Allograft semilunar cardiac valves processing and cryopreservation – morphology in scanning electron microscope

Objective: The most important factors of long term clinical performance of biological heart valve prostheses are methods of processing and cryopreservation. That is why we decided to evaluate the impact of current Allograft Heart Valves (AHV) Bank protocol on valve tissue morphology. Scanning electron microscope (SEM) is a valuable tool for investigation of biological surfaces. In case of cardiac valves it is especially suitable for detection of fine changes in endothelial covering and underlying layers. Material and methods: “Fresh” aortic and pulmonary AHV samples, harvested from “heart-beating” cadaveric donors, were compared with (1) tissue from AHV obtained from non heart-beating donors, (2) samples stored in 4 °C saline for 24 h, (3) antibiotic treated tissue for 24 h at 37 °C and finally (4) cryopreserved valves, stored in liquid nitrogen (−196 °C) for 6–38 months. All samples were dissected, dried with hexamethyldisilazane (HMDS), gold coated, studied and photographed by SEM (Tesla BS 301). Results: Our alternative method of drying samples by the HMDS method proved to be suitable for thin membranes of human semilunar valves. We were able to detect early changes in the endothelium after harvesting, and denudation of the endothelial covering during preservation with and without freezing. Conclusion: SEM (using HMDS drying) along with other methods may be helpful for the morphological control of processing, cryopreservation and liquid nitrogen storage of AHV. According to the current findings we have to avoid washing of AHV in saline after harvesting.     

Correction: Simultaneous confidence regions for closed tests,including Holm‐, Hochberg‐, and Hommel‐related procedures

Algorithm 1 in Guilbaud (2012, p. 327) in Biometrical Journal (DOI: 10.1002/bimj.201100123 ) reproduced a recently detected index error in a theorem concerning a shortcut for rejection decisions for certain multiple‐testing procedures as it was stated in Bernhard et al. (2004, p. 8) in Statistical Papers (DOI: 10.1007/BF02778266 ). This short article provides: (i) the correction to be made to Algorithm 1 and (ii) a brief discussion of the consequences. Although the theoretical developments in Guilbaud (2012) are not affected, the numerical illustrations in Section 7 are affected. A corrected version of that section is given in the Supporting Information.     

Activity of bone morphogenetic protein-7 after treatment at various temperatures: freezing vs. pasteurization vs. allograft

Insufficient bone union is the occasional complication of biomechanical reconstruction after malignant bone tumor resection using temperature treated tumor bearing bone; freezing, pasteurization, and autoclaving. Since bone morphogenetic protein (BMP) plays an important role in bone formation, we assessed the amount and activity of BMP preserved after several temperature treatments, including −196 and −73 °C for 20 min, 60 and 100 °C for 30 min, 60 °C for 10 h following −80 °C for 12 h as an allograft model, and 4 °C as the control. The material extracted from the human femoral bone was treated, and the amount of BMP-7 was analyzed using an enzyme-linked immunosorbent assay. Then, the activity of recombinant human BMP-7 after the treatment was assessed using a bioassay with NIH3T3 cells and immunoblotting analysis to measure the amount of phospho-Smad, one of the signaling substrates that reflect the intracellular reaction of BMPs. Both experiments revealed that BMP-7 was significantly better preserved in the hypothermia groups. The percentages of the amount of BMP-7 in which the control group was set at 100% were 114%, 108%, 70%, 49%, and 53% in the −196, −73, 60, 100 °C, and the allograft-model group, respectively. The percentages of the amount of phospho-Smad were 89%, 87%, 24%, 4.9%, and 14% in the −196, −73, 60, 100 °C, and the allograft-model group, respectively. These results suggested that freezing possibly preserves osteoinductive ability than hyperthermia treatment.     

