Steroids and hematopoiesis. III. The response of granulocytic and erythroid colony-forming cells to steroids of different classes.
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Selected androgenic and nonandrogenic steroids enhance in vitro granulocytic and erythroid colony formation by mouse marrow cells, but do so by influencing either different target cells or cells in different states of cell cycle. Etiocholanolone, a naturally occurring nonandrogenic testosterone metabolite, permits cells not in active cycle to respond to colony-stimulating factor or erythropoietin. Fluoxymesterone, a synthetic androgen, appears to enhance colony growth by increasing the responsiveness of target cells to tropic stimuli. The majority of cells responding to this androgen are in active DNA synthesis. Direct comparison, however, of etiocholanolone-dependent erythroid or granulocytic colony-forming cells demonstrates nonidentity of the target cells. Thus colony-forming units responding to different classes of steroids are in different states of cell cycle and are physically separable. The enhancement of the in vitro response of colony-forming cells to regulating hormones by steroids such as etiocholanolane suggests a mechanism by which such agents may be therapeutically effective in certain cases of marrow failure in man. (+info)
Yops of Yersinia enterocolitica inhibit receptor-dependent superoxide anion production by human granulocytes.
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The virulence plasmid-borne genes encoding Yersinia adhesin A (YadA) and several Yersinia secreted proteins (Yops) are involved in the inhibition of phagocytosis and killing of Yersinia enterocolitica by human granulocytes. One of these Yops, YopH, dephosphorylates multiple tyrosine-phosphorylated proteins in eukaryotic cells and is involved in the inhibition of phagocytosis of Y. enterocolitica by human granulocytes. We investigated whether antibody- and complement-opsonized plasmid-bearing (pYV+) Y. enterocolitica inhibits O2- production by human granulocytes in response to various stimuli and whether YopH is involved. Granulocytes were preincubated with mutant strains unable to express YadA or to secrete Yops or YopH. O2- production by granulocytes during stimulation was assessed by measuring the reduction of ferricytochrome c. PYV+ Y. enterocolitica inhibited O2- production by granulocytes incubated with opsonized Y. enterocolitica or N-formyl-Met-Leu-Phe (f-MLP). This inhibitory effect mediated by pYV did not affect receptor-independent O2- production by granulocytes in response to phorbol myristate acetate, indicating that NADPH activity remained unaffected after activation of protein kinase C. The inhibition of f-MLP-induced O2- production by granulocytes depends on the secretion of Yops and not on the expression of YadA. Insertional inactivation of the yopH gene abrogated the inhibition of phagocytosis of antibody- and complement-opsonized Y. enterocolitica by human granulocytes but not of the f-MLP-induced O2- production by granulocytes or tyrosine phosphorylation of granulocyte proteins. These findings suggest that the specific targets for YopH are not present in f-MLP receptor-linked signal transduction and that other Yop-mediated mechanisms are involved. (+info)
MENT, a heterochromatin protein that mediates higher order chromatin folding, is a new serpin family member.
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Terminal cell differentiation is correlated with the extensive sequestering of previously active genes into compact transcriptionally inert heterochromatin. In vertebrate blood cells, these changes can be traced to the accumulation of a developmentally regulated heterochromatin protein, MENT. Cryoelectron microscopy of chicken granulocyte chromatin, which is highly enriched with MENT, reveals exceptionally compact polynucleosomes, which maintain a level of higher order folding above that imposed by linker histones. The amino acid sequence of MENT reveals a close structural relationship with serpins, a large family of proteins known for their ability to undergo dramatic conformational transitions. Conservation of the "hinge region" consensus in MENT indicates that this ability is retained by the protein. MENT is distinguished from the other serpins by being a basic protein, containing several positively charged surface clusters, which are likely to be involved in ionic interactions with DNA. One of the positively charged domains bears a significant similarity to the chromatin binding region of nuclear lamina proteins and with the A.T-rich DNA-binding motif, which may account for the targeting of MENT to peripheral heterochromatin. MENT ectopically expressed in a mammalian cell line is transported into nuclei and is associated with intranuclear foci of condensed chromatin. (+info)
Transduction of primitive human marrow and cord blood-derived hematopoietic progenitor cells with adeno-associated virus vectors.
