EAC Rosettes Research Articles

(18) Lichen aureus

The clinical course of a patient with acute lymphoid immunoblastic leukaemia and prominent nodular haemorrhagic skin lesions is described. Cytological, cytochemical and electronmicroscopic studies were performed on bone marrow and skin blast cells. The absence of surface immunoglobulins and of the other markers for B lymphocytes (EA and EAC rosettes) and the presence of 30% of spontaneous sheep erythrocyte rosettes excluding an acute leukaemia with Burkitt's tumour cells, suggest that T cells are involved. Complete haematological and cutaneous remission was obtained with prednisone therapy.

Properties of Nine Continuous B‐Cell Lines Established from Enzootic Bovine Leukosis Tumors

We established 9 cell lines from 63 tumor cases of enzootic bovine leukosis and studied their properties. Cells of all lines formed small clumps and floated in culture medium, indicating growth. Four of the 9 cell lines were surface immunoglobulin (SIg)-positive, but the remaining 5 line cells were negative for SIg or, if SIg was detected, the percentage of SIg-positive cells was very low. Tests for the properties of the cells with monoclonal antibodies to lymphocytes revealed that the established line cells are B-lymphocytes. Morphological observation also revealed that they had the morphology of B-lymphoblastic cell. The results of E and EAC rosette assay were negative, but 6 of 8 cell lines were positive for EA rosetting. All the 9 cell lines reacted with MoAb C-143, which recognizes the tumor-associated antigen (TAA) of the EBL tumor cell. All 9 cell lines produced bovine leukosis virus (BLV). These results suggest that the 9 cell lines are tumor cells derived from B-lymphocytes of EBL.

Novel Cell Surface Antigens Expressed on Mouse Alveolar Macrophages

Two new cell surface antigens expressed on mouse alveolar macrophages were defined by rat monoclonal antibodies. One marker, AVM-1, was detected on mouse alveolar macrophages, but it was undetectable on resident peritoneal cells, thioglycollate medium-induced peritoneal cells, and splenic macrophages. Splenic lymphocytes, thymocytes and bone marrow cells were also AVM-1 negative. Anti-AVM-1 monoclonal antibody immunoprecipitated a single polypeptide with a molecular weight of 200,000. Of particular interest was the finding that the anti-AVM-1 antibody could inhibit the formation of EA and EAC rosette on macrophage line cells. A second antigen (AVM-2) was also present on alveolar macrophages, and its molecular weight was 38,000.

Properties of solubilized Fc?-receptors from psoriatic scales

Extracts from psoriatic scales, prepared using Tris-HCl buffer containing ethylenediaminetetraacetatic acid (EDTA) and 2-mercaptoethanol (ME), agglutinated erythrocytes (E) sensitized with IgG antibodies (A) (EA), but not E or E sensitized with F(ab')2-fragments of IgG. The agglutination was inhibited by IgG and Fc fragments of IgG, but not by IgA, IgM or F(ab')2-fragments of IgG. Partially reduced and alkylated IgG did not inhibit the agglutination, indicating that an inter-heavy-chain disulphide-linked Fc region is required for binding of FcR. The extracts inhibited EA, but not E or EAC rosette formation with mononuclear cells. The results strongly indicated that the extract contained functionally active FcR. The agglutinating activity of the extract was not affected by treatment with periodic acid or formaldehyde, whereas heat reduced the activity. Using a monoclonal antibody (B1D6) a functionally active 40 kDa FcR with low affinity for native IgG was purified from the scale extract. The extracts also contained FcR activity not recognized by B1D6.

T and B lymphocytes in horses persistently infected with equine infectious anaemia virus.

