promote IFN-I responses in SLE. Different cell types display distinct susceptibility and characteristics to a certain type of cell death, while different PCDs in various cells have mutual and intricate connections to promote immune dysregulation and contribute to the development of SLE. In this review, we discuss the role of various cell death pathways and their interactions in the pathogenesis of SLE. An in depth understanding of the interconnections among various forms cell death in SLE will lead to a better understanding of disease pathogenesis, shedding light on the development of novel therapeutic targets.

Expression and clinical value of PD-L1 which is regulated by BRD4 in tongue squamous cell carcinoma.

The programmed cell death-ligand-1 (PD-L1) and bromodomain protein 4 (BRD4) are frequently overexpressed in cancer and have even been shown to act synergistically. The aim of this study was to determine their potential oncogenic role .in tongue squamous cell carcinoma (TSCC). We detected significantly higher expression levels of both PD-L1 and BRD4 in TSCC tissues compared to normal tissues (P ≤ .05). In addition, the high levels of PD-L1 were significantly associated with increased tumor lymphatic metastasis (P ≤ .05), tumor staging (P ≤ .01), as well as BRD4 expression (P ≤ .05). Genetic and pharmacological inhibition of BRD4 in TSCC cells not only reduced their growth rate but also PD-L1 levels (P ≤ .05), while overexpression of BRD4 upregulated PD-L1. Bioinformatics analysis showed that c-MYC and CDK9 were interactive partners of both BRD4 and PD-L1. While c-MYC clearly modulated the expression of PD-L1, as well as reversed the inhibitory effects of JQ1, no obvious association was observed between CDK9 and PD-L1. We report a novel regulatory axis consisting of BRD4, PD-L1, and c-MYC that likely drives TSCC progression, and is a potential prognostic marker and/or therapeutic target for TSCC.

Free Flap Transplantation on the repair of defects caused by oral and maxillofacial tumors resection.

OBJECTIVE: To investigate the efficacy of free flap transplantation on the repair of tissue defects after oral and maxillofacial malignant tumor resection and its effects on serum sialic acid (SA) and interleukin-2 (IL-2). METHODS: Fifty-eight patients with oral and maxillofacial tumors were enrolled and set as the observation group. After the tumor resection, free flap transplantation was performed for postoperative repair. The postoperative efficacy, adverse reactions and follow-up indicators were observed. Moreover, 55 patients with benign tumors were enrolled into the control group, and 55 healthy persons were set as the healthy group. The levels of SA and IL-2 of the three groups were detected. RESULTS: In the observation group, 55 patients were successfully repaired (94.83%); 15 patients had adverse reactions after surgery. The follow-up duration was two to four years, and 45 patients survived for three years, with a survival rate of 77.59%. Before treatment, the serum SA level of patients with oral malignant tumor was significantly higher than those of the control group and healthy group, while the IL-2 level was significantly lower than those of the other two groups, and the differences were statistically significant (P<0.05). The serum IL-2 level in the observation group one day and fourteen days after surgery was higher than that before surgery, while the serum SA level was lower than that before surgery; the differences were statistically significant (P<0.05). CONCLUSION: The application of free flap transplantation in the repair of postoperative tissue defects of oral and maxillofacial tumor resection is effective and has less complications, and the determination of both serum SA and IL-2 levels offers important references to recovery of patients with oral and maxillofacial tumors and prognosis evaluation.

Antiviral effect of diammonium glycyrrhizinate on cell infection by porcine parvovirus.

