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Biomedical subjects

R Guerriero

Publications and source records attributed to R Guerriero.

10 recordsLinked to original sources
PubMed1995

Unilineage megakaryocytic proliferation and differentiation of purified hematopoietic progenitors in serum-free liquid culture.

Cellular and molecular analysis of megakaryocytopoiesis has been hampered thus far by the lack of pure and abundant megakaryocyte (MK) cell populations. In this study, hematopoietic progenitor cells (HPCs), stringently purified from peripheral blood, were induced to megakaryocytic differentiation/maturation in serum-free liquid suspension culture treated with a growth factor cocktail (interleukin-3 [IL-3], c-kit ligand, and IL-6) and/or recombinant mpl ligand (mpIL). In particular, (1) the growth factor cocktail induced the growth of a 40% MK population, ie, 4 x 10(4) cells at day 0 generated 2 x 10(5) MK at terminal maturation; (2) further addition of mpIL increased the MK purity level to 80% with a final yield of 4 x 10(5) MKs; (3) treatment with mpIL alone resulted in a 97% to 99% MK population, with a mild increase of cell number (to 1.5 x 10(5) cells). In mpIL-supplemented culture, morphological evaluation indicated the presence of putative mononuclear MK precursors and then of mature polynucleated platelet-forming MKs, peaking at days 5 and 12, respectively. Membrane phenotype analysis showed a gradual decrease of CD34+ HPCs, coupled with an inverse increase of MK-specific antigens (eg, CD61/62/42b) starting before mature MK detection by morphology analysis. In situ hybridization showed the expression of MK-specific von Willebrand gene in both MK precursors and mature MKs. Furthermore, MKs synthesize and secrete low but significant amounts of both IL-6 and granulocyte-macrophage colony-stimulating factor. Comparative culture studies were performed on purified bone marrow CD34+/38hi or CD34+/38lo cells stimulated by mpIL alone. Both populations generated a highly enriched MK progeny (62% and 93% MKs at day 12 of culture, respectively) but showed either little or no proliferation. In conclusion, the purified peripheral blood HPC differentiation culture system allows for growth of a relatively large number of highly purified or "pure" megakaryocytic precursors and then mature MKs, thus providing an in vitro experimental tool to dissect the cellular and molecular basis of megakaryocytopoiesis.

Adult↗
PubMed1990

Blood coagulation factors in human embryonic-fetal development: preferential expression of the FVII/tissue factor pathway.

The expression of a number of blood coagulation factors (F) (FX, FIX, FVIII, FVII, alpha-, beta-, gamma-fibrinogen chains, protein C, and antithrombin III [AT III]) was analyzed at RNA and protein level in 5- to 10-week-old human embryos and fetuses. FX, FIX, and FVII were also analyzed at protein level. Total and poly(A)+ RNA, extracted from embryonic-fetal (FL) and adult liver (AL), were analyzed by dot and Northern blot hybridization with specific cDNA probes. The results indicate that: (1) the size of the messenger RNAs of these factors is equivalent in FL and AL; (2) in the 5- to 10-week period, their abundance in FL increases from 30% to 50% of the adult level except for FIX (from 2% to 10%) and FX (always 100% of the adult value). Western blot analysis of FIX, FX, and FVII in 5- to 10-week soluble liver proteins and 6- to 8-week plasma showed a low level of FIX versus a higher concentration of both FVII and FX, when compared with corresponding adult values, ie, a liver protein level of 10% versus 100% and a plasma concentration level of 10% versus 40%. Although little is known so far on the activity and the functional role of the clotting factors in early human ontogenic development, these studies suggest an activation of FX via the FVII/tissue factor activity rather than the FIXa/FVIIIa phospholipid complex in human embryonic and early fetal life.

Blood Coagulation Factors↗
PubMed1988

Multiple polymorphic sites in factor X locus.

The structure of factor X (FX) gene was analyzed in five FX deficient pedigrees with four different variants of the disease, as well as in 50 normal subjects. Genomic DNA from the deficient patients and the normal controls was digested with 12 restriction endonucleases and hybridized with a FX cDNA probe. The results seemingly exclude gross gene deletions or rearrangements in the deficient patients. A variety of polymorphic sites (ie, EcoRI, HindIII, PstI, PvuII, TaqI) was observed within the FX locus and their relative frequency was established. Intriguingly, a highly polymorphic region for the PvuII endonuclease was identified and located approximately 3 kilobases (kb) from the last 3' exon. These polymorphisms allowed us to analyze the allelic segregation in a FX deficient family and to identify a homozygous subject.

Adult↗
PubMed1985

Intragenic Factor IX restriction site polymorphism in hemophilia B variants.

This study includes 47 normal subjects and 25 hemophilia B patients without inhibitor(s), showing different factor IX coagulant activity and antigen levels. Genomic DNA, digested with various restriction endonucleases, was hybridized with two different factor IX probes, ie, the cDNA and the subgenomic probe for the intragenic TaqI polymorphic site. cDNA restriction patterns suggest absence of gross rearrangements and/or deletions in all hemophilic patients. The frequency of the X chromosome bearing the TaqI polymorphic site is 0.32 +/- 0.09 in hemophilic subjects v 0.36 +/- 0.06 in normal control subjects, the latter value being comparable to that reported for the normal British population. No association between this polymorphism and hemophilia B variants has been observed, thus indicating that a wide spectrum of mutations underlies this blood-clotting disorder and particularly each of its variants.

