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Biomedical subjects

P Hunt

Publications and source records attributed to P Hunt.

At least 19 recordsLinked to original sources
PubMed1995

The Mpl-ligand or thrombopoietin or megakaryocyte growth and differentiative factor has both direct proliferative and differentiative activities on human megakaryocyte progenitors.

Previously, it was believed that megakaryocytopoiesis was regulated by two types of humoral factors: megakaryocyte colony-stimulating factor (MK-CSF), which acts on progenitors inducing their proliferation, and thrombopoietin (TPO), a megakaryocyte(s) (MK) maturational factor that induces platelet formation. The recently cloned Mpl-ligand (Mpl-L) seems to have both properties in vivo and in vitro and has also been called TPO. However, it cannot be excluded that a part of these activities is due to a synergistic effect with growth factors present in the serum or synthesized by accessory cells. To delineate the precise TPO (Mpl-L) biologic activities, we performed serum-free cultures at limiting cell dilution. Target cells were adult human marrow CD34+CD41+ cells, which represent a highly selected population of late MK progenitor or transitional cells. Cells were purified using a flow cytometer equipped with an automatic cloning design unit. We determined that the recombinant molecule had a biologic activity that reached a plateau at 10 ng/mL. At this concentration, a linear relationship between the average MK number per well and the number of cells seeded (between 1 to 50 cells per well) was observed. At one cell per well, 60% of the wells contained a single MK at day 5 of culture. Half of these wells contained only one large MK, whereas the other half contained several MK (up to 25), demonstrating that TPO has direct proliferative biologic activity. In contrast, at limiting dilution, none of the other cytokines tested (stem cell factor [SCF], interleukin-6 [IL-6], and erythropoietin [Epo]) were effective, whereas IL-3 showed a mild effect. However, a combination of SCF plus IL-6 plus IL-3 produced similar results as TPO alone. Addition of the other cytokines to TPO did not enhance the cloning efficiency of the CD34+CD41+ cells but increased twofold the average number of MKs per clone. MKs reached a ploidy of 32N and 64N in the presence of TPO. The mean ploidy value was approximately 6 and was not modified by addition of the other cytokines. At the ultrastructural level, a majority of the MKs showed maturational defects related to an imbalance between the synthesis of alpha-granules and demarcation membranes. However, a fraction (about 30%) had a cytoplasmic maturation that exactly mimicked that of marrow MKs. In addition, proplatelet-shedding MKs were observed in the cultures, even at limiting dilution. Such a result was not observed with any other individual cytokines, including the combination of three cytokines.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigens, CD↗
PubMed1995

Purification and biologic characterization of plasma-derived megakaryocyte growth and development factor.

The isolation and cloning of the ligand for the cytokine receptor, Mpl, have been recently described. In this report we present details of the purification of this novel cytokine (megakaryocyte growth and development factor [MGDF]) from aplastic canine plasma. Two forms of canine MGDF, with apparent molecular weights of 25 kD and 31 kD and sharing a common N-terminal amino acid sequence, were isolated. The sole contaminant detected in purified 25-kD or 31-kD MGDF was canine Ig. Canine MGDF is characterized as a human megakaryocyte colony-stimulating factor that acts synergistically with human recombinant stem cell factor but not interleukin-3. MGDF also appears to be physiologically regulated in response to platelet demand. In canine and murine models, serum levels of MGDF activity peak during the thrombocytopenic periods after irradiation, 5-fluorouracil, or antiplatelet antisera injections. These data indicate that the megakaryocyte-stimulating activity that accumulates in plasma in response to platelet losses is a novel cytokine that functions through an interaction with the Mpl cytokine receptor.

Amino Acid Sequence↗
PubMed1995

Recombinant human megakaryocyte growth and development factor stimulates thrombocytopoiesis in normal nonhuman primates.

Megakaryocyte growth and development factor (MGDF) is a novel cytokine that binds to the c-mpl receptor and stimulates megakaryocyte development in vitro and in vivo. This report describes the ability of recombinant human (r-Hu) MGDF to affect megakaryocytopoiesis in normal nonhuman primates. r-HuMGDF was administered subcutaneously to normal, male rhesus monkeys once per day for 10 consecutive days at dosages of 2.5, 25, or 250 micrograms/kg of body weight. Bone marrow and peripheral blood were assayed for clonogenic activity and peripheral blood counts were monitored. Circulating platelet counts increased significantly (P < .05) for all doses within 6 days of r-HuMGDF administration and reached maximal levels between day 12 and day 14 postcytokine administration. The 2.5, 25.0, and 250.0 micrograms/kg/d doses elicited peak mean platelet counts that were 592%, 670%, and 449% of baseline, respectively. Bone marrow-derived clonogenic data showed significant increases in the concentration of megakaryocyte (MEG)-colony-forming unit (CFU) and granulocyte-erythroid-macrophage-megakaryocyte (GEMM)-CFU, whereas that of granulocyte-macrophage (GM)-CFU and burst-forming unit-erythroid (BFU-e) remained unchanged during the administration of r-HuMGDF. These data show that r-HuMGDF is a potent stimulator of thrombocytopoiesis in the normal nonhuman primate.