Allograft inflammatory factor-1 stimulates chemokine production and induces chemotaxis in human peripheral blood mononuclear cells

Allograft inflammatory factor-1 (AIF-1) is expressed by macrophages, fibroblasts, endothelial cells and smooth muscle cells in immune-inflammatory disorders such as systemic sclerosis, rheumatoid arthritis and several vasculopathies. However, its molecular function is not fully understood. In this study, we examined gene expression profiles and induction of chemokines in monocytes treated with recombinant human AIF (rhAIF-1). Using the high-density oligonucleotide microarray technique, we compared mRNA expression profiles of rhAIF-1-stimulated CD14⁺ peripheral blood mononuclear cells (CD14⁺ PBMCs) derived from healthy volunteers. We demonstrated upregulation of genes for several CC chemokines such as CCL1, CCL2, CCL3, CCL7, and CCL20. Next, using ELISAs, we confirmed that rhAIF-1 promoted the secretion of CCL3/MIP-1α and IL-6 by CD14⁺ PBMCs, whereas only small amounts of CCL1, CCL2/MCP-1, CCL7/MCP-3 and CCL20/MIP-3α were secreted. Conditioned media from rhAIF-1stimulated CD14⁺ PBMCs resulted in migration of PBMCs. These findings suggest that AIF-1, which induced chemokines and enhanced chemotaxis of monocytes, may represent a molecular target for the therapy of immune-inflammatory disorders.     

CCR7 在树突状细胞中的功能及对角膜免疫调节的研究进展

树突状细胞(dendritic cells, DCs)是体内已知的功能最强大的专职抗原提呈细胞,对于诱导机体初始免疫应答尤为重要。趋 化受体因子7 (chemokine receptor 7,CCR7)是一个已知的调节各类免疫细胞向初级、次级淋巴细胞分化,并向外周淋巴器官归巢 的趋化因子受体,其具有自我平衡表达能力,在趋化DCs 从外周组织迁移至次级淋巴器官中起关键作用。随着研究的深入,除了 CCR7 最主要的趋化作用外,更多的功能逐渐被了解。目前,DCs 和CCR7 的相关功能已被应用于诱导角膜移植后的免疫耐受等 眼科领域。就CCR7 在DCs中的功能及其对角膜免疫调节的影响进行综述,探讨其关键作用及可能的治疗靶点。     

兔组织工程同种异体气管支架三种制备方法的比较

目的:探讨深低温冷冻-酶洗法制备的气管支架在去除抗原性、维持生物力学及保护细胞外基质方面的效果。方法:健康新西兰兔24只随机分为气管未处理作为对照A组,深低温冷冻法处理B组,玻璃化法处理C组,深低温冷冻-酶洗法D组,各组样本数均为6。处理后将各组标本行HE染色后光镜观察,戊二醛固定后电镜扫描,并测量气管最大拉伸力、破裂力和变异率等生物力学性能。结果:组织学观察显示对照A组有大量完整的粘膜上皮细胞;B组和C组可见部分气管粘膜上皮细胞;D组标本未见气管粘膜上皮细胞,且细胞核碎裂。电镜显示A、B、C、D组气管支架可见丰富的细胞基质,未暴露胶原纤维。组间两两比较,气管支架的最大拉伸力、最大破裂力和变异率均无统计学差异。结论:综合组织学、扫面电镜和生物力学分析,应用深低温冷冻-酶洗法制备气管支架D组可以有效地去除抗原,维持生物力学性能,并具有较完整的细胞外基质。     

不同移植物对兔前叉韧带重建术后血运变化的影响

目的：比较自体和同种异体半肌腱重建兔前叉韧带对血管生成的影响。方法：选择48 只骨骼发育成熟的新西兰种属兔作为 研究对象,将其随机分为自体组(A 组)和异体组(B 组),每组24 只,分别接受半肌腱重建前叉韧带手术。从手术日期开始,分别记 录手术后第3 周、第6 周、第12 周和第24 周重建韧带的血管生成情况同时对血管生成数量进行定量。结果：手术后两组实验兔 关节均无肿胀,关节囊无炎性反应发生。在手术后第3 周,两组血管生成无明显差异(P>0.05);术后第6 周和第12周,自体移植组 新生血管数量明显高于异体移植组(P<0.05);术后第24 周,异体组新生血管数量明显高于自体组(P<0.05)。结论：在兔前叉韧带的 重建过程中,异体移植物重建的血管生成速度和数量明显慢于自体移植物,提示在临床上选择自体移植物对术后恢复更理想.     

