Cathodoluminescence applied to immunofluorenscence: present state and improved technical prospects by prism spectrometer light selection.

For studies on cathodoluminescence, we equipped a scanning electron microscope with a prism spectrometer and sensitive photomultiplier. The apparatus is described and our initial results are presented on the analyse of cathodoluminescence. The material observed promarily involved studies of immunofluorescent specimens. Humal lymphocytes were labelled with a fluorescent antibody and cryosections of rat kidney with Masugi nephritis were labelled with a fluorescent specific antibody. Our apparatus permitted monochromatic imaging of cathodoluminescence emissions and resulted in much improved micrographs. Some possible improvements of the technique are discussed.     

Cathodoluminescence applied to immunofluorescence: Present state and improved technical prospects by prism spectrometer light selection

Summary For studies on cathodoluminescence, we equipped a scanning electron microscope with a prism spectrometer and sensitive photomultiplier. The apparatus is described and our initial results are presented on the analyse of cathodoluminescence.The material observed primarily involved studies of immunofluorescent specimens. Human lymphocytes were labelled with a fluorescent antibody and cryosections of rat kidney with Masugi nephritis were labelled with a fluorescent specific antibody. Our apparatus permitted monochromatic imaging of cathodoluminescence emissions and resulted in much improved micrographs. Some possible improvements of the technique are discussed.     

Mechanism of hematuria. I. Electron microscopic demonstration of the passage of a red blood cell through a glomerular capillary wall in rat masugi nephritis

Masugi nephritis was induced in Sprague-Dawley rats by an intravenous injection of rabbit anti-rat kidney serum. In the autologous phase of the disease, three of 18 rats manifested continuous hematuria. Ultrastructural examination of renal glomeruli by transmission and scanning microscopy revealed gaps in the basement membranes, and the transcapillary passage of red blood cells through the discontinuous regions in the hematuric rats. Control animals revealed no gaps in the glomerular basement membranes regardless of the method used in their preparation for electron microscopy. These data support the hypothesis that hematuria is a result of the passage of red blood cells through gaps in the glomerular basement membrane in Masugi nephritis.     

Modification of crescentic Masugi nephritis in the rabbit by Bredinin, a new immunosuppressant

Experiments were undertaken to ascertain whether progression of crescentic Masugi nephritis in rabbits could be prevented by the administration of Bredinin (BR). Crescentic glomerulonephritis can be induced with high reproducibility by intramuscular preimmunization on day 1, followed by intravenous injections on days 3 and 5 of nephrotoxic duck gamma-globulin (NTD gamma-gl). 30 rabbits were divided into 3 groups including controls (group 1). Two groups of 10 nephritic rabbits were each treated with 10 mg/kg of BR either after or before the development of proteinuria (groups 2 and 3). In group 3, the onset of proteinuria showed a significant delay and duration of survival was significantly prolonged, compared with controls. Serum antibody titers after day 8 and creatinine levels after day 10, as well as the initial amounts of proteinuria, were also significantly lower during treatment in group 3 than in controls. Histologically, the prominent diffuse intra- and extra-capillary proliferation with monocyte accumulations observed in the control group were markedly diminished in group 3. These results suggest that early treatment in crescentic glomerulonephritis with BR will suppress the production of humoral antibody and prevent progression of the glomerular lesions.     

Localization of laminin in nephritic glomeruli as revealed by a quick-freezing and deep-etching method with immunohistochemistry

Summary The three-dimensional localization of laminin in rat glomeruli at the chronic phase of Masugi nephritis was investigated by a quick-freezing and deep-etching method combined with immunohistochemistry. Light-microscopically, laminin was localized in increased mesangial matrix and thickened glomerular basement membrane. The quick-freezing and deep-etching method revealed that the increased mesangial matrix, which was newly formed in axial portions and areas of mesangial interposition, was composed of fine fibrillar networks. They were revealed with the 3,3-diaminobenzidine tetrahydrochloride (DAB) reaction products of peroxidase-labelled secondary antibody following anti-laminin antibody. However, these reaction products were not uniformly distributed in the newly formed matrix. Although the fibrils organizing lamina densa were also immunostained with anti-laminin antibody, the fibrils connected to mesangial cells, podocytes and endothelial cells had smaller amounts of DAB reaction products for laminin. These results indicate that one of the components of fibrils in the mesangial matrix and lamina densa is laminin, which is heterogeneously distributed in the newly formed matrix.     

