24 results on '"Mostowski HS"'
Search Results
2. Isolation of a circulating CD45-, CD34dim cell population and validation of their endothelial phenotype.
- Author
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Tropea MM, Harper BJ, Graninger GM, Phillips TM, Ferreyra G, Mostowski HS, Danner RL, Suffredini AF, and Solomon MA
- Subjects
- Adult, CD146 Antigen metabolism, Cell Membrane metabolism, Cell Nucleus metabolism, Cell Survival, Dactinomycin analogs & derivatives, Dactinomycin blood, Flow Cytometry, Human Umbilical Vein Endothelial Cells, Humans, Indoles chemistry, Leukocytes, Mononuclear cytology, Microscopy, Microscopy, Fluorescence, Middle Aged, Phenotype, Pilot Projects, Plant Lectins metabolism, Platelet Endothelial Cell Adhesion Molecule-1 metabolism, RNA metabolism, von Willebrand Factor metabolism, Antigens, CD34 metabolism, Cell Separation, Endothelial Cells cytology, Leukocyte Common Antigens metabolism
- Abstract
Accurately detecting circulating endothelial cells (CECs) is important since their enumeration has been proposed as a biomarker to measure injury to the vascular endothelium. However, there is no single methodology for determining CECs in blood, making comparison across studies difficult. Many methods for detecting CECs rely on characteristic cell surface markers and cell viability indicators, but lack secondary validation. Here, a CEC population in healthy adult human subjects was identified by flow cytometry as CD45-, CD34dim that is comparable to a previously described CD45-, CD31bright population. In addition, nuclear staining with 7-aminoactinomycin D (7-AAD) was employed as a standard technique to exclude dead cells. Unexpectedly, the CD45-, CD34dim, 7-AAD- CECs lacked surface detectable CD146, a commonly used marker of CECs. Furthermore, light microscopy revealed this cell population to be composed primarily of large cells without a clearly defined nucleus. Nevertheless, immunostains still demonstrated the presence of the lectin Ulex europaeus and von Willebrand factor. Ultramicro analytical immunochemistry assays for the endothelial cell proteins CD31, CD34, CD62E, CD105, CD141, CD144 and vWF indicated these cells possess an endothelial phenotype. However, only a small amount of RNA, which was mostly degraded, could be isolated from these cells. Thus the majority of CECs in healthy individuals as defined by CD45-, CD34dim, and 7-AAD- have shed their CD146 surface marker and are senescent cells without an identifiable nucleus and lacking RNA of sufficient quantity and quality for transcriptomal analysis. This study highlights the importance of secondary validation of CEC identification.
- Published
- 2014
- Full Text
- View/download PDF
3. Regulatory T cells as central regulators of both autoimmunity and B cell malignancy in New Zealand Black mice.
- Author
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Scaglione BJ, Salerno E, Gala K, Pan M, Langer JA, Mostowski HS, Bauer S, Marti G, Li Y, Tsiagbe VK, and Raveche ES
- Subjects
- Age Factors, Animals, Antibodies blood, Antibodies immunology, Antibodies pharmacology, Antibodies, Antinuclear blood, Ascitic Fluid cytology, Ascitic Fluid immunology, Autoimmune Diseases pathology, B-Lymphocytes cytology, B-Lymphocytes drug effects, B-Lymphocytes immunology, Erythrocytes immunology, Forkhead Transcription Factors genetics, Immune Tolerance immunology, Interferon-alpha blood, Interferon-alpha pharmacology, Interferons genetics, Interferons pharmacology, Interleukin-10 blood, Interleukin-10 genetics, Interleukin-2 Receptor alpha Subunit immunology, Lymph Nodes cytology, Lymph Nodes immunology, Mice, Mice, Inbred C57BL, Mice, Inbred NZB, Poly I-C pharmacology, Spleen cytology, Spleen drug effects, Spleen immunology, T-Lymphocyte Subsets cytology, T-Lymphocyte Subsets drug effects, T-Lymphocyte Subsets immunology, T-Lymphocyte Subsets metabolism, T-Lymphocytes, Regulatory cytology, T-Lymphocytes, Regulatory drug effects, T-Lymphocytes, Regulatory metabolism, ZAP-70 Protein-Tyrosine Kinase genetics, Autoimmune Diseases immunology, Autoimmunity immunology, Leukemia, B-Cell immunology, Leukemia, B-Cell pathology, T-Lymphocytes, Regulatory immunology
- Abstract
Regulatory T cells (Tregs) play an important role in protection against autoimmune disease and are also known to be potent inhibitors of anti-tumor immune responses. The New Zealand Black (NZB) mouse is a murine model for both autoimmune diseases, since high levels of autoantibodies are present, and human CLL, due to the expansion of malignant B-1 cells. In this study, we examined the functional role of CD4(+)CD25(+) Foxp3(+) Tregs in these different manifestations. Flow cytometric analysis showed increased levels of Tregs in NZB mice compared to healthy C57Bl/6 controls. Aged NZB mice that have developed a B-1 cell malignancy identified as IgM(+)CD5(+), have the most pronounced increase in Tregs. Ex vivo treatment of splenocytes from NZB mice with IFN-alpha resulted in a decrease in the frequency of Tregs and malignant B-1 cells. In vivo treatment of both NZB and C57Bl/6 mice with poly (I:C), a potent inducer of IFN-alpha, also led to a decrease in the levels of Tregs and malignant B-1 cells (NZB only) while amplifying autoimmune manifestations. These results indicate that while high levels of Tregs found in NZB mice might suppress a more severe autoimmune disease, they may also contribute to the development of the B cell malignancy.
