Department of Microbiology, Immunology, and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO.
Department of Microbiology, Immunology, and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO.
Exp Hematol. 2020 Jun;86:43-52.e1. doi: 10.1016/j.exphem.2020.05.002. Epub 2020 May 14.
Whole transferrin receptor (TfR) is present in reticulocyte exosomes. Soluble transferrin receptor (sTfR) is cleaved from whole TfR in human plasma, with the remnant cytoplasmic domain (cTfR) remaining membrane associated. In humans, sTfR is a biomarker that can detect iron deficiency in the presence of inflammatory disease. This condition is still a diagnostic dilemma in veterinary species. We aimed to (1) confirm the presence of exosomes and exosome-associated TfR in the serum of dogs, cats, and horses; and (2) to assess and compare the proportion of cTfR to total (cTfR + whole) in exosomal membranes of healthy and diseased dogs and cats and in healthy horses to indirectly predict their anticipated levels of circulating sTfR. We used discarded serum and whole blood samples from canine and feline patients, separated into healthy and diseased groups based on the health status of each patient, and healthy equine participants from a previous study. Ultracentrifugation, followed in some experiments by OptiPrep discontinuous density gradient fractionation, was used to isolate exosomes. Exosomes and associated TfR were identified using TEM and Western blot for TfR, respectively. Densitometry tracings of Western blots of serum exosomes were used to measure the proportion of cTfR to total TfR. Extracellular vesicles compatible with exosomes were successfully isolated and expressed TfR. The proportion of cTfR in dogs was greater than 50%, indicating that a majority of the whole TfR was cleaved to produce sTfR (and remnant cTfR). There was significant interindividual variation and no significant difference between healthy and diseased animals. The proportion of cTfR in cats was very low at 11%, indicating that very little sTfR was likely produced. There was a small yet significant difference between healthy and diseased cats. Healthy horses do not appear to cleave exosome-associated TfR. Diagnosis of iron deficiency in the presence of inflammatory disease remains a challenge in veterinary medicine. Our results indicate that TfR is poorly or unpredictably cleaved in veterinary species, revealing that there are species differences in exosomal TfR handling. These data suggest that development of an assay for the detection and quantification of sTfR in the species investigated may not be warranted.
全转铁蛋白受体（TfR）存在于网织红细胞外体中。可溶性转铁蛋白受体（sTfR）在人血浆中从全 TfR 中切割，残留的细胞质结构域（cTfR）保持膜相关。在人类中，sTfR 是一种生物标志物，可在存在炎症性疾病的情况下检测缺铁。这种情况在兽医物种中仍然是一个诊断难题。我们的目的是：（1）确认犬、猫和马血清中外体和外体相关 TfR 的存在；（2）评估和比较健康和患病犬和猫的外体膜中 cTfR 与总（cTfR+全）的比例，并间接预测其预期的循环 sTfR 水平。我们使用了来自犬和猫患者的废弃血清和全血样本，根据每个患者的健康状况将其分为健康组和患病组，并使用来自先前研究的健康马参与者。使用超速离心，在某些实验中使用 OptiPrep 不连续密度梯度分级分离来分离外体。使用 TEM 鉴定外体，使用 Western blot 鉴定 TfR 分别鉴定相关的 TfR。使用血清外体 Western blot 的密度追踪来测量 cTfR 与总 TfR 的比例。成功分离出与外体相容的细胞外囊泡并表达了 TfR。犬的 cTfR 比例大于 50%，表明大部分全 TfR 被切割产生 sTfR（和残留的 cTfR）。个体间存在显著差异，健康动物和患病动物之间无显著差异。猫的 cTfR 比例非常低，为 11%，表明可能很少产生 sTfR。健康猫和患病猫之间存在微小但显著的差异。健康马似乎不会切割外体相关的 TfR。在存在炎症性疾病的情况下诊断缺铁仍然是兽医医学的一个挑战。我们的结果表明，TfR 在兽医物种中被切割不良或不可预测，这表明外体 TfR 处理存在物种差异。这些数据表明，在研究的物种中开发用于检测和定量 sTfR 的检测可能没有必要。
