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Biomedical subjects

P Hunt

Publications and source records attributed to P Hunt.

At least 91 records · Page 5Linked to original sources
PubMed1994

Identification and cloning of a megakaryocyte growth and development factor that is a ligand for the cytokine receptor Mpl.

A novel megakaryocyte growth and development factor (MGDF) has been identified in aplastic canine plasma, and its cDNAs have been cloned from canine, murine, and human sources. Purified canine MGDF isolated by procedures involving MpI receptor affinity chromatography exists in at least two forms, with apparent molecular masses of 25 kDa and 31 kDa, that share the N-terminal amino acid sequence APP-ACDPRLLNKMLRDSHVLH. Human, dog, and mouse cDNAs for MGDF are highly conserved and encode open reading frames for proteins of 353, 352, and 356 amino acids, respectively, including predicted signal peptides. Canine MGDF and recombinant human MGDF support the development of megakaryocytes from human CD34+ progenitor cell populations in liquid culture and promote the survival of a factor-dependent murine cell line (32D) engineered to express MpI. These biological activities are blocked by the soluble extracellular domain of MpI. These data demonstrate that MGDF is a novel cytokine that regulates megakaryocyte development and is a ligand for the MPI receptor.

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↗
PubMed1993

The effect of the platelet-derived glycosaminoglycan serglycin on in vitro proplatelet-like process formation.

The formation of proplatelet-like processes on megakaryocytes cultured in vitro has been shown to be inhibited by prothrombin, found residually in human serum, which is converted in culture to thrombin. This study reports that another factor found in human serum will counter this inhibition and permit proplatelet-like process formation to occur in vitro even in the presence of inhibitory concentrations of thrombin. The factor was purified from human platelet lysates and identified by amino acid sequence analysis as the proteoglycan serglycin. A similar, if not identical, factor was found at elevated levels in the plasma of thrombocytopenic rabbits. Serglycin probably functions as a proplatelet potentiator by virtue of a tendency to complex with thrombin. Thrombin in complex with serglycin retains its enzymatic properties, but is apparently sterically hindered from interacting with the megakaryocyte cell surface. In preliminary studies, the in vivo administration of serglycin in mice resulted in an increased number of circulating platelets when given in combination with interleukin-6 (IL-6).

Amino Acid Sequence↗
PubMed1993

Megakaryocyte proplatelet-like process formation in vitro is inhibited by serum prothrombin, a process which is blocked by matrix-bound glycosaminoglycans.

The process of platelet shedding from megakaryocytes is incompletely understood, due in part to the impossibility of studying this dynamic process in vivo. Megakaryocytes in situ and in in vitro cultures display extended cytoplasmic processes constricted at platelet-sized intervals which presumably are the structural intermediates between megakaryocytes and platelets. This study describes the establishment of a serum-free culture system of purified guinea pig megakaryocytes in which extensive cytoplasmic process formation can be observed on 21 to 29% of the cells. The addition of as little as 0.05% pooled human serum to the cultures will completely but reversibly block process development. The serum inhibitor was identified as residual prothrombin, which upon contact with megakaryocytes is converted to the serine esterase thrombin. Thrombin directly prevents the formation of new processes and also induces retraction of existing processes. When megakaryocytes are cultured on Matrigel, process formation occurs even in an excess of thrombin. This potentiation of process development in the presence of inhibitory factors is mediated by the glycosaminoglycan content of Matrigel. The physiological implications of these observations are discussed.

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↗
PubMed1992

Determination of the enantiomers of a novel 20,21-dinoreburnamenine derivative in rat plasma and brain by high-performance liquid chromatography using a chiral stationary phase.

A high-performance liquid chromatographic method with solid-phase extraction was developed for the assay of the enantiomers of a novel 20,21-dinoreburnamenine derivative (RU 49041) in rat plasma and brain using a chiral stationary phase (Nucleosil Chiral 2) and ultraviolet detection. The limit of detection was 10 ng/ml (or ng/g) in both tissues and the intra-assay precision was satisfactory (plasma, ca. 5%; brain, ca. 1%). The pharmacokinetic profiles of the two enantiomers were determined following oral administration of the racemate (10 mg/kg). The results show that their pharmacokinetics are very different: whereas both enantiomers appear in the brain, only the 3 alpha,16 beta-enantiomer is detected in plasma.

