Comparative immunoelectron microscopy with monoclonal antibodies on yellow fever virus-infected cells: pre-embedding labelling versus immunocryoultramicrotomy

To study the intra- and extracellular distribution of yellow fever virus 17D (YFV)-specific antigens, pre-embedding immunoelectron microscopy (IEM) and IEM on ultrathin frozen sections were carried out comparatively using monoclonal antibodies (MAB) and YFV-infected cells. In addition, three electron-dense marker systems (IgG-ferritin and IgG-gold and protein A-gold) were compared for their efficiency in detecting bound MAB. Pre-embedding immuno-labelling was performed in microtest plates followed by in situ embedding and immunocryoultramicrotomy was performed using pellets of sucrose-infused cells. In both procedures, cells were prefixed with different concentrations of glutaraldehyde (GA). In pre-embedding IEM virus-specific antigens could be detected on the envelopes of extracellular virions with YFV-neutralizing MAB. Using immunocryoultramicrotomy, neutralizing MAB bound to intracellular mature virions as well as to viral antigens incorporated into cytoplasmic membranes. A concentration of 1% GA destroyed antigenicity entirely, while with 0.25% and 0.1% GA immunoreactivity was retained for more than 3 mth. Some highly reactive MAB labelled antigen significantly in pre-embedding IEM, when used at concentrations of 1 ng/ml. Immunocryoultramicrotomy was 10-100 times less sensitive. On cryosections colloidal gold was the marker of choice, due to the fact that it showed less nonspecific sticking to intracellular components and that it was easily detectable on highly contrasted cryosections. Owing to their higher sensitivity, IgG-ferritin conjugates were preferred in pre-embedding IEM.     

A simplified microwell pseudoreplica for the detection of viruses by electron microscopy and immunoelectron microscopy

Simplified procedures for immunoelectron microscopy (IEM) and electron microscopy (EM) are described. The procedures employ the principle of agar filtration and pseudoreplication. The modification consisted of the use of microwells for storage of gels with or without antiserum (for IEM or EM, respectively) and an array of containers in which pseudoreplication and negative staining were performed. The containers were prepared from 5 ml syringes from which the needle holding parts were cut. This device enabled simultaneous and rapid handling of specimens. With Sindbis virus as a model, our microwell pseudoreplica IEM (MW-PR-IEM) was compared to six other IEM techniques and was found to be the most rapid and sensitive technique. With the MW-PR-IEM technique, the specific minimal detection limit (detection of clumps) was 1.5 x 10(7) virus particles per ml, and the non-specific detection limit (detection of single virions) was 1.8 x 10(6) virus particles per ml.     

Syndecan-1/CD138 expression in normal myeloid,acute lymphoblastic and myeloblastic leukemia cells

Stabilization of cell surface antigens and preservation of ultrastructural integrity are important aspects of immunoelectron microscopical studies. In the present study, 4 anti-syndecan-1/CD138 (B-B2, B-B4, MI15, 1D4) monoclonal antibodies (mAbs) were applied in combination with periodatelysine-paraformaldehyde (PLP) fixation and indirect pre-embedding peroxidase electron microscopical immunocytochemistry to analyse the localization and function of these molecules in normal myeloid cells, acute lymphoblastic leukemia (ALL) cells and acute myeloblastic leukemia (AML) cells. One case of normal human bone marrow, 3 cases of untreated AML and 2 cases of untreated ALL were studied. Samples were immediately fixed for 4 h in freshly-prepared PLP fixative in 0.037 mol/L phosphate buffer, pH 7.4, containing 10 mmol/L sodium metaperiodate, 75 mmol/L lysine, and 2% paraformaldehyde. Expression of syndecan-1 was found at the plasma membrane of all cell types. Staining intensity at the membrane of AML cells was stronger than that on the membrane of normal myeloid and ALL cells. We conclude that anti-syndecan-1/CD138 mAbs in combination with the method described here are a suitable tool for detection of cell surface syndecan molecules in cells originating from progenitor cells that can differentiate in both myeloid and lymphoid cells.     

