Interaction of γ-Polyglutamic Acid/Polyethyleneimine/Plasmid DNA Ternary Complexes with Serum Components Plays a Crucial Role in Transfection in Mice.

Typical examples of non-viral vectors are binary complexes of plasmid DNA with cationic polymers such as polyethyleneimine (PEI). However, problems such as cytotoxicity and hemagglutination, owing to their positively charged surfaces, hinder their in vivo use. Coating binary complexes with anionic polymers, such as γ-polyglutamic acid (γ-PGA), can prevent cytotoxicity and hemagglutination. However, the role of interactions between these complexes and serum components in in vivo gene transfer remains unclear. In this study, we analyzed the contribution of serum components to in vivo gene transfer using PEI/plasmid DNA binary complexes and γ-PGA/PEI/plasmid DNA ternary complexes. In binary complexes, heat-labile components in the serum greatly contribute to the hepatic and splenic gene expression of the luciferase gene. In contrast, serum albumin and salts affected the hepatic and splenic gene expression in the ternary complexes. Changes in physicochemical characteristics, such as increased particle size and decreased absolute values of ζ-potential, might be involved in the enhanced gene expression. These findings would contribute to a better understanding of in vivo non-viral gene transfer using polymers, such as PEI and γ-PGA.

The Effects of Acute Exposure to Ammonia on Oxidative Stress, Hematological Parameters, Flesh Quality, and Gill Morphological Changes of the Large Yellow Croaker (Larimichthys crocea).

Ammonia is considered to be the major chemical pollutant causing fish poisoning in aquaculture. This research aimed to evaluate the impact of acute ammonia exposure on the large yellow croaker's meat quality, gill morphology, liver oxidative stress, and hematological parameters. The fish were exposed to total ammonia nitrogen concentrations of 0, 2.96, 5.92, and 8.87 mg/L for 48 h, respectively. The findings demonstrated that all ammonia-exposed fish had higher liver lactate dehydrogenase and glutamic oxalate transaminase activities. The glucose, blood urea nitrogen, and creatinine levels in 8.87 mg/L total ammonia nitrogen (TAN) were higher than other samples. The total protein, albumin, and triglyceride levels in serum decreased significantly in ammonia-exposed samples. After 48 h of ammonia exposure, superoxide dismutase activities showed a 76.1%, 118.0%, and 156.8% increase when fish were exposed to 2.96, 5.92, and 8.87 mg/L TAN, respectively. Catalase activities and glutathione contents were considerably higher (p < 0.05) in all ammonia-treated samples compared to 0 mg/L TAN. The ammonia-treated gill lamellae become thicker, shorter, and curved. Additionally, the ammonia exposure resulted in the accumulation of free amino acids and the loss of nucleotides. The inosine monophosphate and adenosine monophosphate contents in the flesh were decreased after 12 h of exposure to 2.96, 5.92, and 8.87 mg/L ammonia compared to the control group. Overall, large yellow croakers exposed to ammonia for 6 h presented not only changes in serum composition but also oxidative stress, liver and gill tissue damage and flesh quality deterioration.

Suanzaoren decoction improves depressive-like behaviors by regulating the microbiota-gut-brain axis via inhibiting TLR4/NFκB/NLRP3 inflammation signal pathway.

