Comparison of a Commercial ELISA with the Modified Agglutination Test for Detection of Toxoplasma gondii Antibodies in Sera of Naturally Infected Dogs and Cats

Background

Toxoplasma gondii can infect all warm-blooded animals. Modified agglutination test (MAT) and ELISA are widely used for the detection of T. gondii antibodies. However, there is little information on their acceptability for detecting antibodies in companion animals.

Methods

This study compared ELISA and MAT for their ability to detect T. gondii infection in naturally infected dogs and cats. Blood samples were collected from dogs and cats in different areas of Beijing, China and analyzed by ELISA and MAT. The χ² test and κ analysis were used to evaluate their efficiency and agreement.

Results

For dogs, the seroprevalence of T. gondii antibodies detected by ELISA was 34.7%, which was significantly higher than that detected by MAT (P<0.05). There was no significant difference between ELISA and MAT for detecting T. gondii antibodies in cats. Good agreements between MAT and ELISA were seen in both dogs and cats; however, inconsistent results were demonstrated by κ analysis and in MAT titer assay.

Conclusion

Serum-based ELISA may be more satisfactory for screening test of T. gondii infection in dogs, whereas both methods could be acceptable in cats.     

Long-term treatment of lupus nephritis with cyclosporin A

We evaluated the efficacy and safety of long-term treatment with cyclosporin A (CSA) in type IV lupus nephritis. Seventeen patients with biopsy-proven WHO type IV lupus nephritis were enrolled in a prospective, open study. Twelve of the 17 completed 48 months of treatment with CSA and prednisolone. Three patients required the addition of azathioprine, at 12, 38 and 47 months, respectively, for cutaneous disease flare with refractory rashes. One patient was lost to follow-up at 40 months. The mean +/- SD duration of treatment was 43.2 +/- 10.1 months (range 15.7-48 months). A significant reduction of proteinuria and a significant rise in serum albumin were noted 1 month after initiation of treatment. Improvement was maintained throughout the study except for three patients who relapsed with recurrence of nephrotic syndrome. There were no significant changes in serum creatinine level or creatinine clearances throughout the study. Repeat renal biopsy at 12 months following treatment with CSA showed histological improvement, with WHO type II changes in all 17 patients accompanying significant reduction in activity indices. Patients with baseline haemoglobin (Hgb) levels < 12 g/dl showed significant improvement. Serum C3 and C4 levels were not changed significantly. Corticosteroid-sparing effects were noted. Side-effects included hypertension, gum hypertrophy and mild hirsuitism, but were not serious. Combination therapy using CSA and prednisone is effective and safe for long-term treatment in lupus patients with WHO type IV nephritis.      

应用干细胞分离仪分离脐血与传统手工分离法的效果比较

目的:脐血处理的关键问题是提高干细胞的回收率及实现处理过程的标准化和可重复化,实验对此进行探讨,比较干细胞分离仪与传统羟乙基淀粉手工法分离脐血的效果。方法:实验于2006-12/2007-05在广州医学院附属市一人民医院完成。①脐血来源:39份脐血采自广州医学院附属市一人命医院妇产科健康顺产新生儿脐带,产妇均知情同意。随机数字表法分为仪器分离组17份、手工分离组22份。②实验方法:仪器分离组收集脐血称质量,计算体积,在开始处理前20min缓慢加入相当于20%脐血体积的60g/L羟乙基淀粉。仪器分离组按仪器要求自动分离,分离终体积20mL。手工分离组50g离心5min,压浆板压出全部血浆以及18mL红细胞移至无菌空袋,500g离心13min,自动压浆板压出血浆,保留20mL终体积样本。③实验评估:采用全自动计数仪进行检测有核细胞(白细胞)、红细胞数量。流式细胞仪分析CD34 含量。结果:采用干细胞分离仪处理浓缩脐血,有核细胞回收率为(89.7±3.4)%,CD34 细胞回收率为(98.8±5.1)%,红细胞去除率为(55.2±16.7)%,均比手工分离组分离效果好,差异有显著性意义(P<0.05或0.01)。同时,仪器分离组有核细胞回收率、CD34 细胞回收率的标准差均明显低于手工分离组(3.4vs.15.3;5.1vs.10.3)。结论:相比传统的羟乙基淀粉手工分离法,干细胞分离仪脐血分离浓缩效果理想,且结果标准误小,数据稳定。     

