Biomedical subjects

M B Prystowsky

Publications and source records attributed to M B Prystowsky.

At least 73 records · Page 4Linked to original sources

PubMed1990

Induction of the granulocyte-macrophage colony-stimulating factor (CSF) receptor by granulocyte CSF increases the differentiative options of a murine hematopoietic progenitor cell.

32DC13(G) is an interleukin-3-dependent murine hematopoietic precursor cell line which differentiates into neutrophilic granulocytes upon exposure to granulocyte colony-stimulating factor (G-CSF) but ceases to proliferate and dies when exposed to granulocyte-macrophage (GM)-CSF. Surface receptors for GM-CSF are undetectable on 32DC13(G) cells but can be induced by priming the cells with G-CSF. Exposure of the G-CSF-primed cells to GM-CSF then results in the generation of monocytes as well as granulocytes. The acquired competence to respond to GM-CSF remains irreversibly encoded in the primed cells, although the GM-CSF receptor can be down regulated by interleukin-3. This phenomenon suggests a mechanism by which hematopoietic precursors may obtain additional receptors, thereby increasing their differentiative potential.

Animals↗

PubMed1990

Cell surface vitamin D-binding protein (GC-globulin) is acquired from plasma.

Vitamin D-binding protein (DBP) is an abundant plasma protein. The observation of immunodetectable, cell-associated DBP on peripheral blood mononuclear cells and placental cytotrophoblasts had presented the question of the origin, function, and precise subcellular localization of cell-associated DBP. Using anti-human DBP F(ab')2 with fluorescence-activated cytometric analysis and immunogold electron microscopy, we detected DBP on the plasmalemma of U937 cells, a monoblastic, histiocytic cell line grown in media supplemented with fetal calf serum (FCS). DBP was then removed from FCS by actin affinity chromatography followed by anti-DBP immunoaffinity chromatography. U937 cells in this DBP-free medium exhibited nearly identical growth rates to cells grown in medium containing native FCS. However, in contrast to cells grown with native FCS, those grown for seven to eight generations with DBP-free FCS exhibited less cell-surface DBP as quantified by fluorescence-activated cytometric analysis (73% decrease) and immunoelectron microscopy (88% decrease). DBP mRNA could not be detected in U937 cells, placental tissues, freshly prepared resting and stimulated B and T lymphocytes, or lymphocyte-derived cell lines by Northern analysis. In addition, using the sensitive reverse transcriptase/polymerase chain reaction assay no DBP fragments were detectable in U937 cells. We conclude that U937 cell-associated DBP is exogenously derived from plasma and is located on the plasmalemma. Based upon this conclusion, we postulate that specific binding sites for DBP may exist on the plasma membranes of certain cell types.

Blotting, Northern↗

PubMed1990

Interleukin-2 driven nuclear translocation of prolactin in cloned T-lymphocytes.

The nuclear translocation of PRL is demonstrated at the immunofluorescence and electron microscopic (EM) levels in interleukin-2 (IL2)-stimulated cloned T-cells and concanavalin A-stimulated splenocytes. This translocation occurs 2-10 h after IL2 stimulation, and is reversed by the addition of anti-PRL antiserum into the extracellular culture medium. The nuclear localization of PRL in IL2 stimulated T-cells was confirmed by postembedding immunogold EM. The nuclear uptake of PRL after IL2 stimulation was further documented by EM studies using PRL-colloidal gold conjugates. These studies suggest that the intranuclear PRL is translocated from the extracellular medium via an endosomal/lysosomal pathway over a period of several hours. Finally, the requirement for PRL no later than 6 h after IL2 stimulation is demonstrated through the reversible inhibition of T-cell growth with anti-PRL antiserum.

Animals↗

PubMed1990

Identification of a series of differentiation-associated gene sequences from GM-CSF stimulated bone marrow.