Animals↗
PubMed1995

Transcription factor NF-E2 is required for platelet formation independent of the actions of thrombopoietin/MGDF in megakaryocyte development.

Despite the importance of blood platelets in health and disease, the mechanisms regulating their formation within megakaryocytes are unknown. We generated mice lacking the hematopoietic subunit (p45) of the heterodimeric erythroid transcription factor NF-E2. Unexpectedly, NF-E2-/- mice lack circulating platelets and die of hemorrhage; their megakaryocytes show no cytoplasmic platelet formation. Though platelets are absent, serum levels of the growth factor thrombopoietin/MGDF are not elevated above controls. Nonetheless, NF-E2-/- megakaryocytes proliferate in vivo in response to thrombopoietin administration. Thus, as an essential factor for megakaryocyte maturation and platelet production, NF-E2 must regulate critical target genes independent of the action of thrombopoietin. These findings provide insight into the genetic analysis of megakaryocyte maturation and thrombopoiesis.

Animals↗
PubMed1995

Effects on sheep of transport by road for up to 24 hours.

Five groups of 20 slaughter sheep of approximately 37.9 kg liveweight were transported by road for either three, nine, 15, 18 or 24 hours and three groups were not transported, one of them being deprived of food and water for 24 hours. Before and after transport the liveweight and various blood variables were measured and heart rate and behavioural observations were recorded from subsets of the animals. With increased journey time there was a decrease in liveweight and an increase in the plasma levels of free fatty acids, beta-hydroxybutyrate and urea; however, the changes over 24 hours were similar to those in the group deprived of food and water. In the transported sheep, the heart rate and levels of plasma cortisol and glucose were increased by the stresses of loading and the initial stages of the journey, but after nine hours the sheep appeared, to some extent, to have adapted. They were able to lie down and did not appear to be physically stressed. Measurements of plasma osmolality, total plasma protein and albumin did not indicate that the sheep had become severely dehydrated after 24 hours of transport but upon their return, feeding and drinking activity was greater than that observed before the journey.

Adaptation, Physiological↗
PubMed1995

Measuring irradiated lung and heart area in breast tangential fields using a simulator-based computerized tomography device.

PURPOSE: To illustrate the use of a simulator based computerized tomography system (SIMCT) in the simulation and planning of tangential breast fields. METHODS AND MATERIALS: Forty-five consecutive patients underwent treatment planning using a radiotherapy simulator with computerized tomography attachment. One to three scans were obtained for each patient, calculations were made on the central axis scan. Due to the wide aperture of this system all patients were able to be scanned in the desired treatment position with arm abducted 90 degrees. Using available software tools the area of lung and/or heart included within the tangential fields was calculated. The greatest perpendicular distance (GPD) from the chest wall to posterior field edge was also measured. RESULTS: The mean GPD for the group was 25.40 mm with 71% of patients having GPDs of < or = 30 mm. The mean area of irradiated lung was 1780 sq mm which represented 18.0% of the total ipsilateral lung area seen in the central axis. Seven of the patients with left sided tumors had an average 1314 sq mm heart irradiated in the central axis. This represented 11.9% of total heart area in these patients. CONCLUSIONS: Measurements of irradiated lung and heart area can be easily and accurately made using a SIMCT device. Such measurements may help identify those patients potentially at risk for lung or heart toxicity as a consequence of their treatment. A major advantage of this device is the ability to scan patients in the actual treatment position.

Breast↗
PubMed1995

Platelets generated in vitro from proplatelet-displaying human megakaryocytes are functional.

An in vitro culture system demonstrating the transitions from megakaryocyte progenitors to functional platelets is described. CD34-selected cells from normal human peripheral blood are cultured under conditions that promote megakaryocyte formation. After 8 to 11 days, enriched populations of mature megakaryocytes are replated under conditions that favor the development of proplatelets. Proplatelets express the platelet-specific proteins, glycoproteins Ib and IIb (GPIb and GPIIb), and fibrinogen and also contain microtubule coils equal in size to those found in plasma-derived platelets. In addition, proplatelets have ultrastructural features in common with plasma-derived platelets. Platelet-sized particles from the proplatelet culture supernatants are examined. Ultrastructurally, these particles are identical to plasma-derived platelets. Functionally, these culture-derived platelets aggregate in response to both thrombin and adenosine diphosphate (ADP) plus fibrinogen. This aggregation is specifically inhibited by the addition of a function-blocking anti-GPIIbIIIa antibody. Culture-derived platelets stimulated with agonists also express the activation-dependent antigens P-selectin and functional fibrinogen receptor. This is the first description of an in vitro culture system that sequentially demonstrates megakaryocyte growth, development, and platelet production.