Depression is a common but serious sickness which causes a considerable burden on individuals and society. Recently, it has been well established that the occurrence of depression was related to the microbiota-gut-brain axis. The toll-like receptor 4 (TLR4)/ nuclear factor kappa-B kinase (NFκB)/ NOD-like receptor thermal protein domain associated protein 3 (NLRP3) pathway is closely associated with the regulation of microbiota-gut-brain axis. Suanzaoren Decoction (SZRD), which recorded in Jin Gui Yao Lve in Han dynasty, has been used for treating insomnia and depression for a long time. However, the action mechanism of the depression regulation through the TLR4/NFκB/NLRP3 pathway by SZRD was still unclear. In this study, SZRD was firstly performed on a chronic unpredictable mild stress (CUMS) mice model. The results of behavioral tests showed that SZRD treatment could ameliorate the depressive-like behaviors of CUMS mice effectively. According to our previous researches about the components of SZRD in vitro and in vivo, the identification of serum metabolites in depression model rats was further analyzed qualitatively using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry. 27 prototypes and 44 metabolites were identified. The main types of metabolic reactions are glucuronization, sulfation, and so on. Then, using immunohistochemistry and western blotting to monitor the difference in activation of TLR4/NFκB/NLRP3 signaling pathway in mice brain and colon. The results showed that SZRD treatment could reduce expression levels of related factors. Additionally, the SZRD treatment could also inhibit the histopathological damage in the path morphology of the hippocampus and colon. The results of 16SrRNA demonstrated that SZRD could reduce the dysbiosis of the intestinal flora of depressive mice. The above results provided important information for studying the action mechanism of SZRD in treating depression by regulating microbiota-gut-brain axis via inhibiting TLR4/NFκB/NLRP3 pathway.

Interaction of Lipoplex with Albumin Enhances Gene Expression in Hepatitis Mice.

Understanding the in vivo fate of lipoplex, which is composed of cationic liposomes and DNA, is an important issue toward gene therapy. In disease conditions, the fate of lipoplex might change compared with the normal condition. Here, we examined the contribution of interaction with serum components to in vivo transfection using lipoplex in hepatitis mice. Prior to administration, lipoplex was incubated with serum or albumin. In the liver, the interaction with albumin enhanced gene expression in hepatitis mice, while in the lung, the interaction with serum or albumin enhanced it. In normal mice, the interaction with albumin did not enhance hepatic and pulmonary gene expression. Furthermore, hepatic and pulmonary gene expression levels of albumin-interacted lipoplex were correlated with serum transaminases in hepatitis mice. The albumin interaction increased the hepatic accumulation of lipoplex and serum tumor necrosis factor-α level. We suggest that the interaction with albumin enhanced the inflammation level after the administration of lipoplex in hepatitis mice. Consequently, the enhancement of the inflammation level might enhance the gene expression level. Information obtained in the current study will be valuable toward future clinical application of the lipoplex.

A systematic study of the effect of low pH acid treatment on anti-drug antibodies specific for a domain antibody therapeutic: Impact on drug tolerance, assay sensitivity and post-validation method assessment of ADA in clinical serum samples.

We developed a homogeneous bridging anti-drug antibody (ADA) assay on an electro chemiluminescent immunoassay (ECLIA) platform to support the immunogenicity evaluation of a dimeric domain antibody (dAb) therapeutic in clinical studies. During method development we evaluated the impact of different types of acid at various pH levels on polyclonal and monoclonal ADA controls of differing affinities and on/off rates. The data shows for the first time that acids of different pH can have a differential effect on ADA of various affinities and this in turn impacts assay sensitivity and drug tolerance as defined by these surrogate controls. Acid treatment led to a reduction in signal of intermediate and low affinity ADA, but not high affinity or polyclonal ADA. We also found that acid pretreatment is a requisite for dissociation of drug bound high affinity ADA, but not for low affinity ADA-drug complexes. Although we were unable to identify an acid that would allow a 100% retrieval of ADA signal post-treatment, use of glycine pH3.0 enabled the detection of low, intermediate and high affinity antibodies (Abs) to various extents. Following optimization, the ADA assay method was validated for clinical sample analysis. Consistencies within various parameters of the clinical data such as dose dependent increases in ADA rates and titers were observed, indicating a reliable ADA method. Pre- and post-treatment ADA negative or positive clinical samples without detectable drug were reanalyzed in the absence of acid treatment or presence of added exogenous drug respectively to further assess the effectiveness of the final acid treatment procedure. The overall ADA results indicate that assay conditions developed and validated based on surrogate controls sufficed to provide a reliable clinical data set. The effect of low pH acid treatment on possible pre-existing ADA or soluble multimeric target in normal human serum was also evaluated, and preliminary data indicate that acid type and pH also affect drug-specific signal differentially in individual samples. The results presented here represent the most extensive analyses to date on acid treatment of a wide range of ADA affinities to explore sensitivity and drug tolerance issues. They have led to a refinement of our current best practices for ADA method development and provide a depth of data to interrogate low pH mediated immune complex dissociation.

