Neuraminidase- and benzalkonium chloride-dependent inhibition of basic peptide-induced rat mast cell secretion

Two types of inhibition of basic peptide-induced rat mast cell secretion are reported. Pretreatment of rat peritoneal mast cells with Vibrio comma neuraminidase, an enzyme which cleaves sialic acid from oligosaccharides, led to inhibition of 5-hydroxytryptamine release induced by the basic peptides polylysine, corticotropin_1–24 and a decapeptide sequence of human IgE. Inhibition was similarly observed when mast cells were challenged in the presence of the cationic cell membrane-active substance benzalkonium chloride. It is postulated that both of these experimental procedures inhibit basic peptide-induced secretion by depletion of cell surface negative charge. Sialic acid itself does not act as a specific receptor for basic peptides, since a molar excess of sialic acid in free solution failed to inhibit secretion by binding to basic peptides in the fluid phase.     

IL6Rα function in myeloid cells modulates the inflammatory response during irritant contact dermatitis

Irritant contact dermatitis (ICD) is characterized by epidermal hyperplasia, infiltration of leucocytes into lesional skin and inflammatory cytokine release. The cellular infiltrate during ICD comprises primarily cells of the myeloid lineage. Our group has previously shown that the cytokine IL‐6 confers a protective effect to lesional skin during ICD. How IL‐6Rα function in myeloid cells is involved in the inflammatory response during ICD is, however, unknown. In the present study, utilizing a chemical model of ICD, it is shown that mice with a myeloid‐specific knockout of the IL‐6Rα (IL‐6Rα^Δmyeloid) display an exaggerated inflammatory response to benzalkonium chloride (BKC) and Jet propellant‐8 (JP8) fuel, two well‐characterized irritants relative to littermate control. Results from immunohistochemical and flow cytometric analyses revealed that IL‐6Rα^Δmyeloid mouse skin displayed increased epidermal hyperplasia and inflammatory monocyte influx into lesional skin but lower numbers of resident macrophages relative to littermate controls after irritant exposure. Multiplex immunoassay revealed significantly higher levels of pro‐inflammatory cytokines IL‐1α and TNF‐α, but reduced expression of chemokine proteins including CCL2‐5, CCL7, CCL11, CXCL1 and CXCL10 in IL‐6Rα^Δmyeloid mouse skin relative to littermate control following irritant exposure. These results highlight a previously unknown role of IL‐6Rα function in myeloid cells in modulating the inflammatory response and myeloid population dynamics during ICD.     

In vitro methods of assessing ocular biocompatibility using THP-1-derived macrophages

Macrophages play an important role in the elimination of infections, the removal of debris and in tissue repair after infection and trauma. In vitro models that assess ocular biomaterials for toxicity typically focus on the effects of these materials on epithelial or fibroblast cells. This investigation evaluated known ocular toxins deposited on model materials for their effects on the viability and activation of macrophages. THP-1-derived macrophages were cultured onto silicone films (used as a base biomaterial) deposited with chemical toxins (benzalkonium chloride (BAK), zinc diethyldithiocarbamate (ZDEC) and lipopolysaccharide (LPS)). Utilizing three fluorescent dyes calcein, ethidium homodimer-1 (EthD-1) and annexin V, the viability of macrophages attached to the biomaterial was determined using confocal microscopy. Propidium iodide (PI) staining and alamarBlue® (resazurin) reduction were used to assess cell death and metabolic activity. CD14, CD16, CD33, CD45, and CD54 expression of adherent macrophages, were also evaluated to detect LPS activation of macrophages using flow cytometry. The sensitivity of this test battery was demonstrated as significant toxicity from treated surfaces with ZDEC (0.001–0.01%), and BAK (0.001%–0.1%) was detected. Also, macrophage activation could be detected by measuring CD54 expression after exposure to adsorbed LPS. These in vitro methods will be helpful in determining the toxicity potential of new ocular biomaterials.     

Topical hyaluronan alone promotes corneal epithelial cell migration whereas combination with benzalkonium chloride impairs epithelial wound healing

Abstract

Purpose: To evaluate the effects of topical hyaluronan (HA) on corneal epithelial wound healing when administered with or without benzalkonium chloride (BAC).