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We evaluated the capacity of adeno-associated virus (AAV) vectors to transduce primitive human myeloid progenitor cells derived from marrow and cord blood in long-term cultures and long-term culture-initiating cell (LTC-IC) assays. Single-colony analyses showed that AAV vectors transduced CD34(+) and CD34(+)38(-) clonogenic cells in long-term culture. Gene transfer was readily observed in LTC-ICs derived from 5-, 8-, and 10-week cultures. Recombinant AAV (rAAV) transduction was observed in every donor analyzed, although a wide range of gene transfer frequencies (5% to 100%) was noted. AAV transduction of LTC-ICs was stable, with week-8 and -10 LTC-ICs showing comparable or better transduction relative to week-5 LTC-ICs. Fluorescence in situ hybridization (FISH) analyses performed to determine the fate of AAV vectors in transduced cells showed that 9% to 28% of CD34(+) and CD34(+)38(-) cells showed stable vector integration as evidenced by chromosome-associated signals in metaphase spreads. Comparisons of interphase and metaphase FISH suggested that a fraction of cells also contained episomal vector at early time points after transduction. Despite the apparent loss of the episomal forms with continued culture, the number of metaphases containing integrated vector genomes remained stable long term. Transgene transcription and placental alkaline phosphatase (PLAP) expression was observed in CD34(+), CD34(+)38(-) LTC-ICs in the absence of selective pressure. These results suggest that primitive myeloid progenitors are amenable to genetic modification with AAV vectors. (+info)
A novel spliced form of SH2-containing inositol phosphatase is expressed during myeloid development.
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SH2-containing Inositol Phosphatase (SHIP) is a 145 kD protein expressed in hematopoietic cells. SHIP is phosphorylated on tyrosine after receptor binding by several cytokines and has a negative role in hematopoiesis. We cloned a murine complementary DNA (cDNA) sequence for an isoform of SHIP with an internal 183 nucleotide deletion, encoding a protein 61 amino acids shorter than 145 kD SHIP. This deletion eliminates potential SH3-domain binding regions and a potential binding site for the p85 subunit of Phosphatidylinositol 3-Kinase. Using polyclonal anti-SHIP antibodies, we and others have previously observed a 135 kD SHIP isoform that is coexpressed with 145 kD SHIP. Here, we used monoclonal antibodies raised against the region deleted in the spliced form to show that the product of the novel spliced SHIP cDNA is antigenically identical to the 135 kD SHIP isoform. Like 145 kD SHIP, 135 kD SHIP expression was induced on differentiation of bone marrow cells. After macrophage colony-stimulating factor (M-CSF) stimulation of FDC-P1(Fms) myeloid cells, both 145 and 135 kD SHIP forms were tyrosine phosphorylated and could be coimmunoprecipitated with antibodies to Shc and Grb2. However, experiments showed only a weak association of 135 kD SHIP with p85. A potentially analogous 135 kD SHIP species also appears in human differentiated leukocytes. (+info)
The intracellular serpin proteinase inhibitor 6 is expressed in monocytes and granulocytes and is a potent inhibitor of the azurophilic granule protease, cathepsin G.