The production of terminally differentiated granulocytes and monocytes occurs by a program of orderly and sequential gene expression. This genetic program can be studied in vitro utilizing granulocyte-monocyte colony stimulating factor (GM-CSF) to stimulate proliferation and myeloid cell maturation. We have identified a series of genes expressed during early myeloid differentiation by differential screening of a cDNA library prepared from GM-CSF stimulated murine progenitor cells. From 72 potential early myeloid specific clones, three (B9, C9, C15) were characterized further. The time course of RNA accumulation during GM-CSF stimulated maturation demonstrated a unique pattern for each clone. B9 was expressed predominantly on day 3, and followed a pattern of accumulation similar to that for myeloperoxidase. (Jaffe et al. (1988), Oncogene, 2, 167-174). C9 expression increased gradually to a maximum level on day 3 and then plateaued; it appeared to be equally expressed in granulocytes and monocytes. C15 was expressed at a consistently high level in unstimulated cells and at 1-3 days post GM-CSF stimulation. It then decreased to undetectable levels. Hybridization of these clones to a panel of murine tissue RNAs demonstrated restricted expression of B9 to bone marrow, while C9 was present in most murine tissues, including thymus. C15 expression was relatively restricted to ovary/uterus, liver and adrenal, in addition to bone marrow. Partial DNA sequence analysis suggested that B9 was a novel sequence not previously identified. C9 was identified as thymosin beta-4, and C15 showed extensive homology to lactotransferrin. Thus, screening a bone marrow cDNA library by differential hybridization has successfully yielded a series of DNA sequences regulated during murine myelopoiesis. These include novel sequences (B9), genes previously known to be regulated during myelopoiesis (C15), as well as sequences not recognized previously as being associated with myeloid differentiation (C9).

Animals↗

PubMed1989

The molecular basis of granuloma formation in schistosomiasis. II. Analogies of a T cell-derived suppressor effector factor to the T cell receptor.

During Schistosoma mansoni infection, Ts cells regulate granulomatous modulation via antigenically and genetically restricted suppressor inducer and suppressor effector factors. The T suppressor effector factor (TseF) directly suppresses granuloma formation both in vitro and in vivo. In this study, we probe the molecular basis of these TseF properties. Using techniques of heterodimeric chain reduction with DTT and in vitro functional complementation, chimeric molecules were constructed. By analyzing genetic restrictions, antigenic specificities, and phenotypic markers, the contributions of the component chains to 72 kDa TseF reactivity were determined. One chain bore an Ag receptor and imparted antigenic specificity. The other chain bore an IJ determinant, a TCR beta-chain allotypic determinant, a suppressor effector phenotypic determinant, and imparted functional genetic restriction. Functional activity required covalent, probably sulfhydryl mediated, linkage as succinylation prevented the separated component chains from reconstituting functional activity. Additional studies demonstrated that anti-serum directed against either the T cell receptor or the T3 epsilon-chain could abrogate functional activity. However, TseF bore no T3 epsilon-chain phenotypic marker per se suggesting that TseF effects T lymphocytes via transmembrane signal transduction. These studies suggest that a regulatory network is operative in granuloma modulation. This regulatory network is mediated by a soluble TseF that bears significant structural homologies to the classic TCR.

Animals↗

PubMed1989

Discriminating rejection from CMV infection in renal allograft recipients using flow cytometry.

The ability to distinguish among rejection, cytomegalovirus (CMV) infection, and cyclosporin toxicity in the symptomatic renal allograft recipient remains one of the major issues in clinical transplantation. The practical application of immunologic monitoring of peripheral blood lymphocytes through the use of fluorescently labeled monoclonal antibodies and single-color flow cytometry has been limited by the inability to demonstrate significant correlations between the levels of specific T-cell subset populations and the cause of impaired renal function. In the present study using two-color analysis, we monitored the expression of interleukin-2 receptor (IL-2R) and HLA-DR antigen on the T-cells of a group of 51 renal cadaveric allograft recipients receiving cyclosporin, azathioprine, and prednisone for an average of 4 months after transplantation. We found that the proportion of CD3+ cells coexpressing IL-2R increased above baseline during 12 out of 14 rejection episodes that took place during the course of the study (P less than 10(-6)). Alternatively, we found that the proportion of cells coexpressing HLA-DR antigen on CD2+ cells increased above baseline during 11 out of 11 CMV infections (P less than 10(-6)). There was no correlation between the level of IL-2R+CD3+ cells and CMV infection or between the level of CD2+DR+ cells and rejection. These relationships showed a high degree of sensitivity and specificity when used to discriminate among possible etiologies for decreased renal function in the symptomatic patient.

