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Atherosclerosis (AS) is the most common factor causing many cardiovascular and cerebrovascular diseases and has received considerable attention. The occurrence mechanism of AS is uncertain because it is a choronically pathological process that is influenced by multi-aspects, among which cytokines play the key roles in regulating the processes of the immune system. For example, two key cytokines, namely, IL-10 and MCP-1 (chemokine), which are involved in AS progression with varied levels, can be used for AS status monitoring and early diagnosis of AS-associated diseases. Hence, a new paper-based, surface-enhanced Raman spectroscopy (SERS) sensing platform was established for the detection of these two key cytokines. By combining a nanoporous networking membrane as the substrate and SERS nanotags as the probe for signal reading, together with a sandwich design, sensitive and specific identification and quantification of cytokine targets in human serum were achieved with excellent sensing characteristics. The lowest detectable concentration was determined to be 0.1 pg mL-1 for both IL-10 and MCP-1 in human serum. The assay also exhibits high specificity towards target cytokine detection, with low-nonspecific binding and acceptable cross-reactivity in the presence of other structurally similar targets. Finally, the practicability was validated by performing duplex detection in human serum, which further demonstrates the high specificity of the assay for the detection of target cytokines. Taken together, these promising results illustrate that this developed sensing assay is a candidate for clinical multi-target analysis in real environments.

The cardiovascular system is significantly affected in coronavirus disease-19 (COVID-19). Microvascular injury, endothelial dysfunction, and thrombosis resulting from viral infection or indirectly related to the intense systemic inflammatory and immune responses are characteristic features of severe COVID-19. Pre-existing cardiovascular disease and viral load are linked to myocardial injury and worse outcomes. The vascular response to cytokine production and the interaction between severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and angiotensin-converting enzyme 2 receptor may lead to a significant reduction in cardiac contractility and subsequent myocardial dysfunction. In addition, a considerable proportion of patients who have been infected with SARS-CoV-2 do not fully recover and continue to experience a large number of symptoms and post-acute complications in the absence of a detectable viral infection. This conditions often referred to as ‘post-acute COVID-19’ may have multiple causes. Viral reservoirs or lingering fragments of viral RNA or proteins contribute to the condition. Systemic inflammatory response to COVID-19 has the potential to increase myocardial fibrosis which in turn may impair cardiac remodelling. Here, we summarize the current knowledge of cardiovascular injury and post-acute sequelae of COVID-19. As the pandemic continues and new variants emerge, we can advance our knowledge of the underlying mechanisms only by integrating our understanding of the pathophysiology with the corresponding clinical findings. Identification of new biomarkers of cardiovascular complications, and development of effective treatments for COVID-19 infection are of crucial importance., Graphical Abstract Graphical Abstract

(1) Background: The medical practice of acupuncture involves the insertion of a specialized stainless needle into a specific body point, often called an acupoint, to initiate a perceived phenomenon of de-qi sensation. Therefore, the term “de-qi” describes bodily sensations experienced by the recipient during acupuncture, which may include feelings of soreness, heaviness, fullness, numbness, and migration. However, while acupuncture treatments reportedly result in acupoint activation and an increased release of neurotransmitters or cytokines, detecting these substances released into the acupoint microenvironment is often missed or delayed in clinical and basic practice. (2) Methods: To address this situation, we employed a paper-based enzyme-linked immunosorbent assay method to examine acupoint environmental changes using minute volumes of easily accessible acupoint fluids. (3) Results: Our results indicated that while levels of adenosine triphosphate (ATP), interleukin-1β, interleukin-6, glutamate, substance P, and histamine were all increased in the experimental group following electroacupuncture (EA) treatment, contrary results were observed in the sham EA and transient receptor potential vanilloid 1 (Trpv1−/−) groups. Subsequently, TRPV1 and its associated molecules were augmented in mouse dorsal root ganglion, spinal cord, thalamus, and the somatosensory cortex, then examined by Western blotting and immunofluorescence techniques. Investigations revealed that these elevations were still unobserved in the sham EA or EA in the Trpv1−/− groups. Furthermore, results showed that while administering ATP could mimic EA function, it could be reversed by the ATP P2 receptor antagonist, suramin. (4) Conclusions: Our data provide novel information, indicating that changes in neurotransmitter and cytokine levels can offer insight into acupuncture mechanisms and clinical targeting.

In this study, levels of inflammatory protein biomarkers in venous plasma, plasma derived from capillary blood from the earlobe, and capillary plasma stored as dried plasma spots (DPS) were compared. Samples from 12 male individuals were assessed with a panel of 92 inflammation-related proteins using multiplex proximity extension assay. Correlations between sample types varied greatly between analytes. A high correlation of rho > 0.8 was observed between capillary plasma and DPS for 32 analytes. At this level of correlation, 13 analytes correlated between venous and capillary plasma and 5 analytes in the comparison of venous blood with DPS.

OBJECTIVE: To explore the diagnostic value of a novel test paper, which detect eosinophil cationic protein(ECP) of nasal secretion in allergic rhinitis(AR). METHODS: Nasal secretion and serum samples from 107 patients with allergic rhinitis(AR group) and 40 healthy volunteers(control group) were selected. The nasal symptoms were also evaluated in AR group. The degree of ECP coloration was evaluated by nasal secretion eosinophil cationic protein-myeloperoxid(ECP-MPO) test paper, and the concentration of ECP in nasal secretion and the concentration of cytokines in serum were detected at the same time. The difference and correlation among these indexes were analyzed. The best cutoff value and test efficiency of ECP chromogenic grade and concentration of nasal secretion were calculated by receiver operating characteristic curve(ROC). RESULTS: The concentration of ECP in nasal secretion of AR patients was significantly higher than that of healthy controls(P < 0.05). The color grade of nasal secretion detected by the test paper was positively correlated with the concentration of ECP in nasal secretion(P < 0.05), and there was significant difference among different grades(P < 0.05). There was a satisfying symmetry between the ECP color grade of nasal secretion and the serum specific IgE(sIgE) level as well as a high diagnostic consistency between them(P < 0.05). The area under the curve(AUC) of ECP concentration ROC in nasal secretion was 0.807 2, corresponding to 64% sensitivity and 85% specificity when the cutoff value was set at 0.980 5; when the cutoff value was set at 1, the AUC of nasal secretion ECP color grading was 0.941 9, corresponding to 92% sensitivity and 94% specificity. No clear correlation between the concentration of ECP in nasal secretion and serum cytokines was found(P>0.05). CONCLUSION: The results of this novel test paper is in good agreement with those of serological allergens. It could serve as a preliminary test to evaluate the severity of allergy with satisfactory sensitivity and specificity, and is especially suitable in clinical practice for primary hospital.

Analysis of cytokines levels in human serum is critical as it can be a "symptom diagnostic biomarker" in COVID-19, giving real-time information about human health status. Here, we present the construction and performance of a low-price immunosensor (∼US$0.428 per test) based on microfluidic paper-based system to detect cytokine for predicting the health status of COVID-19 patients. Interleukin-6 (IL-6) was selected as the detection model for the close relationship between IL-6 and COVID-19. The assay, which we integrated into foldable paper system, leverages the magnetic immunoassay, the streptavidin-horseradish peroxidase (HRP) associated with tetramethyl benzidine/hydrogen peroxide (TMB/H

Although the established ELISA-based sensing platforms have many benefits, the importance of cytokine and cancer biomarkers detection for point-of-care diagnostics has propelled the search for more specific, sensitive, simple, accessible, yet economical sensor. Paper-based biosensor holds promise for future in-situ applications and can provide rapid analysis and data without the need to conduct in a laboratory. Electrochemical detection plays a vital role in interpreting results obtained from qualitative assessment to quantitative determination. In this review, various factors affecting the design of an electrochemical paper-based biosensor are highlighted and discussed in depth. Different detection methods, along with the latest development in utilizing them in cytokine and cancer biomarkers detection, are reviewed. Lastly, the fabrication of portable electrochemical paper-based biosensor is ideal in deliberating positive societal implications in developing countries with limited resources and accessibility to healthcare services. Ministry of Education (MOE) Published version The authors would like to acknowledge the financial support for this work funded by Singapore Ministry of Education Academic Research Fund Tier 1 (MOE-2018-T1-001-004).