The percentage of T and B lymphocytes in the peripheral blood of horses chronically infected with equine infectious anaemia (EIA) virus was determined and the results were compared with the percentage of these cells in healthy uninfected horses. Cells with membrane receptors for sheep erythrocytes (T and active T lymphocytes) were determined by E and A rosette techniques, while cells with receptors for the C3b component of complement and those with receptors for mouse erythrocytes (B lymphocytes), were determined by the EAC rosette method. The percentage of Fe positive cells was assayed by the EA rosette test. The majority of peripheral blood lymphocytes (PBL) from both uninfected and EIA-infected horses formed rosettes of each kind with only three erythrocytes indicating a low density of the corresponding receptors on the cell membrane under the condition of the assays used. The percentage of T lymphocytes in the peripheral blood of diseased horses (52.4 +/- 1.6%), as detected by E rosettes, was significantly (p less than 0.01) higher than in control animals (42.4 +/- 3.5%). In clinically healthy horses 8.9 +/- 1.1% of PBL were identified by A rosettes as active T cells, whereas animals with a chronic form of EIA had a much lower (p less than 0.001) percentage of these cells (4.7 +/- 0.7%). In the B lymphocyte subpopulations the percentages of cells bearing Fc and C3b receptors were markedly elevated (p less than 0.001) in EIA-infected horses (24.7 +/- 0.8% and 42.8 +/- 2.2% respectively) as compared to uninfected animals (15.1 +/- 1.4% and 29.6 +/- 1.2% respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Humoral and cellular parameters of the immune system of Cebus apella monkeys. Cross reactivity between monkey and human immunoglobulins

The humoral and cellular immunological parameters of the New World non-human primate Cebus apella were analysed. The study included: serum protein immunoelectrophoretic analysis; cross reactivity between monkey and human immunoglobulins by immunoprecipitation, ELISA and indirect immunofluorescence tests; immunoglobulin quantitation by radial immunodiffusion; and assays with peripheral blood lymphocytes involving tests for E and EAC rosettes and detection of surface markers (surface immunoglobulins and CD4-CD8 antigens). The results obtained showed that (a) at least three immunoglobulins with electrophoretic mobility corresponding to IgG, IgA and IgM which showed cross reactivity with the human ones were present in serum; (b) it was possible to evaluate the relative monkey immunoglobulin concentration using specific antibodies against human immunoglobulins and to obtain absolute values using adequate conversion factors; (c) lymphocytes forming E and EAC rosettes were found in peripheral blood in a similar proportion to that reported in man; (d) lymphocyte surface immunoglobulins were detected using anti-human immunoglobulin serum; (e) it was not possible to demonstrate the presence of T helper and T suppressor/cytotoxic lymphocytes using OK T4 and OK T8 monoclonal antibodies.

MHC antigen expression on bulk isolated macrophage-microglia from newborn mouse brain: induction of Ia antigen expression by γ-interferon

Macrophage-microglia were isolated from primary mixed brain cell cultures of normal newborn mice. They were successfully maintained in vitro for at least 8 weeks. Purity of the cultures was 97–100%, as determined by endocytosis of latex beads, non-specific staining through Fc receptors, EA and EAC rosette formation. These cells were non-specific esterase-positive, but peroxidase-negative. Electron-microscope observations revealed morphological similarities to mature macrophages. Isolated macrophage-microglia seldom incorporated [ 3H]thymidine in vitro. By means of 51Cr release assay, using monoclonal antibodies against mouse major histocompatibility complex (MHC) antigens and complement, we detected class I MHC (H-2) antigen on unstimulated macrophage-microglia, and both class I and class II (Ia) antigens on γ-interferon-treated cells. These observations suggest possible immunoregulatory functions of macrophage-microglia in the central nervous system, as is characteristics of other cells of monocyte lineage.

Characterization of a newly established feline lymphoma-derived cell line (BKD) lacking T and B cell surface markers

A new feline lymphoma-derived cell line, designated BKD, was isolated from an anterior mediastinal tumor. Cells of this line were characterized as lymphoid based on morphology, the lack of intracellular esterase and peroxidase activity, and absence of phagocytic function. In contrast with other established feline lymphoma-derived cell lines, cells of the BKD line lack characteristics of both feline T-cells and B-cells in that they neither form rosettes with guinea pig erythrocytes nor have demonstrable surface or cytoplasmic immunoglobulin. Approximately one third of BKD cells form EAC rosettes, a significant number of rosette forming cells (p = 0.0001) when compared to background sheep E-rosetting activity. In addition, a consistently titratable level of interleukin-2-like activity was produced when BKD cells were coincubated with concanavalin A and phorbol-12-myristate-13-acetate. Chromosome analysis showed that a majority of BKD cells are diploid. This new cell line has been continuously replicating in culture for over one year and produces feline leukemia virus as demonstrated by several analyses.

