Paola Preda

Molecular mechanisms of CD99-induced caspase-independent cell death and cell-cell adhesion in Ewing's sarcoma cells: Actin and zyxin as key intracellular mediators

CD99 is a unique 32-kDa cell surface molecule with broad cellular expression but still poorly understood biological functions. In cancer cells, CD99 is highly expressed in virtually all Ewings sarcoma (ES). Engagement of CD99 induces fast homotypic aggregation of ES cells and caspase-independent apoptosis. In this study, we analysed signal transduction after CD99 engagement on ES cells. Findings obtained with selective inhibitors indicated that only actin cytoskeleton integrity was essential for cell–cell adhesion and apoptosis of ES cells. Indeed, CD99 stimulation induced actin repolymerization, further supporting the role of cytoskeleton in CD99 signaling. Gene expression profiling of ES cells after CD99 engagement showed modulation in the expression of 32 genes. Among the pool of upregulated genes reported to be involved in cell adhesion, we chose to analyse the role of zyxin, a cytoplasmic adherens junction protein found to play a role in the regulation of the actin cytoskeleton. Overexpression of zyxin after CD99 ligation was confirmed by real-time PCR and Western blot. Treatment of ES cells with zyxin antisense oligonucleotides inhibited CD99-induced cell aggregation and apoptosis, suggesting a functional role for this protein. Therefore, our findings indicate that CD99 functions occur through reorganization of cytoskeleton and identify actin and zyxin as the early signaling events driven by CD99 engagement.

Expression of CD86 in acute myelogenous leukemia is a marker of dendritic/monocytic lineage

OBJECTIVE The aim of this study was to determine whether expression of the CD86 costimulatory molecule in acute myeloid leukemia (AML) can identify blast cells committed to the monocytic/dendritic lineage. MATERIAL AND METHODS One hundred ten consecutive AML patients observed at diagnosis were studied by flow cytometry. In selected experiments, in vitro cultures with CD34(+)CD86(+) or CD34(-)CD86(+) blasts were performed in the presence of granulocyte-macrophage colony-stimulating actor (GM-CSF) with or without tumor necrosis factor-alpha (TNF-alpha) or GM-CSF + interleukin-4 (IL-4), respectively, to induce a dendritic differentiation, documented by morphologic and immunophenotypic assays. T-cell alloreactivity to CD86(+) AML cells and leukemic dendritic cells (AML-DC) was tested in mixed leukocyte cultures and anti-leukemic cytotoxic assays. RESULTS CD86 was expressed in 54% AML, whereas CD80 and CD1a were only occasionally positive. CD86(+) AML samples included M5 and M4, but also 47% M0-M1 FAB types, and were more frequently CD14(+) (p < 0.00001) and CD34(-) (p = 0.00005) than CD86(-)AML. Six different patterns of CD86(+) AML were identified, according to CD34 or CD14 total or partial coexpression. Four samples enriched in CD34(+)CD86(+) AML cells differentiated into AML-DC CD86(+), CD80(+), CD40(+), CD11c(+), HLA-DR(++), CD14(+/-) that also were CD1a(+) or CD83(+), after 6 days of in vitro culture with GM-CSF +/- TNF-alpha. CD34(-)CD86(+) AML cells differentiated into AML-DC after 3 to 5 days (n = 5 experiments), and trisomy 8 was found in two AML and AML-DC samples by fluorescence in situ hybridization analysis. Finally, AML-DC induced more potent allo-T-cell proliferation, cytokine release, and anti-leukemic cytotoxicity than CD86(+) blasts. CONCLUSIONS In AML, CD86 is a marker of monocytic/dendritic lineage. Because CD86(+) blasts may differentiate into DC rapidly, CD86(+)AML patients could be optimal candidates for immunotherapy studies, both in autologous and allogeneic settings.

The cellular component in the parietal infiltrate of inflammatory abdominal aortic aneurysms (IAAA)

Eight cases of inflammatory abdominal aortic aneurysm (IAAA) (group I) and a control group of ten cases of atherosclerotic abdominal aortic aneurysm (AAA) with little or no parietal inflammatory infiltrate (group II) were studied; using light microscopy, transmission electron microscopy (TEM), and immunohistochemistry. These were used to define cell composition in the inflammatory process, the degree of cell activation and alteration of connective tissue. Large numbers of B lymphocytes were present in IAAA with preservation of the T4/T8 ratio. In addition, HLA-DR and the IL2-R antigen (specific for activated cells) were widely expressed in the cell population. The interstitial matrix contained deposits of IgG, IgM and C3c together with an increase in type III collagen and a reduction in elastin which appeared fragmented and swollen. This study, therefore, characterised the cellular component of the parietal inflammatory infiltrate in IAAA. The degree of activation shown by these cell elements and the activation of complement suggest that the relevant antigen may have been localised in the aneurysm wall at the time of observation.

