Novel automated three-dimensional genome scanning based on the nuclear architecture of telomeres.

Telomeres, the end of chromosomes, are organized in a nonoverlapping fashion and form microterritories in nuclei of normal cells. Previous studies have shown that normal and tumor cell nuclei differ in their 3D telomeric organization. The differences include a change in the spatial organization of the telomeres, in telomere numbers and sizes and in the presence of telomeric aggregates. Previous attempts to identify the above parameters of 3D telomere organization were semi-automated. Here we describe the automation of 3D scanning for telomere signatures in interphase nuclei based on three-dimensional fluorescent in situ hybridization (3D-FISH) and, for the first time, define its sensitivity in tumor cell detection. The data were acquired with a high-throughput scanning/acquisition system that allows to measure cells and acquire 3D images of nuclei at high resolution with 40 × or 60 × oil and at a speed of 10,000-15,000 cells h(-1) , depending on the cell density on the slides. The automated scanning, TeloScan, is suitable for large series of samples and sample sizes. We define the sensitivity of this automation for tumor cell detection. The data output includes 3D telomere positions, numbers of telomeric aggregates, telomere numbers, and telomere signal intensities. We were able to detect one aberrant cell in 1,000 normal cells. In conclusions, we are able to detect tumor cells based on 3D architectural profiles of the genome. This new tool could, in the future, assist in patient diagnosis, in the detection of minimal residual disease, in the analysis of treatment response and in treatment decisions.

Characterization of cytoplasmically oriented Golgi proteins with a monoclonal antibody.

BALB/c mice were repeatedly immunized with a galactosyl transferase-rich microsomal fraction of rat myeloma cells. Spleen cells were subsequently fused with Sp2/0 mouse myeloma cells, the resulting hybridomas were cloned, and their secreted Ig was screened for reactivity with antigens belonging to the Golgi complex. One such monoclonal antibody, 6F4C5, gave especially intense immunofluorescent staining of the Golgi area of myeloma cells and fibroblasts. It recognized two proteins bands on immunoblots of gel-fractionated cell lysates: a major one with an estimated Mr of 54,000 and a minor one at 86,000. Both proteins were concentrated in microsomal fractions isolated at low ionic strength. They were hydrophilic judging from partitioning of a Triton X-114 cell lysate. Both were cytoplasmically oriented as demonstrated by protease and high KCl treatments of postmitochondrial supernatants and microsomal fractions. Neither was retained by columns of insolubilized wheat germ agglutinin or concanavalin A, which suggests that they are not glycoproteins. Their more detailed location in the Golgi complex was studied by immunoelectron microscopy, using a saponin permeabilization procedure and peroxidase-conjugated reagents. The observed staining was restricted to two or three cisternae in the medial part of the stack. Nevertheless, differential centrifugation experiments indicated that the two antigens may be recovered in distinct subcellular fractions: this may be related to the unexpected observation that rather low salt concentrations strip the antigens from microsomal fraction.

An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy.

Scanning confocal microscopes offer improved rejection of out-of-focus noise and greater resolution than conventional imaging. In such a microscope, the imaging and condenser lenses are identical and confocal. These two lenses are replaced by a single lens when epi-illumination is used, making confocal imaging particularly applicable to incident light microscopy. We describe the results we have obtained with a confocal system in which scanning is performed by moving the light beam, rather than the stage. This system is considerably faster than the scanned stage microscope and is easy to use. We have found that confocal imaging gives greatly enhanced images of biological structures viewed with epifluorescence. The improvements are such that it is possible to optically section thick specimens with little degradation in the image quality of interior sections.

Human myeloma cell line carrying a Philadelphia chromosome.

A new human plasmacytoma cell line (Karpas 707) has been established from a myeloma patient. The cultured cells are negative for Epstein-Barr viral nuclear antigen and free of mycoplasma. They are similar to plasma cells and secrete only lambda light chains. The cells are hypodiploid and contain the Philadelphia chromosome and other abnormalities. This cell line may be suitable for the production of human monoclonal antibodies.

Extramedullary plasmacytoma extensively affecting the sella turcica and paranasal sinuses.

We report a 62-year-old Japanese male who complained of double vision and showed clear boundary mass extending to the clivus, intrasella, suprasella, ethmoidal sinus and sphenoid sinus on neuroimaging. The tumor mass was partially resected via transsphenoidal approach and was diagnosed as the extramedullary plasmacytoma by IgA immunostaining and electron microscopy. Making diagnosis from the imaging findings was difficult in this rare case, but immunohistological and electron microscopic examinations were useful for pathological diagnosis.

Role of intracisternal A-particles and of RNA molecules in plasmacytoma-associated immunodeficiency.

