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Biomedical subjects

A Grossmann

Publications and source records attributed to A Grossmann.

At least 37 records · Page 2Linked to original sources
PubMed1996

Do the preclinical effects of thrombopoietin correlate with its in vitro properties?

In the short time since its cloning, much has been learned of the in vitro properties of thrombopoietin (TPO). In addition to effects on the differentiation of megakaryocytes, TPO has also been shown to stimulate the proliferation of megakaryocytic progenitor cells, colony-forming units-megakaryocytes (CFU-MK), to act in synergy with interleukin 3 or c-kit ligand and erythropoietin (Epo) to stimulate the development of early and the generation of late erythroid progenitor cells, and to affect the rate of entry into the cell cycle and proliferative capacity of hematopoietic stem cells. An important question posed by these observations, for both TPO and for hematopoietic research in general, is whether the in vitro effects of a cytokine are mirrored by its preclinical and clinical biology. The results of recent studies in mice and nonhuman primates will be presented which have attempted to address this issue. In normal animals, TPO increases the numbers of marrow and spleen CFU-granulocyte/erythroid/macrophage/megakaryocyte, CFU-MK, CFU-GM and BFU-E, but its effects in the peripheral blood are limited to marked increases in the platelet count. The reason for widespread progenitor cell effects, yet stable leukocyte and erythrocyte blood counts, is likely the predominant regulatory effects of G-CSF and Epo; in the absence of elevated levels of the lineage-dominant regulator of each of these cell types, expanded progenitor cell numbers are not translated into increased peripheral blood counts. However, in states of increased blood cell demand such as follows myelosuppressive therapy, elevated levels of Epo and G-CSF allow the effects of TPO on erythropoiesis and myelopoiesis to become manifest. The administration of TPO to myelosuppressed animals is associated with not only greatly expanded hematopoietic progenitor cell recovery, but also improvement in platelet, red cell and leukocyte nadir levels and greatly accelerated recovery of all three cell lineages. These results indicate that the panhematopoietic properties of TPO identified by in vitro culture techniques correlate well with its effects in animals. The results of ongoing clinical trials should soon establish whether these conclusions can be extended to patient care.

Animals↗
PubMed1996

Aged murine T-lymphocytes are more resistant to oxidative damage due to the predominance of the cells possessing the memory phenotype.

Glutathione (GSH) is the most important cytosolic antioxidant. Since GSH levels are decreased with age, we hypothesized that T-lymphocytes from old mice would be more sensitive to oxidative stress. T-lymphocytes from young and old mice were exposed to hypoxanthine/xanthine oxidase, and lymphocyte viability, proliferation, GSH content, and calcium signaling were measured. Before exposure, proliferation of T-lymphocytes from young mice was greater than that of old; following exposure, the converse was true. This was in spite of the fact that old mice had lower total GSH levels and greater levels of glutathione disulfide. After oxidative challenge, intracellular calcium responses to anti-CD3 were decreased in naive T-lymphocytes from all mice, while memory lymphocytes were less affected. Higher proportions of memory lymphocytes in old mice resulted in their greater overall preservation of lymphocyte function following oxidative injury, contrary to expectations that lower lymphocyte GSH content with age would increase susceptibility to oxidative stress.

Aging↗
PubMed1996

Protective action of hydroxyethyl rutosides on singlet oxygen challenged cardiomyocytes.