Inflammation, structural, and functional abnormalities within the airways are key features of asthma. Although these processes are well documented, their expression varies across the heterogeneous spectrum of asthma. Type 2 inflammatory responses are characterized by increased levels of eosinophils, FeNO, and type 2 cytokines in blood and/or airways. Presently, type 2 asthma is the best-defined endotype, typically found in patients with allergic asthma, but surprisingly also in nonallergic patients with (severe) asthma. The etiology of asthma with non-type 2 inflammation is less clear. During the past decade, targeted therapies, including biologicals and small molecules, have been increasingly integrated into treatment strategies of severe asthma. These treatments block specific inflammatory pathways or single mediators. Single or composite biomarkers help to identify patients who will benefit from these treatments. So far, only a few inflammatory biomarkers have been validated for clinical application. The European Academy of Allergy & Clinical Immunology Task Force on Biomarkers in Asthma was initiated to review different biomarker sampling methods and to investigate clinical applicability of new and existing inflammatory biomarkers (point-of-care) to support diagnosis, targeted treatment, and monitoring of severe asthma. Subsequently, we discuss existing and novel targeted therapies for asthma as well as applicable biomarkers.

The cardiovascular system is significantly affected in coronavirus disease-19 (COVID-19). Microvascular injury, endothelial dysfunction, and thrombosis resulting from viral infection or indirectly related to the intense systemic inflammatory and immune responses are characteristic features of severe COVID-19. Pre-existing cardiovascular disease and viral load are linked to myocardial injury and worse outcomes. The vascular response to cytokine production and the interaction between severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and angiotensin-converting enzyme 2 receptor may lead to a significant reduction in cardiac contractility and subsequent myocardial dysfunction. In addition, a considerable proportion of patients who have been infected with SARS-CoV-2 do not fully recover and continue to experience a large number of symptoms and post-acute complications in the absence of a detectable viral infection. This conditions often referred to as 'post-acute COVID-19' may have multiple causes. Viral reservoirs or lingering fragments of viral RNA or proteins contribute to the condition. Systemic inflammatory response to COVID-19 has the potential to increase myocardial fibrosis which in turn may impair cardiac remodelling. Here, we summarize the current knowledge of cardiovascular injury and post-acute sequelae of COVID-19. As the pandemic continues and new variants emerge, we can advance our knowledge of the underlying mechanisms only by integrating our understanding of the pathophysiology with the corresponding clinical findings. Identification of new biomarkers of cardiovascular complications, and development of effective treatments for COVID-19 infection are of crucial importance.

Cytokine storm is an acute hyperinflammatory response that may be responsible for critical illness in many conditions including viral infections, cancer, sepsis, and multi-organ failure. The phenomenon has been implicated in critically ill patients infected with SARS-CoV-2, the novel coronavirus implicated in COVID-19. Critically ill COVID-19 patients experiencing cytokine storm are believed to have a worse prognosis and increased fatality rate. In SARS-CoV-2 infected patients, cytokine storm appears important to the pathogenesis of several severe manifestations of COVID-19: acute respiratory distress syndrome, thromboembolic diseases such as acute ischemic strokes caused by large vessel occlusion and myocardial infarction, encephalitis, acute kidney injury, and vasculitis (Kawasaki-like syndrome in children and renal vasculitis in adult). Understanding the pathogenesis of cytokine storm will help unravel not only risk factors for the condition but also therapeutic strategies to modulate the immune response and deliver improved outcomes in COVID-19 patients at high risk for severe disease. In this article, we present an overview of the cytokine storm and its implications in COVID-19 settings and identify potential pathways or biomarkers that could be targeted for therapy. Leveraging expert opinion, emerging evidence, and a case-based approach, this position paper provides critical insights on cytokine storm from both a prognostic and therapeutic standpoint.

OBJECTIVES Sepsis and sterile both release "danger signals' that induce the systemic inflammatory response syndrome (SIRS). So differentiating infection from SIRS can be challenging. Precision diagnostic assays could limit unnecessary antibiotic use, improving outcomes. METHODS After surveying human leukocyte cytokine production responses to sterile damage-associated molecular patterns (DAMPs), bacterial pathogen-associated molecular patterns, and bacteria we created a multiplex assay for 31 cytokines. We then studied plasma from patients with bacteremia, septic shock, "severe sepsis," or trauma (ISS ≥15 with circulating DAMPs) as well as controls. Infections were adjudicated based on post-hospitalization review. Plasma was studied in infection and injury using univariate and multivariate means to determine how such multiplex assays could best distinguish infective from noninfective SIRS. RESULTS Infected patients had high plasma interleukin (IL)-6, IL-1α, and triggering receptor expressed on myeloid cells-1 (TREM-1) compared to controls [false discovery rates (FDR)

The COVID-19 pandemic is an unprecedented healthcare emergency causing mortality and illness across the world. Although primarily affecting the lungs, the SARS-CoV-2 virus also affects the cardiovascular system. In addition to cardiac effects, e.g. myocarditis, arrhythmias, and myocardial damage, the vasculature is affected in COVID-19, both directly by the SARS-CoV-2 virus, and indirectly as a result of a systemic inflammatory cytokine storm. This includes the role of the vascular endothelium in the recruitment of inflammatory leucocytes where they contribute to tissue damage and cytokine release, which are key drivers of acute respiratory distress syndrome (ARDS), in disseminated intravascular coagulation, and cardiovascular complications in COVID-19. There is also evidence linking endothelial cells (ECs) to SARS-CoV-2 infection including: (i) the expression and function of its receptor angiotensin-converting enzyme 2 (ACE2) in the vasculature; (ii) the prevalence of a Kawasaki disease-like syndrome (vasculitis) in COVID-19; and (iii) evidence of EC infection with SARS-CoV-2 in patients with fatal COVID-19. Here, the Working Group on Atherosclerosis and Vascular Biology together with the Council of Basic Cardiovascular Science of the European Society of Cardiology provide a Position Statement on the importance of the endothelium in the underlying pathophysiology behind the clinical presentation in COVID-19 and identify key questions for future research to address. We propose that endothelial biomarkers and tests of function (e.g. flow-mediated dilatation) should be evaluated for their usefulness in the risk stratification of COVID-19 patients. A better understanding of the effects of SARS-CoV-2 on endothelial biology in both the micro- and macrovasculature is required, and endothelial function testing should be considered in the follow-up of convalescent COVID-19 patients for early detection of long-term cardiovascular complications.

The study was focused on assessment of potential health risks of paper-based food contact materials (FCMs) in a step-wise approach using three toxicological bioassays in vitro and chemical analyses of migrating contaminants. 3T3 NRU cytotoxicity test showed high sensitivity to detect basal toxicity of FCMs extracts and served as a first-line test for selection of samples for further testing. The reconstructed human intestine model EpiIntestinal showed more realistic tissue response than cell culture monolayer and higher resistance despite prolonged exposure to the selected 6 samples, i.e. negligible decrease of viability and intestinal penetration, nevertheless an increase of IL-8 after exposure to black printed sample extract. Yeast based assays identified weak agonistic/antagonostic activity to human androgen receptor of the black printed sample. In accordance with the biological effects, the targeted LC and GC analytical methods confirmed the presence of high amounts of phthalates, photoinitiators and PAHs that could justify the hazard of the black printed sample. Heavily printed uncoated FCMs are recognized not to be suitable for direct contact with food. The selected bioassays and chemical analyses might be useful tools to detect targeted biological effects of xenobiotics suspected to contribute to human exposure from food.

Investigation of cellular phosphorylation and signaling pathway has recently gained much attention for the study of pathogenesis of cancer. Related conventional bioanalytical operations for this study including cell culture and Western blotting are time-consuming and labor-intensive. In this work, a paper-based microreactor has been developed to integrate cell culture and subsequent immunoassay on a single paper. The paper-based microreactor was a filter paper with an array of circular zones for running multiple cell cultures and subsequent immunoassays. Cancer cells were directly seeded in the circular zones without hydrogel encapsulation and cultured for 1 day. Subsequently, protein expressions including structural, functional, and phosphorylated proteins of the cells could be detected by their specific antibodies, respectively. Study of the activation level of phosphorylated Stat3 of liver cancer cells stimulated by IL-6 cytokine was demonstrated by the paper-based microreactor. This technique can highly reduce tedious bioanalytical operation and sample and reagent consumption. Also, the time required by the entire process can be shortened. This work provides a simple and rapid screening tool for the investigation of cellular phosphorylation and signaling pathway for understanding the pathogenesis of cancer. In addition, the operation of the paper-based microreactor is compatible to the molecular biological training, and therefore, it has the potential to be developed for routine protocol for various research areas in conventional bioanalytical laboratories.

We report the development of a distance-based paper analytical device combined with a hydrophilic bridge valve (B-dPAD) as a quantitative immunoassay method to monitor human interleukin-6 (IL-6) in human samples. Our device design features (i) a circular sample inlet zone, (ii) a circular capture zone with immobilized anti-IL-6 (anti-Ab

In recent years, a new class of drugs has revolutionized the treatment of autoimmune, allergic, infectious and many more diseases. These drugs are classified into three groups, cytokines, monoclonal antibodies and fusion proteins. Biological drugs have less side effects compared to conventional drugs, and may target special damaged cells, but not all the cells. There may be side effects such as infection, hypersensitivity, hematological disorders, cancer, hepatotoxicity and neurological disorders, but there is not enough evidence or long term studies of the mechanism of action and side effects of these drugs. Patients on biological therapy may need some special consideration in dentistry. This paper is a review regarding the classification, mechanism of action and side effects of these drugs, and dental consideration for patients on biological therapy.

