Stromal cells in hemangioblastoma: neuroectodermal differentiation and morphological similarities to ependymoma

The histogenesis of stromal cells in hemangioblastoma is inconclusive despite a long-term controversy. An immunohistochemical and ultrastructural study was conducted for 17 cases of cerebellar hemangioblastoma. A wide range of immunohistological markers, targeting epithelial, mesenchymal, endothelial and neuroectodermal tissues, was used. In all cases, the microscopic hallmark characterizing hemangioblastomas, that is, lipid-containing stromal cells and a fine capillary network, known as a reticular variant, was noted. Stromal cells showed a variable immunoreactivity for neuroectodermal markers, such as S-100 protein, CD56, CD57, CD99, and neuron-specific enolase. This result, in conjunction with the absence of immunoreactivity for epithelial, mesenchymal, and endothelial markers, likely suggests neuroectodermal differentiation of stromal cells. In three cases, another component, known as a cellular variant, where epithelioid tumor cells were arranged in nests encircled by capillaries and/or in pseudorosette-like structures, was noted. Glial fibrillary acidic protein-immunoreactivity, which was totally absent in cases only showing the reticular pattern, was noted in two of them, suggesting a distinctive sign of glial differentiation in a proportion of hemangioblastomas. Ultrastructurally, microvilli-like projections in intracytoplasmic vacuoles were demonstrated in stromal cells. This result, taken together with the neuroectodermal hypothesis of stromal cells, suggests that hemangioblastomas may occasionally exhibit morphological similarities to ependymomas.     

Fusion and Matrix Protein Gene Sequence Analysis of Paramyxoviruses of Type 1(PMV-1) Isolated from Pigeons in Slovenia

Paramyxoviruses of type 1 (PMV-l) isolated from pigeons were genetically analyzed. A part of the fusion and the matrix protein genes were amplified and sequenced, Typical amino acid sequences associated with virulence were determined at the fusion protein cleavage site in all PMV-1 isolates. All Slovene pigeon PMV-1 strains share high amino acid sequence similarity with other pigeon strains. In the phylogenetic tree, they are clustered together with pigeon PMV-1 isolates with moderate pathogenicity. Phylogenetic analysis obtained from the fusion and the matrix protein gene alignments showed the same branching order. Viruses circulating among pigeons were found to form quite unique lineage of virulent NDV strains.     

Immunolocalization of lamins and nuclear pore complex proteins by atomic force microscopy

The nuclear envelope functions as a selective barrier separating the nuclear from the cytosolic compartment. Nuclear pore complexes (NPCs) mediate nuclear import and export of macromolecules and, therefore, are potential regulators of gene expression. In this study we applied atomic force microscopy (AFM) to visualize the three dimensional (3D) structure of individual NPCs in the absence and presence of two different antibodies, one directed against a pore protein (gp62) and another directed against Xenopus lamin LIII, a component of the nuclear lamina, a filament meshwork localized on the nucleoplasmic side of the nuclear envelope (NE) adjacent to and interacting with NPCs. Using 12-nm gold-labelled secondary antibodies and transmission electron microscopy we could clearly localize the primary single anti-gp62 antibody on NPCs and the primary single anti-LIII antibody between NPCs. Using AFM, the secondary antibodies against anti-gp62 could be detected as particles 7 nm in height on the nucleoplasmic face of NPCs. The secondary antibodies against anti-LIII could be clearly identified between NPCs. The secondary antibodies, attached to a 12-nm colloidal gold particle and visualized on glass, revealed similar shapes and heights as found on NEs. According to the 3D images, the volume of a single gold particle conjugated with secondary antibodies was 10 203 nm³. This volume is equivalent to the volume of 38 IgG molecules associated with one individual gold particle. A similar volume of 11 987 nm³ was calculated from a model assuming that the 150-kDa IgG molecules perfectly cover the spherical gold particle. We conclude that AFM can be used for identifying antibodies or other macromolecules associated with biomembranes.     

Evaluation of endothelial cell migration with a novel in vitro assay system

In this study we introduce a novel in vitro 'oil-drop' assay system for the measurement of endothelial cell (EC) migration, based on the original concept of the Teflon fence assay (Pratt et al., 1984; Am. J. Pathol. 117: 349–354). An aliquot of 15–20,000 human umbilical vein EC (HUVEC) is pipetted through a layer of mineral oil. The cells readily attach, spread and migrate on the surface of a matrix-coated tissue culture dish as a confluent circular monolayer. Migration is measured as the net increase in the total area covered at 24 hours. We have used this system to quantify EC migration on matrices composed of a mixture of type I collagen and either von Willebrand factor (vWF) or fibronectin (FN) in the presence or absence of tumor necrosis factor (TNF). Plating efficiency on both vWF/collagen and FN/collagen, measured by counting cells after attachment and spreading, is about 80%. With this method, migration on vWF/collagen was about 6.4 mm² and 5.3 mm² for TNF-treated and untreated HUVEC, respectively. HUVEC migration on FN/collagen was slightly greater – 6.4 mm² and 6.5 mm² with and without TNF treatment, respectively. During the 24 hour time period, HUVEC numbers increased 30–40% on vWF/collagen, and 60–80% on FN/collagen, with increased proliferation observed with TNF- treatment. EC proliferation could be completely inhibited by 2 mM hydroxyurea. This assay system has proven useful in our studies to quantify cell migration and proliferation.     