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The monocyte and granulocyte azurophilic granule proteinases elastase, proteinase 3, and cathepsin G are implicated in acute and chronic diseases thought to result from an imbalance between the secreted proteinase(s) and circulating serpins such as alpha1-proteinase inhibitor and alpha1-antichymotrypsin. We show here that the intracellular serpin, proteinase inhibitor 6 (PI-6), is present in monocytes, granulocytes, and myelomonocytic cell lines. In extracts from these cells, PI-6 bound an endogenous membrane-associated serine proteinase to form an sodium dodecyl sulfate (SDS)-stable complex. Using antibodies to urokinase, elastase, proteinase 3, or cathepsin G, we demonstrated that the complex contains cathepsin G. Native cathepsin G and recombinant PI-6 formed an SDS-stable complex in vitro similar in size to that observed in the extracts. Further kinetic analysis demonstrated that cathepsin G and PI-6 rapidly form a tight 1:1 complex (ka = 6.8 +/- 0.2 x 10(6) mol/L-1s-1 at 17 degrees C; Ki = 9.2 +/- 0.04 x 10(-10) mol/L). We propose that PI-6 complements alpha1-proteinase inhibitor and alpha1-antichymotrypsin (which control extracellular proteolysis) by neutralizing cathepsin G that leaks into the cytoplasm of monocytes or granulocytes during biosynthesis or phagocytosis. Control of intracellular cathepsin G may be particularly important, because it has recently been shown to activate the proapoptotic proteinase, caspase-7. (+info)
Mechanisms of acute inflammatory lung injury induced by abdominal sepsis.
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Sequestration of neutrophils and release of histotoxic mediators are considered important for the development of pathologic alterations of the lung defined as adult respiratory distress syndrome. Mechanisms of inflammatory lung injury caused by abdominal sepsis were investigated using the colon ascendens stent peritonitis (CASP) model that closely mimics the human disease. In the CASP model, a continuous leakage of intraluminal bacteria into the peritoneal cavity is induced by implantation of a stent in the ascending colon, generating a septic focus. In contrast to the cecal ligation and puncture model of peritonitis, survival of mice following CASP surgery is dependent on IFN-gamma, but independent of tumor necrosis factor (TNF). Here we show that the systemic inflammation induced by CASP surgery results in a rapid and profound increase of lung vascular permeability that was associated with the activation and recruitment of neutrophils to the lung. Activation of circulating granulocytes was characterized by increased production of serine proteinases and reactive oxygen metabolites, as well as elevated expression of cell surface Mac-1. Expression of MIP-2, KC, MIP-1alpha and E-selectin mRNA in lung was strongly increased within 3 h following CASP surgery, whereas up-regulation of IP-10, MCP-1 and P-selectin was delayed. In contrast, induction of RANTES, LIX, ICAM-1 and VCAM-1 mRNA was weak or not detectable after CASP surgery. Importantly, recruitment of leukocytes to the lung was normal in lipopolysaccharide-resistant mice, and was not affected by antibody neutralization of TNF or the chemokines MIP-2 and KC. (+info)
Bone marrow scintigraphy using technetium-99m antigranulocyte antibody in malignant lymphomas.
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BACKGROUND: The purpose of this study was to elucidate the clinical reliability of immunoscintigraphy (IS) to detect infiltration of the bone marrow in patients with malignant lymphoma. PATIENTS AND METHODS: Whole body IS was performed in 103 patients with Hodgkin's disease (HD) or non-Hodgkin's lymphoma (NHL) using Tc-99m labelled anti-NCA-95 which allows visualization of the granulopoietic bone marrow. Of these, 52% were studied prior to any therapy. Findings were compared to posterior iliac crest biopsy as well as MRI and/or follow-up examination. Criteria of marrow infiltration were a positive biopsy, positive follow-up, or positive results of MRI. RESULTS: Comparison of IS and biospy revealed concordant findings in 69 and discordant findings in 34 of 103 patients. Of the 34 patients with discordant results, IS showed lesions suspicious of bone marrow infiltration in 29 patients despite normal biopsy findings. When follow-up and additional examinations were taken into consideration, 10 patients remained with probably false positive and five with false negative IS findings. IS proved to be highly sensitive and specific in patients with HD (100% and 84%, respectively) and high-grade NHL (93% and 84%, respectively). Moderate sensitivity (60%) was found in low-grade NHL. This was possibly due to false negative IS in three to five patients with chemotherapy in contrast to one of five false negative results in patients without chemotherapy. CONCLUSION: Bone marrow scintigraphy using antigranulocyte antibodies is highly sensitive in HD and high-grade NHL. Positive findings in IS subsequent to a negative biopsy should be followed by guided re-biopsy or MRI. (+info)