Introduction : Juvenile idiopathic arthritis (JIA) is a heterogeneous group of inflammatory diseases of joints in children with various and often unfavourable prognosis. It is possible to improve the outcome of the disease in patients with JIA by a correct therapeutic choice made at disease onset – one that enables fast achievement of an inactive disease state and remission. The aim of the investigation was to develop a model/application for automatic calculation of risk of treatment-refractory JIA taking into account the combined action of clinical and cytokine factors. Material and methods: Disease subtype was determined in 105 patients with JIA, as well as the number of poor prognostic factors and disease activity level. Blood serum cytokine levels (IL-1β, IL-4, IL-6, IL-8, IL-10, IL-17, TNF-α, IFN-γ) and their soluble receptors and agonists – interleukin 1 receptor agonist (IL-1Ra), soluble interleukin 2 receptor (sCD25), interleukin 6 (sIL6R), soluble tumour necrosis factor receptor 1 (sTNFR1) – were determined in these patients using immunoenzymatic laboratory methods. Results : The following prognostic factors were taken into account in the study: JIA subtype, disease activity, presence of clinically unfavourable factors and cytokine characteristics. We determined that systemic subtype of JIA, moderate and high disease activity, presence of factors of poor disease course and sCD25 and IL-6 levels are statistically significant factors of treatment-refractory disease course. A Microsoft Excel application was developed for automatic calculation of risk of treatment-refractory JIA in a specific patient based on 10 factors. Conclusions : Use of an application for automatic calculation of risk of treatment-refractory JIA enables prediction of JIA disease course in patients at disease onset and personalization of the treatment protocol.

Objectives: Current studies concentrate on the cytokine network and its role in the pathogenesis of spondyloarthritis (SpA). In this study, we analyzed whether the serum cytokine profile (interleukins: IL-10, IL-11, IL-12, IL-15, IL-17, IL-23 and IL-33) correlates with demographic data, clinical manifestations, disease activity and treatment outcome in a group of patients with axial spondyloarthritis. Material and methods: Forty-nine patients with an established diagnosis of axial spondyloarthritis (aSpA) and 19 healthy volunteers as controls were enrolled in the study. Clinical evaluation included patient’s medical history, 44 joint count, back pain intensity and global disease activity in the preceding week (VAS), the duration of morning stiffness and blood tests. Disease activity was assessed using BASDAI and ASDAS-CRP. Serum concentration of IL-10, IL-11, IL-12, IL-15, IL-17, IL-23 and IL-33 was determined. Results : In patients with aSpA, elevated serum concentration of IL-10, IL-15, IL-17 and IL-23 was detected. In the aSpA group we detected higher values of serum concentration of IL-23 and IL-33 in the subgroup with anterior uveitis (83.1 ±184.0 pg/ml vs. 14.0 ±17.1 pg/ml, p < 0.0001 and 45.5 ±71.9 pg/ml vs. 18.4 ±14.3 pg/ml, p < 0.0001, respectively). Additionally, in the subgroup with peripheral arthritis, elevation of serum concentration of IL-12 (249.3 ±246.9 pg/ml vs. 99.9 ±105.9 pg/ml, p = 0.0001) was detected. Patients with preradiological SpA had higher serum concentration of IL-17 than patients with established diagnosis of AS (6.37 ±8.50 pg/ml vs. 2.04 ±2.98 pg/ml, p = 0.0295). No differences in serum concentration of analyzed cytokines were found between the subgroup with low to moderate disease activity and the subgroup with high to very high disease activity. Conclusions : We report that in aSpA patients, compared to controls, elevated serum concentrations of IL-10, IL-15, IL-17 and IL-23 were observed. Some cytokines may predispose to a more severe course of aSpA.

Inflammatory reaction exerts a pivotal role in secondary damage after cerebral hemorrhage and spinal cord injury. miRNAs can both promote and inhibit inflammatory actions among microglial cells. The objective of the present paper was to figure out whether miR-27b-3p produced regulatory effects during processes of microglial inflammation. Lipopolysaccharides (LPS) were used to prepare microglial activation models. Following miR-27b-3p overexpression and interference, the RNA and protein levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β were subjected to real-time fluorescent quantitative PCR (qPCR) and western blot assays, respectively. Cellular apoptosis was subjected to flow cytometry and miR-27b-3p target genes were visualized using a dual luciferase reporter system for verification. The levels of TNF-α, IL-6, and IL-1β mRNA in miR-27b-3p-overexpressed microglial cells were markedly increased compared to the control. Apoptosis of microglial cells was increased markedly in the overexpressed miR-27b-3p group compared to the negative control. Conversely, a different result was presented in the microglial transfected with miR-27b-3p inhibitors. The downregulation of A20, a miR-27b-3p target gene, mediated levels of TNF-α, IL-6, and IL-1β. Furthermore, A20 reduced microglial apoptosis. These data revealed that miR-27b-3p could mediate not only microglia activation but also neuroinflammation via downregulating A20 expression. Thus, miR-27b-3p is regarded as gene therapy in treating cerebral hemorrhage and spinal cord injury., Graphical abstract

Neurologically deceased organ donors (NDDs) generally display an immune response involving an intense production of pro-inflammatory cytokines referred to as the cytokine storm. The sudden surge of inflammatory mediators in circulation promotes tissue and organ damages and ultimately leads to poor transplant outcome. As microRNAs (miRNAs) are frequently proposed as key regulators of inflammation and are relatively stable in circulation, changes in their profiles could play a role in the onset of the cytokine storm in NDDs. In this proof-of-concept study, we sought to investigate differentially abundant circulating miRNAs in a temporal manner between neurological death and organ recovery and to assess the association between specific miRNAs and levels of inflammatory cytokines in blood. Plasma samples from five NDDs were obtained at multiple time points between organ donation consent and organ recovery. Using a time-course analysis and miRNA sequencing, we identified 32 plasma miRNAs fluctuating between consent and organ recovery (false discovery rate; q-value < 0.1). Eleven miRNAs relatively abundant (>100 reads) and detected in all samples were selected for further biological pathway analysis (miR-486-3p, miR-103a-3p, miR-106b-3p, miR-182-5p, miR-101-3p, miR-10a-5p, miR-125a-5p, miR-146b-5p, miR-26a-5p, miR-423-5p, miR-92b-3p). These miRNAs targeted genes such as c-JUN (TNF signalling pathway) and eEF2 (AMPK pathway), suggesting a potential role in regulation of inflammation. Our results contribute to a better understanding of the miRNAs dynamic after neurological death in organ donors and could potentially be used to predict the related early cytokine storm.Trial registration: ClinicalTrials.gov ID NCT03786991. Registered December 2018

We examined the utility of dried blood on filter paper for the source of cytokine messenger RNA (mRNA). Total RNA was isolated from the dried blood of mice infected with Plasmodium yoelii, and cDNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR). As a reference, we extracted total RNA from peripheral blood collected at the same time as the preparation for dried blood. There was no difference in cytokine mRNA expression between the two sources; the dried blood on filter paper and the peripheral blood. Th1 cells, Th2 cells, and monocytes/macrophages derived cytokine mRNAs in the dried blood from infected mice were detected, and the increase of some of the cytokines mRNAs after infection was also observed. These results suggested that the dried blood on filter paper is satisfactory RNA source for immunological examination in field-based studies.

Long noncoding RNAs (lncRNAs) are known to regulate gene expression; however, in many cases, the mechanism of this regulation is unknown. One novel lncRNA relevant to inflammation and arachidonic acid (AA) metabolism is the p50-associated COX-2 extragenic RNA (PACER). We focused our research on the regulation of PACER in lung cancer. While the function of PACER is not entirely understood, PACER is known to play a role in inflammation-associated conditions. Our data suggest that PACER is critically involved in COX-2 transcription and dysregulation in lung cancer cells. Our analysis of The Cancer Genome Atlas (TCGA) expression data revealed that PACER expression is significantly higher in lung adenocarcinomas than normal lung tissues. Additionally, we discovered that elevated PACER expression strongly correlates with COX-2 expression in lung adenocarcinoma patients. Specific siRNA-mediated knockdown of PACER decreases COX-2 expression indicating a direct relationship. Additionally, we show that PACER expression is induced upon treatment with proinflammatory cytokines to mimic inflammation. Treatment with prostaglandin E2 (PGE2) induces both PACER and COX-2 expression, suggesting a PGE2-mediated feedback loop. Inhibition of COX-2 with celecoxib decreased PACER expression, confirming this self-regulatory process. Significant overlap between the COX-2 promotor and the PACER promotor led us to investigate their transcriptional regulatory mechanisms. Treatment with pharmacologic inhibitors of NF-κB or AP-1 showed a modest effect on both PACER and COX-2 expression but did not eliminate expression. These data suggest that the regulation of expression of both PACER and COX-2 is complex and intricately linked.