1. The effect of a standardized mixture of beta-hydroxyethyl rutosides against oxidative damage in singlet oxygen-challenged isolated cardiac myocytes from adult rats was investigated. The morphology of the myocytes was evaluated as an indicator for cell viability (elongated, rod shaped cells vs. hypercontracted, rounded cells). The determination of the production of thiobarbituric acid reactive substances served as an indicator for lipid peroxidation. 2. Exposure to singlet oxygen which was generated by photo-excitation of rose bengal (10(-7) M) reduced the number of rod shaped (vital) cardiomyocytes by 78.5 +/- 2.5% and increased the production of thiobarbituric acid reactive substances by 1180 +/- 150% in comparison to incubation with control buffer. 3. Coincubation of the cells with beta-hydroxyethyl rutosides (concentration range: 6.7 pg ml-1 to 670 micrograms ml-1) increased the number of rod shape cardiomyocytes after exposure to singlet oxygen in a dose-dependent bell-shaped manner. A significant protective effect was observed at beta-hydroxyethyl rutosides concentrations ranging from 0.67 ng ml-1 to 67 ng ml-1. 4. In spite of their protective action, beta-hydroxyethyl rutosides did not reduce the accumulation of thiobarbituric acid reactive substances, used as an indicator for lipid peroxidation. 5. The data suggest that beta-hydroxyethyl rutosides exert a protective action against oxygen radical-induced damage of cardiac myocytes at very low concentrations without interfering with lipid peroxidation.

Animals↗
PubMed1996

Thrombopoietin expands erythroid, granulocyte-macrophage, and megakaryocytic progenitor cells in normal and myelosuppressed mice.

Thrombopoietin (Tpo), the ligand for the proto-oncogene receptor c-Mpl, increases megakaryocyte size, ploidy, and surface expression of platelet-specific glycoproteins, is inversely related to platelet mass, and is a potent in vivo stimulus of platelet production. However, several features of c-mpl biology, and that of its viral counterpart v-mpl, suggest that the action of Tpo may not be strictly limited to megakaryocytopoiesis. To investigate the possibility that Tpo might affect a multitude of cell lineages, we studied the effects of in vivo administration of the hormone on multiple types of marrow and splenic clonogenic hematopoietic progenitors. We report that Tpo acts to expand BFU-E, CFU-GM, and CFU-Mk and redistribute CFU-E in normal mice and to hasten the recovery of all of these progenitor cell types in myelosuppressed animals. These findings argue that the hematopoietic progenitor cell compartment responds to Tpo as a whole and that the in vivo effects of Tpo administration may be more wide-ranging than previously anticipated.

Animals↗
PubMed1996

Thrombopoietin accelerates platelet, red blood cell, and neutrophil recovery in myelosuppressed mice.

The recent cloning of thrombopoietin (TPO) has allowed us to study its in vivo effects in normal and myelosuppressed mice. Normal Balb/c mice were treated with recombinant human TPO (hTPO) at doses ranging from 1 to 20 kU for 7 days, and complete blood counts (CBCs) and the number of megakaryocytes in the bone marrow were determined. Platelet counts were increased starting on day 5 after mice were treated with hTPO. Platelet counts reached a peak between days 8 and 11 and returned to baseline between days 16 and 20. hTPO treatment increased the number of megakaryocytes in the bone marrow starting on day 3. In normal mice, hTPO treatment did not affect red or white blood cell (RBC or WBC) counts. To test the effects of hTPO in myelosuppressed mice, Balb/c mice were irradiated with 350 cGy total-body irradiation and dosed with 1.2 mg carboplatin, resulting in severe and prolonged thrombocytopenia, anemia, and neutropenia. Treatment with 5-20 kU hTPO for 7 days accelerated the recovery of platelet, RBC, and neutrophil counts in myelosuppressed mice and also significantly improved their nadirs. In addition, bone marrow megakaryocyte numbers recovered 11 days earlier and reticulocyte counts recovered 10 days earlier in hTPO-treated myelosuppressed mice than in controls. These results indicate that TPO can improve hematopoietic recovery in myelosuppressed mice, affecting multiple cell lineages.

Anemia↗
PubMed1995

Stabilization of erythrocytes by aldehydes and suitability of chicken IgY for the detection of potato virus X (PVX) in avidin-biotin enhanced reverse passive haemagglutination.

