Growth history of hepatitis C virus among HIV/HCV co-infected patients in Guizhou Province.

Background: The evolutionary and epidemiological history and the regional differences of various hepatitis C virus (HCV) genotypes are complex. Our aim was to better understand the molecular epidemiology and evolutionary dynamics of HCV among HIV/HCV co-infected individuals in Guizhou Province. This information could contribute to improve HCV prevention and control strategies in Guizhou and surrounding provinces. Methods: The HCV RNA was extracted from the serum of HIV/HCV co-infected patients, and reverse transcription/nested PCR was performed to amplify nucleotide sequences of the C-E1 region. Then, the successfully amplified sequences were selected for phylogenetic analysis. The available C-E1 region reference sequences from the surrounding provinces of Guizhou (Guangxi, Yunnan, Hunan, and Sichuan) were retrieved in GenBank, and the evolutionary analysis by Bayesian Markov chain Monte Carlo (MCMC) algorithm was performed using BEAST software to reconstruct a phylogeographic tree in order to explore their migration patterns. Finally, the epidemiological history of HCV in the Guizhou region was retraced by reconstructing Bayesian skyline plots (BSPs) after excluding sequences from surrounding provinces. Results: Among 186 HIV/HCV co-infected patients, the C-E1 region sequence was successfully amplified in 177 cases. Phylogenetic analysis classified these sequences into six subtypes: 1a, 1b, 3a, 3b, 6a, and 6n. Among them, subtype 6a was the most dominant strain (n = 70), followed by 3b (n = 55), 1b (n = 31), 3a (n = 11), 1a (n = 8), and 6n (n = 2). By reconstructing the phylogeographic tree, we estimated that the 6a strain in Guizhou mainly originated from Yunnan and Guangxi, while the 3b strain emerged due to transmission from the IDU network in Yunnan. Subtypes 1b, 3a, 3b, and 6a, as the major subtypes of HCV in HIV/HCV co-infected individuals in Guizhou, emerged and later grew more rapidly than the national average. Notably, BSPs of the currently prevalent HCV predominant strain subtype 6a in Guizhou have shown a rapid population growth since 2004. Although the growth rate slowed down around 2010, this growth has continued to date. Conclusion: Overall, despite the improvement and implementation of a series of HCV prevention and control policies and measures, a delayed growth pattern may indicate a unique history of the spread of 6a in Guizhou. Its trend as the dominant strain in Guizhou in recent years may continue to increase slowly over subsequent years.

Liproxstatin-1 Alleviates Lung Transplantation-induced Cold Ischemia-Reperfusion Injury by Inhibiting Ferroptosis.

BACKGROUND: Primary graft dysfunction, which is directly related to cold ischemia-reperfusion (CI/R) injury, is a major obstacle in lung transplantation (LTx). Ferroptosis, a novel mode of cell death elicited by iron-dependent lipid peroxidation, has been implicated in ischemic events. This study aimed to investigate the role of ferroptosis in LTx-CI/R injury and the effectiveness of liproxstatin-1 (Lip-1), a ferroptosis inhibitor, in alleviating LTx-CI/R injury. METHODS: LTx-CI/R-induced signal pathway alterations, tissue injury, cell death, inflammatory responses, and ferroptotic features were examined in human lung biopsies, the human bronchial epithelial (BEAS-2B) cells, and the mouse LTx-CI/R model (24-h CI/4-h R). The therapeutic efficacy of Lip-1 was explored and validated both in vitro and in vivo. RESULTS: In human lung tissues, LTx-CI/R activated ferroptosis-related signaling pathway, increased the tissue iron content and lipid peroxidation accumulation, and altered key protein (GPX4, COX2, Nrf2, and SLC7A11) expression and mitochondrial morphology. In BEAS-2B cells, the hallmarks of ferroptosis were significantly evidenced at the setting of both CI and CI/R compared with the control, and the effect of adding Lip-1 only during CI was much better than that of only during reperfusion by Cell Counting Kit-8. Furthermore, Lip-1 administration during CI markedly relieved LTx-CI/R injury in mice, as indicated by significant improvement in lung pathological changes, pulmonary function, inflammation, and ferroptosis. CONCLUSIONS: This study revealed the existence of ferroptosis in the pathophysiology of LTx-CI/R injury. Using Lip-1 to inhibit ferroptosis during CI could ameliorate LTx-CI/R injury, suggesting that Lip-1 administration might be proposed as a new strategy for organ preservation.