A subcellular fraction from murine plasmacytoma cells was shown to suppress the primary antibody response when injected into normal mice. The active subcellular fraction copurified with intracisternal A-particles. The RNA extracted from subcellular fractions enriched in A-particles was also immunosuppressive. This activity was due to a population of RNA molecules that contained polyadenylic acid. Upon fractionation on a sucrose gradient, two populations of immunosuppressive RNA were obtained with sedimentation velocities of 12 to 18S and 40 to 50S. The 40 to 50S RNA was shown to be a thermolabile aggregate of molecules that contained the 12 to 18S RNA molecules. Plasmacytoma-derived material with similar physicochemical characteristics had previously been shown to induce in normal mouse lymphocytes surface immunoglobulins with the plasmacytoma idiotype, supporting the possibility that one of the mechanisms responsible for the development of immunological deficiency is the change of surface immunoglobulins of nonmalignant B-cells.

Electro-acoustic fusion of erythrocytes and of myeloma cells.

Mammalian cells can be concentrated in a sound field. A method is introduced, which combines the reversible aggregation of cells in a sound field with the electrical breakdown of cell membranes to fuse cells, which are in contact. Human red blood cells and mouse myeloma cells are fused by means of that procedure.

Differences in the association of ribosomes with heavy rough and light rough endoplasmic reticulum membranes of MPC-11 cells.

The treatment of total endoplasmic reticulum membranes of mouse plasmacytoma cells with EDTA resulted in an abolition of the heavy rough (HR) subfraction, while there was a large increase in smooth (S) membranes. When HR and light rough (LR) endoplasmic reticulum membranes were treated individually with EDTA and re-centrifuged on discontinuous sucrose gradients it was observed that HR were converted into S membranes, i.e. membranes virtually devoid of ribosomes. LR membranes were not affected to the same extent but there was a shift to a somewhat lower density. A quantitation of ribosomes released by EDTA showed that 95% of 60 S and 72% of 40 S subunits were removed from HR membranes while for LR membranes the corresponding values were 8.5 and 22.6% respectively. Ratios of radioactivity to absorbance at 260 nm calculated for 40 S and 60 S subunits isolated from HR and LR membranes show that 60 S subunits from LR membranes, in contrast to those from HR membranes, equilibrate only slowly with the free pool of ribosomal subunits. The results indicate that the ribosomes associated with HR membranes are 'loosely bound' and those with LR membranes 'tightly bound'. When poly(A)-containing mRNA isolated from HR and LR membranes was translated in vitro and the products analysed for light-chain immunoglobulin content, it was found that the HR fraction was enriched in light-chain mRNA.

Monoclonal antibodies as markers of the endocytic and secretory pathways.

A galactosyltransferase-rich subcellular fraction and wheat germ agglutinin(WGA)-binding microsomal proteins from rat myeloma cells have been used to immunize BALB/c mice. Fusion of the corresponding spleen cells with the Sp2/0 mouse myeloma has lead to the production of hybridomas secreting monoclonal antibodies directed against four proteins of the Golgi complex (GC) and other smooth membranes (SM). Subcellular fractionation of myeloma cells and rat liver, Triton X-114 partitioning, protease treatment and lectin binding studies have permitted us to identify--by immunoblotting--the molecular weight of the proteins involved, their topology and their mode of association with membranes. Morphological analysis has been performed by immunocytochemistry at the light and electron microscopic level. Judging by these criteria, the GCII antigen is a protein of 44 kDa which is loosely associated with the endodomain of Golgi cisternae. GCIII is a detergent-binding glycoprotein of 130 kDa whose epitope is on the endodomain of Golgi cisternae. SMI is a detergent-binding glycoprotein of 58 to 90 kDa found at several stations along the endocytic path: in coated pits, coated vesicles, endocytic vesicles, but not in lysosomes. The epitope recognized by the corresponding antibody faces the ectodomain. When this antibody is added to living cells in culture, it is rapidly internalized. SMII is a detergent-binding glycoprotein of 140 kDa. The epitope recognized is restricted to membranes of Golgi complex cisternae and multivesicular bodies. These reagents should be useful for dissection and perturbation of vesicular traffic.

Ultrastructural analysis of cytoplasmic intermediate filaments and the nuclear lamina in the mouse plasmacytoma cell line MPC-11 after the induction of vimentin synthesis.

We examined cytoplasmic intermediate filaments (IFs) and the nuclear lamina in cells of the mouse plasmacytoma cell line MPC-11 (lacking both IF proteins and lamins A and C) after induction of vimentin synthesis with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) by means of whole-mount immunogold electron microscopy (IEM). The technique of IEM was modified to allow analysis of the cytoskeleton and nuclear lamina of cells grown in suspension culture employing antibodies against vimentin and lamin B. IEM showed that newly synthesized vimentin assembled into IFs which formed anastomosing networks throughout the cytoplasm, radiating primarily from the nucleus. The filaments decorated by gold-conjugated antibodies appeared to make contact with the lipid-depleted nuclear envelope residue either by directly terminating on it or through an indirect link via short fibers of varying diameter. Some filaments terminated on the subunits of the nuclear pore complexes but they did not pass through the pores. In the absence of lamins A and C, lamin B formed a nuclear lamina consisting of a globular-filamentous network anchoring the nuclear pore complexes.