Composite Gastric Carcinoma and Precursor Lesions with Amphicrine Features in Chronic Atrophic Gastritis

A composite carcinoma of the gastric body consisting of endocrine and mucous epithelial cells with interspersed amphicrine cells is reported together with ultrastructural and immunocytochemical documentation of endocrine and nonendocrine differentiation. The tumor was associated with hypergastrinemia related to chronic atrophic gastritis (achlorhydria) and with multiple proliferative lesions, such as intramucosal microcarcinoid (IMC) and endocrine cell proliferations of the micronodular and linear type, which are currently regarded as carcinoid precursor changes. Ultrastructurally, a composite architecture with amphicrine features was demonstrated in the primary tumor, IMC, and liver metastases. On the other hand, the endocrine cell proliferations exclusively contained gastrin and enterochromaffinlike cells. Immunostaining with antibodies to calcitonin documented a number of positive cells both in the primary and in the metastatic sites. This is the first report of mixed exocrine-endocrine-amphicrine components both in a metastasizing carcinoma and in its precursor lesions in a chronic hypergastrinemic state. Unlike previously reported lesions, the endocrine component was unexpectedly composed of calcitonin cells, which are not usually present in the gastric mucosa.

An Ultrastructural and Immunocytochemical Analysis of Human Endothelial Cell Adhesion on Coated Vascular Grafts

Human adult endothelial cells were enzymatically harvested from adipose tissue. Cell viability was established by Trypan blue exclusion and transmission and scanning electron microscopy. Endothelial cells were identified by immunocytochemical investigation at light microscopy, transmission electron microscopy, and scanning electron microscopy. Isolated cells were positive for actin and vimentin, negative for desmin. Factor VIII RA was mainly expressed at cell surface and occasionally disclosed in the cytoplasm. Reactivity for UEA I and J15 was weak or undetectable. Human endothelial cells were seeded and left to adhere for one hour onto different nonvascular substrates (glass, poly-l-lysine, formvar-carbon, fibronectin, Teflon). Scanning electron microscopy defined surface features, suggesting tenacious cell adhesion on the substrate. Different vascular substrates were tested (preclotted Dacron, albumin Dacron, Hemashield Dacron, Gelseal Dacron, ePTFE, fibronectin-ePTFE). Commercially available coated grafts showed qualitative and quantitative differences in cell adhesion. In particular, Gelseal Dacron provided the best quantitative results, even though a wide variability was observed. In contrast, fibronectin-coated ePTFE gave more reliable results and high spreading efficiency. In the short term, coated grafts do not seem to offer greater advantages than fibronectin-coated ePTFE. However, specific incubation times for each coated graft should be selected and the long-term approach (graft culture) should also be attempted.

Electron Microscopic and Immunocyto- chemical Profiles of Human Subcutaneous Fat Tissue Microvascular Endothelial Cells

The ultrastructural and immunocytochemical characteristics of microvascular cells from human subcutaneous fat tissue were studied after the addition of collagenase and Percoll density gradient, respectively. Monoclonal and polyclonal antibodies directed against antigens specific for endothelial cells (factor VIII,Ulex europeaus, CD31, and CD34), pericytes (muscle-specific actin and desmin), adipocytes (S-100 protein), and monocytes-macrophages (MAC 387 and 150.95 protein) were demonstrated by alkaline phosphatase monoclonal antialkaline phosphatase and protein A-gold techniques. In addition, to determine whether the harvesting method interfered with microvascular cell function, DOT immunoassays of factor VIII and CD34 were conducted on solutions recovered at collagenase incubation as well as after nylon filtration and Percoll administration, respectively. After the collagenase step, the vast majority of microvascular cells had the typical ultrastructural and immunophenotypical features of endothelial cells. In sharp contrast, following the Percoll step, only 1% to 18% of microvascular cells stained with factor VIII,Ulex europeaus, and CD31, whereas 90% of them expressed the CD34 antigen. Surprisingly, DOT immunoassay revealed the presence of factor VIII in the washing buffer recovered after the Percoll step only. Consequently the decreased expression of common endothelial cell markers (factor VIII,Ulex europaeus, and CD31) observed at the end of the cell isolation procedure was related to the adverse effects of Percoll on endothelial cell function. The CD34 surface molecule, being highly resistant, is particularly well suited for unequivocal characterization of microvascular cells as true endothelium.