Ultrastructural localization of rotavirus antigens using colloidal gold

Colloidal gold was used to localize six of the ten known proteins of the simian rotavirus SA11 within infected cells by ultrastructural immunocytochemistry. Monospecific or monoclonal antibodies to selected structural and nonstructural proteins were the primary antisera. The major outer capsid glycoprotein, VP7, was associated with nonenveloped particles, with particles de-enveloped by Triton X-100 and with both nuclear and cytoplasmic inclusions. The protease-sensitive outer capsid protein, VP3, was also found on nonenveloped and de-enveloped particles. The major inner capsid protein, VP6, was accessible to antibodies on some of the nonenveloped particles (presumably single-shelled particles) and on the de-enveloped particles. A monospecific antibody to the gene 11 product, believed to be a precursor to a minor structural protein, VP9, reacted strongly with viroplasmic inclusions. Virus particles were weakly labeled by this antibody. NS35, a nonstructural SA11 protein, was found only in the viroplasms. NS29, a nonstructural glycoprotein, was localized to the cytoplasmic side of the endoplasmic reticulum membrane and to the inside of enveloped virus particles. These data support the hypothesis that NS29 facilitates budding of the virus particles and acquisition of the outer capsid layer.     

UCH‐L1 expression of podocytes in diseased glomeruli and in vitro

Ubiquitin C‐terminal hydrolase‐L1 (UCH‐L1), an important member of de‐ubiquitination enzyme families involved in the ubiquitin–proteasome pathway, is expressed mainly in neural and reproductive systems as well as in some tumours. Recently, expression of UCH‐L1 has been discovered in parietal epithelial cells of Bowman's capsules and some tubular epithelia in the kidney. However, whether UCH‐L1 is expressed in the capillary tufts of the glomeruli has not yet become clear. In this study, we used immunohistochemistry, double immunofluorescence labelling, immunoelectron microscopy in kidney biopsy tissues and western blot in cultured rat podocytes to investigate the expression of UCH‐L1 and the regulation of this expression in podocytes. The results demonstrated that UCH‐L1 was expressed in podocytes and its expression was significantly higher in acute proliferative glomerulonephritis (APGN), lupus nephritis (LN), membranous glomerulonephritis (MGN) and IgA nephropathy than that in focal segmental glomerulosclerosis (FSGS), minimal change disease (MCD), minor abnormality and normal kidney tissues (p < 0.05). In in vitro experiments, western blot showed that UCH‐L1 expression significantly increased in the two groups of podocytes co‐cultured with mesangial cells exposed to ATS 50 µl/ml and ATS 50 µl/ml with normal human serum 30 µl/ml, respectively (p < 0.05), while the other groups treated with TGFβ1 1 ng/ml, TNFα 10 ng/ml or IL‐1 10 ng/ml had little rise of UCH‐L1 expression, with no statistical significance compared with normal control (p > 0.05). Further tests indicated that the percentage of PCNA‐positive podocytes in LN, APGN and IgA nephropathy was significantly higher than that in MCD, FSGS and normal control (p < 0.05). These data show for the first time that podocytes do express UCH‐L1 and that its expression can be increased in these immunocomplex‐mediated nephrites. The immune injury is a main cause for stimulating podocytes to express UCH‐L1. The expression of UCH‐L1 may be associated with the regeneration of podocytes as a repair response to immunocomplex‐mediated injury. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Dorsal root projections in the clawed toad (Xenopus laevis) as demonstrated by anterograde labeling with horseradish peroxidase

Horseradish peroxidase was applied to the proximal stumps of severed cervical, thoracic and lumbar dorsal roots in the clawed toad, Xenopus laevis, in order to study the course, distribution and site of termination of dorsal root fibers in the spinal cord and brain stem. The anterograde transport of horseradish peroxidase as applied in the present study proved to be a useful and reliable technique. Results show that on entering the spinal cord, dorsal root fibers segregate into a medially placed component entering the dorsal funiculus and a more laterally situated bundle in the dorsal part of the lateral funiculus. As regards its position the latter bundle presumably represents the anuran homologue of the mammalian tract of Lissauer. Moreover, a small intermediate bundle of fibers directly enters the spinal gray matter. The labeled fibers entering the dorsal funiculus and the tract of Lissauer ascend and descend in the spinal cord, displaying a longitudinal arrangement resembling that of higher vertebrates.In the spinal gray, dorsal root fibers terminate in the dorsal, central and lateral fields of Ebbesson, 25 with the last field being a major terminus for dorsal root fibers originating in the limb-innervating segments. No dorsal root fibers were found to project to the motoneuron fields. A dorsal column nucleus, which is divisible into medial and lateral compartments, is present in the obex region and extends from the level of the second spinal nerve to that of the entrance of the vagus and glossopharyngeal nerves. Dorsal root fibers from the lumbar and all thoracic segments project to the medial compartment of the dorsal column nucleus, whereas those of the cervical enlargement project to the lateral compartment.Although the anuran dorsal column nucleus appears to be less differentiated than that of higher vertebrates, its medial and lateral compartments can be considered to be the forerunners of the mammalian nucleus gracilis and nucleus cuneatus, respectively.     

