Myeloid neddylation targets IRF7 and promotes host innate immunity against RNA viruses.

Neddylation, an important type of post-translational modification, has been implicated in innate and adapted immunity. But the role of neddylation in innate immune response against RNA viruses remains elusive. Here we report that neddylation promotes RNA virus-induced type I IFN production, especially IFN-α. More importantly, myeloid deficiency of UBA3 or NEDD8 renders mice less resistant to RNA virus infection. Neddylation is essential for RNA virus-triggered activation of Ifna gene promoters. Further exploration has revealed that mammalian IRF7undergoes neddylation, which is enhanced after RNA virus infection. Even though neddylation blockade does not hinder RNA virus-triggered IRF7 expression, IRF7 mutant defective in neddylation exhibits reduced ability to activate Ifna gene promoters. Neddylation blockade impedes RNA virus-induced IRF7 nuclear translocation without hindering its phosphorylation and dimerization with IRF3. By contrast, IRF7 mutant defective in neddylation shows enhanced dimerization with IRF5, an Ifna repressor when interacting with IRF7. In conclusion, our data demonstrate that myeloid neddylation contributes to host anti-viral innate immunity through targeting IRF7 and promoting its transcriptional activity.

Simultaneous Determination of Rifamycin Antibiotics and Their Active Metabolites in Human Plasma Using UHPLC-MS/MS to Evaluate Their Impact on Target Peak Concentrations: A Short Communication.

BACKGROUND: Standard and proper antituberculosis (anti-TB) treatment is essential for patients with TB, and rifamycin antibiotics are key components of anti-TB therapy. Therapeutic drug monitoring (TDM) of rifamycin antibiotics can shorten the time to response and complete treatment of TB. Notably, antimicrobial activities of the major active metabolites of rifamycin are similar to those of their parent compounds. Thus, a rapid and simple assay was developed for simultaneous determination of rifamycin antibiotics and their major active metabolites in plasma to evaluate their impact on target peak concentrations. Here, the authors have developed and validated a method for simultaneous determination of rifamycin antibiotics and their active metabolites in human plasma using ultrahigh-performance liquid chromatography tandem mass spectrometry. METHODS: Analytical validation of the assay was performed in accordance with the bioanalytical method validation guidance for industry described by the US Food and Drug Administration and the guidelines for bioanalytical method validation described by the European Medicines Agency. RESULTS: The drug concentration quantification method for rifamycin antibiotics, including rifampicin, rifabutin, and rifapentine, and their major active metabolites was validated. Significant differences in the proportions of active metabolites in rifamycin antibiotics may affect the redefinition of their effective concentration ranges in the plasma. The method developed herein is expected to redefine the ranges of "true" effective concentrations of rifamycin antibiotics (including parent compounds and their active metabolites). CONCLUSIONS: The validated method can be successfully applied for high-throughput analysis of rifamycin antibiotics and their active metabolites for TDM in patients receiving anti-TB treatment regimens containing these antibiotics. Proportions of active metabolites in rifamycin antibiotics markedly varied among individuals. Depending on the clinical indications of patients, the therapeutic ranges for rifamycin antibiotics may be redefined.

Correlation between immunoglobulin index level and disease severity and clinical manifestations in children with atopic dermatitis as a chronic inflammatory.

It was to analyze the clinical characteristics of atopic dermatitis (AD) in children and its relationship with immunoglobulin (IgG, IgA, IgM, IgE) levels. 400 children with AD in the dermatology clinic of Shengli Clinical Medical College of Fujian Medical University, Fujian Provincial Hospital(SCMC-FMU) were the study subjects, and 200 normal children were enrolled as the blank control. The clinical data of the included children were collected, and the serum immunoglobulin levels and other related indicators were measured. The results showed that the IgE level was higher observably in group A when we compared it with group A0, the IgE level was much higher in group B in comparison to group B0, group C showed higher IgE level C than in group C0, and it was the same case in groups D and D0(P < 0.05). The IgM level was decreased greatly in group C than that in normal children in group C0 (P < 0.05), but showed no visible difference among other groups (P > 0.05). The O-SCORAD scores of groups A, B, C, and D were significantly positively correlated with IgE level (P < 0.05). No great correlation was observed between skin dryness and IgE-specific allergen, IgG, IgA, IgM, and IgE levels in groups A - D (P > 0.05). Immunoglobulin deficiency was present in AD children compared with normal children, and IgE could be used as an efficient indicator to assess the severity of AD in children.