NOD1 mediated D. pteronyssinus-induced allergic airway inflammation through RIP2/NF-κB.

BACKGROUND: Dermatophagoides pteronyssinus (D. pteronyssinus) is the main cause of allergic airway inflammation. As the earliest intracytoplasmic pathogen recognition receptors (PRR), NOD1 has been identified as key inflammatory mediator in NOD-like receptor (NLR) family. OBJECTIVE: Our primary aim is to elucidate whether NOD1 and its downstream regulatory proteins mediate D. pteronyssinus-induced allergic airway inflammation. METHODS: Mouse and cell models of D. pteronyssinus-induced allergic airway inflammation were established. NOD1 was inhibited in bronchial epithelium cells (BEAS-2B cells) and mice by cell transfection or application of inhibitor. The change of downstream regulatory proteins was detected by quantitative real-time PCR (qRT-PCR) and Western blot. The relative expression of inflammatory cytokines was evaluated by ELISA. RESULTS: The expression level of NOD1 and its downstream regulatory proteins increased in BEAS-2B cells and mice after treating with D. pteronyssinus extract, followed by the aggravation of inflammatory response. Moreover, inhibition of NOD1 decreased the inflammatory response, which also downregulated the expression of downstream regulatory proteins and inflammatory cytokines. CONCLUSIONS: NOD1 involves in the development of D. pteronyssinus-induced allergic airway inflammation. Inhibition of NOD1 reduces D. pteronyssinus-induced airway inflammation.

Deficiency of HtrA3 Attenuates Bleomycin-Induced Pulmonary Fibrosis Via TGF-ß1/Smad Signaling Pathway.

PURPOSE: Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease characterized by excessive extracellular matrix deposition. No effective treatments are currently available for IPF. High-temperature requirement A3 (HtrA3) suppresses tumor development by antagonizing transforming growth factor ß (TGF-ß) signaling; however, little is known about the role of HtrA3 in IPF. This study investigated the role of HtrA3 in IPF and underlying mechanisms. METHODS: Lung tissues were collected from patients with IPF and mice with bleomycin (BLM)-induced pulmonary fibrosis, and HtrA3 expression was measured in tissue samples. Then, HtrA3 gene knockout mice were treated with BLM to induce pulmonary fibrosis and explore the effects and underlying mechanism of HtrA3 on pulmonary fibrosis. RESULTS: HtrA3 was up-regulated in the lung tissues of patients with IPF and the pulmonary fibrotic mouse model compared to corresponding control groups. HtrA3 knockout decreased pulmonary fibrosis-related protein expression, alleviated the symptoms of pulmonary fibrosis, and inhibited epithelial-mesenchymal transition (EMT) in BLM-induced lung tissue compared with BLM-induced wild-type mice. The TGF-ß1/Smad signaling pathway was activated in fibrotic lung tissue, whereas HtrA3 knockout inhibited this pathway. CONCLUSION: The expression level of HtrA3 is increased in fibrotic lungs. HtrA3 knockout alleviates the symptoms of pulmonary fibrosis probably via the TGF-ß1/Smad signaling pathway. Therefore, HtrA3 inhibition is a potential therapeutic target for pulmonary fibrosis.

Anterior hilum anastomosis versus posterior hilum anastomosis in a mouse lung transplantation model.

