HIV impairs CD34+-derived monocytic precursor differentiation into functional dendritic cells.

Dendritic cells (DCs) perform a basic role in the immune system by allowing the initiation of the primary T-cell-dependent immune response. Given previous indirect evidence that DC maturation and function are impaired by HIV, we have developed an in vitro culture system in order to verify the effect of HIV infection on DC function during the development from hematopoietic progenitors. Considering that monocytic (Mo) differentiating cells efficiently replicate monocytotropic HIV, we examined whether HIV-infected monocytic precursors (MoP) were able to generate functional DCs. CD34+ hematopoietic progenitor cells (HPCs) were induced along Mo differentiative pathway in liquid cultures and at an early stage of culture, MoP were infected with M-tropic BaL HIV strain, and after 2 days they were switched to DC differentiation with GM-CSF and IL-4. Derived DCs were actively infected, as detected by HIV-p24 production. HIV did not significantly affect cell viability, but induced a reduction in cell proliferation and an inefficient functional activity in terms of uptake capability and stimulation of allogenic T cells. These results indicate that HIV-infected MoP lost the capacity to generate functional DCs, and this may represent one of the many mechanisms of immunosuppression exploited by HIV.

Unilineage monocytopoiesis in hematopoietic progenitor culture: switching cytokine treatment at all Mo developmental stages induces differentiation into dendritic cells.

We have developed a new culture system whereby human hematopoietic progenitors purified from adult peripheral blood extensively proliferate and gradually differentiate into >95% pure monocytic (Mo) cells. At all developmental stages treatment with interleukin (IL)-4+granulocyte-macrophage colony-stimulating factor or IL-4+c-Kit-ligand+FLT-3 ligand switched the Mo precursors into dendritic cells (DCs). The switching capacity decreased only at the end of the culture, when most Mo cells matured to macrophages. Moreover, the Mo precursors were highly susceptible to transduction with lentiviral vectors: once switched to DCs, they maintained the transgene expression, as well as the phenotype and function of the DC lineage. Our results provide new insight into the potential role of the Mo lineage as a reservoir of DCs in vivo. Furthermore, the methodology for transduction of Mo precursors provides a tool to generate genetically modified, normally functioning DCs potentially useful for immunotherapy.

Stromal cell-derived factor 1alpha increases polyploidization of megakaryocytes generated by human hematopoietic progenitor cells.

The alpha chemokine receptor CXCR4 has been shown to be expressed on human hematopoietic progenitor cells and during the megakaryocytic differentiation pathway. Stromal cell-derived factor 1 (SDF-1) is the ligand for CXCR4. In this study, the role of SDF-1alpha in megakaryocytopoiesis was investigated. CD34(+) progenitors purified from peripheral blood were grown in serum-free liquid suspension culture supplemented with thrombopoietin to obtain a virtually pure megakaryocytic progeny. In this condition, the addition of SDF-1alpha gives rise to megakaryocytes (MKs) showing an increased DNA content and a rise of lobated nuclei, as compared with untreated cells: at day 5, approximately 20% of the cells already showed the presence of more than one nuclear lobe versus fewer than 5% in the control cells; at day 12, approximately 85% of the cells were of large size and markedly polyploid, whereas approximately 60% of the control cells were polyploid, showed fewer lobes, and were a smaller size. This effect was dose-dependent and did not affect the megakaryocytic proliferation. Experiments with the mitogen-activated protein kinase (MAPK) inhibitor PD98059 suggested a role for MAPK pathway on SDF-1alpha-induced endomitosis. Furthermore, SDF-1alpha induced a significant increase in the number of proplatelet-bearing MKs and promoted the migration of megakaryocytic cells. Treatment with SDF-1alpha caused reduction in CXCR4 abundance on the plasma membrane, seemingly owing to receptor internalization. Furthermore, the presence of SDF-1alpha did not affect the expression of megakaryocytic markers, indicating that differentiation and polyploidization are independently regulated events.

Lineage-specific expression of human immunodeficiency virus (HIV) receptor/coreceptors in differentiating hematopoietic precursors: correlation with susceptibility to T- and M-tropic HIV and chemokine-mediated HIV resistance.