Morphological and functional investigations on macrophages with heterogeneous fluorescent staining.

Four heterogeneous colors of fluorescence were demonstrated in macrophages from rabbit lymph nodes and mouse peritoneal exudate using fluorescent staining. They were bright blue, light blue, bluish green and yellow colors of fluorescence respectively. All of these 4 types of macrophages with heterogeneous colors of fluorescence could form EA or EAC rosettes and were able to phagocytose chicken erythrocytes and ink carbon granules. Among them the macrophages with bright blue color of fluorescence could form large EA or EAC rosettes and showed more rapid and stronger activity to phagocytose chicken erythrocytes, while those with bluish green and yellow colors of fluorescence showed stronger activity to phagocytose ink carbon granules.

Sequential changes of the T Lymphocytes followingE R Rosette formation in guinea pigs. I. Expression of Fc and Complement Receptors

Changes in proportions of the Fc and complement receptor (FcR, CR) positive T lymphocytesfrom guinea pigs following their interaction with rabbit erythrocytes (ER) were studied using EA and EAC rosette forming assays. Significant increases in the percentages of EA and EAC rosette forming cell (RFC) were observed when thymocytes or lymph node cells were assayed after ER rosette formation. Furthermore, T-enriched fraction by the E R monolayer adherence technique also showed similar or somewhat higher increases in the proportions of both EA and EAC RFC than those of unfractionated cells after contact with ER. The double rosette assay by ER with EA or EAC showed that 50–80 % of the Fc and/or complement receptor positive lymphocytes bound rabbit erythrocytes simultaneously. These findings strongly suggest that at least a subset of the guinea pig T cells is altered to express Fc and/or complement receptors on their surfaces following the interaction with E R.

The selective localization of B lymphocytes in the spleen and the role of complement receptors.

The role of complement receptors on the localization of T and B cells in the spleen of mice was studied using short-term homing experiments in cobra venom factor (CoF)-treated animals. The localization ratio of B and T cells in the spleen of CoF-treated mice decreased significantly compared to control recipients. No changes could be found in the relative distribution of resident T and B cells in the spleen or other lymphoid organs of CoF-treated animals and when their spleen or lymph node cells were transferred, the localization pattern was normal. When cells were incubated in serum prior to transfer a disturbed localization ratio in the spleen of untreated recipients was observed. This was due to a blockade of complement receptors as determined by the inability of the incubated cells to form EAC rosettes. No blockade of EAC rosettes and no changes in localization ratios upon transfer could be observed when the cells were incubated in functionally C3-depleted serum. The results suggest a role for the complement-receptor on B cells in the initial localization in the spleen, whereas no influence upon the selective localization in high endothelial venules-bearing organs was found.

Acute undifferentiated leukemia: Induction of partial differentiation by phorbol ester

Expression of lineage-associated surface antigens, was studied in 7 patients with acute undifferentiated leukemia (AUL), 3 patients with acute myeloid leukemia (AML), 4 patients with acute lymphoid leukemia (ALL) and bone marrow from 2 healthy donors, before and after exposure to the differentiating agent 12- O-tetradecanoylphorbol-13-acetate (TPA). The surface antigens were identified by monoclonal antibodies (My4, My8, My9, MO1, B 1, CALLA, T 11) and formation of EA and EAC rosettes. Adherence to plastic was also assessed. Cells from the AML patients responded to TPA with an increase in myeloid antigen positive cells and other markers of differentiation. Four of the AUL patients showed, also, a large increase in the fraction of cells expressing one or more myeloid markers, in correlation with formation of EAC rosettes. In contrast, the percentage of cells expressing myeloid antigens, did not increase in the 4 ALL patients, or in the normal donors. These findings confirm the heterogeneity of undifferentiated leukemias, and suggest the hypothesis that some AUL's can be induced to express markers of early myeloid cells.