Methods: A cultured human corneal epithelial cell line (HCE-T) was subjected to in vitro scratch assays and in situ epithelial migration was evaluated in organ-cultured rabbit corneas. The corneal epithelium of C57BL/6J mice was also evaluated to determine in vivo wound healing. An in vivo imaging system was also used to evaluate the effects of HA on eye drop retention on the ocular surface.

Results: The findings revealed the promotion of HCE-T migration, in situ rabbit corneal epithelial migration, and in vivo wound healing in mouse corneal epithelium by HA. Pre-treatment with HA also protected against delayed epithelial wound healing in BAC in vitro. However, pre-treatment with 3?mg/mL HA did not show a protective effect against BAC in vivo, but instead delayed epithelial wound healing and increased detection of cleaved caspase-3. This suggested that HA promotes the retention of BAC on the ocular surface. The instilled HA was retained after 15?min, at a significantly higher rate than for phosphate-buffered saline.

Conclusions: The combination of HA and BAC impaired wound healing in the corneal epithelium.     

苯扎氯铵改性粘接剂对牙本质-树脂远期粘接强度的影响研究

目的:评估苯扎氯铵(benzalkonium chloride,BAC)改性粘接剂对牙本质-树脂远期粘接强度的影响。方法:将BAC加入到美国3M第八代全酸蚀树脂粘接剂(single bond universal,SBU)中,制成不同浓度的改性粘接剂。将保存于百里香酚溶液中的100颗第三磨牙随机分为10组(n=10),A、A1组:0.25%BAC;B、B1组:0.5%BAC;C、C1组:1%BAC;D、D1:2%BAC;E、E1:对照组。A、B、C、D、E组试件进行剪切强度实验(SBS)并统计断裂模式。A1、B1、C1、D1、E1组试件进行微拉伸实验(μTBS)并统计断裂模式。结果:在剪切强度和微拉伸强度实验中,处理组的强度表现均显著低于对照组(P<0.05),其结果具有统计学意义。D组与C组结果具有统计学差异(P=0.01).D1组与B1组、C1组比较具有统计学意义(P<0.05)。断裂模式均以混合断裂为主,其次是粘接面断裂,内聚断裂最少。结论:全酸蚀粘接模式下,在树脂粘接剂中添加苯扎氯铵并不能改善牙本质与树脂的远期粘接强度,这可能是由于粘接剂的理化性能受损导致。     

普拉洛芬滴眼液中苯扎氯铵的含量测定方法研究

摘要 目的：建立普拉洛芬滴眼液中苯扎氯铵含量的HPLC测定方法。方法：用十八烷基硅烷键合硅胶为填充剂,以0.07mol?L乙酸铵溶液（含三乙胺1%,冰醋酸调节pH至5.0）-乙腈（25：75）为流动相,检测波长为210nm。结果：在选定的色谱条件下,各组分分离度良好,苄基十二烷基二甲基氯化铵、苄基十四烷基二甲基氯化铵分别在3.4～33.8μg?mL-1,0.7～5.5μg?mL-1的浓度范围内线性关系良好（r>0.999）;精密度试验的RSD分别为0.2%和0.7%、稳定性试验的RSD均不大于1.0%;回收率分别为99.9%,101.2%（n=9,RSD<1.0%）。C12与C14的检出限分别为3.4 ng?mL-1和110.5 ng?mL-1。在测定3批样品中,其中C12含量（μg?mL-1）分别为57.195,57.009,56.830;C14含量（μg?mL-1）分别为12.850,12.714,12.937;苯扎氯铵含量（μg?mL-1）分别为70.045,69.723,69.767。结论：本方法专属性强,准确度高,重现性好,能分别控制普拉洛芬滴眼液中苯扎氯铵两种同系物的含量。     

HPLC法测定氯霉素滴眼液中苯扎溴铵含量

目的：建立HPLC测定氯霉素滴眼液中苯扎溴铵含量的方法。方法：采用苯基硅烷键合硅胶为固定相;流动相为0.05 moL· L^-1磷酸二氢钾溶液（用三乙胺调节pH值至5.5）-乙腈（40∶60）;检测波长220 nm;流量1.2 mL· min^-1;柱温40℃;进样量20μL。结果：苯扎溴铵在0.0304～1.218μg范围内呈良好的线性关系（r＝0.9998）;检测限为3.45 ng,定量限为6.9 ng;平均回收率为99.8％（n＝9）,RSD＝1.5％。结论：该方法准确、可靠,可有效控制氯霉素滴眼液中苯扎溴铵的含量。     