Screening microarrays of novel monoclonal antibodies for binding to T-, B- and myeloid leukaemia cells

We have developed a microarray (DotScan) that enables rapid immunophenotyping and classification of leukaemias and lymphomas by measuring the capture of cells by immobilized dots of 82 CD antibodies [Belov, L., de la Vega, O., dos Remedios, C.G., Mulligan, S.P., 2001. Immunophenotyping of leukemia using a cluster of differentiation antibody microarray. Cancer Res. 61, 4483; Belov, L., Huang, P., Barber, N., Mulligan, S.P., Christopherson, R.I., 2003. Identification of repertoires of surface antigens on leukemias using an antibody microarray. Proteomics 3, 2147]. The DotScan technology has been used to investigate the properties of 498 new antibodies submitted to the HLDA8 Workshop. These antibodies have been applied as 10 nl dots to a film of nitrocellulose on a microscope slide to make an HLDA8 microarray. After blocking the remaining nitrocellulose surface, individual arrays were incubated with each of 7 cell types from a human leukaemia cell panel consisting of three cell lines, CCRF-CEM (a T-cell acute lymphocytic leukaemia), MEC-1 (derived from B-cell chronic lymphocytic leukaemia) and HL-60 (a promyelocytic leukaemia), and four leukaemias from patients: a T-cell prolymphocytic leukaemia, a B-cell chronic lymphocytic leukaemia, and two acute myeloid leukaemias. Leukaemia cells were captured by those immobilized antibodies for which they expressed the corresponding surface molecule. Unbound cells were gently washed off, bound cells were fixed to the arrays and dot patterns were recorded using a DotScan array reader and quantified using DotScan data analysis software. The data obtained show the unique expression profiles of the 7 cell types in the leukaemia cell panel obtained with the DotScan microarray, and the differential capture patterns for these 7 cell types screened against the 498 antibodies in the HLDA8 microarray constructed for this study.     

垂体腺瘤中MMP-9及TIMP-1表达与肿瘤生物学行为的关系

目的　探讨MMP 9及其抑制因子TIMP 1在垂体腺瘤中表达与肿瘤生物学行为的关系。方法　应用免疫组化S P法检测上述基因蛋白在 2 3例侵袭性和 2 4例非侵袭性垂体腺瘤组织中的表达。结果　侵袭性垂体腺瘤组中MMP 9的表达和MMP 9表达超过TIMP 1的比例高于非侵袭性腺瘤组 (P <0 0 5 ) ;TIMP 1在侵袭性垂体腺瘤组的表达有降低的趋势 ,但无统计学意义 (P >0 0 5 ) ;MMP 9与TIMP 1表达呈正相关 (P <0 0 5 )。结论　MMP 9表达上调导致其和TIMP 1的表达失衡与垂体腺瘤侵袭性有关 ,MMP 9可作为评估垂体腺瘤侵袭性的分子生物学指标     

红细胞分化调节因子对体外培养的L929和KB细胞的生长抑制作用

从兔网织红细胞提纯的红细胞分化调节因子(erythroid differentiation factor,EDF),能对体外培养的自发转化成纤维细胞系L929及人鼻咽癌细胞系KB产生作用。当EDF剂量为0.10μg/ml时,可引起L929细胞形态发生改变,并有细胞核固缩现象。第2d的细胞生长抑制率为64.86％,软琼脂集落形成率为0;第5d时细胞增殖为负值。~3H-TdR掺入率明显降低。EDF剂量为0.15μg/ml时,对KB细胞生长已有抑制作用。EDF剂量达0.30μg/ml时,生长抑制明显。以上结果证明了EDF对恶性细胞具有增殖抑制作用。这种作用对不同种类细胞敏感性不同,并且与剂量呈正相关。     

The innate immune response under the control of the LXR pathway

Macrophages play essential roles in infection and resolution of inflammation. This review summarizes recent findings that suggest a relevant role for the nuclear receptor liver X receptor (LXR) in the evolution of immune responses. By exerting both positive and negative regulation of specific macrophage gene expression networks, LXRs display anti-inflammatory activities and promote macrophage survival in bacterial infection settings. Agonists that activate the LXR pathway may be used to enhance innate immunity to highly virulent pathogens that otherwise induce macrophage apoptosis as a means to subvert host immune defense.     

HnRNP CBP35-CBP67 interaction during stress response and ageing

Previous studies have demonstrated the existence of nuclear carbohydrate binding proteins in a variety of mammalian cells with molecular masses of 35 000, 67 000, and 70 000 (CBP35, CBP67, and CBP70), which are associated with nuclear ribonucleoprotein (RNP) complexes. CBP35 consists of two domains, an aminoterminal portion that is homologous to certain regions of proteins of the heterogeneous nuclear RNP complex, and a carboxyl-terminal portion homologous to β-galactoside-specific lectins. CBP35 it has been proposed, like the glucose-specific lectin, CBP67, to guide RNP complexes through the nuclear pore. Here we show that the exposure of mature rats to stress induces an increase in nuclear CBP35 bound to CBP67 and retained on immobilized glucose. Nuclear extracts from the livers of old rats displayed no detectable stress response. This CBP35·CBP67 association detected in rat liver is considered with respect to the CBP35·CBP70 association recently observed in HL60 cell nuclear extracts.