Animals↗
PubMed1992

Molecular mechanisms of pattern formation in the vertebrate hindbrain.

During early stages of neural development a series of repeated bulges, termed rhombomeres, form in the vertebrate hindbrain. Studies in the chick have shown that rhombomeres are segments that underlie the patterning of nerves in the hindbrain, and this raises the question of the molecular basis of segment development. Several genes have been found with expression patterns consistent with roles in the formation or differentiation of rhombomeres. The zinc finger gene Krox-20 is expressed in two alternating rhombomeres, r3 and r5, in the mouse hindbrain; these stripes of gene expression are established prior to the morphological appearance of segments. Krox-20 is also expressed in this pattern in the chick and Xenopus, suggesting that it has a conserved role, possibly in the formation of rhombomeres. Four members of the Hox-2 homeobox gene cluster have limits of expression at rhombomere boundaries. Three genes, Hox-2.6, -2.7 and -2.8 have progressively more anterior limits of expression at two-segment intervals, whereas expression of Hox-2.9 is restricted to one rhombomere, r4. The Hox-2 genes are expressed in spatially restricted patterns in early neural crest cells. These findings suggest that the Hox genes have roles in specifying the identity of rhombomeres and of neural crest.

Animals↗
PubMed1992

Analysis of the murine Hox-2.7 gene: conserved alternative transcripts with differential distributions in the nervous system and the potential for shared regulatory regions.

In this study we have investigated the organization and regulation of the mouse Hox-2.7 gene. There are several alternative transcripts some of which are conserved between mouse and humans. By Northern and in situ analysis we are able to identify at least three types of transcripts which are different in size and splicing pattern and have distinctly different boundaries of expression in the nervous system. One subset of the endogenous transcripts has a boundary of expression that corresponds to the adjacent Hox-2.8 gene instead of Hox-2.7. In another type of transcript there is an alternative reading frame which predicts a protein that has homology to an enzyme ATPase and suggests that a non-homeobox containing gene may be located in the Hox-2 cluster. A Hox-2.7-lacZ transgene is expressed in a similar pattern to the endogenous gene in that spatially-restricted domains of expression are seen in the branchial arches, neural tube, paraxial mesoderm (somites), cranial ganglia, neural crest and gut. However, the anterior boundaries of transgene expression only correspond to the subset of Hox-2.7 transcripts which map to the Hox-2.8 boundary. The proximity of a Hox-2.7 promoter to regions which regulate the adjacent Hox-2.6 gene and the expression of transgenic and endogenous transcripts in a Hox-2.8 pattern, suggest that regulatory elements may be shared by neighbouring genes to establish the complete expression pattern.

Amino Acid Sequence↗
PubMed1992

Secondary cytokine production by lymphoid cells used in cellular immunotherapy.

Interleukin-2 (IL-2) has been used extensively in cellular immunotherapy trials as a systemic activator of the immune system as well as an ex vivo stimulant for lymphoid cell function. Despite the measurement of several in vitro and in vivo immunologic parameters related to cellular immunotherapy, determinants of successful cellular immunotherapy remain unknown. To further delineate the consequences of exposing peripheral blood lymphocytes to high concentrations of IL-2, we assessed the supernatants of IL-2-activated peripheral blood lymphocytes for production of tumour necrosis factor (TNF) and interferon-gamma (IFN-gamma). Exposure of normal monocyte-depleted peripheral blood mononuclear cells (PBMC) to IL-2 caused a dose-dependent increase in secretion of TNF and IFN-gamma which increased linearly after 48 h in culture. Analysis of positively selected, highly purified PBMC subpopulations exposed to IL-2 revealed that TNF-alpha and TNF-beta were produced by both CD3+ and CD16+ subpopulations but not by CD22+ cells. These studies were extended to supernatants obtained from PBMC cultures used in the adoptive cellular immunotherapy of patients with advanced cancer. Patients treated with lymphokine-activated killer (LAK) cell immunotherapy were classified as responders (N = 14) or non-responders (N = 17) to therapy. We found no significant difference in the production of TNF between responders and nonresponders (22 +/- 9 U ml-1 vs. 20 +/- 6 U ml-1), P > 0.05. However, LAK cell supernatants harvested from non-responders contained a significantly higher level of IFN-gamma (232 +/- 94 U ml-1) compared with responders (42 +/- 14), P < 0.05. Furthermore, the linear association between IFN-gamma and TNF-alpha production was different between these two response groups (rs = -0.19 for non-responders and rs = 0.48 for responders). These results suggest that secondary cytokine production by adoptively transferred lymphocytesmay play an important role in the host response to cellular immunotherapy.