Human immunodeficiency virus (HIV) entry is mediated not only by the CD4 receptor, but also by interaction with closely related molecules that act as membrane coreceptors. We have analyzed mRNA expression and/or cell membrane exposition of the coreceptors most widely used by diverse HIV-1 strains (CXCR4, CCR5, and CCR3) on purified hematopoietic progenitor cells (HPCs) induced in liquid suspension culture to unilineage differentiation/maturation through the erythroid (E), granulocytic (G), megakaryocytic (Mk), and monocytic (Mo) lineages. Reverse transcriptase-polymerase chain reaction (RT-PCR) and cytofluorimetric analysis showed the presence of both CXCR4 and CCR5 in quiescent HPCs, but failed to detect CCR3-specific transcripts. Chemokine expression in HPC progenies showed that CXCR4 receptor is detected on the majority of MKs from early to late stages of maturation, whereas it is moderately decreased in the Mo lineage. In the G pathway, two distinct cell populations, CXCR4(+) and CXCR4(-), were observed: morphological analysis of the sorted populations showed that the CXCR4(+) cells were largely eosinophils and the CXCR4(-) were granulocytes of the neutrophilic series. Furthermore, in the E pathway, CXCR4 was almost completely absent. CCR5 expression is restricted to Mo cultures, ie, approximately 30% to 80% cells throughout all monocytopoietic differentiation/maturation stages. Finally, CCR3 mRNA is always absent in all the unilineage cultures. Evaluation of CD4 expression by flow cytometry on both quiescent HPCs and differentiating unilineage precursors showed that the CD4 receptor is present on approximately 15% of the starting CD34(+) HPC population, highly expressed in the Mo lineage up to 80% at terminal maturation, present on 20% to 30% of maturing Mks, and not detectable in either the E or G lineage. Expression of CD4 receptor together with CXCR4 and/or CCR5 coreceptor in the four lineages correlates with hematopoietic precursor susceptibility to T-lymphotropic and macrophage (M)-tropic HIV strains infection: (1) CD4(-) G and E cells were resistant to both M-tropic and T-lymphotropic strains; (2) HPC-derived Mks were susceptible to T-tropic, but resistant to M-tropic, infection; (3) Mo differentiating cells efficiently replicate both HIV strains. Furthermore, we showed that the CXCR4 and CCR5 ligands (stromal-derived factor 1 and macrophage-inflammatory protein-1alpha [MIP-1alpha], MIP-1beta and RANTES, respectively) inhibit HIV replication in both maturing Mo and Mk cells. Taken together, our data show a lineage-specific modulation of chemokine receptor/coreceptor during hematopoietic cell differentiation and extend previous observations on the relationship between the expression of HIV receptor/coreceptors, susceptibility, and chemokine-mediated resistance to HIV infection.

Productive human immunodeficiency virus-1 infection of purified megakaryocytic progenitors/precursors and maturing megakaryocytes.

We have evaluated the susceptibility to human immunodeficiency virus (HIV)-1 infection of in vitro grown megakaryopoietic progenitors/precursors and maturing megakaryocytes (MKs), based on the following approach: (1) human hematopoietic progenitor cells (HPCs), stringently purified from peripheral blood and grown in serum-free liquid suspension culture supplemented with thrombopoietin (Tpo), generated a relatively large number of >/= 98% to 99% pure megakaryocytic precursors and then mature-terminal MKs; (2) at different days of culture (ie, 0, 5, 8, 10) the cells were inoculated with 0.1 to 1.0 multiplicity of infection (m.o.i.) of the lymphotropic NL4-3 or 0.1 m.o.i. of the monocytotropic BaL-1 HIV-1 strain; (3) finally, the presence of viral mRNA and proteins was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR)/in situ hybridization and antigen capture assays, respectively, on day 2 to 12 of culture. MKs derived from day 0 and day 5 BaL-1-challenged cells do not support viral replication as assessed by p24 enzyme-linked immunosorbent assay (ELISA) and RT-PCR. On the contrary, HIV transcripts and proteins were clearly detected in all NL4-3 infection experiments by RT-PCR and p24 assay, respectively, with the highest viral expression in day 5 to 8 challenged MKs. In situ hibridization studies indicate that the percentage of HIV+ MKs varies from at least 1% and 5% for day 0 and day 5 infected cells, respectively. Production of an infectious viral progeny, evaluated by the capability of culture supernatants from day 5 NL4-3-challenged MKs to infect C8166 T-lymphoblastoid cell line, was consistently observed (viral titer, approximately 5 x 10(3) tissue culture infectious dose50/mL/10(6) cells). Exposure of MKs to saturating concentration of anti-CD4 OKT4A monoclonal antibody (MoAb), which recognizes the CD4 region binding with the gp120 envelope glycoprotein, markedly inhibited HIV infection, as indicated by a reduction of p24 content in the supernatants: because the inhibitory effect was incomplete, it is apparent that the infection is only partially CD4-dependent, suggesting that an alternative mechanism of viral entry may exist. Morphologic analysis of day 12 MKs derived from HPCs infected at day 0 showed an impaired megakaryocytic differentiation/maturation: the percentage of mature MKs was markedly reduced, in that approximately 80% of cells showed only one nuclear lobe and a pale cytoplasm with few granules. Conversely, megakaryocytic precursors challenged at day 5 to 8 generated fully mature day 10 to 12 MKs showing multiple nuclear segmentation. Thus, the inhibitory effect of HIV on the megakaryopoietic gene program relates to the differentiation stage of cells subjected to the viral challenge. Finally, HPCs treated with 20 or 200 ng/mL of recombinant Tat protein, analyzed at different days of culture, showed an impaired megakaryocytopoiesis comparable to that observed in HIV-infected cells, thus suggesting that Tat is a major mediator in the above described phenomena. These results shed light on the pathogenesis of HIV-related thrombocytopenia; furthermore, they provide a model to investigate the effects of HIV on megakaryocytic differentiation and function.