Zinc, calcium, and magnesium metabolism: effects on plasmacytomas in Balb/c mice.

The effects of different amounts of dietary zinc on the Zn absorption rate and on Zn, calcium and magnesium concentrations in tissues of MOPC 104E tumor-bearing Balb/c mice were determined. The Zn absorption rate was inversely related to the amounts of Zn in their diets and was lower than that of nontumor-bearing control mice fed a laboratory mice chow. Zn concentrations of tumor-bearing mice were also low compared with control mice but tumor Zn concentrations, regardless of the concentrations of Zn in the diets, were higher than those of normal tissues of the host other than the pancreas. Ca concentrations in tumor and tissues of tumor-bearing mice were higher than in control animals but Mg concentrations in tissues of tumor-bearing mice appeared to be similar to those of control mice. Results suggest that tumor-bearing mice have a lower intestinal Zn absorption capacity and a higher Zn uptake rate causing other tissues to become hypozincemic and hypercalcemic.

Autoradiography of phosphatidyl choline.

Saturated choline phosphatides are extracted during conventional tissue processing for electron microscopy. To facilitate autoradiographic subcellular localization of arrhythmogenic myocardial phospholipids, we evaluated tissue processing procedures for preservation of saturated phosphatidyl choline (PC). Suspensions, of a murine plasmacytoma were incubated with negative, unilamellar liposomes containing 14C-choline-labeled PC or 14C-1-palmitate dipalmitoyl PC. Extraction of radioactivity was monitored at each processing step by liquid scintillation spectrometry. Conventional fixation with glutaraldehyde and osmium tetroxide followed by acetone dehydration and Spurr's plastic embedding led to extraction of nearly all radioactivity. However, treatment of cells with 1.5% tannic acid after glutaraldehyde but before osmium tetroxide fixation preserved 93.1 +/- .6% of 14C-choline-labeled PC. Virtually identical results were obtained with dipalmitoyl PC. Autoradiography demonstrated no significant translocation of labeled PC from plasmacytoma cells to unlabeled avian erythrocytes, mixed in equal proportions after fixation but before dehydration and embedding.

Ultrastructural analysis of human plasmacytoma cells prepared on affinity beads after exposure to anti-idiotype antibodies.

Mononuclear cells from a bone marrow infiltrated by plasmacytoma cells were examined by electron microscopy. An analysis was performed according to different cytoarchitectural forms of the endoplasmic reticulum (ER). Six types of plasma cells could be distinguished. The bone marrow cells were treated with an anti-idiotype antiserum from a guinea pig prepared against the patient's monoclonal serum protein and with a FITC conjugated anti-guinea pig antiserum from the rabbit as second layer. Then the cells were passed through an affinity gel column with anti-FITC antibodies. The original preparation and the cells separated on the affinity gel were analysed by electron microscopy. It was found that an ultrastructurally distinct type of plasma cell was enriched 3.5-fold over the original sample by the separation procedure.

Extramedullary plasmacytoma. A form of marginal zone cell lymphoma?

Extramedullary plasmacytoma (EMP), solitary plasmacytoma of bone, and multiple myeloma are related neoplasms, but EMP is clearly a distinct entity. Moreover, there are histologic and clinical similarities between EMP and marginal zone B-cell lymphomas (MZLs) displaying extensive plasma cell differentiation, suggesting a possible histogenetic relationship. The histologic and clinical features of 5 EMPs with extensive plasma cell differentiation were histologically reviewed for features of MZL. The previously diagnosed MZLs, mucosa-associated lymphoid tissue (MALT) type, of 2 patients also were reviewed. All patients were women aged 48 to 79 years. The EMPs originated in the parotid gland, lymph nodes, dura, or small bowel. The initial tumors diagnosed as MALT-type MZL were located in the lung and small bowel. All patients were treated with resection, with or without irradiation therapy. One patient also received systemic chemotherapy. All patients are alive with no evidence of disease. All tumors contained large numbers of plasma cells, constituting between 55% and 90% of the lymphoid cells. Centrocyte-like cells and monocytoid B cells each represented 0% to 25% of the infiltrate. Lymphoepithelial lesions were observed in all of the tumors in sites where epithelium was present. Reactive follicles were found in all of the tumors. EMPs may represent MZLs that have undergone an extensive degree of plasmacytic differentiation.

Cytoplasmic fibrils in plasma cells of a solitary myeloma.

