Role of site-directed mutagenesis and adjuvants in the stability and potency of anthrax protective antigen

Anthrax is a zoonotic infection caused by the gram-positive, aerobic, spore-forming bacterium Bacillus anthracis. Depending on the origin of the infection, serious health problems or mortality is possible. The virulence of B. anthracis is reliant on three pathogenic factors, which are secreted upon infection: protective antigen (PA), lethal factor (LF), and edema factor (EF). Systemic illness results from LF and EF entering cells through the formation of a complex with the heptameric form of PA, bound to the membrane of infected cells through its receptor. The currently available anthrax vaccines have multiple drawbacks, and recombinant PA is considered a promising second-generation vaccine candidate. However, the inherent chemical instability of PA through Asn deamidation at multiple sites prevents its use after long-term storage owing to loss of potency. Moreover, there is a distinct possibility of B. anthracis being used as a bioweapon; thus, the developed vaccine should remain efficacious and stable over the long-term. Second-generation anthrax vaccines with appropriate adjuvant formulations for enhanced immunogenicity and safety are desired. In this article, using protein engineering approaches, we have reviewed the stabilization of anthrax vaccine candidates that are currently licensed or under preclinical and clinical trials. We have also proposed a formulation to enhance recombinant PA vaccine potency via adjuvant formulation.     

Rociletinib (CO-1686) enhanced the efficacy of chemotherapeutic agents in ABCG2-overexpressing cancer cells in vitro and in vivo

Overexpression of adenosine triphosphate (ATP)-binding cassette subfamily G member 2 (ABCG2) in cancer cells is known to cause multidrug resistance (MDR), which severely limits the clinical efficacy of chemotherapy. Currently, there is no FDA-approved MDR modulator for clinical use. In this study, rociletinib (CO-1686), a mutant-selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), was found to significantly improve the efficacy of ABCG2 substrate chemotherapeutic agents in the transporter-overexpressing cancer cells in vitro and in MDR tumor xenografts in nude mice, without incurring additional toxicity. Mechanistic studies revealed that in ABCG2-overexpressing cancer cells, rociletinib inhibited ABCG2-mediated drug efflux and increased intracellular accumulation of ABCG2 probe substrates. Moreover, rociletinib, inhibited the ATPase activity, and competed with [¹²⁵I] iodoarylazidoprazosin (IAAP) photolabeling of ABCG2. However, ABCG2 expression at mRNA and protein levels was not altered in the ABCG2-overexpressing cells after treatment with rociletinib. In addition, rociletinib did not inhibit EGFR downstream signaling and phosphorylation of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK). Our results collectively showed that rociletinib reversed ABCG2-mediated MDR by inhibiting ABCG2 efflux function, thus increasing the cellular accumulation of the transporter substrate anticancer drugs. The findings advocated the combination use of rociletinib and other chemotherapeutic drugs in cancer patients with ABCG2-overexpressing MDR tumors.     

TLC法对众生丸中蒲公英成分的快速鉴别

目的为建立众生丸中蒲公英成分的快速定性鉴别方法,采用薄层色谱法(TLC),基于样品与标准品对照,实现快速定性鉴别。方法通过参考有关文献,经过实验,对众生丸中蒲公英成分的提取方法、提取时间、提取溶剂、展开剂的选择、显色剂的选择对实验结果的影响进行研究。结果以乙酸乙酯为溶剂经超声提取、以三氯甲烷为展开剂,喷以10%硫酸乙醇溶液作为显色剂。薄层色谱鉴别斑点清晰,阴性对照无干扰。结论所建立方法操作简单,具有速度快,分离效果、专属性和重现性好的优点,应用于众生丸中蒲公英成分的快速定性鉴别,可加强对众生丸的质量控制。     

越鞠丸联合盐酸氟西汀胶囊治疗抑郁症临床研究

目的:观察越鞠丸联合盐酸氟西汀胶囊治疗抑郁症的临床效果及对血清脑源性神经营养因子(BDNF)水平的影响。方法:纳入因抑郁症就诊的64例患者,随机分为对照组与治疗组各32例。对照组给予盐酸氟西汀胶囊抗5-羟色胺(5-HT)治疗,治疗组在对照组基础上联合越鞠丸治疗。比较2组治疗前后汉密尔顿抑郁量表(HAMD-24)评分、抑郁症状学快速自评量表(QIDS-SR16)、血清BDNF水平及总体疗效。结果:治疗前后比较,2组HAMD-24评分、QIDS-SR16评分均下降,血清BDNF水平升高(P<0.05),组间比较,治疗组2项评分下降更多,血清BDNF水平升高更多(P<0.05)。治疗后,治疗组总体疗效为90.63%,优于对照组的68.75%,差异有统计学意义(P<0.05)。结论:越鞠丸联合盐酸氟西汀胶囊治疗抑郁症,可更加有效减缓患者抑郁症状,升高血清BDNF水平,增加临床疗效,有效保护患者脑功能,值得临床推广。     

Randomized Crossover Trial of Phosphate-binding Medication on Serum Phosphate Levels in Patients With Aortic Stenosis