Methods are described for the detection of potato virus X (PVX) by reverse passive haemagglutination (RPH) by means of polyclonal antiviral antibodies coupled to sheep red blood cells (sRBC) and the chromic chloride method. The cells were stabilized with pyruvic aldehyde, thus providing a stock suspension for numerous coupling experiments lasting several months. Anti-PVX IgY, which is readily isolated in large amounts from the egg yolk of immunized chickens, was used in an avidin-biotin enhanced RPH assay with stabilized sRBC. With this method the PVX detection rate achieved was comparable to that of RPH assays using fresh non-fixed sRBC. In addition, avidin-coated sRBC could be stored for weeks at 4 degrees C and subsequently used for coupling with biotinylated IgY.

Animals↗
PubMed1995

Influence of age, sex, and dietary restriction on intracellular free calcium responses of CD4+ lymphocytes in rhesus monkeys (Macaca mulatta).

The influence of aging and dietary restriction on increase in intracellular free calcium ([Ca2+]i) of CD4+ lymphocytes from Macaca mulatta was examined after stimulation with anti-CD3 mAb. We used a flow cytometric assay with the dye indo-1 and either direct or reciprocal immunofluorescent staining to identify CD4+ cells. After stimulation with anti-CD3 mAb, intracellular free calcium responses were reduced in CD4+ lymphocytes from old male and female ad libitum fed monkeys compared to young and adult male or female monkeys. Old female monkeys had significantly lower [Ca2+]i than did old male monkeys. The reduced responses were in part related to a decreased percentage of responding cells. Dietary restriction of males over a four-year period did not alter [Ca2+]i response compared to ad libitum fed male monkeys. Female monkeys of all ages (which were restricted only for four months) also had similar [Ca2+]i responses to ad libitum fed controls. Our data suggest that age-related changes in [Ca2+]i responses are similar between humans and M. mulatta, and that over these intervals, no effects of caloric restrictions can be detected.

Aging↗
PubMed1995

Increased expression and subcellular translocation of the mitogen activated protein kinase kinase and mitogen-activated protein kinase in Alzheimer's disease.

The sequential activation of the mitogen-activated protein kinase kinase and its substrate, the mitogen-activated protein kinase is involved in a cascade of protein kinases which link a number of cell surface signals to intracellular changes in enzyme activity and gene expression. In vitro, mitogen-activated protein kinase is able to phosphorylate the microtubule-associated protein tau at Ser-Pro and Thr-Pro sites, thereby generating abnormally hyperphosphorylated tau species that are similar to paired helical filament-tau found in Alzheimer's disease. In the present study, we analysed the levels of immunoreactive mitogen-activated protein kinase kinase and mitogen-activated protein kinase in the temporal cortex (area 22) of patients with Alzheimer's disease by means of enzyme-linked immuno-sorbent assays and compared these changes with the content of abnormally phosphorylated paired helical filament-tau. The levels of immunochemically detected mitogen-activated protein kinase kinase and mitogen-activated protein kinase were both increased in Alzheimer's disease by between 35 and 40% compared with age-matched controls. Elevation of mitogen-activated protein kinase kinase was most pronounced during early stages of Alzheimer's disease and was inversely related to the tissue content of abnormally phosphorylated paired helical filament-tau. Pronounced immunoreactivity of mitogen-activated protein kinase kinase and mitogen-activated protein kinase was present in both tangle bearing neurons and unaffected neurons of the temporal cortex. Immunoreactive neurons were most often localized in the direct vicinity of neuritic plaques. In Alzheimer's disease, the subcellular distribution of mitogen-activated protein kinase kinase and mitogen-activated protein kinase showed a striking translocation from the cytoplasmic to the nuclear compartment. It is suggested that the activation of the mitogen-activated protein kinase cascade which appears to be an early feature of Alzheimer's disease might be critically involved in self-stimulating processes of neurodegeneration and aberrant repair under these conditions.

Aged↗
PubMed1995

Activation of murine T-cells via phospholipase-C gamma 1-associated protein tyrosine phosphorylation is reduced with aging.

