Corneal collagen cross‐linking for infectious keratitis: an update of clinical studies

Collagen cross‐linking (CXL) with ultraviolet light‐activated riboflavin is a corneal surface procedure developed for the treatment of keratoconus and corneal ectasia. With the known microbicidal and corneal stiffening effects of ultraviolet irradiation and photoactivated riboflavin, it has recently been introduced for the management of infectious keratitis, especially for ulcers resistant to antimicrobial therapy or associated with corneal melting. Various authors have attempted to use CXL as an adjunctive, salvage or even as the sole treatment for infectious corneal ulcers. The aim of this review was to provide a summary of the clinical studies in the literature. It is worth noting that there is still no consensus on the treatment protocol of CXL against infectious keratitis. The disparities in outcome measures, treatment protocol and study design can confound the interpretation and hamper the generalization of the study results. Based on current evidence, the role of CXL in infectious keratitis remained unclear despite the reported success in some clinical cases. Further investigations are warranted concerning the efficacy and safety of treating infectious keratitis with CXL.     

Riboflavin concentration in corneal stroma after intracameral injection

AIM:To evaluate the enrichment of riboflavin in the corneal stroma after intracameral injection to research the barrier ability of the corneal endothelium to riboflavin in vivo.METHODS:The right eyes of 30 New Zealand white rabbits were divided into three groups. Different concentrations riboflavin-balanced salt solutions (BSS) were injected into the anterior chamber (10 with 0.5%, 10 with 1%, and 10 with 2%). Eight corneal buttons of 8.5 mm in diameter from each group were dissected at 30min after injection and the riboflavin concentrations in the corneal stroma were determined using high-performance liquid chromatography (HPLC) after removing the epithelium and endothelium. The other two rabbits in every group were observed for 24h and sacrificed. As a comparison, the riboflavin concentrations from 16 corneal stromal samples were determined using HPLC after instillation of 0.1% riboflavin-BSS solution for 30min on the corneal surface (8 without epithelium and 8 with intact epithelium).RESULTS: The mean riboflavin concentrations were 11.19, 18.97, 25.08, 20.18, and 1.13 μg/g for 0.5%, 1%, 2%, de-epithelialzed samples, and the transepithelial groups, respectively. The color change of the corneal stroma and the HPLC results showed that enrichment with riboflavin similar to classical de-epithelialized corneal collagen crosslinking (CXL) could be achieved by intracameral1% riboflavin-BSS solution after 30min; the effect appeared to be continuous for at least 30min.CONCLUSION:Riboflavin can effectively penetrate the corneal stroma through the endothelium after an intracameral injection in vivo, so it could be an enhancing method that could improve the corneal riboflavin concentration in transepithelial CXL.     

Corneal collagen crosslinking in keratoconus and other eye disease

Keratoconus is a condition characterized by biomechanical instability of the cornea, presenting in a progressive, asymmetric and bilateral way. Corneal collagen crosslinking (CXL) with riboflavin and Ultraviolet-A (UVA) is a new technique of corneal tissue strengthening that combines the use of riboflavin as a photo sensitizer and UVA irradiation. Studies showed that CXL was effective in halting the progression of keratoconus over a period of up to four years. The published studies also revealed a reduction of max K readings by more than 2 D, while the postoperative spherical equivalent (SEQ) was reduced by an average of more than 1 D and refractive cylinder decreased by about 1 D. The major indication for the use of CXL is to inhibit the progression of corneal ecstasies, such as keratoconus and pellucid marginal degeneration. CXL may also be effective in the treatment and prophylaxis of iatrogenic keratectasia, resulting from excessively aggressive photo ablation. This treatment has been used to treat infectious corneal ulcers with apparent favorable results. Most recent studies demonstrate the beneficial impact of CXL for iatrogenic ecstasies, pellucid marginal degeneration, infectious keratitis, bullous keratopathy and ulcerative keratitis. Several long-term and short-term complications of CXL have been studied and documented. The possibility of a secondary infection after the procedure exists because the patient is subject to epithelial debridement and the application of a soft contact lens. Formation of temporary corneal haze, permanent scars, endothelial damage, treatment failure, sterile infiltrates, bullous keratopathy and herpes reactivation are the other reported complications of this procedure.     