Objectives: The mouse orthotopic lung transplantation (LTx) model is of enormous research value in lung transplantation. This study compares 2 anastomotic methods (anterior and posterior hilum anastomosis) of mouse LTx in term of difficulty, operation time, and postoperative effects. Methods: Twenty mice received LTx with slipknots for anterior hilum anastomosis (AH group), and 28 received LTx with a microvessel clip for posterior hilum anastomosis (PH group), all by a single surgeon. The operation time was recorded and the grafts were evaluated 24 hours after surgery. Results: The success rates in the recipient animals were 85% (17/20) in AH group and 89% (25/28) in PH group (P > .05). The recipient operation time and back table time in AH group were longer than those in PH group (52.8 ± 5.0 vs 47.3 ± 5.7 minutes, 27.8 ± 3.9 vs 25.3 ± 2.8 minutes, P < .05), but the warm ischemia time did not differ significantly (13.1 ± 2.1 vs 12.2 ± 2.6 minutes, P = .258), meaning that the time discrepancies predominantly originated from the hilum treatment. In AH group, 2 cases failed due to pulmonary venous thrombosis and atelectasis respectively at 24 hours after LTx, but none failed in PH group. No significant difference was observed in the postoperative performance of the successful recipients (thoracic radiographs, macroscopic appearance, oxygenation index, pulmonary compliance, pathologic changes) between the 2 groups. Conclusions: Compared with anterior hilar anastomosis, posterior hilum anastomosis with a microvessel clip is less complicated and less time-consuming in the management of hilar structures and causes fewer postoperative complications.

Survival and outcomes after lung transplantation for connective tissue disease-associated interstitial lung diseases.

BACKGROUND: Lung transplantation (LTx) is the most important treatment for end-stage lung diseases. However, the treatment of connective tissue disease-associated interstitial lung diseases (CTD-ILD) using LTx is still controversial especially for polymyositis/dermatomyositis-associated interstitial lung disease (PM/DM-ILD). METHODS: Patients diagnosed with idiopathic pulmonary fibrosis (IPF) (n=180) and CTD-ILD (n= 36) from 1st January 2015 to 31st December 2019 were recruited into the study. We set polymyositis/dermatomyositis (PM/DM) as a single subgroup, and all the patients underwent LTx at the Wuxi People's Hospital. RESULTS: We found that patients with non-myositis connective tissue-related ILD (NM-CTLD) were younger (p=0.007) and had a higher percentage of females (p=0.000) than patients with IPF. PM/DM-ILD was associated with a higher incidence of primary graft dysfunction (PGD) (p=0.006) and a longer time in the intensive care unit (ICU) (p=0.000). The cumulative survival rates of patients with PM/DM-ILD were significantly lower than those with IPF (log rank, p=0.003). However, there were no significant differences when compared with the cumulative survival rates of patients with NM-CTLD and IPF (log rank, p=0.528). Age- and gender-adjusted Cox proportional hazard analyses indicated that post-LTx PGD (HR 1.498, 95% CI 1.227-1.828, p=0.000) and duration of ICU (HR 1.027, 95% CI 1.007-1.047, p=0.000) were the independent contributors of disease status to survival. Lung infection was the leading cause of post-LTx death in the groups, where the incidence was 65.3% (47/72) in IPF, 66.7% (8/12) in NM-CTLD, and 66.7% (4/6) in PM/DM-ILD. CONCLUSIONS: This study found that patients with NM-CTLD had a similar survival outcome with IPF. However, patients with PM/DM-ILD-performed LTx had a lower survival rate than those with IPF. Key Points • Previous studies have shown that the myopathies associated ILD patients had similar post-LTx outcomes with IPF patients. However, our retrospective analysis indicated that patients with PM/DM-ILD-performed LTx had a lower survival rate than those with IPF. • Patients with NM-CTLD had a similar survival outcome with IPF. • We also found that PM/DM-ILD was associated with a higher incidence of PGD and a longer time in the ICU.

BBOX1-AS1 Accelerates Gastric Cancer Proliferation by Sponging miR-3940-3p to Upregulate BIRC5 Expression.