The influenza A virus (IAV) H1N1pdm09 induces exacerbated inflammation, contributing to disease complications. Inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), favor an inflammatory response that aids viral replication and survival. A pathway by which spontaneous TNF-α production occurs involves either the reduction of Siglec-3 (CD33) levels or the absence of its ligand, sialic acid. Influenza virus uses sialic acid to enter cells by reducing their expression; however, the role of CD33 in IAV H1N1pdm09 stimulation and its relationship with inflammation have not yet been studied. To evaluate the role of CD33 in proinflammatory cytokine production in IAV H1N1pdm09 stimulation, peripheral blood mononuclear cells from healthy subjects were incubated with IAV H1N1pdm09. We observed that the infection caused an increase in the mRNA expression of proinflammatory cytokines such as TNF-α, interleukin (IL)-1β, and IL-6 and a significant reduction in CD33 expression by monocytes at an early stage of infection. Additionally, suppressor of cytokine signaling 3 (SOCS-3) mRNA expression was upregulated at 6 h, and reactive oxygen species (ROS) production increased at 1.5 h. Moreover, a significant reduction in CD33 expression on the cell surface of monocytes from influenza patients or of IAV H1N1pdm09-stimulated monocytes incubated in vitro was observed by flow cytometry. The results suggest that the decrease in CD33 and increase of SOCS-3 expression induced by IAV H1N1pdm09 triggered TNF-α secretion and ROS production, suggesting an additional way to exacerbate inflammation during viral infection. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-021-00663-4.

Stress urinary incontinence (SUI) is defined as involuntary urinary leakage happening in exertion. Nicotinamide phosphoribosyltransferase (Nampt) is seldom researched in the pathogenesis of SUI. Accordingly, the current study set out to elucidate the role of Nampt in SUI progression. Firstly, we determined Nampt expression patterns in SUI patients and rat models. In addition, fibroblasts were obtained from the anterior vaginal wall tissues of non-SUI patients and subjected to treatment with different concentrations of interleukin-1β (IL-1β), followed by quantification of Nampt expressions in fibroblasts. Subsequently, an appropriate concentration of IL-1β was selected to treat anterior vaginal wall fibroblasts. Nampt was further silenced in IL-1β-treated fibroblasts to assess the role of Nampt in autophagy and extracellular matrix (ECM) degradation. Lastly, functional rescue assays were carried out to inhibit autophagy and evaluate the role of autophagy in the mechanism of Nampt modulating IL-1β-treated fibroblast ECM degradation. It was found that Nampt was highly-expressed in SUI patients and rat models and IL-1β-treated fibroblasts. On the other hand, Nampt silencing was found to suppress ECM degradation and promote SUI fibroblast autophagy. Additionally, inhibition of autophagy attenuated the inhibitory effects of Nampt silencing on SUI fibroblast ECM degradation. Collectively, our findings revealed that Nampt was over-expressed in SUI, whereas Nampt silencing enhanced SUI fibroblast autophagy, and thereby inhibited ECM degradation.

Intracerebral hemorrhage (ICH) is a common neurological condition that causes severe disability and even death. Even though the mechanism is not clear, increasing evidence shows the efficacy of atorvastatin on treating ICH. In this study, we examined the impact of atorvastatin on the NOD-like receptor protein 3 (NLRP3) inflammasome and inflammatory pathways following ICH. Mouse models of ICH were established by collagenase injection in adult C57BL/6 mice. IHC mice received atorvastatin treatment 2 h after hematoma establishment. First, the changes of glial cells and neurons in the brains of ICH patients and mice were detected by immunohistochemistry and western blotting. Second, the molecular mechanisms underlying the microglial activation and neuronal loss were evaluated after the application of atorvastatin. Finally, the behavioral deficits of ICH mice without or with the treatment of atorvastatin were determined by neurological defect scores. The results demonstrated that atorvastatin significantly deactivated glial cells by reducing the expression of glial fibrillary acidic protein (GFAP), Ionized calcium binding adapter molecule 1 (Iba1), tumor necrosis factor (TNF)-α, and interleukin (IL)-6 in ICH model mice. For inflammasomes, atorvastatin also showed its efficacy by decreasing the expression of NLRP3, cleaved caspase-1, and IL-1β in ICH mice. Moreover, atorvastatin markedly inhibited the upregulation of toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88), which indicated deactivation of NLRP3 inflammasomes. By inhibiting the activities of inflammasomes in glial cells, neuronal loss was partially prevented by suppressing the apoptosis in the brains of ICH mice, protecting them from neurological defects.

Dehydroevodiamine (DHE) is an effective natural active substance extracted from Euodiae Fructus, which is a widely used herbal drug in traditional Chinese medicine. The focus of this study was to test the possibility of using DHE in the treatment of rheumatoid arthritis (RA) diseases. A rat model of adjuvant-induced arthritis (AIA) was generated using Complete Freund’s Adjuvant (CFA). Body weight changes, arthritis scores, ankle pathology, tumor necrosis factor-alpha (TNF-α), interleukin-1β(IL-1β), interleukin-6 (IL-6), and interleukin-17 (IL-17) secretion, as well as matrix metalloproteinase (MMP) expression in joint tissue, were measured as indicators of viability of DHE medicated AIA rats. Human fibroblast-like synoviocytes (MH7A cells) were connected to check these impacts. The results confirmed that DHE administration had an excellent therapeutic impact on the AIA rat model, substantially relieving joint swelling, inhibiting synovial pannus hyperplasia, and decreasing joint scores. In addition, the serum enzyme-linked immunosorbent assay (ELISA) showed that DHE treatment reduced the expression of pro-inflammatory factors in AIA rats. The immunohistochemical results showed that DHE treatment could reduce the synthesis of MMPs such as matrix metalloproteinase-1(MMP-1) and matrix metalloproteinase-3 (MMP-3) in the ankle tissue of AIA rats. In vitro, DHE inhibited cell proliferation, mRNA transcription, protein synthesis of proinflammatory factors such as IL-1βand IL-6, and matrix metalloproteinases such as MMP-1 and MMP-3. Furthermore, DHE inhibited the phosphorylation levels of p38, JNK, and ERK proteins in TNF-α-treated MH7A cells.This work assessed the effect of DHE in AIA rats and revealed its mechanism in vitro.

Rationale: Corneal neovascularization (CoNV) is a severe complication of various types of corneal diseases, that leads to permanent visual impairment. Current treatments for CoNV, such as steroids or anti-vascular endothelial growth factor agents, are argued over their therapeutic efficacy and adverse effects. Here, we demonstrate that transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) plays an important role in the pathogenesis of CoNV. Methods: Angiogenic activities were assessed in ex vivo and in vitro models subjected to TAK1 inhibition by 5Z-7-oxozeaenol, a selective inhibitor of TAK1. RNA-Seq was used to examine pathways that could be potentially affected by TAK1 inhibition. A gelatin-nanoparticles-encapsulated 5Z-7-oxozeaenol was developed as the eyedrop to treat CoNV in a rodent model. Results: We showed that 5Z-7-oxozeaenol reduced angiogenic processes through impeding cell proliferation. Transcriptome analysis suggested 5Z-7-oxozeaenol principally suppresses cell cycle and DNA replication, thereby restraining cell proliferation. In addition, inhibition of TAK1 by 5Z-7-oxozeaenol blocked TNFα-mediated NFκB signalling, and its downstream genes related to angiogenesis and inflammation. 5Z-7-oxozeaenol also ameliorated pro-angiogenic activity, including endothelial migration and tube formation. Furthermore, topical administration of the gelatin-nanoparticles-encapsulated 5Z-7-oxozeaenol led to significantly greater suppression of CoNV in a mouse model compared to the free form of 5Z-7-oxozeaenol, likely due to extended retention of 5Z-7-oxozeaenol in the cornea. Conclusion: Our study shows the potential of TAK1 as a therapeutic target for pathological angiogenesis, and the gelatin nanoparticle coupled with 5Z-7-oxozeaenol as a promising new eyedrop administration model in treatment of CoNV.