Effect of feeding dried sweet orange (Citrus sinensis) peel and lemon grass (Cymbopogon citratus) leaves on growth performance, carcass traits, serum metabolites and antioxidant status in broiler during the finisher phase.

The current experiment was conducted to evaluate the effects of feeding dried sweet orange peel (SOP) and lemon grass leaves (LGL) as feed additives on broiler growth performance, serum metabolites, and antioxidant status. A total of 192-day-old (Ross 308) broiler chickens were distributed randomly into 4 dietary treatments with 4 replicates per each treatment. The dietary treatments included a control diet without any feed additive (T1), a diet containing 0.8 % SOP (T2), a diet containing 0.8 % LGL (T3), and a diet containing combination of 0.4 % SOP + 0.4 % LGL (T4) was fed during the growth period from 22 to 42 days. Feed intake (FI), body weight gain (BWG), feed conversion ratio (FCR), carcass traits, serum components, and antioxidant status were measured. At the end of the experimental period, the results indicated that supplementation of SOP and LGL alone or in combination did not significantly (P > 0.05) affect BWG, FI, FCR, and carcass characteristics in broiler chickens. Serum total protein was increased significantly (P < 0.05) in T3 and T4 compared to the other treatments. Also, serum globulin increased significantly (P < 0.05) in the treated groups. Serum glucose, low density lipoprotein, triglyceride, and very low density lipoprotein decreased significantly (P < 0.05) in the treatment groups, while cholesterol and high-density lipoprotein decreased in T2 compared to the other groups. Significantly (P < 0.05) higher total antioxidant status was observed in T2 compared to the other treatments. In conclusion, these results indicate that SOP, LGL, and their combination may positively modify some serum components and the antioxidant status without any beneficial effect on growth performance and carcass traits in broiler chickens.

Study on anti-inflammatory efficacy and correlative ingredients with pharmacodynamics detected in acute inflammation rat model serum from Caulis Lonicerae japonicae.

BACKGROUND: Caulis Lonicerae japonicae (CLJ) is often used for the treatment of inflammation such as acute fever, headache, respiratory infection and epidemic diseases. Nevertheless, domestic and foreign researches simply fail to focus on reports of CLJ, especially its anti-inflammatory effects and correlative components. PURPOSE: In this study, we investigated anti-inflammatory effects and serum components of Caulis Lonicerae japonicae (CLJ) in models of acute inflammation and correlative analysis between anti-inflammatory effects and serum components of CLJ in rat serum to analyze the changes in the relative contents of components in serum with time and in the corresponding values to characterize active fractions of CLJ and identify the major active components of CLJ in rats. STUDY DESIGN: Active fractions of CLJ were screened using xylene-induced ear oedema mice model. Anti-inflammatory effects were evaluated using carrageenan-induced paw oedema rat model, and then correlative components in rat serum were demonstrated with HPLC-QTOF/MS/MS method explored. METHODS: HPLC-QTOF/MS/MS was developed to analyze the components absorbed in rat serum after oral administration of CLJ. RESULTS: Ethyl acetate extracts (ECLJ) and n-butanol extracts (BCLJ) of CLJ were preliminarily screened as active fractions of CLJ (EBCLJ) using xylene-induced ear oedema mice model, and effectively inhibited edema and values of interleukin-1 (IL-l), nuclear factor-kappaB (NF-kB), tumor necrosis factor-α (TNF-α) activities and prostaglandinE2 (PGE2) productions using carrageenan-induced paw oedema rat model. 33 peaks were found in total ion current chromatograms of EBCLJ, and 27 components were observed in EBCLJ-treated rat serum, only 11 of which were correlated with anti-inflammatory effects. CONCLUSIONS: The present study contributes to the study on the pharmacodynamic basis of CLJ and provides potent evidence for developing CLJ as a safe and promising natural drug in inflammation treatment.