Adolescent↗

PubMed1989

Delineation of false-positive HIV antibody response in patients with renal failure and history of multiple transfusions.

Four patients with a history of multiple blood transfusions who awaited renal transplantation were tested for human immunodeficiency virus (HIV) infection and found to be positive on enzyme immunoassay (EIA) and negative on Western blot. None of these patients had any clinical evidence of HIV infection. Absorption of these patients' sera with B-lymphoblastoid cell lines (B-LCL) positive for the serologic specificities DR3, DR4 (Dw4, Dw10, Dw14), and DR5 resulted in EIAs that were negative for HIV. Treatment of the B-LCL with an anti-DR monoclonal antibody (L243) interfered with the absorption of the serum sample by B-LCL. This indicates that the initial false-positive EIA results may be due to HLA antibodies. Furthermore, it was shown that these HLA antibodies are not limited in specificity to the HLA type of the host cell used in the preparation of the EIA reagents, but can consist of other DR specificities.

Antibodies, Monoclonal↗

PubMed1989

Myeloperoxidase-positive acute megakaryoblastic leukemia.

Acute megakaryoblastic leukemia (FABM7) is an unusual but well recognized form of acute myelogenous leukemia in which the bone marrow blast cells are phenotypically recognized by the demonstration of cytoplasmic platelet peroxidase or surface staining for the IIb/IIIa platelet-specific glycoprotein. Herein, the authors report a case of acute megakaryoblastic leukemia that satisfies the accepted French-American-British criteria and in which the blast cells also exhibit evidence of myeloid differentiation, including surface MY7 (CD13) by flow cytometry and immunocytochemical positivity for myeloperoxidase. These findings suggest that megakaryoblasts may be closely related to myelomonoblasts, that they have the potential to partially differentiate along multiple phenotypic lines, and that aberrant phenotypes can occur that do not correspond to known stages of normal maturation. The authors illustrate the difficulty in classification of these aberrant phenotypes by standard cytochemical and morphologic criteria.

Cell Membrane↗

PubMed1988

Cyclin mRNA and protein expression in recombinant interleukin 2-stimulated cloned murine T lymphocytes.

Expression of cyclin, a non-histone nuclear protein, during recombinant interleukin 2 (rIL2)-driven cell-cycle progression of cloned T lymphocytes has been assessed. We found that expression of cyclin protein, as detected by immunofluorescence, is tightly associated with proliferation, and not merely S-phase, of L2 cells stimulated with rIL2. Cyclin immunofluorescence was detected in all cell-cycle phases (G1/S/G2/M, as detected by flow cytometry) of proliferating L2 cells. Accumulation of cyclin mRNA levels was induced as early as 1 h after stimulation, was maximal at 25-49 h, and remained elevated throughout stimulation, as detected by Northern blot analysis. A cDNA-encoding murine cyclin was cloned from a cDNA library prepared from IL2-stimulated cloned T cells. The sequence of the 5' end of the murine cyclin cDNA was determined and found to be 88% and 82% similar to the sequences of cDNA clones encoding rat and human cyclin, respectively. The present studies demonstrate that cyclin protein and mRNA accumulation are highly regulated during IL2-induced proliferation of a cloned T cell. These data provide a framework for addressing the molecular mechanisms regulating cyclin gene expression during cellular proliferation.

Animals↗

PubMed1988

Evaluation of B-cell immunity in patients with pretransplant sensitization.

