Transforming growth factor-beta trans-modulates the expression of colony stimulating factor receptors on murine hematopoietic progenitor cell lines

Transforming growth factor beta (TGF-beta) is a potent and selective growth inhibitor of early hematopoietic progenitors and leukemic cells. The cellular mechanism(s) underlying this antiproliferative effect is, however, currently unknown. In the present study, we demonstrate that TGF-beta inhibits the expression of granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL-3), and granulocyte-CSF (G-CSF) receptors on murine factor-dependent and independent hematopoietic progenitor cell lines without a significant change in receptor affinity. A maximum reduction in GM-CSF receptor numbers of 65% to 77% was observed by 96-hour incubation with TGF-beta. The TGF- beta induced trans-down-modulation of GM-CSF receptors was prolonged, noncytotoxic but reversible, and not due to endogenous production of GM- CSF. The TGF-beta induced reduction in CSF receptor numbers preceded TGF-beta's growth inhibitory action. In addition, the ED50 (1 to 10 pmol/L) for TGF-beta's CSF receptor modulatory and antiproliferative effect was similar. The effect of TGF-beta on cell surface CSF receptor expression was specific, because the expression of other cell surface proteins (Ly 5 and Ly 17) was not affected by TGF-beta treatment, and because other growth inhibitors (tumor necrosis factor and interferon) did not affect CSF receptor expression. These data suggest that the downregulation of the growth of hematopoietic progenitor cells by TGF- beta involves reducing the cell surface expression on growth factor receptors.     

Skoura – a genetic island for congenital insensitivity to pain and anhidrosis among Moroccan Jews, as determined by a novel mutation in the NTRK1 gene

Congenital insensitivity to pain with anhidrosis (CIPA) or hereditary sensory and autonomic neuropathy type IV is a rare, autosomal recessive neurologic disorder, characterized by absence of reaction to painful stimuli, mental retardation, self- mutilating behavior, anhidrosis, and recurrent episodes of hyperthermia. Mutations in the neurotrophic tyrosine kinase receptor 1, a receptor phosphorylated by nerve growth factor, have been documented in diverse ethnic groups. We identified the same novel nonsense mutation in two unrelated families of Moroccan Jewish descent, each with two affected siblings. This possible founder mutation may trace to the rural Jewish village in southern Morocco from where both these families originated. Genetic screening for the causative mutation among 300 unrelated Moroccan Jews did not reveal carriers for the causative mutation, thus excluding high risk for CIPA in this ethnic subpopulation.     

Detection of the hemoglobin E mutation using the color complementation assay: application to complex genotyping

The color complementation assay (CCA) is a method of allele-specific DNA amplification by which competitive priming and extension of fluorescently labeled oligonucleotide primers determine the color of DNA amplification product. This diagnostic method precludes the need for radioisotopes, electrophoresis, and multiple high-stringency reaction conditions. The multiplicity of mutant globin genes present in Southeast Asians complicates clinical diagnosis and underscores the importance of DNA-based diagnostic methods. We have applied CCA to distinguish beta A and beta E alleles. Competing 15mer primers were a fluorescein-labeled complement to beta A and a rhodamine-labeled complement to beta E, identical except for their central nucleotides. A common unlabeled primer was used to amplify DNA product, the color of which was determined by the perfectly complementary primer. Color photography and spectrofluorometry, as well as a method of black-white photography that we developed to distinguish fluorescein- and rhodamine- labeled DNA, were used to record results. We applied CCA to define the complex genotype of a Thai woman with thalassemia intermedia, 96% HbE, and 4% HbF whose possible genotypes included several permutations of alpha-thalassemia, beta-thalassemia, and beta E genes. zeta-Globin gene mapping of DNA doubly digested with Bg/II and Asp 718 showed the -alpha 3.7/--SEA genotype, and CCA confirmed homozygous beta E/beta E. The CCA is useful for diagnosing the compound hemoglobin genotypes of Southeast Asians and could be applied also to prenatal diagnosis in this population.     

Differential effect of Serratia protease on platelet surface glycoproteins Ib and V

The effect of a zinc metalloprotease from Serratia marcescens on platelet surface glycoproteins (GP) Ib and V was analyzed. Increasing protease treatments caused progressive loss of GP Ib with appearance of the major fragment, glycocalicin, in the supernatant solution. No GP V was detected in the supernatant solution, and protease-pretreated platelets had the same capacity as control platelets to release fragment 1 of GP V in response to thrombin. The Serratia protease- pretreated platelets did show the lag before thrombin-induced dense granule secretion, characteristic of platelets modified by pretreatment with other nonstimulating proteases. Treatment with Serratia protease gives the only demonstrated selective loss of GP Ib without apparent effect on GP V. It suggests that GP V (1) does not depend on GP Ib for its association with platelets and (2) is not the substrate for protease modification of platelet function.     

Purification and properties of heparin-releasable lipoprotein- associated coagulation inhibitor

The lipoprotein-associated coagulation inhibitor (LACI) is present in vivo in at least three different pools: sequestered in platelets, associated with plasma lipoproteins, and released into plasma by intravenous heparin, possibly from vascular endothelium. In this study we have purified the heparin-relesable form of LACI from post-heparin plasma and show that it is structurally different from lipoprotein LACI. The purification scheme uses heparin-agarose chromatography, immunoaffinity chromatography, and size-exclusion chromatography and results in a 185,000-fold purification with a 33% yield. Heparin- releasable LACI (HRL), as analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis, under reducing conditions, appears as a major band at 40 Kd and a minor band at 36 Kd. Immunoblot analysis suggests that the 36-Kd band arises from carboxyl-terminus proteolysis that occurs during the purification. HRL has a specific activity similar to that of HepG2 or lipoprotein LACI. HRL and lipoprotein LACI combine with lipoproteins in vitro while purified HepG2 LACI does not. I125-labeled HRL, injected into a rabbit, is cleared more slowly than I125-labeled HepG2 LACI, which may be due to attachment to lipoproteins in vivo. Preliminary evidence suggests that HRL is associated with vascular endothelium, possibly by attachment to glycosaminoglycans.     