Preparation of an anti-dexamethasone monoclonal antibody and its use in development of a colloidal gold immunoassay

A lateral flow colloidal gold (CG) immunoassay strip has been developed for detection of dexamethasone (DEX) residues in milk samples. For this purpose, an anti-DEX monoclonal antibody (McAb), based on a DEX succinic anhydride derivative hapten, was prepared and characterized. The McAb showed a high specificity to DEX, the half inhibitory concentration of the antibody was 0.095?ng/mL, its limit of detection (LOD) was 0.017?ng/mL, and its linear range of detection was 0.034–0.265?ng/mL. The developed CG immunoassay had a visual cut-off value of 0.3?ng/mL in phosphate buffered saline (PBS) and 0.5?ng/mL in milk samples. Each test requires 10 min. Analysis of DEX in milk indicated that the results of strip assay had a strong agreement with indirect competitive enzyme-linked immunosorbent assay. Therefore, the CG immunoassay is a sensitive screening method for semi-quantitative and qualitative detection of DEX residues in milk samples.     

Improved detection of HSV by electron microscopy in clinical specimens using ultracentrifugation and colloidal gold immunoelectron microscopy: comparison with viral culture and cytodiagnosis.

Three tests were compared to diagnose herpes virus infection: electron microscopy (EM), viral culture (VC) and cytodiagnosis (Tzanck smear). The study comprised 67 patients with skin or mucous membrane lesions suggestive of herpes simplex virus (HSV) infection. The sensitivity of EM increased 25% after virus concentration by ultracentrifugation. Herpes virus infection was confirmed in 55 of the 67 cases by EM or VC or both. EM detected 53 herpes virus-positive lesion samples of which 14 were not detected by VC; only two lesion samples that were herpes virus-positive in VC were not detected by EM. The sensitivities of EM, VC, and Tzanck smear for the group of 55 herpes virus-positive cases were 96%, 75% and 76%, respectively. The specificity of the Tzanck smear was 83% (prevalence 82%). Colloidal gold immuno-EM was used to rapidly type HSV-1, HSV-2 and varicella zoster virus (VZV) present in skin and mucous membrane lesions in less than 4 h. Immuno-EM was able to detect antiviral antibodies on viral envelopes and viral cores in lesion samples with negative VC. Antiviral antibodies do not interfere with typing of herpes viruses by immuno-EM. It is suggested that formation of viral immune complexes and inactivation of virus particles by antibodies may have caused a negative VC. Improved EM is discussed for its applicability to special cases that cannot rely on VC and cytodiagnosis or when rapid diagnosis is required.     

Retrograde labeling of striato-nigral neurons in the rat by three different tracers

The three tracers horseradish peroxidase (HRP), 4',6-diamidino-2-phenylindol-2HCl (DAPI) and Fast Blue (FB) differ in retrograde labeling of striato-nigral neurons. After a 24 h survival, injection of DAPI into the ventral tegmentum labeled numerous cells throughout the neostriatum, whereas an identical amount of HRP only labeled cells in circumscribed areas of the neostriatum. Injections of FB labeled a substantial number of neostriatal neurons after a survival time of 4 days, but not after 24 h. In addition, differences between retrograde staining of striato-nigral neurons and layer V pyramidal cells of the ipsilateral neocortex, also labeled in these experiments, were found.     

Detection of Norwalk-like virus and specific antibody by immune-electron microscopy with colloidal gold immune complexes.

Direct electron-microscopy (DEM), immune electron microscopy (IEM) and four different procedures of immune electron microscopy with colloidal gold immune complexes were evaluated for the detection of Norwalk-like virus and specific antibody. A solid-phase immune electron microscopy with colloidal gold immune complexes-triple layer method (SPIEMGIC-TLM) is developed for screening patients' specimens for the detection of Norwalk-like virus and its specific antibody. The method demonstrates low non-specific background labelling and is simple, sensitive and easy to perform. A quadruple layer method (SPIEMGIC-QLM), which is a modification of the triple layer method, has been established by adding a cross-linking anti-IgG layer to amplify the reaction and to provide a more sensitive test which is suitable for screening monoclonal antibodies prepared against 32-34-nm Norwalk-like virus isolated in our laboratory.     