The influence of presensitization (blood transfusions) on B-cell immunity as reflected in the serum of two groups of candidates for cadaveric donor renal allografts was examined. The first group initially had a high level of panel-reactive antibody (PRA) greater than 72% but experienced a large decrease in PRA (greater than 70%) 6-34 months prior to transplantation. In contrast, the second group maintained a high PRA (100%) for up to 28 months after sensitization and before transplantation. Three blood Three blood samples from each patient, representing a maximum time span of 34 months, were analyzed. Levels of IgG, IgM isohemagglutinins, and antitetanus antibody were used as indicators of B-cell function. There were no significant differences between the individual values of a single patient with regard to each parameter. However, Group II patients had elevated values of total IgG relative to Group I patients. Total serum IgG-subclass levels (IgG1, IgG2, IgG3, IgG4) were measured and the relationship between a specific IgG subclass and the PRA activity was determined. IgG1 values in Group II were higher than those found for Group I. The other IgG subclasses were all within normal levels and were not significantly different between Group I and Group II. When IgG-subclass typing of PRA was performed, IgG1 accounted for most of the activity in both groups and a fall in PRA-specific IgG1 was associated with the reduced PRA observed in Group I. The data indicate that humoral immunity, as reflected by total and specific immunoglobulin levels, is intact in general in the two groups of presensitized renal allograft candidates examined and that any loss of PRA activity reflects a reduction in a specific immune response.

B-Lymphocytes↗

PubMed1988

Lymphokine regulation of inflammatory processes: interleukin-4 stimulates fibroblast proliferation.

While recent evidence from several laboratories has shown that interleukin-4 (IL-4) can act on a number of cells in addition to B lymphocytes, these have thus far been limited to cells of the hematopoietic lineage. Here we report that murine IL-4 promotes DNA synthesis in both primary and immortalized fibroblasts. Marked stimulation of [3H]thymidine incorporation was observed for primary skin fibroblasts or Balb/c3T3 cells stimulated with HPLC- or immunoaffinity-purified as well as recombinant IL-4. Responses to immunoaffinity and recombinant IL-4 were completely blocked with anti-IL-4 antibody. Similar dose/response relationships were observed for recombinant IL-4 on skin fibroblasts and an IL-4 responsive murine T cell tumor, suggesting that the receptors for this lymphokine on these cells is similar. Together, these results show that IL-4 can cause DNA synthesis by murine fibroblasts presumably through ligand-receptor interactions at the cell surface. Implications of these findings to inflammation during an immune response is discussed.

Animals↗

PubMed1988

Regulation of gene expression in lectin-stimulated or lymphokine-stimulated T lymphocytes. Effects of cyclosporine.

The effects of cyclosporine were examined on gene expression induced in T lymphocytes by mitogenic lectins and interleukin 2 (IL-2). Used at concentrations that inhibited proliferation of human peripheral blood lymphocytes by approximately 90%, CsA suppressed, to different extents, the phytohemagglutinin-stimulated expression of various genes, with levels of mRNAs for IL-2 being inhibited by approximately 100%, c-myc and N-ras by approximately 80%, and c-fos and IL-2 receptors by approximately 50%. Comparisons of the actions of CsA on gene expression in a cloned murine T cell (L2), stimulated with concanavalin A or IL-2, demonstrated that CsA specifically blocked the accumulation of mRNAs for the c-myc and p53 protooncogenes when induced by Con A, but not when induced by IL-2. Taken together, these findings indicate that several pathways can control the expression of a particular gene, and suggest that CsA interferes with only some of these regulatory pathways of gene expression in T lymphocytes.

Cells, Cultured↗

PubMed1988

Volume response of quiescent and interleukin 2-stimulated T-lymphocytes to hypotonicity.

Regulatory volume decreased (RVD) in lymphocytes in response to hyptonically induced swelling is dependent on the membrane permeabilities of K+, Cl-, and H2O. We used electronic cell sizing, cell water determination, and the whole cell patch-clamp method to study these membrane permeabilities in the cloned mouse T-lymphocyte, L2. Quiescent L2 cells express low levels of a voltage-gated K+ channel and show no RVD at 25 degrees C. In contrast, L2 cells stimulated to proliferate with the growth factor interleukin 2 have increased K+ conductance and show RVD in response to hypotonicity. RVD in stimulated cells is blocked by quinine and verapamil at levels that also completely block the voltage-gated K+ conductance. Swollen, unstimulated L2 cells can be induced to shrink by addition of the monovalent cation ionophore gramicidin in the presence of impermeant extracellular organic cations; gramicidin also enhances the rate of RVD in stimulated cells. Additionally, the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) blocks this gramicidin-facilitated RVD. These results suggest that a minimum requisite Cl- permeability is present even in the unstimulated L2 cells, that a necessary and limiting K+ permeability determines the rate of RVD, and that this K+ permeability increases after growth-factor stimulation as predicted from the direct measurement of voltage-gated K+ conductance. The hydraulic permeability is approximately 70% greater in proliferating L2 cells than in quiescent cells. At 37 degrees C, some RVD occurs in unstimulated L2 cells, and stimulated cells show faster and more complete shrinkage. These results are discussed with respect to the underlying membrane permeabilities and their relation to stimulated cell proliferation.