- Published
- 2009
- Full Text
- View/download PDF
4. Virus assay using antibody-functionalized peptide nanotubes.
- Author
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MacCuspie RI, Banerjee IA, Pejoux C, Gummalla S, Mostowski HS, Krause PR, and Matsui H
- Abstract
Robust trace-level detection of viruses is crucial to meet urgent needs in fighting the spread of disease or detecting bioterrorism events. We report a new method for rapid and highly sensitive detection of viruses utilizing fluorescent antibody nanotubes. When viral pathogens were mixed with these antibody nanotubes, the nanotubes rapidly aggregated around the viruses to form a networking structure. Trace quantities of viruses such as herpes simplex virus type 2, adenovirus, vaccinia and influenza type B were detected on attomolar order by changes in fluorescence and light scattering intensities associated with aggregation of dye-loaded antibody nanotubes around viruses. High specificity of each antibody nanotube toward its targeted virus was demonstrated by quantifying concentrations of two different viruses in mixtures. This antibody nanotube assay detects targeted pathogens within 30 minutes after incubation with antibody nanotubes. This antibody nanotube assay could fill a pressing need to detect and quantify viruses both rapidly and sensitively.
- Published
- 2008
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5. Identification of linear heparin-binding peptides derived from human respiratory syncytial virus fusion glycoprotein that inhibit infectivity.
- Author
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Crim RL, Audet SA, Feldman SA, Mostowski HS, and Beeler JA
- Subjects
- Amino Acid Sequence, Animals, Binding Sites, CHO Cells, Chlorates pharmacology, Chlorocebus aethiops, Cricetinae, Cricetulus, Gene Products, gag genetics, Gene Products, gag metabolism, Glycoproteins chemistry, Glycoproteins metabolism, Humans, Lyases pharmacology, Molecular Sequence Data, Peptide Mapping, Peptides chemistry, Peptides metabolism, Respiratory Syncytial Virus, Human drug effects, Respiratory Syncytial Virus, Human metabolism, Vero Cells, Viral Fusion Proteins metabolism, Heparin metabolism, Peptides pharmacology, Respiratory Syncytial Virus, Human pathogenicity, Viral Fusion Proteins chemistry
- Abstract
It has been shown previously that the fusion glycoprotein of human respiratory syncytial virus (RSV-F) interacts with cellular heparan sulfate. Synthetic overlapping peptides derived from the F-protein sequence of RSV subtype A (strain A2) were tested for their ability to bind heparin using heparin-agarose affinity chromatography (HAAC). This evaluation identified 15 peptides representing eight linear heparin-binding domains (HBDs) located within F1 and F2 and spanning the protease cleavage activation site. All peptides bound to Vero and A549 cells, and binding was inhibited by soluble heparins and diminished by either enzymatic treatment to remove cell surface glycosaminoglycans or by treatment with sodium chlorate to decrease cellular sulfation. RSV-F HBD peptides were less likely to bind to glycosaminoglycan-deficient CHO-745 cells than parental CHO-K1 cells that express these molecules. Three RSV-F HBD peptides (F16, F26, and F55) inhibited virus infectivity; two of these peptides (F16 and F55) inhibited binding of virus to Vero cells, while the third (F26) did not. These studies provided evidence that two of the linear HBDs mapped by peptides F16 and F55 may mediate one of the first steps in the attachment of virus to cells while the third, F26, inhibited infectivity at a postattachment step, suggesting that interactions with cell surface glycosaminoglycans may play a role in infectivity of some RSV strains.
- Published
- 2007
- Full Text
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6. CXCL16 influences the nature and specificity of CpG-induced immune activation.
- Author
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Gursel M, Gursel I, Mostowski HS, and Klinman DM
- Subjects
- Antibodies, Blocking metabolism, Antibodies, Blocking physiology, Binding Sites, Antibody, Cell Line, Cell Membrane genetics, Cell Membrane metabolism, Cell Membrane physiology, Cells, Cultured, Chemokine CXCL16, Chemokines, CXC biosynthesis, Chemokines, CXC immunology, Dendritic Cells immunology, Dendritic Cells metabolism, Humans, Intracellular Fluid immunology, Intracellular Fluid metabolism, Membrane Proteins metabolism, Oligodeoxyribonucleotides antagonists & inhibitors, Oligodeoxyribonucleotides classification, Oligodeoxyribonucleotides metabolism, Receptors, Scavenger biosynthesis, Receptors, Scavenger immunology, Subcellular Fractions immunology, Subcellular Fractions metabolism, Toll-Like Receptor 9 biosynthesis, Toll-Like Receptor 9 genetics, Chemokines, CXC physiology, CpG Islands immunology, Receptors, Scavenger physiology
- Abstract
Unmethylated CpG motifs are present at high frequency in bacterial DNA. They provide a danger signal to the mammalian immune system that triggers a protective immune response characterized by the production of Th1 and proinflammatory cytokines and chemokines. Although the recognition of CpG DNA by B cells and plasmacytoid dendritic cells is mediated by TLR 9, these cell types differ in their ability to bind and respond to structurally distinct classes of CpG oligonucleotides. This work establishes that CXCL16, a membrane-bound scavenger receptor, influences the uptake, subcellular localization, and cytokine profile induced by D oligonucleotides. This is the first example of a surface receptor modifying the cellular specificity and nature of the immune response mediated by an intracellular TLR.
- Published
- 2006
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7. Underutilization of the V kappa 10C gene in the B cell repertoire is due to the loss of productive VJ rearrangements during B cell development.