BACKGROUND: Gastric cancer (GC) is one type of the most general malignancies in the globe. Research increasingly suggests long non-coding RNAs (lncRNAs) exert crucial roles in GC. However, the function of BBOX1-AS1 in GC has not been reported yet, it needs more explorations. AIMS: The aim of the study is to figure out the role and related regulation mechanism of BBOX1-AS1 in GC. METHODS: RT-qPCR assay was applied to detect genes expression. The role of BBOX1-AS1 in GC was investigated by cell counting kit-8, colony formation, tunel detection, and western blot assays. The binding ability between miR-3940-3p and BBOX1-AS1 (or BIRC5) by RIP, RNA pull-down and luciferase reporter assays. RESULTS: The expression of BBOX1-AS1 presented significantly upregulation in GC tissues and cells. Moreover, upregulation of BBOX1-AS1 promoted GC cell proliferation, and inhibited GC cell apoptosis. However, downregulation of BBOX1-AS1 led to opposite results. Furtherly, we discovered that BBOX1-AS1 bound with miR-3940-3p and also negatively regulated miR-3940-3p. Besides, it proved that miR-3940-3p interplayed with BIRC5 and negatively regulated BIRC5. Through rescue experiments, we proved that BIRC5 reversed miR-3940-3p-mediated cell proliferation or apoptosis in BBOX1-AS1-dysregulated GC cells. CONCLUSIONS: BBOX1-AS1 accelerates GC proliferation by sponging miR-3940-3p to upregulate BIRC5 expression, which may guide a new direction into the therapeutic strategies of GC.

A novel dendrimer-based complex co-modified with cyclic RGD hexapeptide and penetratin for noninvasive targeting and penetration of the ocular posterior segment.

Noninvasive drug delivery is a promising treatment strategy for ocular posterior segment diseases. Many physiological and anatomical barriers of the eye considerably restrict effective diffusion of therapeutics to the target site. To overcome this problem, a novel cyclic arginine-glycine-aspartate (RGD) hexapeptide and penetratin (PEN) co-modified PEGylation polyamidoamine (PAMAM) was designed as a nanocarriers (NCs), and its penetrating and targeting abilities were evaluated. In this study, we show that PAMAM-PEG (reaction molar ratio 1:32) has a relatively high grafting efficiency and low cytotoxicity. The particle size was within the range of 15-20 nm after modification with RGD and PEN. Cellular uptake of RGD-modified NCs involved significant affinity toward integrin αvß3, which validated the targeting of neovasculature. An in vitro permeation study indicated that modification with PEN significantly improved penetration of the NCs (1.5 times higher). In vivo ocular distribution studies showed that, the NCs (modified with PEN or co-modified with RGD and PEN) were highly distributed in the cornea and retina (p < .001), and modification extended retinal retention time for more than 12 h. Therefore, these NCs appear to be a promising noninvasive ocular drug delivery system for ocular posterior segment diseases.

Brain-targeted intranasal delivery of dopamine with borneol and lactoferrin co-modified nanoparticles for treating Parkinson's disease.

Efficient delivery of brain-targeted drugs is highly important for successful therapy in Parkinson's disease (PD). This study was designed to formulate borneol and lactoferrin co-modified nanoparticles (Lf-BNPs) encapsulated dopamine as a novel drug delivery system to achieve maximum therapeutic efficacy and reduce side effects for PD. Dopamine Lf-BNPs were prepared using the double emulsion solvent evaporation method and evaluated for physicochemical and pharmaceutical properties. In vitro cytotoxicity studies indicated that treatment with dopamine Lf-BNPs has relatively low cytotoxicity in SH-SY5Y and 16HBE cells. Qualitative and quantitative cellular uptake experiments indicated that Lf modification of NPs increased cellular uptake of SH-SY5Y cells and 16HBE cells, and borneol modification can promote the cellular uptake of 16HBE. In vivo pharmacokinetic studies indicated that AUC0-12 h in the rat brain for dopamine Lf-BNPs was significantly higher (p < .05) than that of dopamine nanoparticles. Intranasal administration of dopamine Lf-BNPs effectively alleviated the 6-hydroxydopamine-induced striatum lesion in rats as indicated by the contralateral rotation behavior test and results for striatal monoamine neurotransmitter content detection. Taken together, intranasal administration of dopamine Lf-BNPs may be an effective drug delivery system for Parkinson's disease.

Modification of Three-Phase Drug Release Mode of Octreotide PLGA Microspheres by Microsphere-Gel Composite System.