Cloning, Molecular↗
PubMed1985

Hemophilia B with inhibitor: molecular analysis of the subtotal deletion of the factor IX gene.

The structure of factor IX gene was analyzed in a hemophilia B patient with inhibitor. Genomic DNA, digested with a variety of restriction endonucleases, was hybridized with the cDNA and various genomic factor IX probes. A large subtotal deletion of the gene was observed. The borders of the deletion span from a approximately 125 nucleotide region within the last exon to an unknown domain at least 7.5 kb upstream from the first exon: it thus involves approximately 33 kb of the factor IX locus. The abnormal gene was inherited by the daughter of the propositus, who showed both the normal and the deleted allele.

Chromosome Deletion↗
PubMed1984

Molecular heterogeneity of beta thalassaemia in the Italian population.

Fifty-one subjects originating from Southern Italy and affected by Cooley's anaemia have been studied in order to define the degree of heterogeneity of beta thalassaemia mutations in this high incidence area. Restriction endonuclease mapping has been carried out on genomic DNA by the Southern blot technique both to exclude the existence of gross deletions or rearrangements and to establish the relative frequency of four polymorphic restriction sites (i.e. G gamma and A gamma Hind III, beta Ava II and beta Bam HI) within the gamma delta beta gene region. In 28 subjects unequivocal linkage of the four polymorphic sites has been determined leading to the identification of seven different chromosome haplotypes, six of which had previously been reported associated with specific beta(0) and beta(+) thalassaemia mutations. Globin chain synthesis studies on peripheral blood reticulocytes indicated that subjects carrying the same genotype may behave differently as far as the beta chain production is concerned relative to both the alpha and the non-alpha chains. Thus, beta thalassaemia turns out to be quite heterogeneous even in this limited geographical area. Beta(+) mutations appear to be predominant, particularly those affecting nuclear precursor RNA splicing to mature beta globin mRNA.

Chromosome Mapping↗
PubMed1980

Post-translational control of human hemoglobin synthesis: the role of the differential affinity between globin chains in the control of mutated globin gene expression.

The interactions between beta-thalassemia and the human hemoglobin (Hb) alpha-chain variants, Hb Hasharon, Hb O Idonesia and Hb J Paris, and between alpha-thalassemia and the beta-chain variants, Hb S, Hb C and Hb G San José, which are characterized by preferential decrease of the abnormal Hb level in peripheral bloods, have been studied. Both biosynthesis studies in reticulocytes and determination of the relative affinity of abnormal chains for normal complementary chains by in vivo recombination experiments, involving globin chains previously isolated in their native form, have been carried out in order to provide insights on the molecular events following the synthesis of the mutant chains under conditions of complementary chain deficiency. Furthermore, we have measured the relative affinity for complementary chain of beta D Los Angeles- and alpha J Rovigo-chains, the level of which does not decay in thalassemic carriers, and of alpha Legnano- and beta Osu Christiansborg-chains, which have not yet been observed in association with thalassemias. Our experiments indicated that the differential affinity for beta-chains is not always the major post-translational control mechanism which regulates the level of certain alpha-chain variants in beta-thalassemic heterozygotes, and that preferential removal of abnormal chains by proteolytic enzymes is likely to play an important role in most cases. On the other hand, the low affinity of certain variant beta-chains for alpha-chains may offer an explanation for the low level of certain beta-chain variants in peripheral blood of non-thalassemic carriers, as well as to their decrease under conditions of relative alpha-chain deficiency (alpha-thalassemias).

Globins↗
PubMedDate not supplied

Haemoglobin switching in human embryos: asynchrony of zeta----alpha and epsilon----gamma-globin switches in primitive and definite erythropoietic lineage.

Haemoglobin switching in humans provides a unique model for investigating the mechanisms underlying expression of a developmentally regulated gene family. Numerous studies have focused on the switch from fetal to adult (that is, gamma----beta) globin, but little is known about the embryonic----fetal (that is, zeta----alpha and epsilon----gamma) switches, as well as the transition from 'primitive' yolk sac to 'definitive' liver erythropoiesis. Here we have studied the embryonic----fetal haemoglobin switches in yolk sac, liver and circulating blood erythroblasts from 25 embryos and 6 fetuses. Globin synthesis was also evaluated in purified 'primitive' and 'definitive' erythroblasts. Primitive erythroblasts synthesize essentially zeta and epsilon chains at 5 weeks and alpha- and epsilon-globin with a minor aliquot of zeta and gamma chains at 6-7 weeks, whereas definitive erythroblasts produce alpha and epsilon + gamma + beta-globin at 6 weeks but only alpha and gamma + beta chains from 8 weeks onward. In both lineages the zeta----alpha and the epsilon----gamma switches are asynchronous, the former preceding the latter. Furthermore, zeta- and beta-globin synthesis is restricted to primitive and definitive erythroblasts respectively. These findings are discussed in terms of a monoclonal model for haemoglobin switching in early human ontogeny.

Age Factors↗