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The involvement of toll-like receptor 9 (TLR9), a receptor for bacterial DNA, in septic cardiac depression has not been clarified in vivo. Thus, the aim of the study was to test possible TLR9 inhibitors (H154-thioate, IRS954-thioate, and chloroquine) for their ability to protect the cardiovascular system in a murine model of CpG oligodeoxynucleotide- (ODN-) dependent systemic inflammation. Sepsis was induced by i.p. application of the TLR9 agonist 1668-thioate in C57BL/6 wild type (WT) and TLR9-deficient (TLR9-D) mice. Thirty minutes after stimulation TLR9 antagonists were applied i.v. Survival was monitored up to 18 h after stimulation. Cardiac mRNA expression of inflammatory mediators was analyzed 2 h and 6 h after stimulation with 1668-thioate and hemodynamic parameters were monitored at the later time point. Stimulation with 1668-thioate induced a severe sepsis-like state with significant drop of body temperature and significantly increased mortality in WT animals. Additionally, there was a time-dependent increase of inflammatory mediators in the heart accompanied by development of septic heart failure. These effects were not observed in TLR9-D mice. Inhibition of TLR9 by the suppressive ODN H154-thioate significantly ameliorated cardiac inflammation, preserved cardiac function, and improved survival. This suppressive ODN was the most efficient inhibitor of the tested substances. [ABSTRACT FROM AUTHOR]

In this work the role of trypsin in revealing epidermal cell surface antigens, with the use of immunological markers, was investigated. Two monoclonal antibodies (MCA) were used, the first: D47, belongs to the first cluster of differentiation and recognizes a membrane antigen of human thymocytes; the second HLA-ABC-m3, is an anti-HLA-B27 MCA. Preliminary treatment with various concentrations of trypsin was performed on frozen skin sections and followed by indirect immunofluorescence. D47 reacted with epidermal dentritic cells only after trypsin pretreatment of skin sections. In addition a mild preliminary trypsinization was shown to increase in situ immunoreactivity of MCA HLA-ABC-m3 with epidermal cells. Best results were obtained when trypsin concentrations ranging from 2.5 to 5 μg/ml were applied for 10 mm at 37°C. Preliminary trypsinization may be of interest for a better exposure of some surface antigens to immunohistochemical markers. [ABSTRACT FROM AUTHOR]

Chronic hepatitis B virus (HBV) infection is a global issue and can lead to cirrhosis and hepatocellular carcinoma (HCC). Hepatitis B surface antigen (HBsAg) is an important marker of HBV infection and HBsAg quantification could be a useful tool in clinical practice. This systematic literature review aimed to explore the association between HBsAg titres and long-term disease outcomes and evaluate the relationship between HBsAg titres, or changes in HBsAg titres, and clinical and treatment characteristics in patients with chronic HBV infection. Structured searches were performed in MEDLINE and Embase (January 2000 to 31 March 2023). Eighty-two studies were included, comprising 51% retrospective cohort studies, mostly conducted in Asia (85%). HBsAg levels were shown to predict the long-term development of cirrhosis and HCC in patients who were untreated prior to and during follow-up; however, these data were inconclusive in mixed and treated populations. HBsAg titres were significantly associated with various virological markers including serum HBV DNA, HBcrAg, HBeAg, HBV RNA levels, intrahepatic covalently closed circular DNA (cccDNA) and intrahepatic HBsAg expression. HBsAg titres generally declined over time; this decline was more pronounced in early (HBeAg-positive) than later disease phases (HBeAg-negative). Higher decline in HBsAg levels was consistently associated with subsequent HBsAg seroclearance and a greater decline in total intrahepatic HBV DNA and cccDNA levels. In conclusion, this review showed that HBsAg levels and rates of decline could inform assessment, management and prediction of outcomes in chronic HBV infection. Further studies in broader, more diverse populations and treated patients are needed., (© 2024 GSK plc. Journal of Viral Hepatitis published by John Wiley & Sons Ltd.)

Introduction: Data on the economic burden of chronic hepatitis B infection in Japan are lacking. This study investigated healthcare resource utilization and costs of chronic hepatitis B infection and liver complications in Japan., Methods: This non-interventional study used the Medical Data Vision database. For the first analysis, a population with prevalent chronic hepatitis B infection and absence of liver complications was identified and further stratified by nucleos(t)ide analog treatment history. In the second analysis, patients with prevalent chronic hepatitis B infection and incident liver complications were identified. Patients were followed for 1 year in the first analysis and 2 years in the second analysis. Numbers of all-cause outpatient, inpatient, emergency hospitalizations, medication use, and associated costs per person-year were described across patients without/with nucleos(t)ide analog treatment and in those without/with liver complications., Results: For patients with chronic hepatitis B infection, 75,967 had no liver complications while 17,678 patients had liver complications. All-cause outpatient visits were the largest contributor to healthcare resource utilization and costs, for patients without and with liver complications, and were numerically higher for patients on nucleos(t)ide analog than not. Patients with liver complications had numerically higher all-cause healthcare resource utilization and total costs than patients without complications., Conclusions: Japan has a high economic burden of chronic hepatitis B infection, particularly in patients with liver complications. Optimizing treatment to prevent complications may reduce this burden., (© 2024 The Author(s). Published by S. Karger AG, Basel.)