应用高脂饮食联合维生素D3建立慢性系膜增殖性肾炎血管病变大鼠模型

目的 探讨应用高脂饮食建立慢性系膜增殖性肾炎血管病变模型的方法.方法 雄性Wistar大鼠行单侧肾切除后随机分为单纯肾切除组、单纯肾炎组、单纯高脂组、肾炎高脂组.单纯肾炎组、肾炎高脂组在单侧肾切除后3d尾静脉注射OX7抗体(100 mg/kg),1周后尾静脉连续注射OX7抗体(每次100 mg/kg,1次/周,共3次),单纯肾切除组和单纯高脂组在同一时间尾静脉注射PBS,注射抗体后第2天单纯高脂组、肾炎高脂组腹腔注射维生素D3(6万U/kg,1次/4周),同时给予高脂饲料.分别于第4、8、10周观察各组大鼠的一般情况、体重、血压、尿蛋白、血浆白蛋白、血脂、血钙、肾功能以及肾脏病理改变.结果 模型组(肾炎高脂组)大鼠第8周肾小球外的小动脉出现管壁增厚,管腔变小,平滑肌细胞减少,细胞排列紊乱,纤维组织增生.第10周单纯肾炎组和单纯高脂组肾小球外小动脉管壁轻度增厚,管腔变化不明显,模型组血管病变积分明显高于单纯肾炎组和单纯高脂组(P＜0.05).结论 通过对慢性抗Thy1肾炎大鼠加用高脂饲料并腹腔注射维生素D3的方法,可以成功建立慢性系膜增殖性肾炎血管病变模型.     

A scanning electron microscopic study of rat Masugi nephritis.

Changes in the glomerular capillaries in the first phase of rat Masugi nephritis were studied by scanning electron microscopy. The changes developed immediately after the injection of nephrotoxic rabbit IgG and early endothelial lesions (2 to 6 h) were characterized by an increase in microvilli and a decrease in endothelial pores. The microvilli were fused and produced abundant pored projections (cytofolds). The peripheral endothelium was then lifted off from the glomerular basement membrane (GBM), leaving scattered endothelial fragments on the GBM. The denuded GBM exhibited a rather uniform, thick carpet-like appearance with occasional crater formation. Depositon of fibrin strands was seen associated with endothelial exfoliation. These later dissolved and were converted to a fibrinoid material, consisting of a complex of fragmented, thin fibrils. A parallel study using the electron microscope revealed that the fibrinoid material was removed by emigrating monocytic macrophages. At the stage of resolution (24 to 72 h), the denuded GBM was covered mostly with a regenerating endothelial layer. A possible process of reorganization of the endothelial pores is discussed.     

Interleukin-17 Expression Positively Correlates with Disease Severity of Lupus Nephritis by Increasing Anti-Double-Stranded DNA Antibody Production in a Lupus Model Induced by Activated Lymphocyte Derived DNA

Lupus nephritis is one of the most serious manifestations and one of the strongest predictors of a poor outcome in systemic lupus erythematosus (SLE). Recent evidence implicated a potential role of interlukin-17 (IL-17) in the pathogenesis of lupus nephritis. However, the correlation between IL-17 expression level and the severity of lupus nephritis still remains incompletely understood. In this study, we found that serum IL-17 expression level was associated with the severity of lupus nephritis, which was evaluated by histopathology of kidney sections and urine protein. Of note, we showed that enforced expression of IL-17 using adenovirus construct that expresses IL-17 could enhance the severity of lupus nephritis, while blockade of IL-17 using neutralizing antibody resulted in decreased severity of lupus nephritis. Consistently, we observed an impaired induction of lupus nephritis in IL-17-deficient mice. Further, we revealed that IL-17 expression level was associated with immune complex deposition and complement activation in kidney. Of interest, we found that IL-17 was crucial for increasing anti-double-stranded DNA (dsDNA) antibody production in SLE. Our results suggested that IL-17 expression level positively correlated with the severity of lupus nephritis, at least in part, because of its contribution to anti-dsDNA antibody production. These findings provided a novel mechanism for how IL-17 expression level correlated with disease pathogenesis and suggested that management of IL-17 expression level was a potential and promising approach for treatment of lupus nephritis.     