Cross-linking of the T-cell receptor (CD3) induces activation of tyrosine kinases and the subsequent phosphorylation of intracellular protein substrates. We examined whether early events in signal transduction through CD3 or CD3 x CD4 receptor ligation were altered in aged murine T-lymphocytes. Both calcium mobilization and tyrosine phosphorylation of phospholipase C gamma 1 (PLC gamma 1) were decreased in T-lymphocytes from old mice. In addition, there was less tyrosine phosphorylation of a 35/36 kDa protein both in whole cell lysates and in PLC gamma 1 immunoprecipitates from old mice. This 35/36 kDa phosphoprotein binds specifically to the SH2 domains of PLC gamma 1. Using a fusion protein containing the SH2 domains of PLC gamma 1 and human IgG1 heavy chain, we identified three additional proteins that bind to the SH2 domains which were tyrosine phosphorylated following CD3 x CD4 ligation to a lesser degree with age. The tyrosine phosphorylation of two phosphoproteins binding to a fusion protein consisting of the SH2 domains of GAP (ras GTPase-activating protein) and human IgG1 heavy chain was also reduced with aging. The observed binding to SH2 domains was thiol redox sensitive. Thus, decreases in antioxidants with age may be responsible for inhibitory effects on PLC gamma 1-phosphatidylinositol signaling through redox regulation of tyrosine phosphoproteins.

Aging↗
PubMed1995

Thrombopoietin expands erythroid progenitors, increases red cell production, and enhances erythroid recovery after myelosuppressive therapy.

Thrombopoietin (TPO), the ligand for the receptor protooncogene c-mpl, has been cloned and shown to be the critical regulator of platelet production. Several features of c-Mpl expression, including its presence on erythroid cell lines, and the panmyeloid transformation characteristic of myeloproliferative leukemia (MPL) viral disease led us to investigate whether this receptor-ligand system may play a role in erythropoiesis. We report that although TPO alone did not support the growth of either early or late erythroid progenitors, it acted in synergy with erythropoietin to expand these populations. Moreover, while the effects on erythropoiesis in normal animals were modest, TPO greatly expanded the number of erythroid progenitors and blood reticulocytes and was associated with accelerated red cell recovery in myelosuppressed mice. Together, these data strongly suggest that erythroid progenitors respond to TOP and that this newly cloned cytokine, critical for platelet production, can augment erythropoiesis in states of marrow failure.

Animals↗
PubMed1993

Increased proliferative capacity of CD4+ and CD8+ T lymphocytes from mutant sphha/sphha mice is associated with increased IL-2 receptor expression.

We previously discovered that mutant anemic mice (sphha/sphha) show increased numbers of cycling lymph node T lymphocytes when analyzed by pulse and continuous infusion of tritiated thymidine. We have now further analyzed this in vivo phenomenon by evaluating the in vitro proliferative response of anti-CD3 activated lymphocytes from anemic mice using flow cytometric cell cycle analysis with 5'-bromodeoxyuridine and Hoechst dye. We determined that sorted CD4+ and CD8+ T lymphocytes from anemic mice have significantly greater proliferative capacity when compared with syngeneic control (+/+) mice (P < 0.001). In order to explain this increased growth capacity, we examined whether these cells exhibit differences in cell-surface phenotype (Pgp-1 and IL-2 receptor expression), activation state, or transmembrane signaling, or alterations in accessory cells or cytokines. Increased proliferation of T cells from anemic mice was associated with a larger percentage of T cells expressing IL-2R (p55 or CD25) at 24 and 48 hr after activation. Increased proliferative capacity was not associated with differences in activation state, Pgp-1 phenotype, transmembrane signaling, accessory cells, or cytokines. The mechanism for the abnormally high proliferative rate of T cells from anemic mice remains unclear, but we suggest that this mutant mouse may provide an important model for further studies on the molecular basis of T-cell replication.

Anemia, Hemolytic, Congenital↗
PubMed1993

The effect of 1-chloro-2,4-dinitrobenzene exposure on antigen receptor (CD3)-stimulated transmembrane signal transduction in purified subsets of human peripheral blood lymphocytes.