减压治疗跗跖关节骨折脱位合并前足筋膜间隙综合征：术后足弓形态及感觉运动功能恢复的评估

目的:评估减压治疗跗跖关节骨折脱位合并前足筋膜间隙综合征的疗效和前足感觉及运动功能的康复。方法:于1996-05/2003-04选择第二军医大学长征医院骨科收治的跗跖关节骨折脱位合并前足筋膜间隙综合征患者17例,利用whiteside测压装置进行前足部筋膜间隙内压监测,间隙内压>3.99kPa,均经前足背侧入路进行筋膜间隙切开减压术,同时行复位内固定恢复足的纵弓及横弓。固定48h后,进行功能康复训练,主、被动地活动前足各关节及短波红外线治疗。结果:术后1个月进行第一次随访,前两年每3个月随访1次,以后每半年随访1次。17例患者均随访3.5以上。术后6个月随访时评估疗效,足外形正常,活动范围正常,负重行走无不适,X射线片显示正常11例;足活动轻度受限,不影响行走、工作,可进行适当的体育活动,X射线片显示跗跖关节移位<2mm4例;行走或站立过久时前足部有轻度疼痛4例,其中3例前足增宽,足弓较健侧减小,2例两点辨别觉、痛觉略减退。无一例出现前足僵硬、爪形趾、软组织萎缩及运动功能异常。结论:减压是治疗跗跖关节骨折脱位合并前足筋膜间隙综合征的有效方案,减压后应及时进行进行功能康复训练,并辅以短波红外线治疗,能有效地恢复前足感觉、运动功能。     

Catabolism of lysosome-related organelles in color-changing spiders supports intracellular turnover of pigments

Pigment organelles of vertebrates belong to the lysosome-related organelle (LRO) family, of which melanin-producing melanosomes are the prototypes. While their anabolism has been extensively unraveled through the study of melanosomes in skin melanocytes, their catabolism remains poorly known. Here, we tap into the unique ability of crab spiders to reversibly change body coloration to examine the catabolism of their pigment organelles. By combining ultrastructural and metal analyses on high-pressure frozen integuments, we first assess whether pigment organelles of crab spiders belong to the LRO family and second, how their catabolism is intracellularly processed. Using scanning transmission electron microscopy, electron tomography, and nanoscale Synchrotron-based scanning X-ray fluorescence, we show that pigment organelles possess ultrastructural and chemical hallmarks of LROs, including intraluminal vesicles and metal deposits, similar to melanosomes. Monitoring ultrastructural changes during bleaching suggests that the catabolism of pigment organelles involves the degradation and removal of their intraluminal content, possibly through lysosomal mechanisms. In contrast to skin melanosomes, anabolism and catabolism of pigments proceed within the same cell without requiring either cell death or secretion/phagocytosis. Our work hence provides support for the hypothesis that the endolysosomal system is fully functionalized for within-cell turnover of pigments, leading to functional maintenance under adverse conditions and phenotypic plasticity. First formulated for eye melanosomes in the context of human vision, the hypothesis of intracellular turnover of pigments gets unprecedented strong support from pigment organelles of spiders.