Rheumatoid arthritis (RA) is a chronic inflammatory joint disorder that inflicts damage to the joints of the hands and wrist. The aim of this study was to investigate the protective effect of β-Arrestin-2 (βArr2) on RA in vivo and in vitro. The βArr2 adenovirus (βArr2-Ad) or the control (Con-Ad) was injected into the ankle joint cavity of collagen-induced arthritis (CIA) mice. According to the results, an improvement was shown in the symptoms and pathological injury of RA after an upregulation of βArr2. Correspondingly, the inflammatory response was attenuated, as evidenced by the decreased serum pro-inflammatory cytokines levels and NF-κB pathway-related proteins. Nucleotide-binding domain leucine-rich repeat and pyrin domain containing receptor 3 (NLRP3) inflammasome activation was inhibited in CIA mice treated with βArr2-Ad injection, as reflected by the diminished IL-18 level and declined protein levels of ankle inflammasome components in the ankle joint. Likewise, the anti-inflammatory effect of macrophages was also validated by in vitro experiments. In summary, βArr2 effectively ameliorates ankle inflammation in CIA mice via NF-κB/NLRP3 inflammasome, providing theoretical and clinical basis for RA therapy.Key wordsRheumatoid arthritis; β-arrestin-2; NF-κB; NLRP3.

Background: Toll-like receptor 4 (TLR4) is implicated in neonatal hypoxic-ischemic brain damage (HIBD), but the underlying mechanism is unclear. Hypothesis: We hypothesized that TLR4 mediates brain damage after hypoxic ischemia (HI) by inducing abnormal neuroimmune responses, including activation of immune cells and expression disorder of immune factors, while early inhibition of TLR4 can alleviate the neuroimmune dysfunction. Method: Postnatal day 7 rats were randomized into control, HI, and HI+TAK-242 (TAK-242) groups. The HIBD model was developed using the Rice-Vannucci method (the left side was the ipsilateral side of HI). TAK-242 (0.5 mg/kg) was given to rat pups in the TAK-242 group at 30 min before modeling. Immunofluorescence, immunohistochemistry, and western blotting were used to determine the TLR4 expression; the number of Iba-1+, GFAP+, CD161+, MPO+, and CD3+ cells; ICAM-1 and C3a expression; and interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-10 expression in the hippocampal CA1 region. Result: Significantly increased TLR4 expression was observed in the left hippocampus, and was alleviated by TAK-242. The significant increases in Iba-1+, MPO+, and CD161+ cells at 24 h and 7 days after HI and in GFAP+ and CD3+ T cells at 7 days after HI were also counteracted by TAK-242, but no significant differences were observed among groups at 24 h after HI. ICAM-1 expression increased 24 h after HI, while C3a expression decreased; TAK-242 also alleviated these changes. TNF-α and IL-1β expression increased, while IL-10 expression decreased at 24 h and 7 days after HI; TAK-242 counteracted the increased TNF-α and IL-1β expression at 24 h and the changes in IL-1β and IL-10 at 7 days, but induced no significant differences in IL-10 expression at 24 h and TNF-α expression at 7 days. Conclusion: Early TLR4 inhibition can alleviate hippocampal immune dysfunction after neonatal HIBD.

Purpose: Conventionally, keratoconus (KC) has been considered a noninflammatory corneal ectatic disorder. Recent evidence suggests a possible role of inflammation in the pathogenesis of KC. Hence, we analyzed the levels of inflammatory factors in the tear fluid of Indian KC patients. Methods: Tear fluid samples were collected from age-and sex-matched healthy controls and KC patients (with different grades). The levels of the inflammatory factors in tears were analyzed using cytometric bead array (Human Soluble Protein Flex Set System, BD Biosciences) for levels of interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-12p70, IL-23p40, IL-13, IL-17A, IL-17F, IL-21, interferon-alpha (IFN alpha), IFN., tumor necrosis factor-alpha, CCL2/monocyte chemotactic protein-1, CCL4/macrophage inflammatory protein-1 beta (MIP-1 beta), MIP-1 alpha, CCL5/RANTES, CXCL10/IP10, ICAM1, CD62E, vascular endothelial growth factor and transforming growth factor beta. Results: An increase in Kmax and Kmean, and a decrease in central corneal thickness was observed with increasing grades of KC. Tear analysis showed that most of the tear soluble factors, including cytokines, chemokines, growth factors and cell adhesion molecules were significantly elevated in the KC patients compared to the controls. Conclusion: Our findings suggest that inflammatory factors associated with KC may play a role in its pathogenesis. This opens the potential to explore anti-inflammatory strategies to either halt or delay the progression of KC.

Background Allergen immunotherapy (AIT) is an effective treatment for allergic rhinoconjunctivitis (AR) with or without asthma. It is important to note that due to the complex interaction between patient, allergy triggers, symptomatology and vaccines used for AIT, some patients do not respond optimally to the treatment. Furthermore, there are no validated or generally accepted candidate biomarkers that are predictive of the clinical response to AIT. Clinical management of patients receiving AIT and efficacy in randomised controlled trials for drug development could be enhanced by predictive biomarkers. Method The EAACI taskforce reviewed all candidate biomarkers used in clinical trials of AR patients with/without asthma in a literature review. Biomarkers were grouped into seven domains: (i) IgE (total IgE, specific IgE and sIgE/Total IgE ratio), (ii) IgG-subclasses (sIgG1, sIgG4 including SIgE/IgG4 ratio), (iii) Serum inhibitory activity for IgE (IgE-FAB and IgE-BF), (iv) Basophil activation, (v) Cytokines and Chemokines, (vi) Cellular markers (T regulatory cells, B regulatory cells and dendritic cells) and (vii) In vivo biomarkers (including provocation tests?). Results All biomarkers were reviewed in the light of their potential advantages as well as their respective drawbacks. Unmet needs and specific recommendations on all seven domains were addressed. Conclusions It is recommended to explore the use of allergen-specific IgG4 as a biomarker for compliance. sIgE/tIgE and IgE-FAB are considered as potential surrogate candidate biomarkers. Cytokine/chemokines and cellular reponses provided insight into the mechanisms of AIT. More studies for confirmation and interpretation of the possible association with the clinical response to AIT are needed.

The aim of the present study was to explore the effects of bone marrow mesenchymal stem cells (BMSCs), combined with Atractylodes macrocephala polysaccharide (AMP), in an experimental model of ulcerative colitis. BMSCs were first isolated, cultured, and identified by flow cytometry. A rat model of colitis was established by trinitrobenzene sulfonic acid (TNBS) injection. Rats were treated with BMSCs with or without AMP for 1 or 2 weeks. H&E staining was performed to assess the extent of histological injury. IEC-6 and BMSCs were co-cultured and treated with AMP. Cell migration was measured using the Transwell assay, whilst the levels of cytokines in the rat blood samples were detected using ELISA. In addition, cytokine levels in the cell supernatant were measured by microarray. The results showed that BMSCs were successfully isolated. BMSCs treatment could markedly alleviate injury according to histological analysis and regulate inflammatory cytokine production in this rat model of TNBS-induced colitis, where a higher number of BMSCs was found in the intestinal tract, compared to the model. AMP not only potentiated the effects of BMSCs on preventing TNBS-induced colitis but also promoted BMSC homing to the injured tissue and regulated cytokines. Furthermore, BMSCs and AMP promoted the migration of IEC in vitro and influenced multiple genes. In conclusion, AMP treatment improved the therapeutic effects of BMSCs on ulcerative colitis, potentially providing a novel clinical treatment strategy for colitis.

Sepsis-induced myocardial dysfunction (SIMD) is ubiquitous in septic shock patients and is associated with high morbidity and mortality rates. Heat shock protein 22 (Hsp22), which belongs to the small HSP family of proteins, is involved in several biological functions. However, the function of Hsp22 in lipopolysaccharide (LPS)-induced myocardial injury is not yet established. This study was aimed at investigating the underlying mechanistic aspects of Hsp22 in myocardial injury induced by LPS. In this study, following the random assignment of male C57BL/6 mice into control, LPS-treated, and LPS + Hsp22 treated groups, relevant echocardiograms and staining were performed to scrutinize the cardiac pathology. Plausible mechanisms were proposed based on the findings of the enzyme-linked immunosorbent assay and Western blotting assay. A protective role of Hsp22 against LPS-induced myocardial injury emerged, as evidenced from decreased levels of creatinine kinase-MB (CK-MB), lactate dehydrogenase (LDH), and enhanced cardiac function. The post-LPS administration-caused spike in inflammatory cytokines (IL-1β, IL-6, TNF-α and NLRP3) was attenuated by the Hsp22 pre-treatment. In addition, superoxide dismutase (SOD) activity and B-cell lymphoma-2 (Bcl2) levels were augmented by Hsp22 treatment resulting in lowering of LPS-induced oxidative stress and cardiomyocyte apoptosis. In summary, the suppression of LPS-induced myocardial injury by Hsp22 overexpression via targeting of inflammation, oxidative stress, and apoptosis in cardiomyocytes paves the way for this protein to be employed in the therapy of SIMD.