Anemia, Aplastic↗
PubMed1995

Molecular cloning and expression of DNA encoding ovine interleukin 2.

We have generated DNA encoding the mature form of ovine interleukin 2 (IL-2) by polymerase chain reaction (PCR) using primers complementary to sequences at the 5' and 3' ends of human, murine and bovine IL-2 cDNA. The predicted PCR product of 400 bp was ligated into the yeast Ty-P1 galactose-inducible expression vector pOGS40 which was used to transform yeast spheroplasts. The fusion protein, with a Factor Xa proteolytic cleavage site between ovine IL-2 and the P1 fusion partner, was expressed from galactose-induced transformed yeast. P1:IL-2 fusion protein, which self-assembles into virus-like particles (VLPs) due to the interaction of the P1 protein, was purified from lysates of mechanically disrupted yeast by centrifugation on a discontinuous sucrose gradient. Fusion protein was detected in Western blot analysis with polyclonal antisera raised to recombinant bovine IL-2. Soluble recombinant ovine IL-2 was released from the P1 fusion protein by cleavage with Factor Xa enzyme. After purification recombinant ovine IL-2 was functionally active as shown by its ability to support the proliferation of Con A-activated T cells and was capable of generating maedi visna virus-specific cytotoxic T cells from primed precursor cells. The availability of recombinant ovine IL-2 will greatly help the analysis of the specificity of pathogen-specific cells in the sheep.

Amino Acid Sequence↗
PubMed1995

Restoration of normal Hox code and branchial arch morphogenesis after extensive deletion of hindbrain neural crest.

Among the derivatives of the cephalic neural crest is the ectomesenchyme which subsequently constitutes most of the craniofacial skeleton. There is evidence to suggest that the skeletogenic fate of the hindbrain neural crest is specified before emigration from the neural tube and that Antennapedia class Hox genes are involved in that process. To explore the putative causal link between Hox expression and craniofacial morphology, we produced a specific series of bilateral crest deletions in chick embryos and assessed branchial arch morphology, Hox gene expression, and patterning of skeletal structures in the postoperative embryo. Surprisingly, we found that deletion of the bulk of the rhombencephalic crest and substantial portions of the dorsal rhombencephalon did not prevent normal branchial arch morphogenesis and normal patterns of Hox gene (-A3 and -B4) expression 48 h after operation. Neural crest-like cells have been identified on crest migration pathways at the level of the original ablation, further confirming that ablated cephalic neural crest is replaced by regeneration from the cut edge of the neuroepithelium. Furthermore, in such embryos ectomesenchyme from regenerated crest is able to form a facial skeleton in which the mandible and hyoid apparatus are normal in size and organization. These findings demonstrate that the cranial neuroepithelium has more extensive regenerative capacities than was previously thought, which has important implications for investigations of craniofacial development.

Animals↗
PubMed1995

Functional human platelet generation in vitro and regulation of cytoplasmic process formation.

A recent report from this laboratory described an in vitro system in which CD34+ cells are stimulated to form mature megakaryocytes and, subsequently, cytoplasmic processes also known as pro-platelets that give rise to functional platelets. Thrombin, an important regulator of hemostasis, has been demonstrated to have an inhibitory role in cytoplasmic process formation from both human and guinea pig megakaryocytes. This inhibition can be reversed by antithrombin III (ATIII), an inhibitor of thrombin, in combination with heparin, a cofactor of ATIII and a glycosaminoglycan. Using the described human in vitro system, the role of thrombin and of glycosaminoglycans are investigated. Thrombin receptors are expressed on megakaryocytes, suggesting that the inhibition by thrombin may be direct. Matrigel, a basement membrane matrix containing glycosaminoglycans, is used to compare the frequency and the rate of cytoplasmic process formation. A possible role of glycosaminoglycans in platelet production is discussed.

Antigens, CD↗
PubMed1994

Sequence of the sheep interleukin-10-encoding cDNA.

The ovine interleukin-10 (oIL-10)-encoding cDNA has been cloned and sequenced using gene amplification by the polymerase chain reaction (PCR). We present the complete coding sequence of the ovine IL-10 gene, as well as the predicted amino acid (aa) sequence. The oIL10 DNA coding sequence is 531 nucleotides long and the mature protein product is predicted to be 18,367 Da, consisting of 158 aa, excluding a 19-aa N-terminal hydrophobic signal peptide. The oIL-10 protein is > 77% identical to pig and human IL-10, > 71% identical to rodent IL-10 and > 68% identical to viral IL-10.