The intracellular low-molecular-weight thiol glutathione (GSH) is an important scavenger of free radicals and plays a role in the maintenance of the redox status of protein sulfhydryl groups. We have previously shown that human peripheral blood lymphocytes sorted on their basal GSH content proliferate proportionately to their GSH levels, and that an early event in lymphocyte activation appeared to be dependent on GSH. We have now analyzed transmembrane signal transduction in cells treated with 1-chloro-2,4-dinitrobenzene (CDNB), a GSH-depleting agent. Transmembrane signal transduction was measured as changes in intracellular free calcium and in protein tyrosine phosphorylation after stimulation with anti-CD3 monoclonal antibody. The results show a CDNB dose-dependent reduction in GSH content, the magnitude of intracellular free calcium mobilization, and the extent of tyrosine phosphorylation of several proteins, including phospholipase C-gamma 1. This suggests a role for GSH and/or protein thiol redox status in one of the earliest events controlling the ability of lymphocytes to respond to important proliferative signals in their environment and implies that agents which deplete lymphocyte GSH may be immunosuppressive through effects on CD3/T cell receptor-dependent transmembrane signal transduction.

Antibodies, Monoclonal↗
PubMed1993

Immune function in offspring of nonhuman primates (Macaca nemestrina) exposed weekly to 1.8 g/kg ethanol during pregnancy: preliminary observations.

A preliminary investigation of immune host response was conducted in a group of fetal alcohol-exposed nonhuman primates (Macaca nemestrina) who were part of a broader ongoing study of ethanol teratogenicity. The mothers of the offspring received weekly oral doses of ethanol (1.8 g/kg) for the first 3 or 6 or the entire 24 weeks of gestation. A control group received sucrose solution weekly throughout pregnancy. Four of the 18 ethanol-exposed animals (22%) died or were euthanized after infectious disease or failure to thrive during the first year of life; none of the seven control animals died. This imbalance in survival prompted the present review of immune function in the remaining offspring. Parameters assessed included: (1) white blood cell count (WBC), (2) peripheral blood leucocyte subsets (CD4+, CD8+, CD20+, and CD11c+), (3) T-cell proliferation after activation with phytohemagglutinin (PHA), staphylococcus enterotoxin B (SEB), and tetanus toxoid (TT), (4) phagocytic activity of monocytes, and (5) serum immunoglobulin levels and serum antibody titers after TT vaccination. Mean T-cell proliferation to TT was significantly decreased (p = 0.01) in all ethanol-exposed animals relative to controls, with near-significant decreases (p = 0.06) in response to SEB in the ethanol-exposed animals. Lymphocyte proliferation in response to PHA was not altered. Ethanol-exposed animals had significantly lower TT titers than controls after initial vaccination and booster. WBC, leukocyte subsets, serum immunoglobulins, and monocyte phagocytic activity were not significantly different from control values. These preliminary observations suggest that T-cell proliferation and antigen-specific memory responses may be altered in offspring exposed to weekly doses of ethanol in utero and warrant further evaluation for confirmation.

Animals↗
PubMed1993

Pathologic features associated with decreased longevity of mutant sphha/sphha mice with chronic hemolytic anemia: similarities to sequelae of sickle cell anemia in humans.