Dexmedetomidine (DEX) has been reported to attenuate cecal ligation perforation (CLP)-stimulated acute lung injury (ALI) by downregulating HMGB1 and RAGE. This study aimed to further investigate the specific mechanisms of RAGE and its potential-related mechanisms of DEX on ALI models in vitro and in vivo. The in vitro and in vivo ALI models were established by lipopolysaccharide treatment in MLE-12 cells and CLP in mice, respectively. The effect of DEX on pathological alteration was investigated by HE staining. Thereafter, the myeloperoxidase (MPO) activity and inflammatory cytokine levels were respectively detected to assess the lung injury of mice using commercial kits. The expression levels of HMGB1, RAGE, NF-κB, and pyroptosis-related molecules were detected by RT-qPCR and Western blot. HE staining showed that lung injury, increased inflammatory cell infiltration, and lung permeability was found in the ALI mice, and DEX treatment significantly attenuated lung tissue damage induced by CLP. The MPO activity and inflammatory cytokines (TNF-α, IL-1β, and NLRP3) levels were also significantly reduced after DEX treatment compared with those in the ALI mice. Moreover, DEX activated the HMGB1/RAGE/NF-κB pathway and upregulated the pyroptosis-related proteins. However, the protective DEX effect was impaired by RAGE overexpression in ALI mice and MLE-12 cells. Additionally, DEX treatment significantly suppressed HMGB1 translocation from the nucleus region to the cytoplasm, and this effect was reversed by RAGE overexpression. These findings suggested that DEX may be a useful ALI treatment, and the protective effects on ALI mice may be through the inhibition of HMGB1/RAGE/NF-κB pathway and cell pyroptosis., Graphical abstractDexmedetomidine (DEX) has protective effects on acute lung injury (ALI) in vitro and in vivo. The possible mechanisms may be closely associated with inflammatory response, caspase-1-mediated cell pyroptosis, and high-mobility group protein 1 (HMGB1)/receptor for advanced glycation end products (RAGE)/nuclear factor-κB pathway. Moreover, DEX could promote the HMGB1 translocation from the cytoplasm to the nucleus in lipopolysaccharide-activated MLE-12 cells, whereas the action of RAGE overexpression was opposite.

Perfluorooctanoic acid (PFOA) can accumulate in the livers of humans and animals via the food chain, resulting into liver injury, which is closely related to intestinal flora dysbiosis. Gastrodin has been reported to have hepatoprotective effect. However, whether gastrodin can alleviate PFOA-induced liver injury via modulating gut microbiota remains unclear. Herein, a PFOA-induced liver injury model was established by gavage of PFOA (5 mg/kg body weight) in 2% Tween 80 solution once daily for 6 weeks in mice, and then gastrodin in saline (20 mg/kg body weight) was used once daily for 8 weeks to treat liver damage. The biochemical indexes associated with liver function, oxidative stress, and inflammatory factors were examined. Hematoxylin-eosin staining was used to determine the liver histopathological changes. Besides, 16S rRNA sequencing was used to analyze the difference of gut microbiota between the model and treatment groups. The results showed that gastrodin significantly improved the oxidative stress caused by PFOA. Intestinal flora analysis showed that gastrodin treatment significantly increased the relative abundance of probiotics, such as Lactobacillus, Bifidobacterium, and Bacteroides, while the harmful bacteria, including Desulfovibrio were decreased. Gastrodin treatment also significantly increased the level of short-chain fatty acids (SCFAs), such as butyric acid and isobutyric acid. Spearman correlation analysis showed that the composition changes of gut microbiota and SCFAs increase were both beneficial to alleviate the liver injury caused by PFOA. To sum up, gastrodin can effectively alleviate PFOA-induced liver injury through regulating gut microbiota composition.

Staphylococcus aureus is a skin commensal microorganism commonly colonizing healthy humans. Nevertheless, S. aureus can also be responsible for cutaneous infections and contribute to flare-up of inflammatory skin diseases such as atopic dermatitis (AD), which is characterized by dysbiosis of the skin microbiota with S. aureus as the predominant species. However, the role of major virulence factors of this pathogen such as phenol-soluble modulin (PSM) toxins in epidermal inflammation remains poorly understood. Stimulation of primary human keratinocytes with sublytic concentrations of synthetic and purified PSM α3 resulted in upregulation of a large panel of pro-inflammatory chemokine and cytokine gene expression, including CXCL1, CXCL2, CXCL3, CXCL5, CXCL8, CCL20, IL-1α, IL-1β, IL-6, IL-36γ and TNF-α, while inducing the release of CXCL8, CCL20, TNF-α and IL-6. In addition, using S. aureus culture supernatant from mutants deleted from genes encoding either α-type PSMs or all PSM production, PSMs were shown to be the main factors of S. aureus secretome responsible for pro-inflammatory mediator induction in human keratinocytes. On the other hand, α-type PSM-containing supernatant triggered an intense induction of pro-inflammatory mediator expression and secretion during both topical and basal layer stimulation of an ex vivo model of human skin explants, a physiologically relevant model of pluristratified epidermis. Taken together, the results of this study show that PSMs and more specifically α-type PSMs are major virulence factors of S. aureus inducing a potent inflammatory response during infection of the human epidermis and could thereby contribute to AD flare-up through exacerbation of skin inflammation.

Rotigotine is a non-ergoline dopamine agonist that has been licensed for the treatment of Parkinson’s disease. Cardiovascular diseases are the world’s leading cause of death. Ox-LDL- induced endothelial damages are involved in the initiation and progression of cardiovascular diseases. In this study, we assessed the beneficial properties of Rotigotine on ox-LDL-induced insults to HUVECs to highlight its potential use in the treatment of cardiovascular diseases. Our findings show that Rotigotine suppresses the expressions of low-density lipoprotein receptor (LDL-R), proprotein convertase subtilisin/kexin type 9 (PCSK-9), and sterol regulatory element-binding protein (SREBP-2). It also inhibits ox-LDL-induced cholesterol accumulation in endothelial cells (ECs), improves U937 monocytes adhesion, and decreases the representation of NADPH oxidase (NOX-4) and generation of reactive oxygen species (ROS) in endothelial cells (ECs). Furthermore, Rotigotine inhibited the expressions of both vascular cellular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) in HUVECs and had anti-inflammatory efficacy in ox-LDL-induced cells by inhibiting the expressions of pro-inflammatory cytokines. Notably, Rotigotine inhibits the activation of nuclear factor-kappaB (NF-κB) by preventing nuclear translocation of NF-κB p65 and reducing the luciferase activity of NF-κB reporter. We, therefore, conclude that these effects of Rotigotine on HUVECs suggest that it may play a therapeutic role in cardiovascular diseases.

Bone mesenchymal stem cells (BMSCs) have been used for the treatment of acute uterine injury (AUI)-induced intrauterine adhesion (IUA) via interacting with the endothelial progenitor cells (EPCs), and BMSCs-derived exosomes (BMSCs-exo) may be the key regulators for this process. However, the underlying mechanisms have not been studied. Based on the existed literatures, lipopolysaccharide (LPS) was used to induce AUI in mice models and EPCs to mimic the realistic pathogenesis of IUA in vivo and in vitro. Our data suggested that LPS induced apoptotic and pyroptotic cell death in mice uterine horn tissues and EPCs, and the clinical data supported that increased levels of pro-inflammatory cytokines IL-18 and IL-1β were also observed in IUA patients’ serum samples, and silencing of NLRP3 rescued cell viability in LPS-treated EPCs. Next, the LPS-treated EPCs were respectively co-cultured with BMSCs in the Transwell system and BMSCs-exo, and the results hinted that both BMSCs and BMSCs-exo reversed the promoting effects of LPS treatment-induced cell death in EPCs. Then, we screened out miR-223-3p, as the upstream regulator for NLRP3, was enriched in BMSCs-exo, and BMSCs-exo inactivated NLRP3-mediated cell pyroptosis in EPCs via delivering miR-223-3p. Interestingly, upregulation of miR-223-3p attenuated LPS-induced cell death in EPCs. Collectively, we concluded that BMSCs-exo upregulated miR-223-3p to degrade NLRP3 in EPCs, which further reversed the cytotoxic effects of LPS treatment on EPCs to ameliorate LPS-induced AUI.