Cellular aspects of rabbit Masugi nephritis V. Ultrastructural cytochemistry of monocytic cells

Ultrastructural cytochemistry for nonspecific esterase (NSE) was performed on normal and nephritic rabbit kidneys. In normal glomeruli distinct reaction deposits appearing as electron-dense granules were present in visceral epithelial cells (podocytes) and epithelial cells of Bowman's capsule, but only a few small or minute deposits were seen in some endothelial and mesangial cells. In acute proliferative glomerulonephritis (Masugi nephritis), many reaction deposits occurred in mononuclear cells accumulating in glomerular tufts which presented the characteristic features of monocytic cells. Macrophages which had migrated into subendothelial space as well as epithelioid cells and multinucleated giant cells, all of which are known to be derived from monocytes, also exhibited the reaction product. The NSE granules in mesangial and endothelial cells were much smaller and fewer in number than those in monocytic cells. The present method may contribute to the more precise differentiation of monocytic cells from mesangial and endothelial cells in proliferative glomerulonephritis.     

CD40 Gene Silencing Reduces the Progression of Experimental Lupus Nephritis Modulating Local Milieu and Systemic Mechanisms

Lupus nephritis (LN) is an autoimmune disorder in which co-stimulatory signals have been involved. Here we tested a cholesterol-conjugated-anti-CD40-siRNA in dendritic cells (DC) in vitro and in a model of LPS to check its potency and tissue distribution. Then, we report the effects of Chol-siRNA in an experimental model of mice with established lupus nephritis. Our in vitro studies in DC show a 100%intracellular delivery of Chol-siRNA, with a significant reduction in CD40 after LPS stimuli. In vivo in ICR mice, the CD40-mRNA suppressive effects of our Chol-siRNA on renal tissue were remarkably sustained over a 5 days after a single preliminary dose of Chol-siRNA. The intra-peritoneal administration of Chol-siRNA to NZB/WF1 mice resulted in a reduction of anti-DNA antibody titers, and histopathological renal scores as compared to untreated animals. The higher dose of Chol-siRNA prevented the progression of proteinuria as effectively as cyclophosphamide, whereas the lower dose was as effective as CTLA4. Chol-siRNA markedly reduced insterstitialCD3+ and plasma cell infiltrates as well as glomerular deposits of IgG and C3. Circulating soluble CD40 and activated splenic lymphocyte subsets were also strikingly reduced by Chol-siRNA. Our data show the potency of our compound for the therapeutic use of anti-CD40-siRNA in human LN and other autoimmune disorders.     

An ultrastructural study of proliferative nephritis induced experimentally by a monoclonal antibody against mesangial cells: replacement of mesangial cells by cells of the monocyte-macrophage system

An experimental nephritis accompanied by transient proteinuria can be produced by an intravenous injection of the monoclonal antibody, 1-22-3, raised against isolated rat glomeruli. The present study deals with the ultrastructural changes in the glomeruli in rats after the injection of this antibody. At 2 h after injection, all the mesangial cells had completely degenerated and neutrophils invaded most mesangial areas. Monocytes occupied the vacant mesangial areas at 24 h and gradually increased in number over the next 4 days. At 4 and 6 days, macrophage-like cells, possibly derived from monocytes, underwent frequent mitosis, resulting in a remarkable proliferation of these cells. The interpretation of these cells as macrophages was strongly supported by the fact that they contained previously injected latex particles in large numbers. From 2 to 4 weeks after injection, the macrophage-like cells gradually transformed into cells indistinguishable from normal mesangial cells. In the present experimental nephritis where all mesangial cells were initially destroyed, cells of the monocyte-macrophage system appear to play a leading role in the pathogenesis of the ensuing proliferative glomerulonephritis, and represent the source of the replacing mesangial cells.     