- Author
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Fitzsimmons SP, Clark KJ, Mostowski HS, and Shapiro MA
- Subjects
- Animals, B-Lymphocytes cytology, B-Lymphocytes immunology, Bone Marrow Cells cytology, Bone Marrow Cells immunology, Bone Marrow Cells metabolism, Cell Differentiation genetics, Cell Differentiation immunology, Cell Separation, Female, Immunoglobulin Variable Region biosynthesis, Immunoglobulin kappa-Chains biosynthesis, Male, Mice, Mice, Inbred BALB C, Multigene Family immunology, Recombination, Genetic immunology, Spleen cytology, Spleen immunology, Spleen metabolism, Transcription, Genetic immunology, B-Lymphocytes metabolism, Gene Rearrangement, B-Lymphocyte, Genes, Immunoglobulin, Immunoglobulin Variable Region genetics, Immunoglobulin kappa-Chains genetics
- Abstract
The V kappa10 family of murine light chain Ig genes is composed of three members, two of which (V kappa 10A and V kappa 10B) are well used. V kappa 10C, the third member of this family, is not detected in any expressed Abs. Our previous work showed that V kappa 10C is structurally functional and can recombine, but mRNA levels in spleen were extremely low relative to those of V kappa 10A and V kappa 10B. Furthermore, while the V kappa 10C promoter was efficient in B cells, it was shown to work inefficiently in pre-B cell lines. Here, we extend our analysis of the V kappa 10 family and examine V kappa 10 gene accessibility, their representation in V kappa cDNA phage libraries, and the frequency and nature of rearrangements during different stages of B cell development. We demonstrate that V kappa 10C is under-represented in V kappa cDNA libraries, but that the frequency of its sterile transcripts in pre-B cells surpasses both V kappa 10A and V kappa 10B, indicating that the gene is as accessible as V kappa 10A and V kappa 10B to the recombination machinery. We also demonstrate that V kappa 10C recombines at a frequency equal to that of V kappa 10A in pre-B cells and has a normal nonproductive to productive recombination ratio. As B cells develop, however, both the frequency of V kappa 10C rearrangements and the presence of productive rearrangements decline, indicating that these cells are in some fashion being eliminated.
- Published
- 2000
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8. Fas ligand induction in human NK cells is regulated by redox through a calcineurin-nuclear factors of activated T cell-dependent pathway.
- Author
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Furuke K, Shiraishi M, Mostowski HS, and Bloom ET
- Subjects
- Antibodies, Monoclonal pharmacology, Calcineurin Inhibitors, Cells, Cultured, Culture Media, Conditioned, DNA-Binding Proteins antagonists & inhibitors, Enzyme Activation drug effects, Enzyme Activation immunology, Fas Ligand Protein, Humans, Hydrogen Peroxide pharmacology, Interleukin-2 pharmacology, Intracellular Fluid metabolism, Killer Cells, Natural drug effects, Killer Cells, Natural enzymology, Ligands, Membrane Glycoproteins antagonists & inhibitors, Membrane Glycoproteins genetics, NFATC Transcription Factors, Oxidation-Reduction, Peroxides metabolism, RNA, Messenger antagonists & inhibitors, RNA, Messenger metabolism, Receptors, IgG immunology, Sulfhydryl Compounds metabolism, Time Factors, Transcription Factors antagonists & inhibitors, Calcineurin physiology, DNA-Binding Proteins physiology, Killer Cells, Natural immunology, Lymphocyte Activation immunology, Membrane Glycoproteins biosynthesis, Nuclear Proteins, T-Lymphocytes immunology, Transcription Factors physiology, fas Receptor metabolism
- Abstract
Fas ligand (FasL) on cytotoxic lymphocytes is important for mediating apoptosis of activated lymphocytes and other target cells. We have reported that NK cell functions, such as proliferation, cell death, and killing activity, are subject to regulation by cellular redox status. Here, we report that expression of FasL protein and mRNA in activated NK cells is also regulated by redox. Ligation of CD16 on IL-2-preactivated NK cells resulted in reduction of intracellular peroxide level as well as induction of FasL expression. This CD16-induced FasL expression was suppressed by oxidative stress, including thiol deprivation or treatment with hydrogen peroxide (H2O2). Addition of thiol-reducing compounds, such as L-cystine, 2-ME, or N-acetyl cysteine, restored FasL expression. These data suggest that CD16 stimulation requires cellular reducing status for FasL induction in NK cells. Because FasL gene activation following CD16 cross-linking is regulated by the NF of activated T cells (NFAT), we examined the effect of oxidative stresses on NFAT activation. Electrophoretic mobility shift assays revealed that both thiol insufficiency and H2O2 treatment suppressed DNA-binding activity of NFAT and that addition of thiol-reducing compounds reversed or even enhanced it. Furthermore, these oxidative stresses inhibited activity of calcineurin, a serine/threonine phosphatase that regulates NFAT activation. These results suggest that suppression of calcineurin and NFAT activation is a mechanism by which oxidative stress inhibits FasL induction in activated NK cells and further support the hypothesis that thiol-reducing compounds might be required for maintenance of optimal NK functions under physiologic oxidative conditions.