Immunogold labeling of chromosomes for scanning electron microscopy: A closer look at phosphorylated histone H3 in mitotic metaphase chromosomes of Hordeum vulgare

High-resolution detection of phosphorylated histone H3 at serine 10 in mitotic barley chromosomes for scanning electron microscopy was shown using a novel application of indirect immunogold labeling with Nanogold. This method permits localization and quantification of signals in a three-dimensional context. Because the chromosome structure is well preserved, characterization of binding sites (chromomeres, parallel matrix fibers, solenoids), currently in the realm of nanometer decades, is possible. Quantification and three-dimensional localization of labels is possible with stereoscopic analysis. Limitations of the method pertain to the challenges in preservation of chromosome ultrastructure, accessibility of immunoreactants into the fixed chromatin and unspecific labeling. The differences between silver and gold enhancement and the current status of labeling efficiency are addressed. This revised version was published online in July 2006 with corrections to the Cover Date.     

胶体金免疫电镜技术鉴定一株抗HFRS病毒囊膜糖蛋白和核蛋白双特异性McAb

采用超薄切片和胶体金免疫电镜技术鉴定抗HFRS病毒McAb 3G1,结果发现其既对成熟病毒颗粒和囊膜颗粒标记阳性,又对颗粒状包涵体及丝状颗粒包含体标记阳性,从而提示该株McAb可能具有HFRS病毒囊膜糖蛋白和核蛋白双特异性。     

Nucleus tegmenti pedunculopontinus: Efferent connections with special reference to the basal ganglia,studied in the rat by anterograde and retrograde transport of horseradish peroxidase

The efferent connections of the brain stem nucleus tegmenti pedunculopontinus were studied in the rat using the techniques of anterograde and retrograde transport of the enzyme horseradish peroxidase, laying particular emphasis on that part of pedunculopontinus which receives direct descending projections from the basal ganglia and related nuclei. In a preliminary series of experiments horseradish peroxidase was injected into either the entopeduncular nucleus or the subthalamic nucleus and, following anterograde transport of enzyme, terminal labelling was identified in nucleus tegmenti pedunculopontinus, surrounding the brachium conjunctivum in the caudal mesencephalon.In a subsequent series of experiments, horseradish peroxidase was injected into that region of nucleus tegmenti pedunculopontinus which receives entopeduncular and subthalamic efferents and its efferent projections were studied by anterograde transport of the enzyme. The results indicate that nucleus tegmenti pedunculopontinus gives rise to widely distributed efferent projections which terminate rostrally in mesencephalic, diencephalic and telencephalic structures and caudally in the pontine tegmentum. In the mesencephalon, terminal labelling was found in the pars compacta of the ipsilateral substantia nigra and sometimes in the adjoining ventral tegmental area. Labelling was also found in the ipsilateral half of the periaqueductal grey. In the diencephalon terminal labelling occurred bilaterally in the subthalamic nucleus and ipsilaterally in the intralaminar nuclei of the thalamus. Further rostrally, terminal labelling was particularly evident in the ipsilateral pallidal complex, especially in the caudal two-thirds of the entopeduncular nucleus and the ventral half of the caudal third of the globus pallidus. Caudal to pedunculopontine injection sites dense labelling was observed in the reticular formation of the pontine tegmentum.In a final series of experiments, confirmation of apparent pedunculopontine efferent projections was sought using the retrograde transport of horseradish peroxidase. Enzyme was injected into sites possibly receiving pedunculopontine efferents and the peribrachial area of the brain stem was examined for retrograde cell labelling. In this way, pedunculopontine projections were confirmed to the globus pallidus, entopeduncular nucleus, subthalamic nucleus, substantia nigra, parafascicular nucleus and pontine reticular formation. Injections into the globus pallidus or subthalamic nucleus gave rise to retrograde cell labelling bilaterally in pedunculopontinus. In addition, retrograde transport studies alone demonstrated projections from pedunculopontinus to the cerebral cortex and to the spinal cord.It is concluded that the nucleus tegmenti pedunculopontinus has reciprocal relationships with parts of the basal ganglia and some functionally related nuclei (in particular, the pallidal complex, subthalamic nucleus and substantia nigra). These connections support the view that nucleus tegmenti pedunculopontinus is likely to be involved in the subcortical regulation and mediation of basal ganglia influences upon the lower motor system. This suggests a potential role for pedunculopontine afferent and efferent pathways in the pathophysiology of basal ganglia related disorders of movement.     

