Defective Function of Renal Lysosomes in Mice with the Chediak-Higashi Syndrome

Morphologically abnormal lysosomes demonstrated in individuals with the Chediak-Higashi syndrome (CHS) suggested a defect in the function of these abnormal lysosomes. To gain direct experimental evidence of such a defect, horseradish peroxidase (HRP) was injected intravenously into CHS and control mice, the mice killed at varying intervals and the kidneys studied by ultrastructural cytochemistry. No morophologic difference was observed in the absorption and uptake of HRP by proximal convoluted tubules in the two groups of mice. In CHS mice, however, some of the HRP fused with enlarged lysosomes. By 48 hours after injection, the lysosomes of normal mice had digested all but trace amounts of HRP, whereas large amounts were still present in CHS mice at this time. In CHS mice, moderate amounts were still present at 72 hours and trace amounts 96 hours post injection. This slowed rate of digestion of HRP by lysosomes of the proximal convoluted tubule cells of CHS mice suggests a similar defect in all cells in CHS individuals in which there is a lysosomal degradation of protein or other matter obtained by endocytosis. Such a defect may explain some manifestations of impaired host defense observed in CHS.     

Fate of Exogenous Peroxidase in Renal Lysosomes of Mice with Chediak-Higashi Syndrome

The beige mouse is a homolog of Chediak-Higashi syndrome, a disorder which is characterized by the presence of enlarged (anomalous) lysosomes in many cell types. In kidney, anomalous lysosomes are present in cells of the proximal convoluted tubules. In this study, the degradation of injected horseradish peroxidase (HRP) in lysosomes was studied in both the convoluted (S1-S2) and straight (S3) segments of the proximal tubules of beige and control (C57 B1) mice. Tissues were removed at intervals from 18 hours to 7 days after HRP injection. Peroxidase activity was visualized for light and electron microscopy by incubating sections in diaminobenzidine medium. No differences in the rate of degradation of HRP were demonstrable between anomalous lysosomes in S1-S2 cells of beige kidney and those in controls. In both animals, HRP was demonstrable in these lysosomes at 18 and 36 hours but not at 48 hours after injection. By electron microscopy, reaction product appeared as a flocculent precipitate distributed uniformly throughout the lysosome. In contrast to those of S1-S2 cells, lysosomes of beige S3 cells degraded HRP much more slowly than did those of control mice. In controls, HRP was demonstrable in S3 lysosomes at 18 hours but not at 48 hours after injection. In beige mouse kidney HRP was demonstrable in many S3 lysosomes at 48 hours, and it persisted in some lysosomes as long as 5 days after injection. These findings indicate that beige S3 lysosomes are defective in degrading protein. As reported recently, these lysosomes are also markedly enlarged and altered in content. They appear to arise as part of a renal lesion of unknown pathogenesis which is confined to the S3 segments of the proximal tubules. The slower rate of degradation of protein appears to be another manifestation of the alteration in these lysosomes.     

Infection and Immunoglobulin Concentrations in Chediak-Higashi Mice

The Chediak-Higashi syndrome (CHS) has been reported in man, cattle, mink, and mice. CHS humans and cattle have an increased incidence of pyogenic infections, whereas CHS mink are more susceptible to Aleutian disease. Age- and sex-matched groups of CHS mice (mutant strain) and C57 Bl/6N (parent strain) were challenged with Candida albicans, Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus intravenously and Streptococcus pneumoniae intraperitoneally. A significant increase (P < 0.001) in mortality rate for the CHS mice was demonstrated with all five challenge organisms. Heterozygous mice challenged with C. albicans had a mortality rate essentially the same as C57 Bl/6N mice. The serum immunoglobulin concentrations of CHS mice were found to be the same or greater than the control or heterozygous mice. CHS mice have an increased susceptibility to pyogenic infections, which is not due to immunoglobulin deficiency. These mice may provide a useful laboratory model for the study of increased susceptibility to infection.     