Hepatitis B virus (HBV) middle surface antigen (MHBs) mutation or deletion occurs in patients with chronic HBV infection. However, the functional role of MHBs in HBV infection is still an enigma. Here, we reported that 7.33% (11/150) isolates of CHB patients had MHBs start codon mutations compared with 0.00% (0/146) in acute hepatitis B (AHB) patients. Interestingly, MHBs loss accounted for 11.88% (126/1061) isolates from NCBI GenBank, compared with 0.09% (1/1061) and 0.00% (0/1061) for HBV large surface antigen (LHBs) loss and HBV small surface antigen (SHBs) loss, respectively. One persistent HBV clone of genotype B (B56, MHBs loss) from a CHB patient was hydrodynamically injected into BALB/c mice. B56 persisted for >70 weeks in BALB/c mice, whereas B56 with restored MHBs (B56M+) was quickly cleared within 28 days. Serum cytokine assays demonstrated that CXCL1, CXCL2, IL-6 and IL-33 were significantly increased during rapid HBV clearance in B56M+ mice. Furthermore, the enhancers and promoters of B56 were proved to be required for B56 persistence in mice. Ablating MHBs expression improved the persistence of a new clone (HBV1.3, genotype B) which was recreated by using enhancers and promoters of B56. These data demonstrated that MHBs deletion can promote the persistence of specific HBV variants in a hydrodynamic mouse model. MHBs re-expression restored a rapid clearance of HBV, which was accompanied by cytokine responses including the elevation of CXCL1, CXCL2, IL-6 and IL-33.

Macrophage infiltration is a hallmark pathological change observed in early stage myocardial ischemia/reperfusion (MI/R) injury and one of the main causes of myocardial damage. Here, we investigated the effects of p-Coumaric acid (p-CA) on macrophage polarization following MI/R injury and its mechanisms. In vitro, p-CA decreases the expression of LPS/IFN-γ-induced M1 macrophage markers (TNF-α, IL-6, iNOS and CCL2) and increases IL-4-induced M2 macrophage markers (IL-10, CD206, Arg1 and Mrc) in mouse bone marrow-derived macrophages (BMDMs). Additionally, p-CA elevated indoleamine 2, 3-dioxygenase (IDO) protein expression levels, M2 macrophage polarization and M2 macrophage markers through IL-4. In contrast, repression of IDO attenuated p-CA functions regulating BMDMs through IL-4. In vivo, IDO expression was downregulated in mouse hearts subjected to MI/R injury. Treatment of p-CA increased IDO expression in the hearts of MI/R mice. Functionally, p-CA decreases M1 macrophage markers, the number of M1 macrophages and inflammation around heart tissue following MI/R injury. Importantly, p-CA reduces cardiomyocyte apoptosis caused by MI/R. Altogether, our study identified that p-CA modulates macrophage polarization by promoting IDO expression and that p-CA attenuates macrophage-mediated inflammation following MI/R by promoting M2 macrophage polarization through IDO.

Endoplasmic reticulum stress (ERS)-mediated autophagy is indispensable for modulation of replication and pathogenesis of numerous mammalian viruses. We have previously shown that classical swine fever virus (CSFV) infection induces ERS-mediated autophagy for maintaining viral replication both in vivo and in vitro, however, the underlying mechanism remains unclarified. Here we found that CSFV infection activates the PERK pathway-dependent complete autophagy to promote viral replication in cultured PK-15 and 3D4/2 cells. Likewise, our results also suggested the essential roles of the IRE1/GRP78-mediated complete autophagy in CSFV replication in vitro. Furthermore, we suggested that CSFV infection induces activation of the PERK and IRE1 pathway for potential immunoregulation via promoting transcription of proinflammatory cytokine (IFN-γ and TNF-α) genes in the CSFV-infected cells. Finally, pharmacological treatment of PERK- or IRE1-pathway regulators, and the corresponding SiRNAs interventions did not affect the viabilities of the cells, excluding the potential interference elicited by altered cell viabilities. Taken together, our results suggest that CSFV infection induces complete autophagy through activation of the PERK and IRE1 pathway to facilitate viral replication in cultured cells, and modulation of proinflammatory cytokines may be a potential mechanism involved in this event. Our findings will open new horizons for molecular mechanisms of sustainable replication and pathogenesis of CSFV, and lay a theoretical foundation for the development of ERS-autophagy-targeting therapeutic strategies for clinical control of CSF.

Severe viral pneumonia caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is characterized by a hyperinflammatory state typified by elevated circulating pro-inflammatory cytokines, frequently leading to potentially lethal vascular complications including thromboembolism, disseminated intracellular coagulopathy and vasculitis. Though endothelial infection and subsequent endothelial damage have been described in patients with fatal COVID-19, the mechanism by which this occurs remains elusive, particularly given that, under naïve conditions, pulmonary endothelial cells demonstrate minimal cell surface expression of the SARS-CoV-2 binding receptor ACE2. Herein we describe SARS-CoV-2 infection of the pulmonary endothelium in postmortem lung samples from individuals who died of COVID-19, demonstrating both heterogeneous ACE2 expression and endothelial damage. In primary endothelial cell cultures, we show that SARS-CoV-2 infection is dependent on the induction of ACE2 protein expression and that this process is facilitated by type 1 interferon-alpha (IFNα) or -beta(β)—two of the main anti-viral cytokines induced in severe SARS-CoV-2 infection—but not significantly by other cytokines (including interleukin 6 and interferon γ/λ). Our findings suggest that the stereotypical anti-viral interferon response may paradoxically facilitate the propagation of COVID-19 from the respiratory epithelium to the vasculature, raising concerns regarding the use of exogenous IFNα/β in the treatment of patients with COVID-19. Supplementary Information The online version contains supplementary material available at 10.1007/s10456-021-09823-4.

IL-23 is a cytokine member of the IL-12 superfamily. These heterodimeric cytokines offer broad immune regulatory activity with potential effector function in inflammatory arthritis. IL-23 is a pro-inflammatory cytokine secreted by dendritic cells and macrophages. It plays a key role in both innate and adaptive immunity. By promoting and maintaining T cell differentiation into Th17 T cells, IL-23 is a key player in the pathogenesis of rheumatic diseases. Data from pre-clinical IL-23 knockout models show the major importance of IL-23 in development of arthritis. The induction and maintenance of type 17 cells, which secrete IL-17A and other pro-inflammatory cytokines, contributes to local synovial inflammation and skin inflammation in PsA, and perhaps in RA. Commensurate with this, therapeutic strategies targeting IL-23 have proven efficient in PsA in several studies, albeit not yet in RA.

Rheumatoid arthritis (RA) is a chronic, multi-factorial disease characterized by Synovial hyperplasia, chronic inflammation, and autoimmune reaction. Fascin1 overexpression has been implicated in cancer, immune, and inflammatory diseases. However, the relationship between Fascin1 and rheumatoid arthritis (RA) has not yet been determined. We investigated whether Fascin1 could modulate pro-inflammatory cytokine secretion and the proliferation, apoptosis, and invasion/migration of fibroblast-like synoviocytes (RA-FLSs). Fascin 1 was suppressed with a short interfering (si)RNA approach. Functional analysis contained MTT assay, flow cytometry,Transwell™ assays, wound healing, Quantitative polymerase chain reaction and western blotting were used to detect cell proliferation,apoptosis ratio, invasion/ migration, the mRNA and protein expression of the realted markers, respectively. Overexpression of fascin1 was observed in RA-FLSs group compared with control group. Fascin1 expression positively correlated with changes in the expression of RA disease activity markers (RF, CRP, and DAB28, respectively). We also observed a significant positive correlation between Fascin1 and STAT3 mRNA levels in RA- FLSs.Fascin1 silencing attenuated the expression of pro-inflammatory cytokines; reduced FLS proliferation in vitro; and increased apoptosis ratio and bax, cleaved PARP, and caspase-3 expression. si- Fascin1 transfection delayed RA-FLS invasion/migration and reversed the epithelial- mesenchymal transition. These data suggest that Fascin1 exerts positive effects on the proliferation, cell cycle, and invasion/migration of RA-FLSs by activating signal transducer and activator of transcription 3 signaling.After all, Fascin1 contributed to RA development.