血府逐瘀汤中芍药苷在大鼠体内的药代动力学特性

目的: 研究血府逐瘀汤中芍药苷在大鼠体内的药代动力学特性。 方法: 采用HPLC测定大鼠血清中芍药苷含量,Diamonsil C₁₈色谱柱(4.6 mm×250 mm,5 μm),柱温30 ℃,流动相乙腈-水(16 :84),流速1.0 mL ·min^-1,检测波长230 nm,进样量20 μL,以核黄素为内标物。 结果: 芍药苷质量浓度在0.01~1.0 mg ·L^-1线性关系良好。定量限0.01 mg ·L^-1。平均日内、日间精密度分别为3.3%,3.9%,平均回收率101.3%。大鼠灌胃血府逐瘀汤后芍药苷的药峰浓度(C_max)(0.363±0.080) mg ·L^-1,达峰时间(T_max)(0.276±0.084) h,吸收速率常数(K_a) (30.905±10.114) h^-1,末端消除速率(K_e)(1.638±0.181) h^-1,半衰期(t_1/2)(0.501±0.038) h,清除率/绝对生物利用度(CL/F)(69.846±4.624) L ·h^-1 ·kg^-1,表观分布容积/绝对生物利用度(V_z/F)(36.521±12.287) L ·kg^-1,药时曲线下面积(AUC_0-t)(0.356±0.024) mg ·L^-1 ·h。 结论: 血府逐瘀汤中芍药苷在大鼠体内具有吸收速率快、分布容积大、生物半衰期短的药代动力学特性,为研究药物配伍对芍药苷药动学的影响和血府逐瘀汤发挥药效的作用机制提供参考。     

利用动物模型评价核黄素光化学法灭活红细胞病毒的效果

本研究旨在评估核黄素光化学法(核黄素终浓度为150μmol/L,光照时间为20分钟,光照强度为40 000Lux)灭活红细胞中病毒的有效性。将HCMV作为指示病毒加入红细胞中。30只BALA/c小鼠设为实验组(n=10)、病毒对照组(n=10)、可见光对照组(n=5)和红细胞对照组(n=5);实验组小鼠注射经核黄素光化学法灭活处理的红细胞;病毒对照组小鼠注射未经灭活处理的红细胞;可见光对照组小鼠注射经可见光照射的红细胞;红细胞对照组小鼠注射正常红细胞。取各组小鼠进行体外病毒分离,PCR检测HCMV UL83基因,间接免疫荧光鉴定PP65抗原。结果表明:病毒对照组和可见光对照组病毒分离、PCR及间接免疫荧光检测均为阳性,而实验组和红细胞对照组所有结果均为阴性。结论:核黄素光化学法灭活红细胞病毒是有效的。     

跨角膜上皮核黄素-紫外线胶原交联的研究进展

角膜胶原交联(CXL)是一种用于治疗圆锥角膜的新技术,其主要特点是利用370nm波长的紫外线A照射核黄素产生的光敏反应来诱导角膜胶原纤维间的交联,以此提高角膜硬度,增强角膜生物力学稳定性。该方法目前已被应用于多种角膜疾病的治疗,并取得了较理想的临床疗效。但最近也有文献报道经典交联方法术前去角膜上皮所引起的一些术后不良反应和并发症。针对去角膜上皮引起的术后不良反应和并发症有学者提出了跨角膜上皮核黄素-紫外线胶原交联法。本文主要对角膜胶原交联的基本原理、操作方法、去角膜上皮的影响及近年来跨上皮交联方法的研究与应用情况进行综述。     

Pathogen reduction of buffy coat platelet concentrates using riboflavin and light: comparisons with pathogen-reduction technology-treated apheresis platelet products