Detection of Coxiella burnetii in cow's milk by PCR-enzyme-linked immunosorbent assay combined with a novel sample preparation method.

The use of an adequate concentration of Triton X-100 enhanced immunomagnetic separation of Coxiella burnetii from milk. PCR-enzyme-linked immunosorbent assay (PCR-ELISA) could detect coxiellas more sensitively than could conventional PCR. PCR-ELISA is therefore thought to be suitable for the simultaneous assay of a large number of samples. However, the number of cows from which raw milk tested positive for coxiellas by PCR-ELISA was inconsistent with that found with the antibody to coxiella by indirect immunofluorescence assay. The inconsistency is thought to be associated with the differences in the infectious route, infectious dose, or the timing of yielding the antibody and the period of duration of the antibody.     

Combined treatment of autoimmune MRL/Mp-lpr/lpr mice with cholera toxin plus irradiation. Combined treatment of autoimmune MRL/l mice

MRL/Mp-lpr/lpr (MRL/1) mice spontaneously develop autoimmune diseases like systemic lupus erythematosus (SLE) from 2 months of age, accompanied by massive lymphadenopathy. Such mice of 2 months of age were treated with 1 microgram cholera toxin (CT) every 7 days and/or with 400 rad of one-shot 60Co irradiation. CT treatment alone markedly improved nephritis as evaluated by proteinuria and moderately suppressed lymphadenopathy and anti-DNA antibody production, while irradiation alone prominently improved lymphadenopathy but showed little effect on both nephritis and anti-DNA antibody production. On the other hand, when mice were treated with the combination of CT plus irradiation, autoimmune nephritis as well as anti-DNA production and lymphadenopathy were almost completely inhibited. Taken together, each agent exerts the improvement effect at the different points from each other in an abnormal immunological circuit displayed in MRL/1 mice. This kind of combined treatment may be applicable to the clinical use for autoimmune diseases.     

Severe Nephrotoxic Nephritis following Conditional and Kidney-Specific Knockdown of Stanniocalcin-1

Background

Inflammation is the hallmark of nephrotoxic nephritis. Stanniocalcin-1 (STC1), a pro-survival factor, inhibits macrophages, stabilizes endothelial barrier function, and diminishes trans-endothelial migration of leukocytes; consistently, transgenic (Tg) overexpression of STC1 protects from nephrotoxic nephritis. Herein, we sought to determine the phenotype of nephrotoxic nephritis after conditional and kidney-specific knockdown of STC1.

Methods

We used Tg mice that, express either STC1 shRNA (70% knockdown of STC1 within 4d) or scrambled shRNA (control) upon delivery of Cre-expressing plasmid to the kidney using ultrasound microbubble technique. Sheep anti-mouse GBM antibody was administered 4d after shRNA activation; and mice were euthanized 10 days later for analysis.

Results

Serum creatinine, proteinuria, albuminuria and urine output were similar 10 days after anti-GBM delivery in both groups; however, anti-GBM antibody delivery to mice with kidney-specific knockdown of STC1 produced severe nephrotoxic nephritis, characterized by severe tubular necrosis, glomerular hyalinosis/necrosis and massive cast formation, while control mice manifested mild tubular injury and crescentic glomerulonephritis. Surprisingly, the expression of cytokines/chemokines and infiltration with T-cells and macrophages were also diminished in STC1 knockdown kidneys. Staining for sheep anti-mouse GBM antibody, deposition of mouse C₃ and IgG in the kidney, and antibody response to sheep IgG were equal.

Conclusions

nephrotoxic nephritis after kidney-specific knockdown of STC1 is characterized by severe tubular and glomerular necrosis, possibly due to loss of STC1-mediated pro-survival factors, and we attribute the paucity of inflammation to diminished release of cytokines/chemokines/growth factors from the necrotic epithelium.     