Animals↗

PubMed1988

Myeloperoxidase and oncogene expression in GM-CSF induced bone marrow differentiation.

DNA synthesis, morphology, specific RNA accumulation and rates of specific protein synthesis in GM-CSF stimulated bone marrow progenitor cells were studied. DNA synthesis increased markedly for 64 hours and then gradually decreased to 5% maximal activity by 160 hours. Morphologic examination 40 to 64 hours after stimulation revealed an increasing proportion of immature myeloid cells. After this proliferative peak, cells differentiated into segmented neutrophils and monocytes/macrophages; only mature forms were present by 160 hours. Accumulation of mRNA for c-myb and c-myc was maximal at 40 hours just prior to maximal [3H]thymidine incorporation, while maximal accumulation of histone type 3 (H3) was coincident with maximal [3H]thymidine incorporation at 64 hours. As proliferation decreased and differentiation proceeded, levels of mRNA for c-myb and H3 decreased markedly, while levels of RNA for c-myc decreased gradually and remained elevated above day 0 levels. Levels of c-fos mRNA fluctuated slightly during the first 64 hours of culture and increased 13-fold by 160 hours when mature cells were present. Similarly, beta-2 microglobulin mRNA increased steadily to maximal levels at 112 to 160 hours which were 15-fold higher than day 0 levels. Myeloperoxidase (MPO) mRNA was present in maximal amounts at 40 to 64 hours after stimulation with GM-CSF as the number of immature myeloid cells peaked. Immunoprecipitation of MPO from pulse-labeled cell lysates demonstrated a 7-fold rise in synthetic rate of MPO of 64 hours and a 28-fold decline by 160 hours when only 5% immature myeloid cells were present. Thus, MPO protein synthesis closely follows MPO mRNA accumulation. Immunoprecipitation of lactoferrin, a marker of myeloid secondary granules, demonstrated a gradual 5-fold increase in synthetic rate as the cells matured. Taken together, these data show that maximal expression of the early myeloid differentiation enzyme myeloperoxidase in GM-CSF stimulated normal bone marrow cells occurs during peak proliferation of immature myeloid cells.

Animals↗

PubMed1988

Defective T cell-mediated, isotype-specific immunoglobulin regulation in B cell chronic lymphocytic leukemia.

The consistent occurrence of T cell abnormalities in patients with B cell chronic lymphocytic leukemia (B-CLL) suggest that the non-neoplastic host T cells may be involved in the pathogenesis of this B cell neoplasm. Because potential defects of immunoglobulin regulation are evident in B-CLL patients, we investigated one aspect of this by studying the T cell-mediated immunoglobulin isotype-specific immunoregulatory circuit in B-CLL. The existence of class-specific immunoglobulin regulatory mechanisms mediated by Fc receptor-bearing T cells (FcR + T) through soluble immunoglobulin binding factors (IgBFs) has been well established in many experimental systems. IgBFs can both suppress and enhance B cell activity in an isotype-specific manner. We investigated the apparently abnormal IgA regulation in a B-CLL patient (CLL249) whose B cells secrete primarily IgA in vitro. Enumeration of FcR + T cells showed a disproportionate increase in IgA FcR + T cells in the peripheral blood of this patient. Our studies showed that the neoplastic B cells were not intrinsically unresponsive to the suppressing component of IgABF produced from normal T cells, but rather the IgABF produced by the CLL249 host T cells was defective. CLL249 IgABF was unable to suppress IgA secretion by host or normal B cells and enhanced the in vitro proliferation of the host B cells. Size fractionation of both normal and CLL249 IgABF by gel-filtration high-performance liquid chromatography (HPLC) demonstrated differences in the ultraviolet-absorbing components of IgABF obtained from normal T cells v that from our patient with defective IgA regulation. Such T cell dysfunction may not be restricted to IgA regulation, since we have found similar expansion of isotype-specific FcR + T cells associated with expansion of the corresponding B cell clone in other patients with B-CLL. These data suggest that this T cell-mediated regulatory circuit could be significantly involved in the pathogenesis of B-CLL.