Chediak-Higashi 综合征的相关基因及蛋白质

Chediak-Higashi综合征(CHS)是一种罕见的常染色体隐性遗传疾病,其致病基因——溶酶体转运调节因子基因定位于染色体1q42.1-42.2。该基因在转录过程中可形成两种大小不同的转录本。两种转录体在各组织中的表达不同,提示它们的生物学性质也不相同。其编码的蛋白——溶酶体转运调节因子(ly-sosomal-trafficking regulator)属于一个大的BEACH蛋白家族,与家族中其他成员有着相似的结构和功能。BEACH家族蛋白的鉴别和相关动物模型的研究使我们认识到BEACH的蛋白质参与了多种不同的生化途径,BEACH蛋白突变或缺乏可以导致囊泡转运缺陷。     

The Chediak-Higashi Syndrome: Fine Structure of Giant Inclusions in Freeze-Fractured Neutrophils

The technic of freeze-fracture and etching has been used in the present study to examine the fine structure of giant inclusions in circulating leukocytes from a patient with the Chediak-Higashi syndrome (CHS). The surface granularity of the membranes enclosing the giant inclusions differed slightly from that of normal sized organelles. Two types of giant granules were distinguished in replicas of freeze-fractured CHS neutrophils. The difference in fine structure suggests that one variety is a massively enlarged, but essentially unaltered primary lysosome, while the other develops as a result of continued fusion of small organelles with huge inclusions throughout the stages of polymorphonuclear leukocyte maturation.     

The Chediak-Higashi Syndrome: Quantitation of a Deficiency in Maximal Bactericidal Capacity

The maximum bactericidal capacity of neutrophils from a patient with the Chediak-Higashi syndrome (CHS) was measured by a quantitative assay in which the neutrophils were challenged with increasing multiples of Staphylococcus aureus, 502A. At various bacterial challenges from 0.5 to 65 bacteria per neutrophil, the CHS cells killed normal numbers of bacteria in 60 minutes. However, at higher ratios with a mean of 118 bacteria per neutrophil, the percentage of bacteria killed in 60 minutes by CHS neutrophils (8.1 ± 2.3%) was significantly less (P < 0.001) than that killed by normal neutrophils (44 ± 2.3%). The CHS neutrophils lagged in their ability to kill low challenges of bacteria (0.5 or 1.2 bacteria per neutrophil) only at 20 minutes. A hydrogen-peroxide-producing strain of Streptococcus faecalis was killed normally by the CHS neutrophils at 60 minutes, with all ratios of challenge up to 114 to 1. Electron microscopic examination of 60-minute specimens from high ratios of challenge that were stained for myeloperoxidase activity revealed a failure of many bacteria-laden phagosomes to display this marker of degranulation. The results of this study indicate that the maximal bactericidal capacity of CHS neutrophils is saturated by a significantly lower challenge of bacteria than is required to saturate normal cells. This appears to be the result of sequestration of a significant portion of these cells' bactericidal resources in the giant granules that do not participate appreciably in degranulation.     

Leukocyte Dysfunction in the Bovine Homologue of the Chediak-Higashi Syndrome of Humans

The increased susceptibility to pyogenic infections noted in cattle with the Chediak-Higashi syndrome trait has been related to an impairment of leukocyte function at the cellular level. Investigations of the relationship of abnormal granule formation to increased susceptibility to infection, conducted with cell suspensions containing high concentrations of polymorphonuclear leukocytes, revealed a bactericidal defect that was associated with abnormal intracellular killing and not due to defective particle ingestion. The in vitro bactericidal defect was associated with a metabolic anomaly in the hexose monophosphate shunt, but not with an alteration in the capacity to reduce nitroblue tetrazolium dye. Ultrastructural histochemical studies of phagocytosis and phagolysosome formation in polymorphonuclear leukocytes suggest that the impairment in bactericidal capacity is correlated also with either a delay or failure of primary granules to degranulate.     

Chediak-Higashi Syndrome: Variable Cytochemical Reactivity of Giant Inclusions in Polymorphonuclear Leukocytes

Neutrophils from the peripheral blood of patients with the Chediak-Higashi syndrome (CHS) contain large numbers of giant granules. Previous studies have demonstrated that the huge neutrophil organelles contain hydrolytic enzymes and are, therefore, massive lysosomes. The basis for the inability of giant granules to react with phagocytic vacuoles containing foreign particulates taken up by CHS neutrophils has not been determined. In the present investigation we have examined the ultrastructural cytochemistry of the huge inclusions in neutrophils from 3 patients. Electron-dense reaction product of the acid phosphatase reaction stained the giant inclusions and a few normal-sized azurophilic granules in the process of fusion with them. The amount of reaction product deposited in the huge organelles and its distribution, however, were extremely variable, even in the same cell. Myeloperoxidase staining revealed a similar variability in the intensity and distribution of its electrondense reaction product. The findings suggest that giant primary granules in circulating CHS neutrophils retain characteristics similar to newly formed azurophilic lysosomes in promyelocytes and myelocytes. Dilution of enzyme reaction products and variability in their localization indicate that most of the giant primary granules undergo transformation or incorporation into huge secondary lysosomes, which are virtually unable to participate in the degranulation reaction after uptake of foreign particulates by CHS neutrophils.     