A colony of sphha/sphha mice with congenital hemolytic anemia and an abnormality in erythrocyte spectrin assembly was screened to determine the cause of premature death. Sphha/sphha mice have decreased life span, with 50% of animals dying by 6 months of age. The phenotype of these mutant mice includes moderate anemia (hematocrit: 21 to 28%), reticulocytosis, leukocytosis, lymphocytosis, extensive extramedullary hematopoiesis in spleen and liver, lymph node hyperplasia and membranoproliferative glomerulonephritis. With increased surveillance of this mouse colony, 20 clinically sick anemic mice were evaluated (complete blood counts and cultures of blood), euthanized and necropsied. Compared with anemic mice without clinical signs of disease, sick anemic mice had significantly higher white blood cell counts with only 4 (20%) of 20 animals being severely anemic (hematocrit: 4 to 8%). Blood from 11 (45%) of 20 animals was culture-positive for Pasteurella pneumotropica, Enterococcus, and/or Escherichia coli. In addition to the usual lesions in sphha/sphha mice, sick anemic mice had pneumonitis (95%) with thrombosis and infarction (80%) of one or more organs (spleen, myocardium, pancreas, liver, or bone marrow). The thrombotic tendency that accompanies the chronic hemolytic anemia in sphha/sphha mice, as well as the other clinicopathologic changes in these mutant mice, bears a striking resemblance to some poorly understood sequelae in human patients with sickle cell anemia. This mouse model may be useful in studying the pathophysiology of complications associated with sickle cell anemia in humans.

Anemia, Hemolytic↗
PubMed1992

Sulfhydryl oxidation down-regulates T-cell signaling and inhibits tyrosine phosphorylation of phospholipase C gamma 1.

Early events in both T-cell receptor (CD3)- and CD4-induced signal transduction pathways include tyrosine phosphorylation of protein substrates, the generation of phosphatidylinositol-phosphate breakdown products, and the mobilization of intracellular Ca2+. Oxidative stress in T cells mediated by sulfhydryl-reactive nonpolar maleimides was shown previously to down-regulate both receptor-mediated Ca2+ mobilization and interleukin 2 production. Here we show that N-ethylmaleimide suppresses both CD3- and CD4-induced Ca2+ responses in human T cells correlating with a reduction in the level of phospholipase C gamma 1 (PLC gamma 1) tyrosine phosphorylation. The inhibition of tyrosine phosphorylation of PLC gamma 1 and additional protein substrates was observed at concentrations of N-ethylmaleimide above 20 microM, whereas lower concentrations of oxidant appeared to increase tyrosine kinase activity following cell stimulation. Sulfhydryl oxidation did not directly affect the catalytic activity of PLC gamma 1, since immunopurified enzyme from N-ethylmaleimide-treated T cells was fully active. Although N-ethylmaleimide treatment of T cells did not cause a direct effect on total pp56lck kinase activity measured in vitro, the interaction between CD4 and pp56lck was oxidation-sensitive in vivo. However, CD3-induced signaling was inhibited at N-ethylmaleimide concentrations lower than that required for CD4/pp56lck dissociation, suggesting that CD3-associated tyrosine kinase activity involves acutely sensitive regulatory thiols. In addition to chemically induced sulfhydryl oxidation, naturally regulated cellular redox states appear to dictate the potential for T-cell responsiveness, since degranulating human peripheral blood neutrophils inhibited CD3-induced Ca2+ mobilization in T lymphocytes. These data indicate that signal transduction in T cells involves the activation of PLC gamma 1 by tyrosine phosphorylation through an oxidation-sensitive intermediate between surface receptors and tyrosine kinases, perhaps including the interaction between CD4 and pp56lck.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1991

Altered lymphocyte populations in sphha/sphha mice with chronic hemolytic anemia.

Lymphocyte kinetics and phenotype were examined in mutant anemic sphha/sphha mice that manifest a lifelong lymphocytosis which accompanies their chronic hemolytic anemia. Anemic mice have significant increases in CD4+, CD8+, and sIgM+ lymphocytes in peripheral blood. Pulse and continuous infusion studies with [3H]TdR suggest that this apparent lymphoid expansion is not due to increased production of lymphocytes in bone marrow or thymus but rather to a redistribution of lymphocytes from the spleen to other peripheral lymphoid tissue sites as well as increased proliferation of T and B lymphocytes in lymph nodes. This murine model could be useful to examine lymphocyte perturbations that may accompany chronic hemolytic anemia in humans.

Anemia, Hemolytic, Congenital↗