Cells, Cultured↗
PubMed1992

Cruciate thinning of the zona pellucida for more successful enhancement of blastocyst hatching in the mouse.

Implantation rates remain low following human in-vitro fertilization (IVF). Suboptimal culture conditions may limit the ability of embryos to hatch as blastocysts, and artificial opening of the zona pellucida has been proposed as a means to promote subsequent hatching (assisted hatching). Such techniques must have minimal adverse effects on the embryos, while maximizing the potential for an embryo to hatch fully as a blastocyst. In a mouse model, we compared embryonic development after zona drilling, and cruciate thinning of the zona (CTOZ) intended to simulate the natural thinning of the zona pellucida. Using acidic Tyrode's solution both zona drilling and cruciate-thinning were performed on day 3 morulae. On day 4 the rates of complete hatching of blastocysts were 0/165, 24/172 and 72/175 in control, zona drilled and thinned groups respectively (P less than 0.0001). On day 5 the rates of complete hatching in the same groups were 20/165, 54/172 and 120/175 respectively (P less than 0.00001) and by day 6, 66/165, 74/172 and 130/175 respectively (P less than 0.00001). The rate of arrest at the morula stage was 24/172 versus 8/175 in the zona drilled and thinned groups respectively (P less than 0.005, whilst the rate of arrest at the blastocyst stage was 21/172 versus 14/175 respectively (NS). Hence cruciate thinning of the zona appears less detrimental at the morula stage than zona drilling, but eventual rates of arrest at the blastocyst stage were comparable. Both techniques significantly increased the rate of hatching, but zona drilling did not guarantee complete hatching.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Hox codes and positional specification in vertebrate embryonic axes.

We have compared the ways in which vertebrate Hox genes are used in the patterning of three distinct embryonic contexts, the branchial region, the somites, and the limb. We have identified common features of the three systems, but have suggested on the basis of their differences (in both embryological properties and use of Hox genes) that it is better to consider each as an independent system for regional specification. Nevertheless, there are sufficient common features to expect that exploitation of the distinct experimental advantages of each system will provide important insights to the mode of operation of the others.

Animals↗
PubMed1991

A distinct Hox code for the branchial region of the vertebrate head.

The branchial region of the vertebrate head forms through complex interactions involving rhombomeric segments, neural crest and branchial arches. It is though that aspects of their patterning mechanisms are linked and involve Hox-2 genes, whose overlapping and spatially restricted expression domains represent a combinatorial code for generating regional diversity. Vertebrates possess four Hox clusters of Antennapedia class homeobox genes, related to each other by duplication and divergence from a common ancestral complex. In consequence, at equivalent positions in different clusters there are highly related genes known as subfamilies or paralogous groups. As Hox-2 genes cannot fully account for patterning individual rhombomeres, we investigated whether offsets in expression limits of paralogous genes could account for the generation of regional diversity. We report here that, with the exception of the labial subfamily, paralogues show identical expression limits in rhombomeres, cranial ganglia and branchial arches, providing a combinatorial Hox code for the branchial region that seems to be different in organization to that of the trunk.

Alleles↗