In order to obtain sustained release of biodegradable microspheres, the purpose of this study was to design and characterize an injectable octreotide microsphere-gel composite system. The octreotide microspheres were prepared by phase separation method, which used PLGA as a carrier material, dimethyl silicone oil as a phase separation reagent, and n-heptane-Span 80 as a hardener. In addition, we used poloxamer 407 (PL 407) and poloxamer 188 (PL 188) as the thermosensitive gel matrix material. The composite system was obtained by scattering octreotide microspheres in a poloxamer gel. In vitro data showed that the release time of the composite system could last for about 50 days. Because of the blocking and control actions of the poloxamer gel, the initial burst release was significantly reduced and the plateau phase was eliminated. Pharmacokinetic data showed that the burst release of the composite system was significantly less than that of the microspheres, i.e., Cmax1 was reduced by about half. From day 2 to day 50, higher plasma concentration levels and more stable drug release behavior were exhibited. In addition, the good biocompatibility of the composite system in vivo was also demonstrated by hematoxylin-eosin (HE) staining. Therefore, the octreotide microsphere-gel composite system will be a new direction for hydrophilic polypeptide/protein-loaded sustained release dosage forms with high pharmacological activity.

Afatinib-loaded immunoliposomes functionalized with cetuximab: A novel strategy targeting the epidermal growth factor receptor for treatment of non-small-cell lung cancer.

Afatinib, a selective and irreversible inhibitor of tyrosine kinase, was approved for the treatment of advanced non-small-cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) overexpression in 2013. Cetuximab (CTX), an anti-EGFR monoclonal antibody, is co-administered with afatinib to improve efficacy. Unfortunately, dose-related adverse reactions caused by combination therapy have affected patient compliance, and have resulted in treatment discontinuation in severe cases. In the present study, afatinib was encapsulated in "liposomes" (LPs) to achieve longer circulation in the blood and an enhanced permeability-and-retention effect in tumors. Concomitantly, CTX was designed to bind to drug-loaded LPs to form "immuno-LPs" for tumor-cell selectivity and therapeutic activity. In vitro, the cellular internalization rate of immuno-LPs was significantly higher than that of LPs (p < 0.05). In vivo, a markedly increased area under the curve and prolonged terminal half-life were detected in rats injected with the two LP formulations, indicating that LP encapsulation protected afatinib from binding to hemoglobin to control the risk of idiosyncratic drug reactions. Compared with free afatinib and LPs, immuno-LPs exhibited strongly enhanced drug delivery and antitumor efficacy in an NSCLC xenograft model, with stronger tumor selectivity and potentially fewer side-effects. Hence, EGFR-targeting immuno-LPs appear to be promising for NSCLC treatment.

Determination of tyrosine kinase inhibitor afatinib in rat plasma using LC-MS/MS and its application to in vivo pharmacokinetic studies of afatinib liposomes.

A sensitive quantitative liquid chromatography-tandem mass spectrometry assay for afatinib in rat plasma was developed and validated using afatinib dimaleate as a standard. The analyte was determined to be ionized using the positive ion multiple reaction monitoring mode through electrospraying on an AB Sciex Triple Quad™ 4500 system. Protein precipitation was used for sample preparation, and an Agilent Eclipse XDB-CN column (100 × 2.1 mm, 3.5 µm) was applied for chromatographic separation. The mobile phase was water and methanol (15:85, v/v) containing 0.1% formic acid with a flow rate of 0.5 mL/min. The linear range was 0.5-200 ng/mL, with r2 = 0.9994 ± 0.0004 calculated from linear regression, with 1/x2 as the weighting factor for the calibration. The average recovery of the plasma samples was stable and reproducible. The analyte is sufficiently stable for handling and analysis. The pharmacokinetic study and comparison were performed by analyzing plasma concentrations in rats administered afatinib solution or prepared afatinib liposomes using the developed determination method. The Cmax of the afatinib liposomes was nearly 400-fold higher than that of the afatinib solution. These results indicate that liposome-encapsulation protected afatinib from endogenous protein binding, thereby reducing the risk of idiosyncratic drug reactions by protein adducts. Thus, Afatinib liposomes seem to be a promising strategy for the treatment of non-small cell lung cancer.

[Formulation and Efficacy of Liposome-encapsulated Afatinib for Therapy of Non-small Cell Lung Cancer].