Amino Acid Sequence↗
PubMed1994

Enrichment and characterization of peripheral blood-derived megakaryocyte progenitors that mature in short-term liquid culture.

A population of peripheral blood-derived cells that mature into megakaryocytes within four to eight days of liquid culture is described. This population was enriched from normal leukapheresis units by counterflow centrifugal elutriation and CD34 selection. The cells were incubated in suspension with known megakaryocyte growth or maturation factors. Megakaryocytes were identified within the cultures with antibodies to platelet-glycoproteins (Ib and IIb) and cytologically classified as stage I-IV cells. Plasma from aplastic dogs (APK9) or human recombinant interleukin 3 (IL-3) were the only culture additives which reproducibly resulted in megakaryocyte development. The activity present in APK9 was relatively megakaryocyte-specific while IL-3 was not. The phenotype of the short-term megakaryocyte progenitor cell population was determined by FACS and found to be CD34bright but not CD34dull or CD34-. The population was further characterized as CD34+/CD38+ and CD34+/HLA-DR+. Both CD34+/CD41- and CD34+/CD41+ populations contained megakaryocyte progenitor cells, although megakaryocytes that developed from the latter population were fewer in number. In summary, we have developed an enrichment protocol that, when coupled with a liquid culture assay system, enabled us to characterize short-term megakaryocyte progenitors from peripheral blood. These cells may be important for early platelet recovery in transplantation settings.

Anemia, Aplastic↗
PubMed1994

New classes of antimuscarinic agents endowed with selective antispasmodic properties. 1-Arylsulfonyl pyrrolidin-2-ones and 2-thiones, 1-arylsulfonyl piperidin-2-ones and 2-thiones and 1-arylsulfonyl hexahydro-2H-azepin-2-one.

A series of 1-arylsulfonylpyrrolidin-2-ones (and 2-thiones), 1-aryl sulfonylpiperidin-2-ones (and 2-thiones) and 1-arylsulfonyl hexahydro-2H-azepin-2-one were synthesized and submitted to a battery of binding assays. The compounds showed little or no affinity for the receptors tested other than muscarinic receptors labelled either with [3H]pirenzepine or with [3H]quinuclidinyl benzilate. When tested in the isolated guinea pig ileum, they antagonized the contractions induced by acetylcholine and behaved as competitive muscarinic antagonists. After parenteral administration in mice, most compounds inhibited carbachol-induced diarrhoea but were less effective in counteracting salivation and lacrimation and showed little or no mydriatic action, thus displaying selectivity at the intestinal level. The reference drugs tested, atropine, butyl scopolamine and cimetropium bromide were far less selective. maximal in vivo activity was obtained by introducing diethylamino or 1-piperidino or 1-hexahydroazepinyl groups in the 4-position of the phenyl ring while the enlargement of a 5- to a 6-membered lactam ring or its conversion into a thiolactam had a less marked effect. The most interesting compounds were further evaluated for their ability to antagonize carbachol-induced colonic hypermotility in the rat and arecoline-induced analgesia in mice. The effect on gastric acid secretion in the rat was also investigated. The overall in vivo data showed that compounds 14, 15, 26 and 27, i.e. those bearing a 1-hexahydroazepinyl group in the 4-position of the phenyl ring, were the most potent and selective compounds.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Evidence that stem cell factor is involved in the rebound thrombocytosis that follows 5-fluorouracil treatment.

The mechanisms responsible for 5-fluorouracil (5FU)-induced rebound thrombocytosis are not completely understood. SI/SI(d) mice, which do not undergo rebound thrombocytosis in response to 5FU, provide a genetic approach to the study of this phenomenon. Recent reports by several groups that the SI locus encodes a protein known variably as stem cell factor (SCF), mast cell growth factor, or kit ligand, suggests the possibility that the lack of wild-type SCF in SI/SI(d) mice is responsible for their defective response to 5FU-induced thrombocytopenia. It is shown in this report that SCF-treated SI/SI(d) mice are as capable as their wild-type littermates in undergoing rebound thrombocytosis. W/Wv mice, mutated at the locus encoding the SCF receptor, also do not undergo rebound thrombocytosis, but are not responsive to SCF treatment. In normal mice, it is shown by RNA solution hybridization that SCF mRNA expression is increased during the 5FU-induced platelet nadir period. It is also shown by autoradiography that maturing megakaryocytes express SCF receptors, and that in vivo administration of SCF significantly raises the numbers of megakaryocytes, as well as circulating platelet counts. Taken together, these data indicate that SCF may be an important regulator of platelet production under both normal and physiologically disturbed situations.

Animals↗