Anti-psoriasis activities of hydroxytyrosol on HaCaT cells under psoriatic inflammation in vitro.

BACKGROUND: Psoriasis is a prevalent chronic inflammatory dermatosis, which can significantly impact life quality of patients. The treatment of psoriasis is no cure and novel therapeutic options are urgently needed. Hydroxytyrosol (HT) possesses multiple biological activities, such as anti-inflammatory and anti-proliferation properties, suggesting its potential to counteract hallmarks of psoriasis. However, its role in the regulation of psoriasis remains unknown. OBJECTIVE: In the current study, we explored the anti-proliferative activity and anti-inflammatory responses of HT in psoriatic keratinocytes in vitro. METHODS: We used M5 cytokines cocktail, which includes tumor necrosis factor (TNF)-α, oncostatin-M, interleukin (IL)-17A, IL-1α, and IL-22, to simulate HaCaT cells to establish the cell model of psoriasis and explore the effects of HT on psoriasis in vitro. RESULTS: This study showed that HT exerted potent anti-inflammatory effect via influencing the expression of IL-6, IL-8, and TNF-α in M5-induced cell model of psoriasis. Moreover, it suppressed the expression of antimicrobial proteins in psoriatic keratinocytes. Additionally, it inhibited cell proliferation in psoriasis cell model. CONCLUSIONS: Altogether, our results suggested that HT has anti-psoriasis effects in vitro and HT may be a promising therapeutic agent in psoriasis treatment.

Population Pharmacokinetic Modeling of Lenvatinib in Chinese Patients With Advanced Hepatocellular Carcinoma Using Real-World Data.

Lenvatinib is a novel oral angiogenesis inhibitor approved in China for the treatment of unresectable hepatocellular carcinoma (HCC) without prior systemic treatment. We described the population pharmacokinetics of lenvatinib in Chinese patients with advanced HCC and explore the potential patient characteristics associated with lenvatinib pharmacokinetics using real-world data. A total of 266 samples, provided by 127 Chinese patients with advanced HCC, were analyzed by nonlinear mixed-effects modeling. Monte Carlo simulation was conducted to assess impact of covariates on the exposure to lenvatinib. The clearance of lenvatinib in Chinese patients with advanced HCC was 5.3 L/h, and alkaline phosphatase, total bilirubin, and sex were identified as important covariates associated with it. The clearance of Child-Pugh class B patients (4.82L/h) was significantly lower than that of Child-Pugh class A patients (5.53 L/h), and the systemic exposure increased with the increase of alkaline phosphatase and total bilirubin. There were sex differences in the pharmacokinetic characteristics of lenvatinib. The clearance of women was significantly lower than that of men (4.61 vs 5.6 L/h; P < .001), and the area under the plasma concentration-time curve of women was ≈20% higher than that of men. In this study, a population pharmacokinetic model of lenvatinib was established, which can be used to simulate clinical trials or various dosing scenarios. Our findings provide important new insights for optimizing the use of lenvatinib in patients with advanced HCC.

Development and evaluation of a new test kit for determination of immunosuppressants in blood by UHPLC-MS/MS.