- Published
- 1999
9. Production of IL-10 by human natural killer cells stimulated with IL-2 and/or IL-12.
- Author
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Mehrotra PT, Donnelly RP, Wong S, Kanegane H, Geremew A, Mostowski HS, Furuke K, Siegel JP, and Bloom ET
- Subjects
- Cells, Cultured, Flow Cytometry, Humans, Interleukin-10 genetics, Killer Cells, Natural metabolism, Lymphocyte Activation drug effects, Polymerase Chain Reaction, RNA, Messenger analysis, Interleukin-10 biosynthesis, Interleukin-12 pharmacology, Interleukin-2 pharmacology, Killer Cells, Natural drug effects
- Abstract
Human NK cell activity can be augmented in vitro by stimulation with IL-2 or IL-12, both of which also induce the production of IFN-gamma, TNF-alpha, and granulocyte-macrophage CSF by NK cells. For the first time, we demonstrate that freshly purified NK cells stimulated with IL-2 proliferated and produced IL-10 in a dose-dependent manner. IL-10 mRNA expression, as detected by semiquantitative reverse transcription-PCR, reached peak levels at 24 h. IL-10 protein was detectable on day 2 and further increased on days 3 and 6 as measured by ELISA. However, IL-12 alone induced neither substantial proliferation nor detectable IL-10 production by fresh NK cells, but it synergized with IL-2 in inducing IL-10 mRNA expression and protein synthesis. IL-10 production by activated NK cells was confirmed by intracytoplasmic cytokine staining by three-color immunofluorescence of CD16+ and/or CD56+ NK cells with anti-IL-10 antibody. IL-10 production by NK cells was further confirmed in the NK-like cell line, YT, which constitutively expressed IL-10 mRNA and protein. IL-12 alone did not induce NK proliferation, but it inhibited IL-2-induced proliferation. Neutralization of endogenously produced IL-10 with anti-IL-10 antibodies did not overcome the inhibition of IL-2-induced proliferation by IL-12. Together, these results demonstrate that IL-2 and IL-12 synergize to induce IL-10 production by human NK cells and that IL-12 inhibits IL-2 induced NK cell proliferation by an IL-10-independent mechanism.
- Published
- 1998
10. IL-12 administered in vivo to young and aged mice. Discrepancy between the effects on tumor growth in vivo and cytotoxic T lymphocyte generation ex vivo: dependence on IFN-gamma.
- Author
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Horvath-Arcidiacono JA, Mostowski HS, and Bloom ET
- Subjects
- Adjuvants, Immunologic administration & dosage, Age Factors, Animals, Cell Separation, Female, Interferon-gamma biosynthesis, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Mice, Knockout, Neoplasm Transplantation, Tumor Cells, Cultured, Graft Rejection immunology, Interferon-gamma immunology, Interleukin-12 administration & dosage, Interleukin-12 immunology, T-Lymphocytes, Cytotoxic immunology
- Abstract
Because IL-12 restores allogeneic cytotoxic T lymphocyte (CTL) activity by T cells of aged mice in vitro, we initially assessed whether IL-12 could overcome age-related deficits when given to aged mice in vivo. Growth of P815(H-2(d)) was enhanced in aged compared with young BALB/c (H-2(d)) mice and tumor growth was curtailed by IL-12 in both age groups. Unexpectedly, secondary CTL stimulated ex vivo with P815 were reduced in IL-12-treated mice compared with controls. Primary CTL generated ex vivo across MHC differences in IL-12 treated BALB/c and C57BL/6 young mice were reduced by 90-99%, were dose- and time-dependent, and were associated with reduced allo-stimulated NK-like activity and [3H]thymidine incorporation. IFN-gamma was elevated in sera and in supernatants from allo-stimulated cultures from IL-12-treated mice, while IL-4 was reduced in such supernatants, suggesting that, despite reduced CTL, IL-12 was associated with increased Th1- and reduced Th2-type cytokine production. IL-12 also induced splenomegaly, primarily due to increased numbers of cells lacking markers of mature T, B and NK cells, or macrophages, or polymorphonuclear leukocyte morphology. IFN-gamma mutant mice exhibited reduced splenic enlargement in response to IL-12, suggesting that the splenomegaly was due, in part, to IFN-gamma production. However, reduced CTL generation was not due entirely to dilution of CTL precursor cells because spleen cellularity and size increased 3-fold while CTL activity decreased 10- to 100-fold, and CTL generation normalized to CD8(+) T effector cells was still significantly reduced in IL-12-treated mice. Interestingly, purified CD4(+) and CD8(+) T cells from IL-12-treated normal mice exhibited greater proliferative and cytolytic activities respectively compared with controls. Thus, effector T cells in IL-12-treated mice were not impaired, but exhibited augmented responsiveness, suggesting that IL-12 induced complex interactions among spleen cell populations and that these effects, in part, are mediated by IFN-gamma.
- Published
- 1996
- Full Text
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11. Mitogenic stimulation of human lymphocytes mediated by a cell surface elastase.
- Author
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Packard BZ, Mostowski HS, and Komoriya A
- Subjects
- Cell Division, Cell Line, Cell Membrane enzymology, Humans, Leukocyte Elastase analysis, Lymphocytes, Tumor-Infiltrating enzymology, Mitogens isolation & purification, Monitoring, Immunologic, Signal Transduction drug effects, Tumor Cells, Cultured drug effects, Dipeptidyl Peptidase 4 analysis, Lymphocytes, Tumor-Infiltrating drug effects, Mitogens pharmacology, Pancreatic Elastase analysis, Proteins pharmacology, Serpins pharmacology
- Abstract
A ca. 45-kDa protein which was recently identified and purified to homogeneity from a solid tumor cell line as a T-cell mitogen was found to have significant sequence similarities with human monocyte/neutrophil elastase inhibitor (EI) [1]. Since EI is a known substrate for elastase, a determination of whether a cell surface expressed elastase-like molecule might be the binding protein for this 45-kDa factor and mediate mitogenic signal transduction was undertaken. First, the surface of tumor infiltrating lymphocytes, TIL 660, the indicator cell line used for the purification of this mitogen, was shown to stain positively with an anti-elastase antibody using flow cytofluorometry for quantitation. Then, after observing an inverse correlation between cell surface staining and the proliferative status of the TILs, behavior which might be expected of a growth factor receptor upon activation, mitogenic signal transduction was attempted through the elastase-like molecules of the lymphocytes' plasma membrane with the anti-elastase antibody in the role of mitogen. A greater than 4-fold mitogenic stimulation was observed when this antibody was covalently linked to latex beads; in contrast, addition of the soluble form of the same antibody did not result in any increase in [3H]thymidine incorporation into the cells' DNA. Hence, these data support induced clustering of an elastase-like molecule on the lymphocyte surface as a mediator of mitogenesis and suggest that the binding protein for mitogenic signal transduction induced by the 45-kDa protein, a member of the serine protease inhibitor (serpin) superfamily of proteins, is a molecule with structure similar to a serine protease.