Genetically-encoded biosensors based on a single fluorescent protein are widely used to visualize analyte levels or enzymatic activities in cells, though usually to monitor relative changes rather than absolute values. We report photochromism-enabled absolute quantification (PEAQ) biosensing, a method that leverages the photochromic properties of biosensors to provide an absolute measure of the analyte concentration or activity. We develop proof-of-concept photochromic variants of the popular GCaMP family of Ca 2+ biosensors, and show that these can be used to resolve dynamic changes in the absolute Ca 2+ concentration in live cells. We also develop intermittent quantification, a technique that combines absolute aquisitions with fast fluorescence acquisitions to deliver fast but fully quantitative measurements. We also show how the photochromism-based measurements can be expanded to situations where the absolute illumination intensities are unknown. In principle, PEAQ biosensing can be applied to other biosensors with photochromic properties, thereby expanding the possibilities for fully quantitative measurements in complex and dynamic systems., (© 2022. The Author(s).)

Composed of a reversibly photoswitchable unit allosterically linked to a sensing module, reversibly photoswitchable sensors (rs-sensors) represent a new and attractive strategy to quantitatively read-out analyte concentrations. However, their kinetic response to illumination is complex, and much attention is required from the design to the application steps. Here, we exploit a generic kinetic model of rs-sensors which enables us to point to key thermokinetic parameters, such as dissociation constants and kinetic rates for exchange toward the analyte, and cross-sections for photoswitching. The application of the model allows to evaluate the robustness of the analyzed parameters and to introduce a methodology for their reliable use. Model and methodology have been experimentally tested on a newly reported calcium sensor based on a reversibly photoswitchable green fluorescent protein allosterically linked to a calcium-sensing module integrating calmodulin and an RS20 peptide.

Fluorophores with dynamic or controllable fluorescence emission have become essential tools for advanced imaging, such as superresolution imaging. These applications have driven the continuing development of photoactivatable or photoconvertible labels, including genetically encoded fluorescent proteins. These new probes work well but require the introduction of new labels that may interfere with the proper functioning of existing constructs and therefore require extensive functional characterization. In this work we show that the widely used red fluorescent protein mCherry can be brought to a purely chemically induced blue-fluorescent state by incubation with β-mercaptoethanol (βME). The molecules can be recovered to the red fluorescent state by washing out the βME or through irradiation with violet light, with up to 80% total recovery. We show that this can be used to perform single-molecule localization microscopy (SMLM) on cells expressing mCherry, which renders this approach applicable to a very wide range of existing constructs. We performed a detailed investigation of the mechanism underlying these dynamics, using X-ray crystallography, NMR spectroscopy, and ab initio quantum-mechanical calculations. We find that the βME-induced fluorescence quenching of mCherry occurs both via the direct addition of βME to the chromophore and through βME-mediated reduction of the chromophore. These results not only offer a strategy to expand SMLM imaging to a broad range of available biological models, but also present unique insights into the chemistry and functioning of a highly important class of fluorophores., Competing Interests: The authors declare no conflict of interest.

"Smart fluorophores", such as reversibly switchable fluorescent proteins, are crucial for advanced fluorescence imaging. However, only a limited number of such labels is available, and many display reduced biological performance compared to more classical variants. We present the development of robustly photoswitchable variants of enhanced green fluorescent protein (EGFP), named rsGreens, that display up to 30-fold higher fluorescence in E. coli colonies grown at 37 °C and more than 4-fold higher fluorescence when expressed in HEK293T cells compared to their ancestor protein rsEGFP. This enhancement is not due to an intrinsic increase in the fluorescence brightness of the probes, but rather due to enhanced expression levels that allow many more probe molecules to be functional at any given time. We developed rsGreens displaying a range of photoswitching kinetics and show how these can be used for multimodal diffraction-unlimited fluorescence imaging such as pcSOFI and RESOLFT, achieving a spatial resolution of ∼70 nm. By determining the first ever crystal structures of a negative reversibly switchable FP derived from Aequorea victoria in both the "on"- and "off"-conformation we were able to confirm the presence of a cis-trans isomerization and provide further insights into the mechanisms underlying the photochromism. Our work demonstrates that genetically encoded "smart fluorophores" can be readily optimized for biological performance and provides a practical strategy for developing maturation- and stability-enhanced photochromic fluorescent proteins.