In this study, we have developed and validated a new test kit that applied pure solvents instead of blood samples for determination of immunosuppressant drugs in blood by ultra high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). We have used commercially available quality control samples to verify this new test kit, and the results showed that the performance of our new kit was comparable with commercially available kits by immunoassays. The new test kit is pure solvent-based, which saves the cost and can be directly loaded without pre-treatment, thus shortening the detection time. The agreement between pure solvent-based kit and blood-based kit was also evaluated by Bland-Altman plot and two-tailed Student's T-test. For 97.08% of the 137 Tacrolimus clinical samples, the concentration difference between these two kits was laid within 20% of the mean concentration. The concentrations of 12 cyclosporine samples and 15 sirolimus samples obtained by both kits showed no significant difference evaluated by the two-tailed Student's T-test. These results further demonstrated the good agreement between these two types of test kits. Meanwhile, our pure solvent-based test kit could be stored stably for 14 weeks at -30 °C. Therefore, the newly developed pure solvent-based test kit can be used for detecting the whole blood immunosuppressant concentration and therapeutic drug monitoring of immunosuppressants.

Quantification of sorafenib, lenvatinib, and apatinib in human plasma for therapeutic drug monitoring by UPLC-MS/MS.

Sorafenib, lenvatinib, and apatinib, as multi-targeted tyrosine kinase inhibitors with anti-proliferative and anti-angiogenic effects, are widely used for systemic therapy in advanced hepatocellular carcinoma patients. Nevertheless, insufficient efficacy or adverse effects often appear due to the significant inter-individual variability of plasma concentration for these drugs. In order to carry out therapeutic drug monitoring of these drugs and then ensure the effectiveness and safety of the medical treatment, the first method allowing to quantify sorafenib, lenvatinib, and apatinib simultaneously in human plasma was developed in this study. The analysis was performed by UPLC-MS/MS system and the chromatographic separation was achieved on a C18 column using a gradient elution of water-acetonitrile in 3.5 min. This method presented satisfactory results in terms of specificity, precision (coefficient of variation of intra-day and inter-day:1.4-6.6 %), accuracy (92.6-105.4 %), matrix effects (96.9-107.2 %), extraction recovery (90.5-99.4 %), as well as stability in human plasma and even whole blood under certain conditions. This sensitive, rapid and simple method was successfully applied to the analysis of sorafenib, lenvatinib and apatinib for therapeutic drug monitoring in hepatocellular carcinoma patients, and it was expected to be applied to further study about clarifying the concentration- efficacy and concentration-toxic relationship of sorafenib, lenvatinib, and apatinib in hepatocellular carcinoma patients.

Comparison of sample preparation methods, validation of an UPLC-MS/MS procedure for the quantification of cyclosporine A in whole blood sample.

Current main methods for therapeutic drug monitoring (TDM) of cyclosporine A (CsA) are immunoassays and liquid chromatography tandem mass spectrometry. The sample pretreatment of these methods is mainly based on extraction of drug which is bound to erythrocytes by divalent heavy metal ions (such as zinc and copper). Although these methods are effective for whole blood drug extraction and measurement, the pollution of heavy metals in sample pretreatment process will have potential negative impact on environment and human health. To overcome the pollution problem, in this study we have developed and validated an UPLC-MS/MS method for CsA determination in whole blood samples using physical pretreatment method. According to the characteristics of erythrocytes, a series of physical pretreatment methods, including sonication, freeze-thaw and osmotic burst, have been developed and evaluated. The results showed that the osmotic burst method was an effective way for drug extraction from erythrocytes. The lower limit of quantitation for CsA was 25 ng/mL, the within-run and between-run coefficient of variations were both less than 11.6 %. The agreement of the UPLC-MS/MS methods using these two sample pretreatment was evaluated by Bland-Altman plot and the two-tailed Student's T-test. Comparison studies show that the effect of erythrocyte fragmentation by osmotic burst is similar to that of zinc sulfate method. The CsA measurement of 103 whole blood samples obtained by these two UPLC-MS/MS assays were no significant difference. These results demonstrate that the sample pretreatment by osmotic burst method is an eco-friendly and precise method for detecting the whole blood CsA concentration and therapeutic drug monitoring of CsA.

Rapid and highly sensitive quantification of the anti-tuberculosis agents isoniazid, ethambutol, pyrazinamide, rifampicin and rifabutin in human plasma by UPLC-MS/MS.