BACKGROUND AND OBJECTIVES: A pathogen-reduction technology (PRT) system using riboflavin and light has been developed for the treatment of platelet concentrates (PC) obtained by either buffy coat preparation (BCPC) or apheresis procedures (APPC). The aim of this study was to evaluate the effects of the treatment process on in vitro cell quality and on riboflavin conversion in PC. MATERIALS AND METHODS: BCPC were prepared with the Compomat G4 from whole blood which had been stored overnight after collection. APPC were obtained using the TRIMA apheresis procedure. Both PC products had been stored for 18-24 h prior to PRT treatment. BCPC and APPC were treated with PRT on day 2 and day 1 of shelf-life, respectively. The treated PCs were then maintained for an additional 5 days after the PRT treatment. A panel of cell quality assays and high-performance liquid chromatography (HPLC) analysis were performed. RESULTS: Cell counts and plasma lactate dehydrogenase (LDH) levels during storage indicated that PRT did not induce significant cell lysis. Acceleration of a decrease in glucose and an increase in lactate was observed for treated PCs, but no significant differences were observed between treated BCPC and APPC. The pH of treated samples remained above 7.0, although was lower than that of the control. Platelet morphology of BCPC and APPC was well preserved. P-selectin expression indicated significant platelet activation when compared with control PC (BCPC on day 6: 39% vs. 12%; APPC on day 5: 35% vs. 18%). Both P-selectin expression and microparticle formation were not significantly different between treated BCPC and APPC during storage. The JC-1 assay also displayed no loss of mitochondria integrity during the storage of treated products. Approximately 20% of riboflavin converted into photoproducts, including lumichrome. CONCLUSIONS: PRT treatment had an effect on the development of the normal platelet storage lesion at a level which seems tolerable for clinical usage.     

A comparison of methods of pathogen inactivation of FFP

Introduction Current methods for pathogen inactivation of plasma involve four major processes using solvent–detergent (SD), methylene blue (MB), amotosalen and riboflavin as additives. Three of these methods involve the use of visible or ultraviolet light. Methods A comparison of the four methods was made using publications in Medline, Pubmed, Embase and Biosis to obtain data on the logistics of use, the quality of the plasma proteins and the effectiveness of pathogen inactivation. Results Three of the methods, MB, amotosalen and riboflavin, are designed for use in a blood bank; the SD method is generally applied at a centralized manufacturing centre and involves large plasma pools. All methods result in a reduction in protein values with the per cent retention of FVIII activity in the range of 67–78% and fibrinogen of 65–84%. Protein S and alpha₂‐antiplasmin are lower following solvent–detergent treatment. Alterations in fibrinogen structure have been reported with methylene blue. Discussion Three of the methods are designed for small volume use in a blood bank. All four methods have some effect on the coagulant proteins; however, the final concentrations are within regulated limits. While there is variability in the effectiveness against pathogens, direct comparison is difficult because of the methodologies used. Nonetheless, all are effective in inactivating HIV and other lipid‐enveloped pathogens. Clinical studies on the effectiveness of these products are surprisingly sparse, and no randomized clinical trials have yet been performed with amotosalen or riboflavin plasmas.     

Pathogen inactivation of Leishmania donovani infantum in plasma and platelet concentrates using riboflavin and ultraviolet light

BACKGROUND AND OBJECTIVES: Leishmania is transmitted by the bite of the phlebotomine sandfly or by transfusion of infected blood products. Leishmaniasis currently poses a significant problem in several parts of the world, and is an emerging problem in others. The Mirasol PRT technology is based on the use of riboflavin and ultraviolet light to generate chemical reactions in the nucleic acids of pathogens, which prevents replication and leads to inactivation. The intent of this study was to examine the ability of the Mirasol PRT System to kill the Leishmania parasite in human plasma and platelet concentrates. MATERIALS AND METHODS: In visceral Leishmaniasis, amastigotes are present in the blood and in the reticuloendothelial system within monocytes. For each unit of plasma or platelets treated, isolated mononuclear cells obtained from 100 ml of normal donor whole blood were incubated with 1.0 x 10(8) Leishmania donovani infantum promastigotes to produce amastigote-laden macrophages. The infected macrophages were added to 250 ml of human plasma or to 250 ml of platelet concentrates. Infected units were cultured pretreatment in 10-fold serial dilutions to determine the limits of detection. Thirty millilitres of 500 microM riboflavin was added to each unit, which was then illuminated with 5.9 J/cm2 of ultraviolet light (6.24 J/ml). After treatment and after 2 months of frozen storage, plasma units were cultured in 10-fold serial dilutions. Platelets were cultured on the day of treatment and on day 5 of storage post-illumination. RESULTS: A 5 log reduction of Leishmania was demonstrated in five of six units of plasma, and a 7 log reduction of Leishmania was demonstrated in one plasma unit. A 5 log reduction of Leishmania was demonstrated in five of six units of platelets, and a 6 log reduction of Leishmania was demonstrated in one unit. CONCLUSIONS: There is no donor screen for Leishmania and other pathogens constantly emerging in our blood supply. The Mirasol PRT System for Platelets and Plasma is an effective means of killing Leishmania and other emerging pathogens in these blood products.