Background: Rhinovirus infections are the dominant cause of asthma exacerbations, and deficient virus induction of IFN-α/β/λ in asthmatic patients is important in asthma exacerbation pathogenesis. Mechanisms causing this interferon deficiency in asthmatic patients are unknown., Objective: We sought to investigate the expression of suppressor of cytokine signaling (SOCS) 1 in tissues from asthmatic patients and its possible role in impaired virus-induced interferon induction in these patients., Methods: We assessed SOCS1 mRNA and protein levels in vitro, bronchial biopsy specimens, and mice. The role of SOCS1 was inferred by proof-of-concept studies using overexpression with reporter genes and SOCS1-deficient mice. A nuclear role of SOCS1 was shown by using bronchial biopsy staining, overexpression of mutant SOCS1 constructs, and confocal microscopy. SOCS1 levels were also correlated with asthma-related clinical outcomes., Results: We report induction of SOCS1 in bronchial epithelial cells (BECs) by asthma exacerbation-related cytokines and by rhinovirus infection in vitro. We found that SOCS1 was increased in vivo in bronchial epithelium and related to asthma severity. SOCS1 expression was also increased in primary BECs from asthmatic patients ex vivo and was related to interferon deficiency and increased viral replication. In primary human epithelium, mouse lung macrophages, and SOCS1-deficient mice, SOCS1 suppressed rhinovirus induction of interferons. Suppression of virus-induced interferon levels was dependent on SOCS1 nuclear translocation but independent of proteasomal degradation of transcription factors. Nuclear SOCS1 levels were also increased in BECs from asthmatic patients., Conclusion: We describe a novel mechanism explaining interferon deficiency in asthmatic patients through a novel nuclear function of SOCS1 and identify SOCS1 as an important therapeutic target for asthma exacerbations., (Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.)

Background: Defective rhinovirus (RV)-induced interferon (IFN)-β and IFN-λ production and increased RV replication have been reported in primary human bronchial epithelial cells (HBECs) from subjects with asthma. How universal this defect is in asthma is unknown. Additionally, the IFN subtypes induced by RV infection in primary HBECs have not been comprehensively investigated., Objective: To study RV induction of IFN-α, IFN-β and IFN-λ and RV replication in HBECs from subjects with atopic asthma and healthy controls., Methods: HBECs were obtained from subjects with asthma and healthy controls and infected with RV16 and RV1B, and cells and supernatants harvested at 8, 24 and 48h. IFN proteins were analysed by ELISA and IFN mRNA and viral RNA expression by quantitative PCR. Virus release was assessed in cell supernatants., Results: IFN-β and IFN-λ were the only IFNs induced by RV in HBECs and IFN-λ protein induction was substantially greater than IFN-β. Induction of IFN-λ1 mRNA by RV16 at 48h was significantly greater in HBECs from subjects with asthma; otherwise there were no significant differences between subjects with asthma and controls in RV replication, or in induction of type I or III IFN protein or mRNA., Conclusions: IFN-λ and, to a lesser degree, IFN-β are the major IFN subtypes induced by RV infection of HBECs. Neither defective IFN induction by RV nor increased RV replication was observed in the HBECs from subjects with well controlled asthma reported in this study.

Defective Rhinovirus induced interferon-β and interferon-λ production has been reported in bronchial epithelial cells from asthmatics but the mechanisms of defective interferon induction in asthma are unknown. Virus infection can induce interferon through Toll like Receptors (TLR)3, TLR7 and TLR8. The role of these TLRs in interferon induction in asthma is unclear. This objective of this study was to measure the type I and III interferon response to TLR in bronchial epithelial cells and peripheral blood cells from atopic asthmatics and non-atopic non-asthmatics. Bronchial epithelial cells and peripheral blood mononuclear cells from atopic asthmatic and non-atopic non-asthmatic subjects were stimulated with agonists to TLR3, TLR4 & TLRs7-9 and type I and III interferon and pro-inflammatory cytokine, interleukin(IL)-6 and IL-8, responses assessed. mRNA expression was analysed by qPCR. Interferon proteins were analysed by ELISA. Pro-inflammatory cytokines were induced by each TLR ligand in both cell types. Ligands to TLR3 and TLR7/8, but not other TLRs, induced interferon-β and interferon-λ in bronchial epithelial cells. The ligand to TLR7/8, but not those to other TLRs, induced only type I interferons in peripheral blood mononuclear cells. No difference was observed in TLR induced interferon or pro-inflammatory cytokine production between asthmatic and non-asthmatic subjects from either cell type. TLR3 and TLR7/8,, stimulation induced interferon in bronchial epithelial cells and peripheral blood mononuclear cells. Interferon induction to TLR agonists was not observed to be different in asthmatics and non-asthmatics.