Adult↗

PubMed1988

Partial characterization of a fibroblast-stimulating factor produced by cloned murine T lymphocytes.

T cells may regulate tissue fibrosis through the elaboration of soluble factors that stimulate fibroblast growth. The authors previously identified a factor produced by cloned Schistosoma mansoni antigen-specific T cells which served as a competence factor for murine fibroblasts. In the present report, they further characterize this fibroblast-stimulating factor (FsF) and differentiate it from a number of other T-cell-derived lymphokine activities. Crude supernatants from concanavalin-activated cloned T cells were fractionated by gel filtration, ion exchange, or reversed-phase high-pressure liquid chromatography. FsF has an apparent molecular weight of 17,000 and could be differentiated from colony-stimulating factor (CSF), interleukin-3 (IL-3), and interferon (IFN) on the basis of chromatographic characteristics. Highly purified or recombinant IL-2, IL-3, CSF, and IFN had no significant effect on fibroblast proliferation. Furthermore, a monoclonal anti-B-cell-stimulating factor-1 antibody only partially blocked the fibroblast proliferation induced by T-cell supernatants.

Animals↗

PubMed1987

Proliferating cell nuclear antigen/cyclin is an interleukin 2-responsive gene.

Previously, we have shown that a protein designated p36 is synthesized at a high rate during interleukin 2-driven proliferation of a cloned T lymphocyte, L2. Biosynthesis of p36 increases 1000-fold during the initial mid-G1 phase of the cell cycle and remains high while the cells proliferate. In this report, we show that p36 has the same migration pattern by two-dimensional gel electrophoresis as proliferating cell nuclear antigen (PCNA)/cyclin and that antiserum to PCNA/cyclin selectively immunoprecipitates p36. In addition, by indirect immunofluorescence, PCNA/cyclin accumulates in the nucleus of interleukin 2-stimulated L2 cells during proliferation and is not detectable prior to the initial S phase or after proliferation ceases. These data indicate that PCNA/cyclin expression is induced by interleukin 2 and that PCNA/cyclin accumulation is closely associated with T lymphocyte proliferation.

Autoantigens↗

PubMed1987

Flow cytometry in hairy cell leukemia before and during interferon alfa-2b therapy.

Mononuclear cells from 15 patients with hairy cell leukemia were studied before and during therapy with interferon alfa-2b (IFN) by regular peripheral blood differential counts and flow cytometry, using a panel of monoclonal antibodies (Moab). Seven leukemic phase patients (Group 1) had a mean leukocyte count of 48,870/microliter at entry with a mean absolute hairy cell (HC) count of 40,100/microliter. After 3 months on IFN, both parameters decreased significantly (WBC 3,500/microliter; HC count 130/microliter). In eight patients with a cytopenic form of the disease (Group 2) the mean leukocyte count rose from 2950/microliter to 3890/microliter while the mean absolute HC count decreased from 300/microliter to 120/microliter. The morphologic shifts correlated well with changes in the Moab reaction pattern. In Group 1 the activity of all Moab decreased significantly. In Group 2, only cells expressing Leu 3a and Leu 11a (a marker of natural killer cells) showed a significant shift, the latter increasing from 170/microliter to 360/microliter. This increase in natural killer cell antigen expression was not obvious based on routine morphologic observations alone. We show that flow cytometry may be a useful adjunct in monitoring the response of HCL to therapy. Changes in populations of cells that may be difficult to discriminate on morphologic grounds alone may be observed.

Antibodies, Monoclonal↗