A 56-year-old woman had solitary plasma-cell myeloma in the right humerus. Six weeks after local irradiation therapy, she died from pulmonary alveolar hemorrhage and bilateral pleural effusion with malignant plasma-cell infiltration of the right atrium. Ultrastructural studies of the plasma cells from the myocardium demonstrated large bundles of prominent, non-branching, solid cytoplasmic fibrils in many cells. Each fibril had a width of 66--132 A, periodicity of 30--60 A, and a beaded appearance. These features suggest that the fibrils may be protein material, possibly an immunoglobulin.

Cardiac plasmacytoma.

A 69-year-old white woman presented with a 3-month history of progressive dyspnea, orthopnea, fatigue and weakness. Clinical, diagnostic imaging and echocardiographic investigations suggested an occult primary cancer with metastasis to the heart. The patient's condition deteriorated gradually, and she died 2 months later. At autopsy, a malignant tumor encasing the heart and a 1-cm solitary tumor nodule in the lower lobe of the left lung were found. Histologic and electron microscopic studies revealed a plasmacytoma predominantly involving the epicardium and a small solitary plasmacytoma located in the left lung. The two tumors were further confirmed by immunohistochemical studies that showed monoclonal IgG expression and kappa light chain restriction.

IgM lambda cytoplasmic crystals in three cases of immunocytoma: a clinical, cytochemical, and ultrastructural study.

Endoplasmic reticulum-associated crystals were seen in 1-10% of the bone-marrow lymphocytes and in the lymphocytes of the peripheral blood in three cases of immunocytoma. Their crystalline nature and their location in the cisternae of the rough endoplasmic reticulum was proved by ultrastructural study. IgM lambda in the crystals was demonstrated by fluorescent and peroxidase-labelled antibody methods. The crystals did not stain with the PAS reaction, suggesting that the immunoglobulin was not bound to a carbohydrate group. A defect in glucosyltransferase activity with failure to modify the immunoglobulins could explain the absence of the PAS reaction and the accumulation of immunoglobulin in crystalline before reaching the Golgi region.

Multiple primary cutaneous plasmacytomas.

BACKGROUND: Cutaneous plasmacytoma is an uncommon tumor and is mostly seen in the context of end-stage multiple myeloma. Only 20 cases of primary cutaneous plasmacytoma have been documented. A significant proportion of these patients went on to develop systemic disease with a poor prognosis. In a number of patients, however, the abnormal clone of plasma cells may arise in the skin and never progress to multiple myeloma involving the bone marrow. OBSERVATIONS: We describe a patient who developed multiple primary cutaneous plasmacytomas after a possible insect bite reaction. The monoclonality of the tumor cells is demonstrated using immunohistochemical techniques. He has been treated vigorously with chemotherapy and local radiotherapy and remains well 3 years after diagnosis. Bone marrow has been harvested for use as an autologous bone marrow transplant in the event of systemic relapse. CONCLUSIONS: Unlike previous reports of this rare entity, this case documents the monoclonality of tissue plasma cells with immunohistochemical techniques. As cutaneous plasmacytomas have been reported with an early significant mortality, unlike extramedullary plasmacytomas elsewhere, we have advocated combination chemotherapy and cryopreservation of uninvolved bone marrow for future autologous bone marrow transplantation should systemic myelomatosis develop in the patient.

Revised seminology of the different mononuclear cells under scanning electron microscopy.

Scanning electron microscopy shows that the thymus cell surface is not smooth but slightly undulated; this type of surface characteristic is found on the cells of several types of T-lymphocyte tumors such as T-cell lymphosarcoma, mycosis fungoides, and T-immunoblastic lymphosarcoma. The cell surface of Frabricius' bursa is covered with numerous short villi. They are found on the cells of several kinds of B-lymphocyte tumors, including B-cell chronic lymphoid leukemia, B-prolymphocytic lymphosarcoma, and B-immunoblastic lymphosarcoma. Mycosis fungoides cells frequently have a characteristic shape. Normal and myeloma plasma cells are similar in shape but their surface is covered with numberous characteristic tiny balls. Immunoblasts are twice the size of so-called small lymphocytes. In the blood and in the lymphocyte population transformed by mitogens, cell surfaces vary from completely smooth (with no undulations as in thymic cells) to villous, with short to very long villi. These variations are visible even when a constant technique and the critical-point drying method are used. Their significance, however, is not known.

Intracellular crystalline deposits in lymphoplasmacellular disorders.

Crystalline immunoglobulin intracellular inclusions may be found in a range of B-cell neoplasias, including MALT (mucosa-associated lymphoid tissue) lymphoma. The crystals are regarded as an outcome of impaired cell secretory activity leading to their accumulation. At times the deposits may be so profound as to obscure the diagnosis and may even lead to misdiagnosis. Examples of the crystals are presented along with a brief literature review.