With the increased cases of multidrug- or rifampicin-resistant tuberculosis and co-infection with HIV globally, it is difficult to achieve ideal clinical responses because of poor drug absorption and drug-drug interactions. Herein, a bioanalytical UPLC-MS/MS method was developed and validated to quantify five anti-TB agents in human plasma samples for detecting blood drug concentrations to improve therapeutic effects. To overcome the matrix effects, stable isotope labeled analogue of each analyte was used for internal standardization. A simple single-step protein precipitation by acetonitrile was employed for the sample preparation, then the analytes including rifampicin, rifabutin, pyrazinamid, ethambutol, isoniazid and their isotope labeled internal standards (ILISs) were implemented on an HILIC silica column with a gradient mode. The linear range for each analyte was covering the peak drug concentration (Cmax) in the 20 times diluted plasma samples. The coefficient of variation of intra- and inter-day precision was less than 17.0 %, and the accuracy ranged between 91.5 and 110.0 %. The extraction recoveries of all agents were ≥90.2 %, and the matrix effects with internal standard-normalization for all agents were 97.1-110.0 %. The optimal blood sampling time was designed basing on the results of stability validation. This UPLC-MS/MS method with a run time of 3.5 min was successfully applied to routine therapeutic monitoring of the five anti-TB agents in patient plasma.

MLN4924 suppresses lipopolysaccharide-induced proinflammatory cytokine production in neutrophils in a dose-dependent manner.

Neddylation is a ubiquitination-like pathway. It has been reported that neddylation inhibition with the pharmacological agent MLN4924 potently uppresses lipopolysaccharide (LPS)-induced proinflammatory cytokine production, including tumor necrosis factor (TNF)-α and interleukin (IL)-6, by preventing the degradation of phosphorylated inhibitor of κB (p-IκB) in macrophages. However, whether neddylation serves a similar role in neutrophils remains unknown. In the present study MLN4924 treatment led to the accumulation of P-IκBα in neutrophils as well as the decreased production of TNF-α, IL-6 and IL-1ß in response to LPS, in a dose-dependent manner. The viability of neutrophils was only marginally affected in the same conditions, without statistical significance. Furthermore, the nuclear factor (NF)-κB inhibitor JSH-23 mimicked the effects of MLN4924 in neutrophils, and the inhibitory effects of MLN4924 on LPS-induced proinflammatory cytokine production diminished in the presence of JSH-23. Thus, the results of the present study suggest that neddylation inhibition suppresses neutrophil function by suppressing the NF-κB signaling pathway.

Neddylation is required for herpes simplex virus type I (HSV-1)-induced early phase interferon-beta production.

Type I interferons such as interferon-beta (IFN-ß) play essential roles in the host innate immune response to herpes simplex virus type I (HSV-1) infection. The transcription of type I interferon genes is controlled by nuclear factor-κB (NF-κB) and interferon regulatory factor (IRF) family members including IRF3. NF-κB activation depends on the phosphorylation of inhibitor of κB (IκB), which triggers its ubiqitination and degradation. It has been reported that neddylation inhibition by a pharmacological agent MLN4924 potently suppresses lipopolysaccharide (LPS)-induced proinflammatory cytokine production with the accumulation of phosphorylated IκBα. However, the role of neddylation in type I interferon expression remains unknown. Here, we report that neddylation inhibition with MLN4924 or upon UBA3 deficiency led to accumulation of phosphorylated IκBα, impaired IκBα degradation, and impaired NF-κB nuclear translocation in the early phase of HSV-1 infection even though phosphorylation and nuclear translocation of IRF3 were not affected. The blockade of NF-κB nuclear translocation by neddylation inhibition becomes less efficient at the later time points of HSV-1 infection. Consequently, HSV-1-induced early phase IFN-ß production significantly decreased upon MLN4924 treatment and UBA3 deficiency. NF-κB inhibitor JSH-23 mimicked the effects of neddylation inhibition in the early phase of HSV-1 infection. Moreover, the effects of neddylation inhibition on HSV-1-induced early phase IFN-ß production diminished in the presence of NF-κB inhibitor JSH-23. Thus, neddylation contributes to HSV-1-induced early phase IFN-ß production through, at least partially, promoting NF-κB activation.