- Published
- 1995
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12. Expression of a constitutive mutant of iron regulatory protein 1 abolishes iron homeostasis in mammalian cells.
- Author
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DeRusso PA, Philpott CC, Iwai K, Mostowski HS, Klausner RD, and Rouault TA
- Subjects
- Ferritins biosynthesis, Humans, Iron Regulatory Protein 1, Iron-Regulatory Proteins, Mutation, RNA metabolism, Receptors, Transferrin analysis, Tumor Cells, Cultured, Homeostasis, Iron metabolism, RNA-Binding Proteins physiology
- Abstract
Iron regulatory proteins (IRPs) are iron-sensing proteins that bind to RNA stem-loop sequences known as iron-responsive elements (IREs) when cells are depleted of iron. Although IRPs have been shown to bind to IREs derived from ferritin and transferrin receptor (TfR) mRNAs in vitro, there has not been a direct demonstration of the impact of a recombinant IRP on the expression of endogenous IRE-containing transcripts. In this study, we evaluate the impact of expression of C437S, a mutant of IRP1 that binds IREs regardless of cellular iron status, on the regulation of biosynthesis of ferritin and TfR. Despite being made iron-replete, cells expressing C437S continue to synthesize and express high amounts of TfR, while the synthesis of ferritin is repressed. Thus, a single mutant IRP can prevent the usual homeostatic changes in ferritin and TfR biosynthesis. Cells expressing the mutant protein would therefore be predicted to be unable to defend against iron overload. Preliminary results show that cells treated with iron have diminished cell survival when C437S is expressed, and we have thus created a tissue culture model system for the study of iron toxicity.
- Published
- 1995
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13. Does the age-related change in CD44-defined T-cell subsets have functional significance for cytotoxic T lymphocyte generation?
- Author
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Bloom ET, Mostowski HS, and Horvath JA
- Subjects
- Animals, Antibodies, Monoclonal, Female, Flow Cytometry, Hyaluronan Receptors, Lymphocyte Culture Test, Mixed, Male, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Aging immunology, Carrier Proteins immunology, Receptors, Cell Surface immunology, Receptors, Lymphocyte Homing immunology, T-Lymphocyte Subsets immunology, T-Lymphocytes, Cytotoxic immunology
- Abstract
CD44 or Pgp-1 is a transmembrane leukocyte adhesion-related glycoprotein which is often expressed in greater density on the membranes of memory T lymphocytes (CD44hi) compared to naive T cells (CD44lo). The proportion of Pgphi or CD44hi cells among T cells is increased with advancing age. We examined the relevance of this alteration for the age-related decrease in the generation of allospecific CTL activity. The findings confirm the age-related increase in the frequency of CD44hi cells in spleens of aged mice of several strains, but also show interstrain variability in the magnitude of the increase (bm1 > C57BL/6 > BALB/c). In contrast, we found that after allo-stimulation, the proportion of cells bearing the memory phenotype is decreased in cells from aged mice, particularly within the CD8+ T cell subset. To determine if these observations reflected an alteration in the frequency or responsiveness of naive T cells, enriched populations of spleen cells depleted of CD44hi cells were prepared from spleen cells of young and aged mice, and stimulated in mixed lymphocyte culture. Enrichment for cells expressing the naive phenotype did not restore the ability of T cells from aged mice to generate allospecific CTL. Together, these findings suggest that (1) the age-related increase in frequency of splenic T cells expressing memory phenotype and concordant decrease in phenotypically naive cells, does not explain the age-related decrease in the ability to generate primary allo-CTL, and (2) naive cells from aged mice exhibit intrinsically compromised ability to generate CTL in response to primary alloantigenic stimulation.
- Published
- 1994
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14. Effects of IL-12 on the generation of cytotoxic activity in human CD8+ T lymphocytes.
- Author
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Mehrotra PT, Wu D, Crim JA, Mostowski HS, and Siegel JP
- Subjects
- Cells, Cultured, Drug Synergism, Humans, Interleukin-12, Interleukin-2 pharmacology, Interleukin-4 pharmacology, Lymphocyte Activation drug effects, Recombinant Proteins pharmacology, T-Lymphocytes, Cytotoxic drug effects, CD8 Antigens analysis, Cytotoxicity, Immunologic drug effects, Interleukins pharmacology, T-Lymphocytes, Cytotoxic immunology
- Abstract
We have studied the effects of human rIL-12 on the proliferation and generation of cytotoxic activity in human CTL precursors. Purified human blood CD8+ T lymphocytes were stimulated overnight with immobilized alpha-CD3 and cultured 3 to 4 additional days under various conditions. The addition of IL-12 resulted in a marked (10- to 20-fold), dose-dependent, augmentation of cytotoxicity per cell with a smaller (2-fold) increase in cell number. IL-12 augmentation of proliferation and cytotoxicity of CD8+ T cells was not inhibited by a mAb to the p55 subunit of the IL-2 receptor (alpha-Tac) at a concentration sufficient to block the activity of exogenously added IL-2, indicating that the activity of IL-12 did not require IL-2. Addition of IL-12 at the time of alpha-CD3 activation or 1 day later was highly effective at augmenting cytotoxicity, whereas delayed addition of IL-12 (day 2 or 3) resulted in a smaller increase in CTL activity with no increase in cell number. IL-12 at all doses tested synergized with low dose IL-2 in inducing the proliferation and differentiation of CD8+ T cells. The synergistic effect was not blocked by adding neutralizing serum to IFN-gamma. In contrast to this synergistic effect, IL-12 significantly inhibited the proliferation observed in the presence of higher concentrations of IL-2 (4,500 and 13,500 pg/ml). An inhibitory effect of IL-12 was also observed when IL-12 was added to CD8+ T lymphocytes 3 days subsequent to activation with alpha-CD3 and IL-2. This broad set of potent effects of IL-12 on CD8+ T cell responses suggests that IL-12 may play an important immunoregulatory role on CTL development in vivo and may be a useful tool for manipulating this process in vivo for investigational and immunotherapeutic purposes.