Rationale: Respiratory virus infections are associated with chronic obstructive pulmonary disease (COPD) exacerbations, but a causative relationship has not been proven. Studies of naturally occurring exacerbations are difficult and the mechanisms linking virus infection to exacerbations are poorly understood. We hypothesized that experimental rhinovirus infection in subjects with COPD would reproduce the features of naturally occurring COPD exacerbations and is a valid model of COPD exacerbations., Objectives: To evaluate experimental rhinovirus infection as a model of COPD exacerbation and to investigate the mechanisms of virus-induced exacerbations., Methods: We used experimental rhinovirus infection in 13 subjects with COPD and 13 nonobstructed control subjects to investigate clinical, physiologic, pathologic, and antiviral responses and relationships between virus load and these outcomes., Measurements and Main Results: Clinical data; inflammatory mediators in blood, sputum, and bronchoalveolar lavage; and viral load in nasal lavage, sputum, and bronchoalveolar lavage were measured at baseline and after infection with rhinovirus 16. After rhinovirus infection subjects with COPD developed lower respiratory symptoms, airflow obstruction, and systemic and airway inflammation that were greater and more prolonged compared with the control group. Neutrophil markers in sputum related to clinical outcomes and virus load correlated with inflammatory markers. Virus load was higher and IFN production by bronchoalveolar lavage cells was impaired in the subjects with COPD., Conclusions: We have developed a new model of COPD exacerbation that strongly supports a causal relationship between rhinovirus infection and COPD exacerbations. Impaired IFN production and neutrophilic inflammation may be important mechanisms in virus-induced COPD exacerbations.

Langerhans cells (LC) are CD4-positive antigen-presenting cells within the human epidermis and thus potentially may be infected by the causative agent of AIDS (acquired immunodeficiency syndrome), called HIV (human immunodeficiency virus). Because CD4 antigens have been demonstrated to be decreased in HIV-infected lymphocytes, we wondered whether some immunologic markers of LC might be modified in seropositive patients. For this purpose, LC, obtained from clinically unaffected skin of ARC (AIDS-related complex) and AIDS patients, were subjected to anti-CD4 (OKT4) and anti-HLA class II monoclonal antibodies (anti-DR: BL1 and anti-DQ). The density of the antigenic sites/LC recognized by the antibodies was evaluated by employing the electron microscopic immunogold labeling procedure. The density of CD4 molecules/cell measured in LC of ARC and AIDS patients by direct count of gold particles bound to the cell membrane was found to be dramatically decreased among AIDS LC, whereas a small subset of ARC LC strongly expressed this antigen. In contrast, the density of HLA class II (DR and DQ) antigenic determinants was found unchanged in comparison with that of healthy donors. In addition to the quantitative modifications of the CD4 molecule expression by ARC and AIDS LC, the observation in these cell populations of several surface protrusions suggesting viral buds affords evidence that LC are a target for HIV.

In this work the reactivity of 16 monoclonal antibodies raised against different HLA class I specificities was tested with human skin of healthy donors of known HLA typing. By indirect immunofluorescence, six antibodies reacted strongly with keratinocytes carrying the corresponding alloantigens. The reactivity of 3 other antibodies which was weak or absent using indirect immunofluorescence, was enhanced by various amplification systems such as avidin-biotin-peroxidase method, biotin-streptavidin-fluorescein complex and especially preliminary trypsin treatment that revealed alloantigens masked in the epidermis. The immunostaining of 4 antibodies was negative regardless of the method used. Some of the antibodies we tested cross-reacted with cytoplasmic antigens of keratinocytes. This study has allowed to select a battery of monoclonal antibodies which can specifically detect alloantigens on keratinocytes and will be useful for the recognition the cell origin in allografting experiments.

Human keratinocytes may be grown in vitro into living epithelia devoid of Langerhans cells and MHC class II antigens. These epithelia have been shown to be usable as epidermal allografts in patients with dermal wounds, without any apparent sign of rejection in the 12-month follow-up study. To evidence a progressive replacement by recipient cells of the grafted keratinocytes, we employed anti-MHC class I antigen monoclonal antibodies directed against tissue specificities expressed by either donors or recipients. At 2 and 4 weeks after grafting, some small epithelial cell islets from the recipient phenotype were clearly identified among cells from a donor origin by indirect immunofluorescence. At 6 months, all keratinocytes present at the grafted areas were labelled by antibodies directed toward recipient specificities only. This replacement may be related to the fact that, when placed on such superficial dermal wounds, the allografts are likely colonized by epithelial cells proliferating from residual recipient dermal appendages.