BACKGROUND: Afatinib, a second-generation irreversible epidermal growth factor inhibitor receptor for the development of non-small cell lung cancer and secondary drug resistance, has low bioavailability and adverse reactions due to current oral administration. The aim of this study was to prepare a novel drug delivery system, afatinib liposome, and to establish a method for the determination of encapsulation efficiency. METHODS: Four different preparation methods were used to prepare afatinib liposomes, and the optimal preparation process was determined by comparing the encapsulation efficiency and particle size. RESULTS: It has been verified that sephadex microcolumn centrifugation can be used to purify afatinib liposomes, and UV spectrophotometry can be employed to determine the entrapment efficiency of liposomes. Among different preparation methods, the encapsulation efficiency of afatinib liposomes prepared by ammonium sulfate gradient method was 90.73% and the average particle size was 108.6 nm. CONCLUSIONS: Ammonium sulfate gradient method can be successfully applied to prepare afatinib liposomes that performed higher encapsulation efficiency and smaller particle size. The UV spectrophotometry employed to determine the liposome encapsulation efficiency was easy operation and with high accuracy.

LC-MS/MS method for the determination of the prodrug aripiprazole lauroxil and its three metabolites in plasma and its application to in vitro biotransformation and animal pharmacokinetic studies.

A sensitive and selective LC-MS/MS method for determination of the prodrug aripiprazole lauroxil (AL) and the three metabolites (N-hydroxymethyl aripiprazole [NHA], aripiprazole [ARP], and dehydro aripiprazole [DHA]) in plasma was developed using ARP-d8 as an internal standard. The analytes were determined on an AB Sciex Triple Quad™ 4500 system using positive ion electrospray ionization and selected multiple reaction monitoring mode. Solid phase extraction was applied for sample preparation for AL, ARP, and DHA, and protein precipitation for NHA. Chromatographic separation was performed on an Agilent Eclipse XDB-CN column (100 × 2.1 mm i.d., 3.5 µm) using the mobile phase of water and acetonitrile (25:75, v/v) containing 0.1% formic acid with a flow rate of 0.5 mL/min. The linear ranges for AL, NHA, ARP, and DHA were 0.5-50 ng/mL, 1.0-50 ng/mL, 0.5-50 ng/mL, and 0.05-5.0 ng/mL, respectively. The average recovery in the plasma sample was stable and reproducible. The precision and accuracy of the intra- and inter-run were within assay variability criteria limits. The developed method was suitable for in vitro biotransformation studies in plasma and animal pharmacokinetic studies after intramuscular injection of AL formulations.

Meta-analysis for evaluating the accuracy of endoscopy with narrow band imaging in detecting colorectal adenomas.

BACKGROUND AND AIM: The aim of this study was to determine whether the use of the narrow band imaging (NBI) system could enhance the accuracy of adenoma detection during an endoscopic examination of the colon and rectum. METHODS: MEDLINE, EMBASE, and the Cochrane Library databases were searched along with a hand search of abstracts from relevant conferences up to June 2011. The rates of adenoma and flat adenoma detection, and withdrawal time were analyzed using Review Manager 4.2. RESULTS: A total of 3049 subjects in eight trials were included. Meta-analysis revealed that there was no statistically significant difference in the rates of adenoma detection between the NBI group and the white light colonoscopy group (pooled relative risk [RR]: 1.09, 95% confidence interval [CI]: 1.00-1.19, P = 0.05). However, after exclusion of high-definition television modalities, the rate of adenoma detection by NBI was significantly higher than that by white light, particularly for patients with one adenoma (pooled RR 1.36, 95%CI 1.07-1.71, P = 0.02). Endoscopy with the NBI system significantly increased the rate of flat adenoma detection (pooled RR 1.96, 95%CI 1.09-3.52, P = 0.02). However, endoscopy with NBI had longer withdrawal time than that with white light (pooled weighted mean difference: 0.90, 95%CI: 0.38-1.42, P = 0.0006). CONCLUSIONS: Endoscopy with NBI seems to improve the detection of flat adenomas, particularly with high-definition technology, but prolongs the withdrawal time. These results indicate that endoscopy routinely using the NBI system for the surveillance of adenomas may be recommended after the technique is further modified.