Background Abnormalities of inflammation have been implicated in the pathophysiology of depression and suicide, based on observations of increased levels of proinflammatory cytokines in the serum of people who were depressed and died by suicide. More recently, abnormalities in cytokines and innate immunity receptors such as toll-like receptors have also been observed in the postmortem brains of people who were depressed and died by suicide. In addition to toll-like receptors, another subfamily of innate immunity receptors known as NOD-like receptors containing pyrin (NLRPs) are the most widely present NOD-like receptors in the central nervous system. NLRPs also form inflammasomes that play an important role in brain function. We studied the role of NLRPs in depression and suicide. Methods We determined the protein and mRNA expression of NLRP1, NLRP3 and NLRP6 and the components of their inflammasomes (i.e., adaptor molecule apoptosis-associated speck-like protein [ASC], caspase1, caspase3, interleukin [IL]-1β and IL-18) postmortem in the prefrontal cortex of people who were depressed and died by suicide, and in healthy controls. We determined mRNA levels using quantitative polymerase chain reaction, and we determined protein expression using Western blot immunolabelling. Results We found that the protein and mRNA expression levels of NLRP1, NLRP3, NLRP6, caspase3 and ASC were significantly increased in people who were depressed and died by suicide compared to healthy controls. Limitations Some people who were depressed and died by suicide were taking antidepressant medication at the time of their death. Conclusion Similar to toll-like receptors, NLRP and its inflammasomes were upregulated in people who were depressed and died by suicide compared to healthy controls. Innate immunity receptors in general - and NLRPs and inflammasomes in particular - may play an important role in the pathophysiology of depression and suicide.

Short-chain fatty acids (SCFAs) are a product of intestinal bacteria metabolism. Our previous study has found that intestinal bacteria in patients with Alzheimer’s disease (AD) can promote the activation of NLRP3 inflammasome and mediate neuroinflammation. In this study, we mainly explored the regulation of intestinal microenvironmental immunity by intestinal bacterial metabolite SCFAs and the mechanism of NLRP3 activation. First, wild-type (WT) and APP/PS1 mice were intervened with SCFAs. As a result, the proportion of double-negative T cells (CD3+CD4−CD8−, DNTs) in the intestine was increased, SCFAs could promote the expression of intestinal NLRP3 and inflammatory factors (IL-18, IL-6 and TNF-α). Moreover, SCAFs could also promote the level of inflammatory factors in the cerebrospinal fluid (CSF) of mice and aggravate the cognitive impairment in AD mice. CD3+ T cells isolated from the spleen were pre-treated with SCFAs, followed by detection of the proportion of DNTs. Consequently, SCFAs could promote the formation of DNTs, activate OX40 signal and simultaneously up-regulate the protein expression of Bcl-2, Bcl-xl and Survivin. Knockdown of OX40 could inhibit SCFAs-induced differentiation of DNTs. The co-culture of DNTs and intestinal macrophages showed that DNTs could activate Fas/FasL-TNF-α signal and induce the activation of NLRP3 inflammasome. In AD mouse models, treatment with Fas and TNFR1 inhibitors could significantly inhibit SCFAs-induced NLRP3 activation and inflammatory factors, while attenuate the inflammatory response in the brain tissue of mice and improve the cognitive ability of mice, however, without significant effect on the level of DNTs. The present study showed that SCFAs can promote the formation of DNTs through OX40. DNTs could induce the activation of NLRP3 inflammasome and the release of inflammatory factors in macrophages through Fas/FasL-TNF-α signals, thereby increasing the level of inflammatory factors in the central nervous system. When Fas and TNFR1 were inhibited by suppressing the functions of DNTs and macrophages, the activation of NLRP3 was inhibited. DNTs are affected by SCFAs, which is a new mechanism of neuroinflammation in AD.

An oral antiviral against SARS-CoV-2 that also attenuates inflammatory instigators of severe COVID-19 is not available to date. Herein, we show that the apoA-I mimetic peptide 4 F inhibits Spike mediated viral entry and has antiviral activity against SARS-CoV-2 in human lung epithelial Calu3 and Vero-E6 cells. In SARS-CoV-2 infected Calu3 cells, 4 F upregulated inducers of the interferon pathway such as MX-1 and Heme oxygenase 1 (HO-1) and downregulated mitochondrial reactive oxygen species (mito-ROS) and CD147, a host protein that mediates viral entry. 4 F also reduced associated cellular apoptosis and secretion of IL-6 in both SARS-CoV-2 infected Vero-E6 and Calu3 cells. Thus, 4 F attenuates in vitro SARS-CoV-2 replication, associated apoptosis in epithelial cells and secretion of IL-6, a major cytokine related to COVID-19 morbidity. Given established safety of 4 F in humans, clinical studies are warranted to establish 4 F as therapy for COVID-19., GRAPHICAL ABSTRACT

Several cytokines involved in inflammatory pathologies signal via the Janus kinase-signal transducer and activator of transcription pathway. Four JAKs are known: JAK1, JAK2, JAK3 and TYK2. The specific activation of JAKs and STATs determines the biological effects of each cytokine. JAK1 is involved in the signalling of ‘γc’ receptor cytokines (IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21), pro-inflammatory cytokines including IL-6, as well as IFN. The critical position of JAK1 downstream of these cytokines suggests that JAK1-selective inhibitors are comparable to non-selective ones, without the unwanted consequences of JAK2- or JAK3-blockade. JAK inhibition has led to a better understanding of the biology of synovial inflammation and bone homeostasis. Moreover, the efficacy of non-selective JAK inhibitors and novel JAK1-selective drugs in RA supports a role for JAK1 in its pathogenesis. JAK1-selective drugs are also showing promise in axial spondyloarthritis, suggesting that they may target additional regulatory pathways that impact cytokines such as TNF and IL-17A, which do not use JAKs. Additionally, evidence now supports a JAK1 predominance in the signalling of IL-6 and oncostatin M, and indirectly, of TNF in synovial fibroblasts, macrophages and endothelial cells. Notably, bone homeostasis is also dependent on cytokines relying on JAK1 signalling to promote receptor activator of NF-κB ligand expression in osteoblasts and T cells, contributing to osteoclastogenesis. Here, the contribution of JAK1 over other kinases is unclear. While beneficial effects of JAK inhibitors on bone erosion are supported by preclinical and clinical data, effects on new bone formation in axial spondyloarthritis requires additional study. CME: This supplement is CME Accredited. To receive a CME certificate of participation, you should: Read all the papers in the supplement Register or log in at www.paradigmmc.com/962 to complete and submit the post activity assessment. You must answer 70% of the questions correctly to earn credit. You will have unlimited opportunities to successfully complete the assessment. You will receive a maximum of 7.0 AMA PRA Category 1 Credits(TM) upon successful completion of the assessment.

Background: In vitro stimulation of whole blood or isolated blood cells with specific antigens is used for several purposes. Immediately following incubation with antigens, samples have to be centrifuged to stop the reactions by remaining cells and the supernatant refrigerated or analysed directly to preserve the analytes of interest, which makes samples difficult to prepare outside laboratories. We have tested whether spotting whole blood on filter paper after activation can be used in one of the tests for Mycobacterium tuberculosis infection (MTI), the QuantiFERON®-TB Gold In Tube test (QFT), where the spotting technique can make it suitable for use in locations without facilities like a centrifuge and a refrigerator. Materials and methods: Samples from 22 individuals undergoing screening for MTI and 10 healthy controls were incubated, centrifuged and IFN-γ measured by Enzyme-linked immunosorbent assay (ELISA), as described in the kit insert. In parallel, activated blood was spotted on filter paper (Schleicher & Schuell) and dried. The dried blood spot samples were analysed for 21 inflammatory markers with an in-house assay based on Luminex technology. Results: Our multiplex measurements of inflammatory markers in samples from suspected MTI patients confirmed the IFN-γ findings in the QFT. IL-2, GM-CSF, IL-5, and IL-1β were also found as useful markers for MTI. We were not able to distinguish between active tuberculosis and latent MTI. Conclusion: Applying blood on filter paper after incubation makes in vitro stimulation tests feasible in locations where heat and electricity is unavailable.

Dried blood spot samples (DBSS) on filter paper offer several advantages compared to conventional serum/plasma samples: they do not require any phlebotomy or separation of blood by centrifugation; they are less invasive; they allow sample stability and shipment at room temperature; and they pose a negligible risk of infection with blood-borne viruses, such as HIV, HBV and HCV, to those who handle them. Therefore dried blood spot samples (DBSS) on filter paper can be a quick, convenient and inexpensive means of obtaining blood samples for biomarker discovery, disease screening, diagnosis and treatment monitoring in non-hospitalized, public health settings. In this study, we investigated for the first time the potential application of dried blood spot samples (DBSS) in protein expression profiling using antibody array technology. First, optimal conditions for array assay performance using dried blood spot samples (DBSS) was established, including sample elution buffer, elution time, elution temperature and assay blocking buffer. Second, we analyzed dried blood spot samples (DBSS) using three distinct antibody array platforms, including sandwich-based antibody arrays, quantitative antibody arrays and biotin-label-based antibody arrays. In comparison with paired serum samples, detection of circulating proteins in dried blood spot samples (DBSS) correlated well for both low- and high-abundance proteins on all three antibody array platforms. In conclusion, our study strongly indicates the novel application of multiplex antibody array platforms to analyze dried blood spot samples (DBSS) on filter paper represents a viable, cost-effective method for protein profiling, biomarker discovery and disease screening in a large, population-based survey.