Immunosuppression by human seminal plasma.

Immunosuppressive property of normal human seminal plasma has been demonstrated on various in vitro immune parameters such as, blast transformation of lymphocytes with Phytohemagglutinin (P), Concanavalin A and Lipopolysaccharide, E & EAC rosette formation and mixed lymphocyte culture. The immunosuppression appears to be a T-cell mediated phenomenon. Analysis of semen samples obtained from 86 donors, comprising azoospermic, oligospermic, normospermic and polyspermic individuals, suggests that immunosuppression of the seminal plasma has no correlation with either the sperm density or the protein content of seminal plasma. The possible implication of the immunosuppressive activity in the development of acquired immuno-deficiency syndrome is also discussed.

Multiple sclerosis, HLA, and lymphocyte surface markers.

The frequencies of lymphocytes of different types in the blood of 96 multiple sclerosis patients in different phases of the disease were examined by rosetting, in relation to the HLA antigens present. In the total series of patients, there is a significant deficit of A2, and this is also suggested in the active and progressive cases, but there is no evidence of association of particular HLA haplotypes with the different phases of the disease. There was little variation among the patient categories in the frequency of T cells of different types, but patients in relapse showed a higher proportion of cells showing surface IgG. In general, there is little association of cell surface markers with HLA type, but when B8 is present there appear to be fewer E and EAC rosettes in patients in exacerbation, fewer E and IgG surface markers in remission, and more of the IgG markers in progression.

Analysis of T-cell subsets in B-cell chronic lymphocytic leukemia: a correlation with the stage of disease.

T-cell subsets were determined by the Leu monoclonal antibodies in the peripheral blood and/or bone marrow of 52 patients with B-cell chronic lymphocytic leukemia (B-CLL) not on therapy at the time of study. The diagnosis of B-CLL required that the leukemic cells expressed surface receptors for "la-like" antigen, Fc fragment of IgG, mouse red blood cells (MRBC), C3-coated red cells (EAC), and low density of monoclonal surface immunoglobulin. The Leu-3a+/2a+ ratio was applied to define the balance between the helper/suppressor subsets in the residual T-lymphocytes. Most patients showed a decrease in the Leu-3a+/2a+ ratios at all stages of disease. The decrease in ratio was mainly related to a decrease in the Leu-3a+ T-cell subset. The more advanced stages of B-CLL were associated with lower Leu-3a+/2a+ ratio, higher total white cell and percent lymphocyte counts. There was no correlation between the proportion of EAC or MRBC rosetting cells and stages of B-CLL. This analysis further suggests that B-CLL is an immunosuppressed state that becomes more pronounced in the advanced stages and is characterized by a progressive decrease in the Leu-3a+ (helper) T-cell subset.

Prognostic Significance of Receptors for the Third Component of Complement and Heavy Chain Phenotype in Diffuse B-Cell Lymphomas

Abstract As part of a larger study to determine the prognostic significance of cell marker phenotype in diffuse lymphomas, 51 patients with monoclonal B-cell lymphoma were further characterized by receptors for C3 (EAC rosettes) as well as heavy and light chain phenotypes. Patients with greater than 10% EAC rosette-forming cells were found to have a statistically significant longer survival than those with less than 10% EAC rosettes (p = 0.005). A similar trend in survival duration was found for patients whose cells expressed mu heavy chain on their surfaces when compared to those with gamma heavy chain on their cell surfaces (p = 0.05). No difference was observed for light chain phenotype. No correlation was observed between these prognostic groups and any of the three most frequently used histologic classifications (i.e., Rappaport, Lukes, Kiel).