Chediak-Higashi Syndrome: Variable Cytochemical Reactivity of Giant Inclusions in Polymorphonuclear Leukocytes

Neutrophils from the peripheral blood of patients with the Chediak-Higashi syndrome (CHS) contain large numbers of giant granules. Previous studies have demonstrated that the huge neutrophil organelles contain hydrolytic enzymes and are, therefore, massive lysosomes. The basis for the inability of giant granules to react with phagocytic vacuoles containing foreign particulates taken up by CHS neutrophils has not been determined. In the present investigation we have examined the ultrastructural cytochemistry of the huge inclusions in neutrophils from 3 patients. Electron-dense reaction product of the acid phosphatase reaction stained the giant inclusions and a few normal-sized azurophilic granules in the process of fusion with them. The amount of reaction product deposited in the huge organelles and its distribution, however, were extremely variable, even in the same cell. Myeloperoxidase staining revealed a similar variability in the intensity and distribution of its electrondense reaction product. The findings suggest that giant primary granules in circulating CHS neutrophils retain characteristics similar to newly formed azurophilic lysosomes in promyelocytes and myelocytes. Dilution of enzyme reaction products and variability in their localization indicate that most of the giant primary granules undergo transformation or incorporation into huge secondary lysosomes, which are virtually unable to participate in the degranulation reaction after uptake of foreign particulates by CHS neutrophils.     

Chediak-Higashi Syndrome: Immunological responses to Epstein-Barr virus studies in gene heterozygotes

Immunologic studies were performed in five fathers and nine mothers of patients with Chediak-Higashi Syndrome (CHS). Antibody response to Epstein-Barr virus capsid antigen was higher than in normal controls. Antibodies to diffuse component of the early antigen were not detected and serum antibodies to the restricted component of the early antigen were observed in 64% of the subjects studied. Low natural killer activity and increased proportions of OKT8 positive cells were increased. These data indicate that immunologic alterations similar to those seen in CHS patients can be observed in their asymptomatic parents.     

缺血再灌注损伤对小鼠肾脏血管内皮生长因子受体3表达的影响

研究肾脏缺血再灌注( ischemia-reperfusion, IR)对小鼠肾脏血管内皮生长因子受体3( vascular endothelial growth factor receptor 3, VEGFR3)表达的影响。方法30只雄性C57BL/6小鼠随即分为5组：假手术组( Sham组)﹑缺血再灌注0﹑6、12﹑24 h组( IR 0 h组﹑IR 6 h组﹑IR 12 h组﹑IR 24 h组),每组6只。缺血再灌注组用无创性动脉夹夹闭左侧肾带,置于32℃温箱后1 h松开血管夹,随后切除右肾。 Sham组操作同上,但不夹闭左侧肾蒂。再灌注0﹑6﹑12﹑24 h后处死小鼠,收集肾脏及小鼠外周血标本。测定血肌酐(Cr)和尿素氮(BUN)水平。 PAS染色后显微镜下观察肾脏病理学变化, Western印迹和免疫组织化学法检测肾脏组织血管内皮生长因子受体3的表达。结果与Sham组相比较,再灌注0 h时小鼠血肌酐和尿素氮无明显升高,但缺血再灌注6﹑12﹑24 h后血肌酐和尿素氮呈显著性上升。 IR各组肾组织病理损伤也随着再灌注时间的延长而逐渐加重,可见肾小管上皮细胞明显肿胀坏死,蛋白管型形成,刷状缘脱落。随着缺血再灌注时间的进展, VEGFR-3蛋白表达量增加,免疫组织化学染色结果显示VEGFR3主要分布在肾脏皮质髓质交界处的肾小管。结论 VEGFR3在肾脏缺血再灌注损伤后表达量上升,且表达部位主要分布于肾脏皮髓质交界处的肾小管,因此VEGFR3可能参与调控肾脏缺血再灌注损伤。     

Alteration of Lysophosphatidylcholine-Related Metabolic Parameters in the Plasma of Mice with Experimental Sepsis

Plasma concentration of lysophosphatidylcholine (LPC) was reported to decrease in patients with sepsis. However, the mechanisms of sepsis-induced decrease in plasma LPC levels are not currently well known. In mice subjected to cecal ligation and puncture (CLP), a model of polymicrobial peritoneal sepsis, we examined alterations in LPC-related metabolic parameters in plasma, i.e., the plasma concentration of LPC-related substances (i.e., phosphatidylcholine (PC) and lysophosphatidic acid (LPA)), and activities or levels in the plasma of some enzymes that can be involved in the regulation of plasma LPC concentration (i.e., secretory phospholipase A2 (sPLA2), lecithin:cholesterol acyltransferase (LCAT), acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT), and autotaxin (ATX)), as well as plasma albumin concentration. We found that levels of LPC and albumin and enzyme activities of LCAT, ATX, and sPLA2 were decreased, whereas levels of PC, LPA, and LPCAT1–3 were increased in the plasma of mice subjected to CLP. Bacterial peritonitis led to alterations in all the measured LPC-related metabolic parameters in the plasma, which could potentially contribute to sepsis-induced decrease in plasma LPC levels. These findings could lead to the novel biomarkers of sepsis.     