How and why animals produce their colors are fundamental questions in biology. In cases where pigments are involved, they are usually synthesized and stored in specialized intracellular organelles (1). Bagnara et al. (2) postulated in 1979 that all pigment organelles of vertebrates derive from a common primordial organelle. Since then, an increasing body of evidence has shown that pigment organelles, from mammal melanosomes to snake pterinosomes, belong to the lysosome-related organelle (LRO) family (3, 4). LROs intersect the endolysosomal system (5), which forms a complex and active network of membrane-bound compartments produced by endocytic and secretory pathways (6). Studying the subcellular aspect of LROs in relation to pigmentation is therefore a critical step to understand animal coloration.Detailed investigations of intracellular processes and trafficking leading to the biogenesis of pigment LROs have been largely performed on mammalian melanosomes (3, 5). They revealed that skin melanocytes divert components of the endolysosomal system to progressively generate melanosome precursors derived from endosomes, bearing intraluminal vesicles (ILVs) and amyloid fibrils, and then mature pigmented melanosomes that are transferred to keratinocytes. Melanosome formation is controlled by a range of genes involved in the endolysosomal system of mammals (3, 5). The finding of homologous genes controlling pterinosomes, iridisomes, and ommochromasomes of snakes and insects, among others, has led to a general model of pigment organelle formation (3, 4, 7). However, coloration involves not only pigmentation phases but also, bleaching phases that lead to pigment removal, a process that is far less understood, even for melanosomes (8).While bleaching can result from death of pigment-containing cells, such as during peeling (9), this process is also compatible with pigment cells remaining alive (10). The latter phenomenon questions how pigment cells accommodate both the production and the removal of pigment organelles, as well as whether recycling pathways connecting the two phases exist. The occurrence of within-cell melanosomal degradation as a common physiological process required for melanin turnover has long been debated (8, 11–13). Here, turnover is defined as a pigmentation–depigmentation–repigmentation cycle at the cellular scale, without requiring the reuse of organellar materials. Evidence for physiological degradations of pigment LROs remains scarce [but see the studies on differences in human skin pigmentation (14, 15)], which we attribute to the rarity of biological systems displaying active and concomitant production and removal of pigments within a single cell and a lack of studies focusing on this phase.Color-changing crab spiders can dynamically match the flower color on which they hunt (16, 17). They do so by reversibly changing their body coloration over the course of a few days, up to weeks, between white and yellow (18). This slow morphological color changes results from the metabolism of yellow pigments, thought to be ommochromes, in integument cells (10, 18, 19). Ommochromes are pigments deriving from tryptophan through the formation of colored kynurenines and transient precursors (7, 20). They are involved in various color-changing species, including dragonflies and cephalopods (21–23). The crab spider is the best understood nonmodel system among the ones displaying morphological color changes in terms of pigment organelle metabolism, ranging from locusts to planarians (24, 25). During yellowing (i.e., anabolic phase), pigments are deposited within specialized intracellular organelles, whose intracellular origin remains undetermined (19). During bleaching, pigment organelles are thought to undergo an autocatalytic process and to recycle their membrane for another cycle of yellowing (10). However, ultrastructural and chemical evidence for this recycling process is scarce, and we do not fully comprehend how both anabolism and catabolism of ommochromes and pigment organelles of crab spiders fit within intracellular trafficking pathways (7). Testing the hypothesis that these pigment organelles are members of the LRO family may help position them into the endolysosomal system, which in return, might provide insights into the roles of the endolysosomal system in both pigmentation and bleaching phases, as well as in reversibility.LROs are defined as being morphologically distinct from lysosomes, containing a subset of cell type–specific contents necessary for their function and being in many instances secretory organelles. However, LROs also share features with lysosomes such as the presence of lysosomal-associated membrane proteins, hydrolases, and an acidic pH (at least temporary), and some can be accessible via the endocytic pathway (3, 5). In practice, the assignment of organelles to the LRO family is based on genetic defects associated with human diseases, such as Hermansky–Pudlak, Griscelli, and Chediak–Higashi syndromes (3–5). However, such genetic dependence cannot be tested in spiders because they are in most cases not genetically tractable. Therefore, other markers of their secretory/endocytic nature should be assessed. LROs often bear ultrastructural signatures of their intracellular origin, such as endosomal/Golgi connections, ILVs, physiological amyloid fibrils, and membrane tubulations (6). Conversely, LROs act as important regulators of metals by storing and releasing them in a dynamic manner (26). Specific techniques are required to retain such labile signatures as membrane tubulations and metals. For relatively thick tissues, this is best done by high-pressure freezing (HPF), which avoids artifacts produced by chemical fixatives (27). As an example, electron microscopy (EM) combined with HPF successfully revealed the morphological details of LROs like melanosomes (28) and Weibel–Palade bodies (29, 30) and their close contacts with other organelles (28). Given the submicrometer size of pigment organelles (19), detection of metals in their lumen can only be tackled by highly sensitive and spatially resolved nanoimaging methods (31), like scanning Synchrotron X-ray fluorescence (SXRF). SXRF is a state-of-the-art chemical imaging technique to map multiple trace elements with subpart per million sensitivity down to a few tens of nanometers resolution (32–34). Therefore, to reveal the intracellular origin of pigment organelles and their catabolism, we exploited the combination of EM and SXRF on white, yellow, and bleaching crab spiders fixed in a near-native state by HPF (SI Appendix discusses the overall fixation quality; SI Appendix, Fig. S1).