- Published
- 1993
15. Pore-forming protein in individual cytotoxic T lymphocytes: the effect of senescence provides a probe for understanding the lytic mechanism.
- Author
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Horvath JA, Mostowski HS, Okumura K, and Bloom ET
- Subjects
- Animals, CD4 Antigens analysis, CD8 Antigens analysis, Cells, Cultured, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Perforin, Pore Forming Cytotoxic Proteins, T-Lymphocyte Subsets chemistry, T-Lymphocytes, Cytotoxic chemistry, Aging immunology, Cytotoxicity, Immunologic, Membrane Glycoproteins, Membrane Proteins analysis, T-Lymphocytes, Cytotoxic immunology
- Abstract
The senescent decline of cytolytic T lymphocyte (CTL) activity was examined (a) to learn more about the effect of aging on the immune system, and (b) to probe the mechanism of cell-mediated cytolysis. The effect of age on the generation of pore-forming protein (Pfp) was examined at the cellular level in a murine model using CTL stimulated in allogeneic mixed lymphocyte culture (MLC). Pfp expression was analyzed by immunocytochemistry and enzyme-linked immunosorbent assay (ELISA). Immunocytochemical analyses of Pfp in MLC-stimulated splenic T cells from a large number of mice revealed that although stimulated cells from aged mice exhibited fewer Pfp-producing cells than those from young, the diminution in the proportion of Pfp+ cells was small compared to the age-related decrease in lytic activities (approximately 2-fold vs. approximately 7.4-fold, respectively). Time-course analysis disclosed similar kinetics for the generation of Pfp+ cells among responding cells from young and aged mice. No significant age-related difference in the proportion of Pfp+ cells was observed in MLC-stimulated lymph node cells despite a large and significant difference in lytic activity (approximately 6.5-fold). Purified CD8+ T cells demonstrated a large age-related difference in CTL activity (approximately 3-11-fold) and accounted for virtually all the Pfp. Although little difference in the proportion of Pfp+ CD8+ T cells could be detected between age groups, stimulated CD8+ cells or whole splenic T cells from old mice consistently exhibited a striking reduction in both the intensity of Pfp staining and the apparent numbers of granules per cell. This difference in Pfp was examined by ELISA and total Pfp levels were found to be approximately 12-fold greater in CTL generated from splenic T cells of young compared to aged mice. The results demonstrate that Pfp levels are reduced in CTL from aged compared to young mice at the level of the individual cells and suggest the possibility that a threshold level of Pfp may be required for potency of effector cell function.
- Published
- 1992
- Full Text
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16. Cytokine-induced enhancement of ICAM-1 expression results in increased vulnerability of tumor cells to monocyte-mediated lysis.
- Author
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Webb DS, Mostowski HS, and Gerrard TL
- Subjects
- Antibodies, Monoclonal immunology, Cell Adhesion Molecules immunology, Humans, Intercellular Adhesion Molecule-1, Lymphocyte Function-Associated Antigen-1 physiology, Tumor Cells, Cultured, Up-Regulation, Cell Adhesion Molecules biosynthesis, Cytokines pharmacology, Cytotoxicity, Immunologic, Monocytes immunology, Neoplasms immunology
- Abstract
Pretreatment of the human melanoma cell line, A375, and the human colon carcinoma cell line, HT-29, with certain cytokines was found to increase the vulnerability of these cells to monocyte-mediated killing. This activity was found to correlate with increased expression of intercellular adhesion molecule-1 (ICAM-1) on the tumor cells and was blocked by anti-ICAM-1 antibodies. Both IFN-gamma and TNF induced large increases in the ICAM-1 expression on both cell lines and increased the susceptibility of the tumor cells to monocyte-mediated killing. IFN-alpha and IL-1 beta, however, induced only small increases in ICAM-1 expression and enhanced the lysis of the A375 cells but not the HT-29 cells by monocytes. These differences may be the result of a higher basal expression of ICAM-1 found on the A375 cells when compared with the HT-29 cells. These data indicate that regulation of ICAM-1 expression on tumor cells can alter the vulnerability of these cells to lysis by monocytes.
- Published
- 1991
17. Re-evaluation of the involvement of the adhesion molecules ICAM-1/LFA-1 in syncytia formation of HIV-1-infected subclones of a CEM T-cell leukemic line.