Evaluation of pulmonary toxicity of benzalkonium chloride and triethylene glycol mixtures using in vitro and in vivo systems

Benzalkonium chloride (BAC) is a widely used disinfectant/preservative, and respiratory exposure to this compound has been reported to be highly toxic. Spray‐form household products have been known to contain BAC together with triethylene glycol (TEG) in their solutions. The purpose of this study was to estimate the toxicity of BAC and TEG mixtures to pulmonary organs using in vitro and in vivo experiments. Human alveolar epithelial (A549) cells incubated with BAC (1‐10 μg/mL) for 24 hours showed significant cytotoxicity, while TEG (up to 1000 μg/mL) did not affect cell viability. However, TEG in combination with BAC aggravated cell damage and inhibited colony formation as compared to BAC alone. TEG also exacerbated BAC‐promoted production of reactive oxygen species (ROS) and reduction of glutathione (GSH) level in A549 cells. However, pretreatment of the cells with N‐acetylcysteine (NAC) alleviated the cytotoxicity, indicating oxidative stress could be a mechanism of the toxicity. Quantification of intracellular BAC by LC/MS/MS showed that cellular distribution/absorption of BAC was enhanced in A549 cells when it was exposed together with TEG. Intratracheal instillation of BAC (400 μg/kg) in rats was toxic to the pulmonary tissues while that of TEG (up to 1000 μg/kg) did not show any harmful effect. A combination of nontoxic doses of BAC (200 μg/kg) and TEG (1000 μg/kg) promoted significant lung injury in rats, as shown by increased protein content and lactate dehydrogenase (LDH) activity in bronchoalveolar lavage fluids (BALF). Moreover, BAC/TEG mixture recruited inflammatory cells, polymorphonuclear leukocytes (PMNs), in terminal bronchioles and elevated cytokine levels, tumor necrosis factor α (TNF‐α), and interleukin 6 (IL‐6) in BALF. These results suggest that TEG can potentiate BAC‐induced pulmonary toxicity and inflammation, and thus respiratory exposure to the air mist from spray‐form products containing this chemical combination is potentially harmful to humans.     

双氯芬酸钠滴眼液抑菌剂剂量筛选及抑菌效力考察

目的研究双氯芬酸納滴眼液的抑菌效力,探索抑菌剂的合理添加量。方法按《中国药典》2015年版四部,以大肠埃希菌、金黄色葡萄球菌、铜绿假单胞菌、白色念珠菌和黑曲霉为实验菌株,测定10家企业双氯芬酸钠滴眼液的抑菌效力,并对硫柳汞、羟苯乙酯和苯扎氯铵3种抑菌剂的最低有效量进行研究。结果10家企业中没有一家抑菌效力达到A级,有3家企业:达到B级,另外7家均未达到B级。抑菌剂剂量筛选实验中,当双氯芬酸钠滴眼液含硫柳汞质量农度达到0.01 mg·mL^-1,羟苯乙酯质量浪浓度达到0.3 mg·mL^-1时,抑菌效力可达到B级,苯扎氯铵质量农浓度达到0.01 mg·mL^-1时,抑菌效力即可达到A级。结论市售双氯芬酸钠滴眼液的抑菌效力不容乐观,建议企业合理添加抑菌剂,对抑菌剂的理化指标和生物指标一同评价保证药品质量安全可靠。     

药源性眼损伤快速评价的斑马鱼模型

目的 苯扎溴铵和羟苯乙酯是广泛应用于眼用制剂的防腐剂,其在眼表的积累是引起眼用制剂药物毒性的重要因素.目前还没有一种简单、快速的方法来检测防腐剂的体内毒性.本研究以苯扎溴铵和羟苯乙酯为探针药物建立一种快速筛选和评价化合物眼毒性的斑马鱼模型和方法.方法 体式显微镜下活体观察斑马鱼幼鱼给药后眼部形态的变化;采用斑马鱼行为学...