The Effects of Inflammation on Lipid Accumulation in the Kidneys of Children with Primary Nephrotic Syndrome

This study aimed to characterize the relationship between inflammation and lipid accumulation in children with primary nephrotic syndrome (PNS). Local expression of interleukin-1β (IL-1β), transforming growth factor-β1 (TGF-β1), low-density lipoprotein receptor (LDLr), sterol regulatory element binding protein-2 (SREBP-2), SREBP cleavage-activating protein (SCAP), and apolipoprotein B100 (apoB100) was analyzed by immunohistochemistry in kidney tissues obtained from children with PNS. Renal histopathology was evaluated by hematoxylin and eosin and periodic acid-Schiff staining. Serum levels of IL-1β and TGF-β1 were measured by enzyme-linked immunosorbent assays. Expression of IL-1β, TGF-β1, LDLr, SREBP-2, SCAP, and apoB100 was higher in samples from patients with non-minimal change necrotic syndrome (NMCNS) compared to both controls and patients with minimal change necrotic syndrome. Deposition of apoB100 was significantly correlated with expression of IL-1β, TGF-β1, LDLr, SREBP-2, and SCAP and with the glomerulosclerosis index, but not with plasma lipid levels. Expression of IL-1β and TGF-β1 was significantly correlated with expression of LDLr, SREBP-2, and SCAP. These findings suggest that inflammation leads to lipid accumulation in the kidney through disruption of the expression of proteins in the SCAP/SREBP-2/LDLr signaling pathway, which may underlie glomerulosclerosis and tubulointerstitial fibrosis in NMCNS.     

LYST蛋白表达量在Chediak-Higashi综合征临床诊断中的应用

目的 探讨LYST基因突变导致的蛋白差异表达在罕见病Chediak-Higashi综合征(Chediak-Higashi syndrome,CHS)中的临床诊断应用价值.方法 利用免疫印迹(Western blotting,WB)检测CHS患儿、患儿直系家属以及健康志愿者的外周血白细胞中LYST蛋白表达量.结果 患儿白细胞中LYST蛋白出现两种截短体(p.R1104X、p.N2535KfsX2),呈高表达,而患儿直系家属以及健康志愿者白细胞中未检测到LYST蛋白表达.结论 CHS患儿外周血白细胞LYST截短体表达量较健康人显著增加.因此,免疫印迹检测LYST截短体的高表达可以作为辅助临床诊断CHS的一种新方法.     

Heterophile antibodies in sera of patients with Chediak-Higashi syndrome

Sera of 6 patients with Chediak-Higashi syndrome and sera of their mothers were studied for heterophile antibodies. Sera of 5 patients as well as 5 sera of their mothers contained antibodies against trypsinized bovine erythrocytes, tissue sediments of guinea pig kidney or high molecular weight glycoprotein (HMWGP) of bovine erythrocyte stromata. The antibodies combining with HMWGP in enzyme immunoassay belonged to IgM and IgG classes. Although none of the sera had significant titer of agglutinins against sheep erythrocytes, on the basis of absorption and inhibition studies, these antibodies seemed to belong to the Hanganutziu-Deicher group of antibodies.     

Infrared Thermometry for Acute Radiation Syndrome in Mice

Bulletin of Experimental Biology and Medicine - Experiment on female ICR CD-1 mice showed that non-contact infrared thermometry can be used for short-term and medium-term prognosis of animal death...     

Xenotransplantation of Embryonic Precursors of Human Myogenesis for the Correction of Dystrophinopathy in Mice with Hereditary Muscular Dystrophy

Human embryonic myogenic precursors were transplanted into muscles of mdx mice with hereditary dystrophin-deficient muscular dystrophy. Transplantation induced the synthesis of human dystrophin. The number of dystrophin-positive fibers progressively decreased, however, some of them were preserved even 5 months after transplantation. Our results indicate that xenogeneic transplantation of embryonic myogenic precursors compensates the genetic defect in dystrophin-deficient mice.