Modeling the consequence of increased host tolerance toward avian brood parasitism

Avian brood parasites greatly reduce the reproductive success of their hosts. Empirical studies have demonstrated that some hosts have evolved defenses against parasitism like an ability to recognize and reject parasitic eggs that are dissimilar to their own eggs. Detailed mechanisms of how hosts recognize parasitism still remain unknown, but recent studies have shown that the host’s recognition, in many cases, is based on discordance of the eggs in a clutch, and that hosts are more error-prone when the nest is multiply parasitized, i.e., hosts tend to accept more multiple parasitism than single parasitism. In an area in Hungary, the great reed warbler Acrocephalus arundinaceus, one of the main hosts of the common cuckoo Cuculus canorus, is heavily parasitized and the parasitism rate has been kept at quite a high level for decades. Previous mathematical models suggest that such a high parasitism rate can be maintained because the focal host population behaves as a sink where few hosts can reproduce but immigration from outside replenishes the loss of host reproduction in the sink population. Here, we explore the consequences of the increased host tolerance towards multiple parasitism which has been overlooked in the previous studies using a simple model. Our model analysis shows that the increased host tolerance can dramatically contribute to both the parasite abundance and the parasitism rate being kept at a high level. We suggest that such a host behavior, combined with host immigration, can be an important factor responsible for the observed severe parasitism.     

Recognition of scent marks in solitary bees to avoid previously visited flowers

Knowing how floral visitors forage efficiently among flowers is important to understanding plant-pollinator interactions. When bees search for rewarding flowers, they use several visual cues to detect the available floral resources. In addition to these cues, bees can recognize scent marks, which are olfactory cues left on flowers foraged by previous visitors. This behavior is well known in social bees, such as honeybees and bumblebees. Although solitary bees do not need to give information about which flowers were foraged to conspecifics, several pieces of evidence have indicated the use of scent marks. However, it is unknown whether the behavior is widely used in many different bee species. We investigated whether four different solitary bees, Colletes patellatus (Colletidae), Andrena prostomias (Andrenidae), Osmia orientalis (Megachilidae), and Tetralonia mitsukurii (Apidae), can recognize flowers that have been foraged previously by visitors within 3 min. All four bees showed rejection responses to flowers foraged by conspecifics. However, our results showed that responses to foraged flowers varied among bee species. The tendency of A. prostomias and T. mitsukurii to reject the foraged flowers was pronounced, while in C. patellatus and O. orientalis it was weak. In both A. prostomias and T. mitsukurii, the rejection rate of flowers foraged by conspecifics decreased as the time lag after the last visit increased. Both bees visited the flowers from which pollen or nectar had been artificially removed. We suggest that A. prostomias and T. mitsukurii would recognize scent marks left by previous visitors, while the other two bees would not recognize them so strongly. It is likely that the decision to use scent marks is dependent either on the richness of resources or on the complexity of floral structure.     

Combination of Ferulic Acid,Ligustrazine and Tetrahydropalmatine attenuates Epithelial-mesenchymal Transformation via Wnt/β-catenin Pathway in Endometriosis

Previously the potential therapeutic action of ferulic acid, ligustrazine and tetrahydropalmatine (FLT) are discovered with unclear mechanism in rat autograft endometriosis. However, the effect of FLT on endometrial cells and allograft endometriosis is still unclear. This study is designed to elucidate the influence of FLT on epithelial-mesenchymal transformation in allograft endometriosis and endometrium cells. In vivo, fluorescent xenogeneic endometriosis model was established. In vitro, invasion and metastasis were analyzed after treating FLT. Epithelial-mesenchymal transformation and Wnt/β-catenin pathway were inspected in vitro and in vivo. Activator or inhibitor of Wnt/β-catenin signaling was performed to inspect mechanism of epithelial-mesenchymal transformation. In vivo, FLT not only decreased fluorescent intensity and volume of ectopic lesion, but also ameliorated pathological morphology. E₂ and PROG levels in serum were reduced by FLT. In endometrial cells, FLT significantly inhibited the invasion and metastasis. Meantime, epithelial-mesenchymal transformation was reversed, accompanied by suppression of Wnt/β-catenin pathway. In-depth study, activation of Wnt/β-catenin pathway lead to promotion of epithelial-mesenchymal transformation, which was reversed by FLT. FLT prevented fluorescent allograft endometriosis and endometrium cells, which was related to suppress epithelial-mesenchymal transformation through inactivating Wnt/β-catenin pathway. The findings disclose molecular mechanism of epithelial-mesenchymal transformation in endometriosis by FLT, and contribute to further application.