- Author
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Gruber MF, Webb DS, Gerrard TL, Mostowski HS, Vujcic L, and Golding H
- Subjects
- Antibodies, Monoclonal immunology, Cell Adhesion Molecules biosynthesis, Cell Adhesion Molecules immunology, Cell Fusion, Clone Cells, Humans, Intercellular Adhesion Molecule-1, Interferon-gamma pharmacology, Lymphocyte Function-Associated Antigen-1 biosynthesis, Lymphocyte Function-Associated Antigen-1 immunology, RNA-Directed DNA Polymerase metabolism, Tumor Cells, Cultured, Tumor Necrosis Factor-alpha pharmacology, Cell Adhesion Molecules physiology, Giant Cells microbiology, HIV-1 physiology, Lymphocyte Function-Associated Antigen-1 physiology
- Abstract
The role of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and LFA-1 in human immunodeficiency virus type 1 (HIV-1)-induced cell fusion was investigated in subclones of a T-cell leukemic cell line (CEM) with differing abilities to form syncytia. Addition of monoclonal antibodies 84H10 directed against ICAM-1 and MHM23 directed against the common beta subunit of LFA-1 (CD18) resulted in greater than 50% suppression of syncytia formation in cultures of these clones infected with cell-free virus. Two subclones, 2G5-144-84 and 2G5-1, were deficient in their ability to form syncytia and expressed reduced amounts of LFA-1 compared with the parental line. The expression of ICAM-1 but not LFA-1 was upregulated on the clones following treatment with interferon-gamma (IFN gamma); however, this did not overcome the delay in syncytia formation observed in these cells. The syncytia-positive subclones 1B11-39 and 17D-9 expressed high levels of LFA-1. Basal expression of ICAM-1 was upregulated on these cells by treatment with tumor necrosis factor-alpha (TNF alpha), which also accelerated and enhanced syncytia formation. However, anti-ICAM-1 and anti-LFA-1 (CD18) antibodies did not reverse the TNF alpha-induced enhancement of syncytia formation of HIV-1-infected clones 1B11-39 and 17D-9. Under conditions of low viral expression, adhesion molecules may contribute to syncytia formation if adequate levels of both receptor and ligand in the ICAM-1/LFA-1 complex are expressed.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1991
- Full Text
- View/download PDF
18. Regulation of human cytotoxic T lymphocyte development by IL-7.
- Author
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Hickman CJ, Crim JA, Mostowski HS, and Siegel JP
- Subjects
- Antigens, Differentiation, T-Lymphocyte analysis, CD8 Antigens, Concanavalin A pharmacology, Dose-Response Relationship, Drug, Esterases metabolism, Humans, Immunity, Cellular, Immunologic Memory, In Vitro Techniques, Influenza A virus immunology, Interleukin-7 pharmacology, Lymphocyte Activation, Lymphocyte Culture Test, Mixed, Time Factors, Cytotoxicity, Immunologic drug effects, Interleukin-7 physiology, T-Lymphocytes, Cytotoxic physiology
- Abstract
The effects of IL-7 on the generation of human CTL in alloantigen-, virus-, and lectin-stimulated systems were examined. Addition of IL-7 at the onset of cultures resulted in marked (up to 80-fold) augmentation of cytotoxicity accompanied by smaller (1.5- to 4-fold) increases in total lymphocyte number. Studies of CTL development in purified lectin-stimulated CD8+ T cell populations demonstrated that IL-7 could act directly on the CD8+ lymphocyte subset to augment cytotoxicity. In MLC, the IL-7-induced enhancement of cytotoxicity was found to be mediated primarily by the CD8+ subpopulation of lymphocytes. Late addition of IL-7 (day 5 of 7) resulted in an increase in cytolytic activity that was associated with little or no increase in total or activated CD8+ lymphocyte number indicating that IL-7 may act as a differentiation factor for human CTL. A role for endogenous IL-7 in CTL development was suggested by the observation that addition of neutralizing antiserum to IL-7 to MLC at initiation (or 5 days thereafter) resulted in decreased levels of cytotoxicity. These results indicate that IL-7 can exert major up-regulatory effects on human CTL development and suggest that these effects are both proliferative and differentiative.
- Published
- 1990
19. A bioassay for the measurement of human interleukin-4.
- Author
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Siegel JP and Mostowski HS
- Subjects
- Antigens, CD analysis, Antigens, Differentiation, B-Lymphocyte analysis, Biological Assay, Burkitt Lymphoma, Clone Cells, Cytokines pharmacology, Flow Cytometry, Humans, In Vitro Techniques, Interferons pharmacology, Receptors, Fc analysis, Receptors, IgE, Tumor Cells, Cultured, Tumor Necrosis Factor-alpha pharmacology, Interleukin-4 analysis
- Abstract
We have developed a bioassay for human IL-4 based upon its ability to upregulate CD23 (low affinity IgE receptor) expression. Ramos, a B lymphocyte line derived from a Burkitt lymphoma, was repetitively subcloned yielding a clone, Ramos.G6.C10, which is several fold more sensitive to this effect of IL-4. In microtiter plates cells were cultured for 48 h in the presence of dilutions of recombinant human IL-4 or samples, and then stained with murine anti-human CD23 and goat anti-mouse IgG-FITC. IL-4 induced an eight-fold increase (60 channel shift) in fluorescence intensity as measured by flow cytometry. Significant effects were observed at an IL-4 concentration of 50-100 pg/ml and increased with concentrations up to 800 pg/ml. Inter- and intra-assay coefficients of variation were 10% and 11% respectively. The bioassay showed good specificity for IL-4; however, tumor necrosis factors alpha and beta, at optimal concentrations, gave readings barely at the threshold of detection.
- Published
- 1990
- Full Text
- View/download PDF
20. IL-4 and granulocyte-macrophage colony-stimulating factor selectively increase HLA-DR and HLA-DP antigens but not HLA-DQ antigens on human monocytes.