Porcine parvovirus (PPV) can cause reproductive failure in swine, resulting in economic losses to the industry. Antiviral effects of diammonium glycyrrhizinate (DG) have been reported on several animal viruses; however, to date it has yet to be tested on PPV. In this study, the antiviral activity of DG on swine testis (ST) cell infection by PPV was investigated using an empirically determined, non-toxic concentration of DG and three different experimental designs: (1) pre-treatment of virus prior to infection; (2) pre-treatment of cells prior to infection; and (3) direct treatment of virus-infected cells. The results showed that DG possesses potent inhibitory effects on PPV when the virus was treated before incubation with ST cells and that virus infectivity decreased in a dose-dependent manner. Results were confirmed by indirect immunofluorescence assays and real-time quantitative PCR. In addition, deoxycholate was used as a control to exclude the possibility that DG acted as a detergent to inhibit PPV infectivity. The study clearly indicates that DG has a direct anti-PPV effect in vitro.

Identifying the importance of PCK1 in maintaining ileal epithelial barrier integrity in Crohn's disease.

BACKGROUND: Crohn's disease (CD) is marked by disruption of intestinal epithelial barrier, with unclear underlying molecular mechanisms. This study aimed to investigate key genes regulating the intestinal barrier in CD patients. METHODS: Differential gene expression analysis and gene set enrichment analysis were conducted to identify potential key genes involved in CD within the GEO database. Single-cell RNA sequencing from ileum samples in GSE134809 of 59,831 inflamed and uninflamed cells from 11 CD patients and microarray data from ileal tissues in GSE69762 (3 controls and 4 CD patients) and GSE75214 (11 controls and 51 CD patients) with GSE179285 (49 uninflamed and 33 inflamed from CD patients) as the validation set. Protein-protein interaction and logistic regression analyses identified key downregulated genes in CD. A key gene was then investigated through immunohistochemistry of ileal tissues from 5 CD patients and in the Caco-2 cell line with RNA interference and treatment with IFN-γ and TNF-α to stimulate inflammation. RESULTS: Single-cell RNA-seq identified 33 genes and microarray identified 167 genes with significant downregulation in inflamed CD samples. PCK1 was identified and validated as one of the most promising candidate genes. Reduced PCK1 expression was evident in inflamed ileal tissues. In vitro, knockdown of PCK1 resulted in decreased cell viability, increased apoptosis, and reduced nectin-2 production, while combination of IFN-γ and TNF-α significantly reduced PCK1. CONCLUSIONS: PCK1 is downregulated in inflamed ileal tissues of CD patients and may be a key factor in maintaining epithelial integrity during inflammation in Crohn's disease.

Lipid Metabolism: Immune Regulation and Therapeutic Prospectives in Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a heterogeneous disease characterized by the production of abnormal autoantibodies and immune complexes that can affect the organ and organ systems, particularly the kidneys and the cardiovascular system. Emerging evidence suggests that dysregulated lipid metabolism, especially in key effector cells, such as T cells, B cells, and innate immune cells, exerts complex effects on the pathogenesis and progression of SLE. Beyond their important roles as membrane components and energy storage, different lipids can also modulate different cellular processes, such as proliferation, differentiation, and survival. In this review, we summarize altered lipid metabolism and the associated mechanisms involved in the pathogenesis and progression of SLE. Furthermore, we discuss the recent progress in the role of lipid metabolism as a potential therapeutic target in SLE.

Development of reverse transcription loop-mediated isothermal amplification for rapid detection of porcine epidemic diarrhea virus.

In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed for detection of porcine epidemic diarrhea virus (PEDV). Six primers were designed to amplify the nucleocapsid (N) gene of PEDV. The optimization, sensitivity, and specificity of the RT-LAMP were investigated. The results showed that the optimal reaction condition for RT-LAMP amplifying PEDV N gene was achieved at 63°C for 50 min. The RT-LAMP assay was more sensitive than gel-based RT-PCR and enzyme-linked immunosorbent assay. It was capable of detecting PEDV from clinical samples and differentiating PEDV from Porcine transmissible gastroenteritis virus, Porcine rotavirus, Porcine pseudorabies virus, Porcine reproductive and respiratory syndrome virus, and Avian infectious bronchitis virus.

Reverse transcription loop-mediated isothermal amplification for rapid detection of transmissible gastroenteritis virus.