Suppression of murine lupus nephritis by administration of an anti-idiotypic antibody to anti-DNA

The suppression of pathogenic antibodies to DNA in NZB/NZW f1 female mice was achieved by repeated inoculation of the mice with a monoclonal anti-idiotypic antibody (anti-Id). The anti-Id, an IgG1, kappa, was directed against a major cross-reactive idiotype (Id) on NZB/NZW IgG antibodies to DNA. One hundred micrograms of the anti-Id were inoculated i.p. every 2 wk, beginning at 6 wk of age (nondiseased mice--no circulating anti-DNA or proteinuria) or 20 wk of age (diseased mice--all with circulating anti-DNA, one-third with proteinuria). As controls, littermates received an IgG, kappa non-DNA-binding myeloma or no treatment. In the young mice, nephritis and anti-DNA antibodies appeared at the same time in all groups, and their circulating antibodies to DNA did not bear the target Id. In the older (20-wk-old) mice, survival was significantly prolonged because of delay in the onset of nephritis; the total quantities of antibodies to DNA were diminished, and the target Id, initially present on circulating IgG, was deleted. These benefits were transient; the suppression of antibodies was followed by the appearance of large quantities of anti-DNA that did not bear the major Id. Therefore, although administration of anti-Id was effective in reducing an undesirable antibody response after the target Id was present on circulating antibodies, the benefits were limited, probably by Id "switch" or by increased synthesis of pathogenic antibodies bearing a minor Id.     

Isolation and characterization of circulating immune complexes from rats with experimental membranous nephropathy

Circulating immune complexes (CIC) were isolated from serum from controls and rats with active Heymann nephritis (n = 31) by two methods. CIC detected by the fluid phase Clq binding assay were precipitated from serum using Clq and polyethylene glycol. CIC were also isolated by sequential chromatography with anion exchange and lectin affinity supports. The isolated material was analyzed by PAGE and immunoblotting. The immune complex material isolated by both methods from rats with Heymann nephritis contained the same 60/65-kDa tubular Ag. By immunoblotting, the 60/65-kDa tubular Ag-bound antibodies from rats with active Heymann nephritis, but not antibodies to gp330. Antibody bound to the 60/65-kDa tubular protein in the CIC was isolated. This antibody bound to a similar Ag in glomerular eluates from rats with active Heymann nephritis when tested by immunoblotting. These observations suggest that glomerular immune deposits and CIC in rats with Heymann nephritis contain the same tubular Ag. The 60/65-kDa Ag was isolated from CIC by HPLC using anion exchange and hydrophobic interaction columns. Rats immunized with this Ag developed Heymann nephritis. These studies suggest that CIC contribute to the development of glomerular subepithelial immune deposits in this model of membranous nephropathy. These studies do not exclude the participation of other Ag-antibody systems in Heymann nephritis, including gp330. This report describes methods for isolation and characterization of Ag-antibody components of CIC that might be useful to studies of other immune complex-mediated diseases.     

Effect of ACE inhibitors on proteinuria and renal functioning in primary glomerulopathies]

Angiotensin converting enzyme inhibitors was given to 16 patients with glomerular nephritis in whom a complete remission of nephrotic syndrome could not be achieved with immunosuppressive-anti-inflammatory therapy. Captopril in the daily dose of 25-75 mg and enalapril in the daily dose of 10 mg were administered for 1-36 months (mean 12.6 months). Daily proteinuria decreased by 40-80% comparing with baseline value in 2/3 of patients. Total protein and albumin serum levels increased simultaneously. No changes in blood creatinine were noted in patients with initially normal renal functioning except one patient. Renal functioning was stable in 50% of patients with increased blood creatinine levels (mean 200 mumol/L). Blood creatinine was increasing in the remaining patients.     