Detection of villous conidia of Conidiobolus coronatus in a blood sample by scanning electron microscopy investigation

Conidiobolus coronatus is a major insect pathogen belonging to the fungal order Entomophthorales, causing a rare subcutaneous infection of the nasofacial region, resulting in swelling of predominantly the nose, mouth, and perinasal tissue. Later in the course of the infection firm, painless, subcutaneous nodules develop that are attached to the underlying tissues but not to the skin. No morphological studies are available in the literature on the morphology of C. coronatus in vivo and all morphological studies have been conducted on in vitro cultures. Here the authors report on the ultrastructural pathology as seen with a scanning electron microscope (SEM) of villous conidia of C. coronatus, detected in a 37-year-old woman who presented to the casualty department at Pretoria Academic Hospital, South Africa with left-sided facial pain and headache. The diagnosis of C. coronatus was confirmed by LightCycler real-time flourescence PCR technique. Research shows that typically diagnosis of the pathogen is established only on histological examination, and in over 85% of cases cultures for the causative organism is negative. This pathogen has not previously been found in a blood sample and the authors present for the first time the morphology of C. coronatus in blood using the SEM.     

Transient expression of type I collagen in glomeruli with anti-Thy-1 antibody-induced mesangial proliferative lesions

Glomerular expression of extracellular matrices at the protein and mRNA levels was examined in rats with mesangial proliferative glomerulonephritis induced by the intravenous administration of a monoclonal anti-rat Thy-1 antibody. In close association with the mesangial proliferative lesion, type I collagen was imrnunostained at day 8 but not demonstrated at day 28 in the glomeruli of the kidneys. Type I collagen mRNA expression prominently increased in the nephritic glomeruli at day 4, prior to the appearance of type I collagen protein. In addition, fibronectin expression was also elevated in the diseased glomeruli at both the protein and mRNA levels. These results indicated that glomerular, probably mesangial cells, change their phenotypes during this disease, to synthesize abnormal extracellular matrices that lead to the progression of glomerular sclerosis.     

Detection of spirochaetal microorganisms by focus floating microscopy in necrobiosis lipoidica in patients from central Europe

Aims:  Necrobiosis lipoidica (NL) is a chronic inflammatory skin disease with unknown aetiology. The aim was to determine the presence of spirochaetal microorganisms in NL.
Methods and results:  Focus-floating microscopy (FFM) is a modified immunohistochemical technique that was developed to detect borrelial spirochaetes within tissue sections. It has proven to be more sensitive for the detection of spirochaetes than polymerase chain reaction (PCR). Fifty-six cases of NL as well as 44 negative and 33 positive controls were investigated for the presence of Borrelia within tissue specimens. Using FFM, Borrelia could be detected in 42 cases (75.0%) and were seen significantly more often in histologically active inflammatory-rich (38/41, 92.7%) than in inflammatory-poor (4/15, 26.7%) cases of NL ( P  < 0.001). Seven cases investigated with a Borrelia- specific PCR (23s-RNA) remained negative. In contrast, FFM was positive in 30 of 33 (90.9%) positive controls of acrodermatitis chronica atrophicans and 15 of the positive controls (45.5%) were also positive with PCR, whereas no negative controls revealed any microorganisms.
Conclusions:  Detection of spirochaetes in NL points to a specific involvement of B. burgdorferi or other similar strains in the development of or trigger for this disease.     

重组汉坦病毒核衣壳蛋白抗原用于胶体金标记免疫层析法检测肾综合征出血热抗体

目的制备高表达量、高活性的HV NP重组抗原,以此为基础建立一个可同时检测HFRs患者血清中IgM和lgG抗体的快速胶体金标记诊断试剂盒。方法从TA-A537中扩增出截短的HV S基因片段P6-119,定向克隆至原核表达载体pET-30a中进行原核表达,采用亲和层析法纯化重组蛋白。以胶体金标记重组抗原,应用金标快速免疫层析法同时检测HFRS患者血清中IgM和IgG抗体。结果金标快速免疫层析法可同时检测HFRS患者血清中IgM和IgG抗体,与IFA比较,IgG抗体检测的敏感性和特异性分别为94.9%、100%。与ELISA比较,IgM抗体检测的敏感性和特异性均为100%。结论截短的重组汉坦病毒NP蛋白表达量高且具有良好的抗原性。以此为基础建立的金标快速免疫层析法显示出很高的敏感性和特异性,且具有简便、快速等优点,非常适用于各种层次尤其是缺乏实验条件和专业人员的基层医疗单位对HFRS疑似患者作出早期诊断。