Transmissible gastroenteritis virus (TGEV) is the causative agent of porcine transmissible gastroenteritis, and sensitive detection methods are required for preventing the disease. In this article, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) was developed to detect TGEV. Three pairs of primers targeting the nucleocapsid (N) gene of TGEV were synthesized and used in the RT-LAMP. The optimization, sensitivity, and specificity of the RT-LAMP were evaluated. Our results showed that the RT-LAMP amplified the N gene with high specificity, efficiency, and rapidity at isothermal condition. The optimal reaction condition was achieved at 60°C for 30 min. The RT-LAMP assay was more sensitive than gel-based RT-PCR and PCR. It had a higher sensitivity than enzyme-linked immunosorbent assay (ELISA) using the equal virus templates. In addition, the established RT-LAMP differentiated TGEV from porcine epidemic diarrhea virus, porcine rotavirus, porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus, and avian infectious bronchitis virus. The approach is suitable for detecting TGEV for field diagnostics or in less-equipped laboratories due to its convenience and simplicity.

Site-specific PEGylation of interleukin-2 enhances immunosuppression via the sustained activation of regulatory T cells.

The preferential activation of regulatory T (Treg) cells by interleukin-2 (IL-2), which selectively binds to the trimeric IL-2 receptor (IL-2R) on Treg cells, makes this cytokine a promising therapeutic for the treatment of autoimmune diseases. However, IL-2 has a narrow therapeutic window and a short half-life. Here, we show that the pharmacokinetics and half-life of IL-2 can be substantially improved by orthogonally conjugating the cytokine to poly(ethylene glycol) (PEG) moieties via a copper-free click reaction through the incorporation of azide-bearing amino acids at defined sites. Subcutaneous injection of a PEGylated IL-2 that optimally induced sustained Treg-cell activation and expansion over a wide range of doses through highly selective binding to trimeric IL-2R led to enhanced therapeutic efficacy in mouse models of lupus, collagen-induced arthritis and graft-versus-host disease without compromising the immune defences of the host against viral infection. Site-specific PEGylation could be used more generally to engineer cytokines with improved therapeutic performance for the treatment of autoimmune diseases.

A Serological Survey of Borrelia burgdorferi Infection in Sheep in Northeast China Regions Through Outer Surface Protein C-Based Enzyme-Linked Immunosorbent Assay.

Borrelia burgdorferi as a causative agent of Lyme disease is transmitted by Ixodes spp. ticks to humans and animals. Sheep is considered a natural reservoir for B. burgdorferi and plays a pivotal role in disease transmission and the expansion of natural foci. An epidemiological investigation of B. burgdorferi in sheep is essential for prevention and control of Lyme disease. In this study, we developed a recombinant outer surface protein C (OspC)-based ELISA for serological study of B. burgdorferi in sheep with a specificity and sensitivity of 84.4% and 86.2%, respectively. A total of 972 collected serum samples from the Northeast China regions in 2015 and 2016 were determined with positive rates of 5.8% and 12.2%, respectively. Thus, specific pathogen-free sheep were infected with B. burgdorferi SZ strain to study on the secretion of specificity antibody against OspC. It revealed that specific antibody was detected on day 5 postinoculation and sustained in a high level for ∼28 days, the peak occurred at ∼13 days. Taken together, the result indicated that the established ELISA is capable for clinical diagnosis and epidemiological study on B. burgdorferi in sheep at the early stage of infection and detecting the specific antibody during the secretion period.

Porcine IL-12 plasmid as an adjuvant improves the cellular and humoral immune responses of DNA vaccine targeting transmissible gastroenteritis virus spike gene in a mouse model.