Mechanisms of T and B cell collaboration in the in vitro production of anti-DNA antibodies in the NZB/NZW F1 murine SLE model

B/W mice spontaneously develop IgG antibodies to DNA that cause lethal immune nephritis. T and B cell interactions in the in vitro anti-DNA antibody response of B/W mice were investigated, and two distinct families of helper T cells that drive these responses were defined. First, the anti-DNA antibody-forming cell (AFC) response was found to be increased in B/W mice with nephritis and was inhibited with the monoclonal antibody anti-L3T4, suggesting a major role for helper T cells. Purified splenic T cells from mice with nephritis were able to augment both the IgG and the IgM anti-DNA AFC response of young B/W B cells. T helper cells were cloned from spleens of NZB/W F female mice with high titer anti-DNA antibodies and nephritis. The cloned T cells augmented both IgG and IgM anti-DNA AFC responses of young B/W B cells. Four clones--27.9, 30.7, 30.8, and 30.10--were selected for further study. These cells proliferated, in the context of syngeneic (H2d/z) antigen-presenting cells (APC) but not to allogeneic APC. Analysis of the mechanism of T helper cell clone-mediated augmentation of anti-DNA AFC revealed two populations: "cognate" T helper cells, which specifically augment anti-DNA AFC (30.7 and 30.10), and non-antigen-specific T helper cells (27.9 and 30.8), which augment the response of B cells of differing specificity by a bystander mechanism, probably through increased release of B cell growth and differentiation factors.     

Combined treatment of autoimmune MRL/Mp-lpr/lpr mice with cholera toxin plus irradiation

MRL/Mp-lpr/lpr (MRL/1) mice spontaneously develop autoimmune diseases like systemic lupus erythematosus (SLE) from 2 months of age, accompanied by massive lymphadenopathy. Such mice of 2 months of age were treated with 1g cholera toxin (CT) every 7 days and/or with 400 rad of one-shot⁶⁰Co irradiation. CT treatment alone markedly improved nephritis as evaluated by proteinuria and moderately suppressed lymphadenopathy and anti-DNA antibody production, while irradiation alone prominently improved lymphadenopathy but showed little effect on both nephritis and anti-DNA antibody production. On the other hand, when mice were treated with the combination of CT plus irradiation, autoimmune nephritis as well as anti-DNA production and lymphadenopathy were almost completely inhibited. Taken together, each agent exerts the improvement effect at the different points from each other in an abnormal immunological circuit displayed in MRL/1 mice. This kind of combined treatment may be applicable to the clinical use for autoimmune diseases.     

The effect of antibody protein dose on the uniformity of tumor distribution of radioantibodies: An autoradiographic study

Summary The inaccessibility of radiolabeled antibody to poorly vascularized regions of solid tumors may reduce the therapeutic efficacy of these macromolecules. Theoretical mathematical models have predicted that increasing the protein dose administered would reduce the heterogeneity of radioantibody distribution. This investigation was undertaken to evaluate this hypothesis in experimental animal models. We have utilized the technique of macroautoradiography to demonstrate an increase in tumor penetration of the lower-affinity¹²⁵I-labeled NP-4 or higher-affinity Immu-14 anti-carcinoembryonic antigen (anti-CEA) mAbs into small (60.25—0.4 g) and large (0.8–1.5 g) GW-39 and LS174T human colonic xenografts, grown subcutaneously in the nude mouse, when 400 µg unlabeled antibody is administered simultaneously with 10 µg (100 µCi) radioantibody. Further increases in protein to 800 µg result in a reduction in total tumor uptake of the antibody. These differences in mAb distribution could be visualized as early as 1 day after antibody injection. Improved mAb penetration was also achieved for the Mu-9 anti-CSAp (anti-mucin) antibody using 800 µg unlabeled antibody. An irrelevant antibody (AFP-7-31) was found to be homogeneously distributed 3 days after injection, even at a low protein dose. Attempts to improve mAb penetration by increasing the protein dose in the GS-2 colorectal tumor, a model that has low NP-4 accretion as a result physiological barriers separating antibody from antigen, were not successful. These results suggest that a more homogeneous distribution of radioantibody can be achieved by carefully selecting a dose of unlabeled antibody to coadminister. Work is currently in progress to determine the effect of improved tumor distribution of radioantibody on the therapeutic potential of a single dose of radioantibody.This work has been supported in part by USPHS grants CA-37895, CA 39841, and RR-05 903 (Division of Research Resources), National Institutes of Health, Department of Health and Human ServicesResearch fellow of the Dutch Cancer Society