Filamentous Inclusions in Nonneoplastic and Neoplastic Pancreas: An Ultrastructural and Immunogold Labeling Study

Filamentous inclusions (FI) are unusual, irregularly shaped cytoplasmic inclusions, which are mostly found in acinar cell carcinomas of the pancreas and are consequently thought to be an abnormal zymogen granule type. This study describes identical inclusions in acinar, centroacinar, and small duct epithelial cells from nonneoplastic pancreas, as well as those found in tumor cells from a mixed acinar-endocrine pancreatic carcinoma. An ultrastructural and immunogold labeling demonstration indicates that these inclusions are aggregates of intermediate filaments immunoreacting with the anti-cytokeratin AE1/AE3 mixture and with V9 clone anti-vimentin monoclonal antibodies. Their pleomorphic appearance, variable immunoreactivity, and frequent association with lipid droplets and secondary lysosomes, mostly of the angulate type, led to the hypothesis that the FI undergo a degenerative remodeling pathway similar to that proposed for hepatic Mallory bodies. A survey of the literature on FI and human tumors suggests that they are a variably expressed ultrastructural feature of tumor cells originating from exocrine cell-containing tissues, namely the pancreas and gastrointestinal tract.

First evidence of a pathogenic insertion in the NOTCH3 gene causing CADASIL

CADASIL (OMIM 125310) is an increasingly recognised adult-onset autosomal-dominant vascular disease that is characterised by recurrent transient ischaemic attacks and strokes (43% of patients), vascular dementia (6%), migraine with aura (40% of patients) and psychiatric disturbances (9% of patients); epilepsy has been reported in 2–10% of subjects.1 All patients revealed prominent signal abnormalities on brain magnetic resonance imaging (MRI)—leukoencephalopathy on T2- and small subcortical infarcts on T1-weighted images.2 The pathological hallmark of CADASIL is a non-amyloid and non-arteriosclerotic angiopathy, which predominantly affects the small penetrating brain arteries. Vascular lesions are characterised by degeneration and loss of smooth-muscle cells and by the presence of granular osmiophilic material (GOM) accumulating within the smooth-muscle-cell basement membrane and the surrounding extracellular matrix. Examination of several peripheral organs revealed vessel changes, including the presence of GOM deposits, providing evidence that CADASIL is a systemic arteriopathy.3 It has been reported that CADASIL is caused by single missense mutations, small in-frame deletions or splice-site mutations in the NOTCH3 gene encoding a transmembrane receptor (http://www.hgmd.cf.ac.uk/ac/gene.php?gene = NOTCH3). Almost all previously reported mutations resulted in an odd number of cysteine residues within one of the 34 epidermal growth factor (EGF)-like repeats in the …

Stromal cells in primary myelofibrosis: ultrastructural observations.

SummaryThe bone marrows of five patients with primary myelofibrosis at different stages of the disease have been studied. In the myelofibrotic bone marrow, associated with “reticulum cells”, two other cell types have been identified, namely fibroblast-like and myofibroblast-like reticulum cells, as well as a spectrum of transitional forms.Our findings suggest that reticulum cells may represent a reserve stromal cell pool (i.e. primitive reticulum cells) able to modulate themselves and to transform differently according to functional requirements.Some suggestions regarding the functional significance of fibroblastlike and myofibroblast-like reticulum cells in primary myelofibrosis are suggested.

Hereditary sensory and autonomic neuropathy with ataxia and late onset

We report two brothers affected by a dominantly inherited form of hereditary sensory and autonomic neuropathy (HSAN), characterized by clinical features of sensory ataxia, and by late onset in the 6th decade. Sural nerve biopsy in the proband showed almost complete loss of myelinated fibers, and relative sparing of unmyelinated fibers. This family showed an atypical presentation of HSAN, which is usually characterized by acrodystrophic manifestations of infantile or juvenile onset. Although a few reports of HSAN presenting with late onset and/or ataxia appeared, this is the first report of a family with dominant HSAN characterized by late onset sensory ataxia.