Transmissible gastroenteritis (TGE), caused by transmissible gastroenteritis virus (TGEV), is a highly infectious disease in pigs. Vaccination is an effective approach to prevent TGEV infection. Here, we evaluated the potential of TGEV S1 as a DNA vaccine and porcine interleukin (pIL)-12 as an adjuvant in a mouse model. A DNA vaccine was constructed with the TGEV S1 gene to induce immune response in an experimental mouse model; pIL-12 was chosen as the immunological adjuvant within this DNA vaccine. The pVAX1-(TGEV-S1) and pVAX1-(pIL-12) vectors were transfected into BHK-21 cells and expressed in vitro. Experimental mice were separately immunized with each of the recombinant plasmids and controls through the intramuscular route. The lymphocytes isolated from the blood and spleen were analyzed for proliferation, cytotoxic activities, and populations of CD4+ and CD8+ cells. The titers of TGEV S1 in an enzyme-linked immunosorbent assay (ELISA) and TGEV neutralizing antibodies and the concentrations of interferon (IFN)-γ and IL-4 were also analyzed in the serum. The plasmids pVAX1-(TGEV-S1) and pVAX1-(pIL-12) could be expressed in BHK-21 cells, and the combination of pVAX1-(TGEV-S1) and pVAX1-(pIL-12) could induce a significant increase in all markers. pIL-12 could act as an immunological adjuvant in the DNA vaccine for TGEV-S1. Furthermore, the DNA vaccine prepared using TGEV-S1 and porcine IL-12 could induce excellent humoral and cellular immune responses.

The efficacy and safety of antithrombotic therapy in patients with positive antiphospholipid antibodies receiving invasive procedures: experience from a single tertiary center.

OBJECTIVES: To evaluate the efficacy and safety of antithrombotic prophylaxis and to explore potential risk factors for thrombotic/bleeding events in patients with positive antiphospholipid (aPL) antibodies receiving invasive procedures. METHOD: All aPL-positive patients who underwent invasive procedures in Peking Union Medical College Hospital, from January 2002 to April 2018, were retrospectively enrolled. Demographic features, clinical features, antiphospholipid antibody profiles, types of invasive procedures, and antithrombotic management, as well as complications and outcomes, were systematically reviewed and recorded. RESULTS: A total of 111 aPL-positive patients with 130 invasive procedures were enrolled. One hundred nine (83.8%) cases were on regular antithrombotic therapy which started at least 1 month prior to the invasive procedures, with 58 (44.6%) receiving anticoagulation therapy, 27 (20.8%) receiving antiplatelet therapy, and 24 (18.5%) receiving both. During the periprocedural period, the median time free of antithrombotic therapy was 2.5 days (interquartile range 1.5-6.0 days). Two (1.5%) periprocedural thrombotic events and 18 (13.8%) bleeding events were identified. Large open/laparoscopic surgeries of the thorax and abdomen were associated with a higher risk of bleeding (OR 3.46, 95% CI 1.24-9.67, p = 0.014). All bleeding events were manageable and not life-threatening. CONCLUSIONS: Aggressive antithrombotic therapy was associated with fewer thrombotic events in aPL-positive patients receiving invasive procedures, but might contribute to an increased bleeding rate, especially in large open surgeries. This study justifies more caution in prophylactic antithrombotic therapy in periprocedural aPL-positive patients.

[Dental pulp stem cells modified by HIF-1α can differentiate into blood vessels].