PurposeAortic stenosis is a common cause of valvular heart disease with no means of prevention. The recognized association between aortic stenosis and serum phosphate raises the possibility of preventing progression of the disorder by using phosphate-binding drugs, but there is uncertainty whether such treatment lowers serum phosphate levels in patients without diagnosed renal failure. This pilot study was conducted to answer this question in patients with aortic stenosis.MethodsA randomized, double-blind, crossover trial of the phosphate-binding drug sevelamer was conducted in 72 patients. Patients were prescribed sevelamer 0.8 g (low-dose), sevelamer 2.4 g (high-dose), and matching placebo, 3 times daily with food; each regimen lasted 6 weeks and was allocated at random. Serum phosphate levels were measured at the end of each treatment period, and within-person levels were compared.FindingsSixty-one patients completed the 3 treatment periods. There was no significant difference in the mean end-treatment phosphate levels across all patients (3.38, 3.36, and 3.31 mg/dL with placebo, low-dose sevelamer, and high-dose sevelamer, respectively). Post hoc analysis showed a reduction in phosphate levels with increasing sevelamer dose in the highest baseline phosphate quartile group; a 0.3 mg/dL reduction (mean, 4.09 mg/dL with placebo, 3.95 mg/dL with low-dose sevelamer, and 3.79 mg/dL with high-dose sevelamer; P_trend = 0.027).ImplicationsSevelamer had no overall statistically significant effect in lowering serum phosphate levels, but a reduction was observed in patients with phosphate levels in the highest quartile group of the population distribution. This hypothesis-generating result requires confirmation in an independent study. If confirmed, a trial of sevelamer in preventing the progression of aortic stenosis may be justified in patients with high phosphate levels. ISRCTN Registry identifier: ISRCTN17365679.     

Faster bone union progression and less sclerosis at the osteotomy margin after medial opening-wedge high tibial osteotomy using highly porous β-tricalcium phosphate granules versus allogeneic bone chips: A matched case–control study

BackgroundThis study compared bone union progression using highly porous (80% porosity) β-tricalcium phosphate (β-TCP) granules or allogeneic bone chips in the gap created by medial opening-wedge high tibial osteotomy (MOWHTO).MethodsThe study population consisted of 54 patients who received MOWHTO with locking plate fixation: 27 patients using highly porous β-TCP granules, and 27 age- and sex-matched patients using allogeneic bone chips. Bone union progression was evaluated 1, 3, 6, and 12 months postoperatively. The presence of radiographic sclerosis at the osteotomy margin was also assessed.ResultsAmong all patients, the highest degree of bone union observed 12 months postoperatively was grade 4. As postoperative time passed, bone union progression of highly porous β-TCP granules increased linearly and was statistically significant compared with that of cancellous allogeneic bone chips (P = 0.014). The presence of radiographic sclerosis at the osteotomy margin was significantly less common in the β-TCP group than in the allograft group (P = 0.003) and was the strongest predictor of delayed progress of bone union (odds ratio = 6.16, P = 0.006).ConclusionsPatients who underwent MOWHTO using highly porous β-TCP granules had faster new bone remodeling, less radiographic sclerosis at the osteotomy margin, and no inferior clinical outcome compared with allogeneic bone chips, as determined at the 1-year follow up. The presence of radiographic sclerosis at the osteotomy margin in patients undergoing MOWHTO using allogeneic bone or synthetic bone substitute may indicate delayed progress of bone union.     

Reduction of BMP6‐induced bone formation by calcium phosphate in wild‐type compared with nude mice

Nude mice have been extensively used to investigate the potency of tissue engineering strategies for bone repair. However, the contribution of pro‐inflammatory and proregenerative stimuli of the host for the process of new bone formation and integration remains poorly understood. In this study, ectopic bone formation was investigated in nude (Nu) versus wild‐type (WT) mice. Calcium phosphate (CaP) scaffolds (CopiOs [Zimmer] and Bio‐Oss [Geistlich]) were loaded with different concentrations of rhBMP6 (40, 120, and 240 ng/mm³ rhBMP6) and implanted subcutaneously in Nu (BALB/c and NMR1) and WT (BALB/c and c57BL/6) mice. CaP scaffolds loaded with rhBMP6 did not form bone in WT mice. However, in Nu mice, 40 ng/mm³ rhBMP6 was sufficient to generate relevant volumes of new bone at 6 weeks after implantation. Looking into potential underlying mechanisms, TNF‐α blocking antibodies were injected intraperitoneally but could not restore bone formation. Also, mouse periosteal cells (mPDCs) seeded in CopiOs loaded with rhBMP6 did not significantly improve the outcome. Abrogation of bone formation was associated with dense cellular infiltration, in particular with the presence of CD3+ T‐lymphocytes. To probe a correlation between calcium ions and impaired bone formation in WT mice, type 1 collagen gels were loaded with rhBMP6 and calcium chloride and injected subcutaneously. These gels generated new bone in WT mice despite the increased percentage of CD3+ cells at Day 3 after implantation as compared with control gels. Overall, this study illustrated the negative effect of the inflammatory host response on the bone‐forming capacity of rhBMP6 coated on bioceramic scaffolds.