Prognostic significance of receptors for the third component of complement an heavy chain phenotype in diffuse B-cell lymphomas

As part of a larger study to determine the prognostic significance of cell marker phenotype in diffuse lymphomas, 51 patients with monoclonal B-cell lymphoma were further characterized by receptors for C3 (EAC rosettes) as well as heavy and light chain phenotypes. Patients with greater than 10% EAC rosette-forming cells were found to have a statistically significant longer survival than those with less than 10% EAC rosettes (p = 0.005). A similar trend in survival duration was found for patients whose cells expressed mu heavy chain on their surfaces when compared to those with gamma heavy chain on their cell surfaces (p = 0.05). No difference was observed for light chain phenotype. No correlation was observed between these prognostic groups and any of the three most frequently used histologic classifications (i.e., Rappaport, Lukes, Kiel).

Non-Hodgkin's lymphomaの免疫機能的分類に関する研究

Twenty-six patients with non-Hodgkin's lymphoma (NHL) born in Ehime Prefecture were classified immunofunctionally by testing E, EA and EAC rosettes, surface immunoglobulin, Ia antigen and terminal trasferase (TdT) of lymphoma cells. Geographical distribution of the immunofunctional cell type, clinical stage and PPD and PHA skin test values was examined. According to cell type, there were those types of patients: 13 cases of the T-cell type (50%), 10 cases of the B-cell type (38%) and 3 cases of the Null-cell type (12%). There was no true histiocyte type. In the geographic study, the percentage of the T-cell type in the Matsuyama district was less than that in Kyushu island, but more than that in the northeast part of Japan. The number of T-cell type patients was much larger in the southwest part of the prefecture. B-cell type patients in stage I or II and T-cell type patients in stage III or IV were predominant. Rare cases of the T-cell type with EA or EAC rosetting were observed. Cells from Null-cell type patients and a T-cell type patient with diffuse lymphoblastic histology were TdT-positive, but others were negative. PPD and PHA skin tests showed a much weaker reaction in the T-cell type than in the B- and Null-cell types.

Decrease of E and EA but Not EAC Rosette Formation after Incubation of Blood Lymphocytes in Cerebrospinal Fluid

Peripheral blood lymphocytes (PBL) were incubated in autologous cerebrospinal fluid (CSF) or Hanks' balanced salt solution (HBSS) for between 5 and 60 min at 37 degrees C. Incubation in CSF caused an increase in cell death as measured by trypan blue inclusion. Incubation in CSF also resulted in a decreased percentage of E and EA rosette-forming cells (RFC)--but not of EAC-RFC--as compared with preincubation levels. Incubation in HBSS did not cause significant changes in rosette formation. Although incubation in CSF resulted in decreased E and EA rosette formation by PBL, lymphocytes isolated from CSF showed increased E and EAC rosette formation as compared with PBL.

Immunological Behaviour of Meningeal Exudate Cells in Experimental Allergic Encephalomyelitis

Publisher Summary This chapter presents a study to characterize the inflammatory infiltrate in the central nervous system (CNS) of guinea pigs with experimental allergic encephalomyelitis (EAE). In the study, inflammatory cells found over the surface of the brain and within the leptomeninges were isolated by washing and subsequently identified, and their immunological specificity was determined. By light microscopy, approximately 60% were lymphocytes, 30% were cells of the mononuclear phagocyte series and 5% were neutrophils. E, EA, and EAC rosetting techniques were used to determine the proportions of T-lymphocytes and cells with Fc and C3 receptors respectively. The percentage of phagocytic cells in the meningeal exudate was also determined. The immunological specificity of the meningeal inflammatory cells was determined using the lymphocyte transformation test and compared with another inflammatory cell population from the same animal derived from an oil-induced peritoneal exudate. The meningeal exudates cells from animals with acute EAE responded only to the mitogen concanavalin A (Con A) and not to the antigens myelin basic protein (MBP) and tuberculin (PPD). In contrast, the PECs proliferated in the presence of both antigens and the mitogen.