PURPOSE: To investigate the method of differentiating dental pulp stem cells (DPSCs) modified by HIF-1α into blood vessels. METHODS: DPSCs were extracted from teeth samples from 20 patients and were identified by Strol-1 and CD146. DPSCs were divided into experimental group and control group according to DPSCs were modified by HIF-1α not or. HIF-1α-mRNA expression was detected by RT-PCR. HIF-1α, VEGF, SDF-1, Ang-2 and PDGF expression were detected using Western blot in different time after culture for 1 d, 4 d, 7 d and 14 d. Statistical analysis was carried out with SPSS 16.0 software package. RESULTS: Most DPSCs appeared round, oval under phase-contrast microscopy. CD146 and Strol-1 showed green fluorescence. HIF-1α and HIF-1α-mRNA expression became higher with time passing and the difference was statistically significant (P<0.05). Compared with the control group, HIF-1α protein and mRNA increased obviously in the experimental group 1d, 4d, 7d and 14d after transfection, and the difference was statistically significant (P<0.05). The level of VEGF, SDF-1, Ang-2 and PDGF in the control group was changed unconspicuously, and the expression was not different at different times (P>0.05). The level of VEGF, SDF-1, Ang-2 and PDGF in the exprimental group increased, and the difference was statistically significant between different time points(P<0.05). Compared with the control group, the level of VEGF, SDF-1, Ang-2 and PDGF in the experimental group was higher 1 d, 4 d, 7 d and 14 d after transfection, respectively, and the difference was statistically significant(P<0.05). CONCLUSIONS: DPSCs modified by HIF-1α gene can successfully induce vascular differentiation in vitro, which provides foundation for further angioplasty.

Evaluation on the efficacy and immunogenicity of recombinant DNA plasmids expressing spike genes from porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus.

Porcine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PDEV) can cause severe diarrhea in pigs. Development of effective vaccines against TGEV and PEDV is one of important prevention measures. The spike (S) protein is the surface glycoprotein of TGEV and PEDV, which can induce specific neutralization antibodies and is a candidate antigen for vaccination attempts. In this study, the open reading frames of the TGEV S1 protein and in addition of the S or S1 proteins of PEDV were inserted into the eukaryotic expression vector, pIRES, resulting in recombinant plasmids, pIRES-(TGEV-S1-PEDV-S1) and pIRES-(TGEV-S1-PEDV-S). Subsequently, 6-8 weeks old Kunming mice were inoculated with both DNA plasmids. Lymphocyte proliferation assay, virus neutralization assay, IFN-γ assay and CTL activity assay were performed. TGEV/PEDV specific antibody responses as well as kinetic changes of T lymphocyte subgroups of the immunized mice were analyzed. The results showed that the recombinant DNA plasmids increased the proliferation of T lymphocytes and the number of CD4+ and CD8+ T lymphocyte subgroups. In addition, the DNA vaccines induced a high level of IFN-γ in the immunized mice. The specific CTL activity in the pIRES-(TGEV-S1-PEDV-S) group became significant at 42 days post-immunization. At 35 days post-immunization, the recombinant DNA plasmids bearing full-length S genes of TGEV and PEDV stimulated higher levels of specific antibodies and neutralizing antibodies in immunized mice.

Immune responses induced by DNA vaccines bearing Spike gene of PEDV combined with porcine IL-18.

Porcine epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemic diarrhea, a highly contagious enteric disease of swine. The Spike (S) protein is one of the main structural proteins of PEDV capable of inducing neutralizing antibodies in vivo. Herein, we generated three distinct DNA constructs in the eukaryotic expression plasmid pVAX1; one encoding the S protein [pVAX1-(PEDV-S)], the second encoding the N-terminal fragment (S1) [pVAX1-(PEDV-S1)] containing potent antigenic sites, and the third expressing the porcine interleukin-18 (pIL-18) [pVAX1-(IL-18)]. Immunofluorescence assays in BHK-21 cells demonstrated successful protein expression from all 3 constructs. Kunming mice were injected separately with each of these constructs or with a pVAX1-(PEDV-S1)/pVAX1-(IL-18) combination, an attenuated PEDV vaccine, or vector only control. Animals were examined for T lymphocyte proliferation, anti-PEDV antibodies, IFN-γ and IL-4 protein levels, and cytotoxic T cell function in mouse peripheral blood and spleen. In all cases, results showed that pVAX1-(PEDV-S) and the combination of pVAX1-(PEDV-S1) with pVAX1-(IL-18) induced the strongest responses; however, pIL-18 had no adjuvant effects when given in combination with pVAX1-(PEDV-S1).