Effects of recombinant canine stem cell factor, a c-kit ligand, and recombinant granulocyte colony-stimulating factor on hematopoietic recovery after otherwise lethal total body irradiation

The effects of recombinant canine stem cell factor (rcSCF) on hematopoiesis were studied in normal dogs and in dogs given otherwise lethal total body irradiation (TBI) without marrow transplant. Results were compared with previous and concurrent data with recombinant granulocyte colony-stimulating factor (rG-CSF). Four normal dogs received 200 micrograms rcSCF per kilogram body weight daily either by continuous intravenous infusion for 28 days (n = 2) or by subcutaneous (SC) injection in two divided doses for 20 days (n = 2). All dogs showed at least a twofold increase in peripheral blood neutrophil counts starting approximately 7 days after the initiation of treatment. Hematocrit level and monocyte, lymphocyte, eosinophil, reticulocyte, and platelet counts were not elevated. Marrow sections after rcSCF treatment showed panhyperplasia. The only toxicity was facial edema during the first few days of rcSCF administration, presumably caused by mast cell stimulation. Ten dogs were given 400 cGy TBI at 10 cGy/min from two opposing 60Co sources. They were given no marrow infusion and received 200 micrograms/kg/d rcSCF SC in two divided doses for 21 days starting within 2 hours of TBI. Five of the 10 dogs showed complete and sustained hematopoietic recovery and survived as compared with 1 of 28 control dogs not receiving growth factor (P < .005). RcSCF treatment allowed for hematopoietic recovery in two of seven dogs administered 500 cGy of TBI but in none of five dogs given 600 cGy of TBI. Results with rcSCF are similar to those obtained with rG-CSF. The rate of neutrophil recovery in rcSCF-treated dogs after 400 cGy TBI was not different from that of rG-CSF-treated dogs (P = .65), but the rate of platelet recovery was faster (P = .06) in the rcSCF-treated animals. Combined treatment with rcSCF and rcG-CSF after 500 cGy TBI did not result in strongly improved survival as compared with results obtained with either factor alone.     

Synergistic cytotoxic effect of azidothymidine and recombinant interferon alpha on normal human bone marrow progenitor cells

Azidothymidine (AZT) and interferon alpha (IFN-alpha) are among the drugs showing strong in vitro activity against the human immunodeficiency virus type-1 (HIV-1). Each drug, however, has significant toxicity against normal marrow progenitor cells that frequently proves dose-limiting in patients. In this study, AZT and recombinant IFN-alpha 2a (rIFN-alpha 2a) were tested as single agents and in combination against normal myeloid (CFU-GM) and erythroid (BFU- E) colony forming cells in a standard methylcellulose culture assay. The data were analyzed using a quantitative computerized analysis based on the median-effect principle and the isobologram equation as described by Chou and Talalay (Adv Enz Regul 22:27, 1984). The ED90 for BFU-E and CFU-GM inhibition was then compared with previously measured in vivo plasma levels of each drug and the ED90 for the anti-HIV-1 effect in vitro. We demonstrate that (a) the drugs are strongly synergistic in inhibiting marrow progenitor cell growth and that this synergism occurs at drug levels that are within the range of measured plasma levels in phase I clinical trials, (b) BFU-E are more sensitive than CFU-GM to the inhibiting effects of AZT, rIFN-alpha 2a or both drugs in combination, (c) the drug concentrations in combination that synergistically inhibit bone marrow progenitors are much higher than those required to inhibit HIV-1 replication in vitro, and (d) the anti- HIV-1 effect for the combination of AZT and rIFN-alpha 2a was clearly superior to the effect of AZT or rIFN-alpha 2a alone as indicated by the combination index and the dose-reduction index. These data suggest that substantially lower doses of AZT and rIFN-alpha than those currently being tested in clinical trials might not only maintain a strong synergistic anti-HIV-1 effect but might also avoid significant hematologic toxicity.     

Evaluation of the anticonvulsant and muscle relaxant effects of the methanol root bark extracts of Annona senegalensis

ObjectiveTo investigate the potentials of the root bark of Annona (A.) senegalensis in the control of seizure and related hypnotic and motor incoordination effects in mice using experimental models.MethodsThe methanol extract (ME) of the root bark of A. senegalensis was studied in mice using pentylenetetrazole (PTZ) induced convulsions, phenobarbitone induced sleeping time and motor coordination test on rota-rod performance. Acute toxicity and lethality (LD₅₀) test as well as phytochemical analysis were also carried out.ResultsThe extract (200, 400, 800 mg/kg) exhibited a non-dose dependent significant (P <0.05) delay in the onset of both tonic and clonic phases of seizure induced by PTZ (60 mg/kg, s.c.) as well as offered a 100% protection (200 mg/kg) in mice from PTZ induced seizures. The extract significantly (P <0.05) decreased the latency and increased the duration of phenobarbitone induced sleeping time. At 200 mg/kg, the extract exhibited a significant (P <0.05) motor incoordination. The acute toxicity test revealed an oral LD₅₀ of 1 296 mg/kg, while the phytochemical studies showed the presence of alkaloids, resins, glycosides, carbohydrate, reducing sugar, flavonoids, terpenoids, saponins and tannins.ConclusionThe extract of A. senegalensis possessed anticonvulsant activity with pronounced hypnotic and muscle relaxant effects.