- Author
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Gerrard TL, Dyer DR, and Mostowski HS
- Subjects
- Biological Factors pharmacology, Cytokines, Dose-Response Relationship, Drug, Granulocyte-Macrophage Colony-Stimulating Factor, Humans, In Vitro Techniques, Interferon-gamma pharmacology, Monocytes metabolism, Recombinant Proteins, Colony-Stimulating Factors pharmacology, Growth Substances pharmacology, HLA-DP Antigens metabolism, HLA-DQ Antigens metabolism, HLA-DR Antigens metabolism, Interleukin-4 pharmacology, Monocytes immunology
- Abstract
A number of cytokines were tested for their ability to modulate HLA-DR Ag expression on normal human monocytes. IL-4, granulocyte-macrophage (GM)-CSF as well as IFN-gamma were able to increase HLA-DR Ag expression on monocytes. IFN-alpha was also able to augment HLA-DR Ag expression, but to a lesser degree. Macrophage-CSF, granulocyte-CSF, TNF-alpha, TNF-beta, and IL-6 were not able to augment HLA-DR Ag expression. There were distinct patterns in the ability of different cytokines to augment class II histocompatibility Ag expression. IL-4 and GM-CSF selectively increased only HLA-DR and HLA-DP, but did not increase HLA-DQ antigens on monocytes. IFN-gamma, however, was able to augment the expression of HLA-DR, HLA-DP, and HLA-DQ Ag. Combinations of IFN-gamma with either IL-4 or GM-CSF did not show any synergy for the augmentation of any of the class II antigens on monocytes.
- Published
- 1990
21. The activation, proliferation, and differentiation of human B lymphocytes.
- Author
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Ambrus JL Jr, Jurgensen CH, Bowen DL, Tomita S, Nakagawa T, Nakagawa N, Goldstein H, Witzel NL, Mostowski HS, and Fauci AS
- Subjects
- B-Lymphocytes cytology, Cell Differentiation, Cell Division, DNA Replication, Humans, Mitogens, B-Lymphocytes immunology, Lymphocyte Activation
- Published
- 1987
- Full Text
- View/download PDF
22. Human monoclonal anti-keyhole limpet hemocyanin antibody-secreting hybridoma produced from peripheral blood B lymphocytes of a keyhole limpet hemocyanin-immune individual.
- Author
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Lane HC, Shelhamer JH, Mostowski HS, and Fauci AS
- Subjects
- Animals, Antibodies, Monoclonal biosynthesis, Antibody-Producing Cells immunology, Humans, Hybridomas immunology, Mice, Mice, Inbred BALB C, Antibodies, Monoclonal immunology, B-Lymphocytes immunology, Hemocyanins immunology, Hybridomas metabolism
- Abstract
A human IgMk monoclonal antibody, 2F7, of predetermined specificity, has been produced by the fusion of human peripheral blood lymphocytes with the nonsecreting mouse myeloma line SP-1. The heterohybridoma has remained stable for over 8 mo, with culture supernatants containing up to 30 micrograms/ml of specific IgM. The antibody has been shown to be capable of inducing a blastogenic response in the absence of antigen in the peripheral blood lymphocytes of normal subjects immune to the antigen. The ability to choose an antigen, immunize a human subject to that antigen, and then use the peripheral blood lymphocytes from that subject to produce antigen-specific human monoclonal antibodies should be of great value in a wide variety of investigative, diagnostic, and therapeutic endeavors.U
- Published
- 1982
- Full Text
- View/download PDF
23. Characterization of a monoclonal antibody that defines an immunoregulatory T cell subset for immunoglobulin synthesis in humans.
- Author
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Haynes BF, Mann DL, Hemler ME, Schroer JA, Shelhamer JH, Eisenbarth GS, Strominger JL, Thomas CA, Mostowski HS, and Fauci AS
- Subjects
- Antibody Specificity, Clone Cells immunology, Epitopes, Humans, Isoantibodies, Membrane Proteins immunology, Myeloma Proteins immunology, Rosette Formation, Antibody Formation, Antigens, Surface analysis, Lymphocyte Cooperation, T-Lymphocytes immunology
- Abstract
This study characterizes a monoclonal antibody (3A1), and partially characterizes the cell surface antigen and the functional peripheral blood T cell subset that it defines. The 3A1 antigen is present on the surface of several human T cell lines (HSB-2, CEM, MOLT-4, and others) in various amounts but is absent from the T cell line YT4E and all human B cell lines tested. Immunoprecipitation of an HSB-2 extract with 3A1 yielded one specific band with a molecular weight of approximately 40,000 in the presence of reducing agent. With directly fluoresceinated 3A1 antibody, fluorescence-activated cell sorter analysis showed that 85% of peripheral blood E-rosette-positive T cells were positive for the 3A1 antigen. After E-rosette-positive cells had been separated into 3A1+ and 3A1- cell suspensions, the 3A1+ cells helped autologous peripheral blood B cell suspensions toward pokeweed mitogen-driven proliferation and intracytoplasmic Ig production, whereas 3A1-T cells did not. Further, addition of 3A1- cells from some but not all normal subjects to cocultures of 3A1+ cells and B cells actively suppressed intracytoplasmic Ig production. However, the 3A1+T cell subset could be activated by concanavalin A to maximally suppress B cell Ig synthesis in vitro. Thus, the 3A1 antibody defines a major functional subject of peripheral blood T cells and should provide a useful marker for the study of human T cell function.
- Published
- 1980
- Full Text
- View/download PDF
24. Characterization of a monoclonal antibody (4F2) that binds to human monocytes and to a subset of activated lymphocytes.
- Author
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Haynes BF, Hemler ME, Mann DL, Eisenbarth GS, Shelhamer J, Mostowski HS, Thomas CA, Strominger JL, and Fauci AS
- Subjects
- Chemical Phenomena, Chemistry, Clone Cells immunology, Humans, Immunoglobulin G, Isoelectric Focusing, Lymphocytes immunology, Molecular Weight, Myeloma Proteins immunology, Antibodies immunology, Binding Sites, Antibody, Lymphocyte Activation, Monocytes immunology
- Published
- 1981
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