Etienne de Harven

Feline Leukemia Virus: Occurrence of Viral Antigen in the Tissues of Cats with Lymphosarcoma and Other Diseases

Felinie leutkemia viruts antigen is demonstrable by immunodiffusion with rabbit precipitating antiserum to purified felinie leukemia virus. The felinie leukemia virus antigen was found in the tissues of 25 of 33 cats with lymphosarcoma and of 5 of 13 cats with infectious peritonitis. Its presence was correlated with the occurrence of felinie leukemia virus demonstrable by electron microscopy. The one clinically normal cat giving a positive test for feline leukemia virus antigen belonged to a household in which two cats had developed lymphosarcoma. With the exception of a dog with lymphosarcoma induced by feline leukemia virus, the antigent was absent from lymphosarcoma and nonlymphomatous tumors of other species (man, dog, cow, goat, or pig).

Cell surface antigens, serial sectioning of single cells as an approach to topographical analysis.

The topographical distribution of H-2 antigens on the surfaces of C57BL/6 mouse thymocytes and lymph-node lymphocytes was investigated by a new technique. Single cells were reacted with visually labeled antibody directed against H-2 antigen. Each cell was processed for electron microscopy individually and was serially sectioned. Models constructed from serial electron micrographs provided detailed views of the entire cell surface and showed that H-2 antigen occurs on thymocytes in small isolated regions and on lymphocytes in large interconnected regions.

Regulation of expression of a human lymphoid cell surface marker by iron

Abstract Spontaneous rosette formation of human peripheral blood lymphocytes with sheep erythrocytes was inhibited by pretreatment of cells with Fe-citrate, fully saturated TF (but not native TF), fully saturated LF, or native LF. The inhibition was observed within 5 min of incubation; it was relatively temperature dependent, and not mediated by the presence of macrophages, since it occurred after depletion of adherent cells from the suspension. The inhibitory effect of iron was reversed by further treatment of the cells with two highly specific iron-chelating agents, DFX and SHAM, or culture in medium supplemented with 15% FCS. Alternate expression or inhibition of the receptor was observed when the cells were cultured sequentially in low- or high-iron medium for periods of 24 hr. To our knowledge, this is the first demonstration of regulation of expression of a surface marker in mammalian cells by iron. Iron is known to play a similar regulatory role of membrane receptors in bacteria.

Ultrastructural Studies of the Megakaryoblastic Leukemias of Down Syndrome

The ultrastructure of the leukemic cells in transient leukemia (six cases), myelodysplasia (five cases) and acute megakaryoblastic leukemia (one case) in patients with Down syndrome were studied. The cells were identified to be of megakaryocytic lineage by virtue of the expression of platelet glycoprotein GpIIIa, detected by immunogold labelling. In all patients, some of the leukemic cells had ultrastructural features of megakaryocytes, including ectoplasmic protrusions, demarcation membranes, and alpha granules. Differentiation was greatest in the cells of patients with transient leukemia. These studies provide a detailed assessment of the ultrastructural features of the leukemic cells in the megakaryoblastic leukemias of Down syndrome.

Functional and Morphological Characterization of Immunomagnetically Selected CD34+ Hematopoietic Progenitor Cells

We evaluated the potential of immunomagnetically selected (miniMACS) progenitor cells to give rise to colony‐forming cells and their precursors, detected as long‐term culture‐initiating cells (LTC‐IC), as well as their capacity to expand in liquid cultures. A 90% mean purity, a 43.2% yield and a 55.8‐fold enrichment were achieved from normal bone marrow. When corrected for enrichment, the mean number of committed progenitor cells and the frequency of LTC‐IC (evaluated by means of limiting dilution assay [LDA]) were not statistically different in low density mononuclear cells or in the CD34‐enriched fractions. In five cases CD34+ selected cells grown in a stroma‐free long‐term bone marrow culture system with the addition of stem cell factor, interleukin 3, interleukin 6 and GM‐CSF every 48 h, showed a 15 (±15) and 31 (±21) mean colony forming unit‐granulocyte/macrophage fold increase on cultures at days 7 and 14. However, when corrected for enrichment, the expansion capability of these cells was significantly lower than that of the unseparated fraction, particularly after the first week. Immediately after separation, electron microscopy revealed that the CD34+ selected fraction contained more than 45% of well‐differentiated myeloid cells (MPO+ ), with iron beads preferentially clustered at one pole of the cell surface and sometimes already endocytosed in pinocytic vesicles. After 24 h and 48 h incubation at 37°C, the majority of the cells showed no iron particles, but about 30% of the cells were iron‐labeled phagocytic cells. The percentage of apoptotic cells with internalized iron was negligible. These data show that immunomagnetically separated CD34+ cells may have a slightly impaired short‐term expansion capability, but give rise to both committed and more primitive progenitor cells. During the separation, the iron beads are internalized, rapidly processed in the cytoplasm and do not seem to interfere with in vitro growth.

Ultrastructural features of CD34+ hematopoietic progenitor cells from bone marrow, peripheral blood and umbilical cord blood.

Hematopoietic progenitor cells from different sources have been widely characterized, but their ultrastructural morphology has never been described in detail. In this study, imunomag-netically separated CD34+ cells from normal bone marrow (BM), mobilized peripheral blood (PBSC) and human umbilical cord blood (CB) were studied by transmission electron microscopy (TEM) using a cytochemical method which reveals endogenous myelo-peroxidase (MPO) activity. This technique is particularly suited for detecting early signs of the myeloid commitment. The CD34+ cells from PBSC were morphologically very homogeneous and 94.7 ± 4.5% of these cells were MPO−: these ultrastructural features are generally considered typical of immature cells. The CD34+ BM cells were instead more heterogeneous, with 24.6 ± 7.4% showing intense MPO activity. The ultrastructural characteristics of CB cells fell between those observed in PBSC and BM, but there was a high percentage of morphologically immature cells with no evidence of MPO activity (about 83%). The number of apoptotic cells within samples from different sources was also examined both by TEM and flow cytometry. The percentage of apoptotic cells was 0.7% in PBSC, 2.3% in BM, 2.9% in CB from vaginal delivery and 11.6% in CB from cesarean section. These observations confirm the relative phenotypic immaturity of CB in comparison with BM cells; they also suggest that CB collected after cesarean section may be associated with reduced stem cells viability.

Hairy Cell Leukemia, an Ultrastructural Study with Double Immunogold Labeling

Two cases of hairy cell leukemia were studied using a novel method of sequential double immunogold labeling for both scanning and transmission electron microscopy. Characteristic ribosome lamellae complexes were observed in one case only. The antigens expressed on the surface of hairy cells were those recognized by anti-CD 11c and anti-CD20 murine monoclonal antibodies. All hairy cells, in both cases, co-labeled for the corresponding antigens, as demonstrated by recognizing colloidal gold markers of distinct sizes (5 and 20 nm, or 15 and 40 nm) under SEM and TEM. The specificity of the double labeling method seems to be most satisfactory. Immuno-electron microscopy enables us to characterize the integrated ultrastructural and antigenic phenotype of hairy cells at the single cell level. This represents an unique analytical approach which may contribute significantly in further studies aimed at a better understanding of the pathogenesis of hairy cell leukemia.