A splice variant of Dp71 lacking the syntrophin binding site is expressed in early stages of human neural development.

Dp71, a 71 kDa C-terminal isoform of dystrophin, is the major product of the DMD gene in brain. Two alternatively spliced transcripts of Dp71 were amplified by RT-PCR from different areas of human fetal neural tissue. Both transcripts were spliced out of exons 71 and 78. The shorter transcript was also alternatively spliced of exons 72-74, a region comprising the coding sequence for the binding site to syntrophin, one component of the dystrophin-associated protein complex. Results indicate that alternatively spliced forms of Dp71 are regulated during human neural development.

Human immunodeficiency virus (HIV)-resistant CD4+ UT-7 megakaryocytic human cell line becomes highly HIV-1 and HIV-2 susceptible upon CXCR4 transfection: induction of cell differentiation by HIV-1 infection.

Recent findings have shown that the expression of the seven trans-membrane G-protein-coupled CXCR4 (the receptor for the stromal cell-derived factor [SDF]-1 chemokine) is necessary for the entry of T-lymphotropic human immunodeficiency virus (HIV) strains, acting as a coreceptor of the CD4 molecule. In the human system, the role of CXCR4 in HIV infection has been determined through env-mediated cell fusion assays and confirmed by blocking viral entry in CD4+/CXCR4+ cells by SDF-1 pretreatment. We observed that the human megakaryoblastic CD4+ UT-7 cell line fails to express CXCR4 RNA and is fully resistant to HIV entry. Transfection of an expression vector containing the CXCR4 c-DNA rendered UT-7 cells readily infectable by different T-lymphotropic syncytium-inducing HIV-1 and HIV-2 isolates. Interestingly, HIV-1 infection of CXCR4 expressing UT-7 cells (named UT-7/fus) induces the formation of polynucleated cells through a process highly reminiscent of megakaryocytic differentiation and maturation. On the contrary, no morphologic changes were observed in HIV-2-infected UT-7/fus cells. These findings further strengthen the role of CXCR4 as a molecule necessary for the replication of T-lymphotropic HIV-1 and HIV-2 isolates and provide a useful model to study the functional role of CD4 coreceptors in HIV infection.

Anti-HIV viral interference induced by retroviral vectors expressing a nonproducer HIV-1 variant.

A Hut-78 cell clone (F12) harboring a nonproducer human immunodeficiency virus (HIV-1) variant shows complete resistance to HIV-1 or HIV-2 superinfection. The F12-HIV provirus produces an altered HIV-1 protein pattern and cannot generate even immature viral particles. We demonstrated that HeLa CD4+ cells transfected with the F12-HIV genome resist HIV superinfection through a CD4-independent mechanism. As F12-HIV appears to be a useful system to induce anti-HIV intracellular immunization, we constructed various retroviral vectors containing the F12-HIV genome, modified by elimination of the F12 3'LTR and part of its nef gene, inserted 'antisense' with respect to the Moloney murine leukemia virus 5' LTR. Here we show that recombinant retroviral particles carrying the N2/F12-HIV nef- (as) construct can stably transduce both CEMss human cells and primary human peripheral blood lymphocytes, inducing the expression of the F12-HIV genome. These results could open the way to an anti-AIDS gene therapy strategy based on F12-HIV-induced intracellular immunization.

A nonproducer, interfering human immunodeficiency virus (HIV) type 1 provirus can be transduced through a murine leukemia virus-based retroviral vector: recovery of an anti-HIV mouse/human pseudotype retrovirus.

The expression of a human immunodeficiency virus (HIV) type 1 provirus (F12-HIV) cloned from a nonproducer, chronically infected CD4 down-regulated Hut-78 cell clone (F12) does not lead to the formation of viral particles and, upon transfection in HeLa CD4+ cells, confers resistance to HIV superinfection without affecting the CD4 receptor exposure. In an attempt to transfer the anti-HIV properties of F12-HIV into human primary cell, we constructed a Moloney murine leukemia virus-based retroviral vector containing an F12-HIV genome lacking the 3' long terminal repeat and part of the nef gene, which was expressed under the control of its 5' long terminal repeat. The F12-HIV genome was inserted in the orientation opposite to that of the murine leukemia virus transcriptional unit and was designated the N2/F12-HIV nef-antisense vector. Lymphoblastoid CEMss cells, as well as human peripheral blood lymphocytes, were successfully transduced by the recombinant retrovirus emerging from the producer PA317 clones. CEMss clones expressing the F12-HIV nef-antisense vector became resistant to HIV superinfection even at the highest utilized multiplicity of infection (10(5) 50% tissue culture infective doses per 10(6) cells). In transduced CEMss cells the viral interference induced by the F12-HIV expression is not due to CD4 HIV receptor down-regulation. Nonproducer, interfering HIV proviruses transduced into retroviral vectors may, therefore, provide an alternative strategy for the protection of CD4+ human primary cells from HIV infection, which strategy may be used in designating a safe and efficient gene therapy protocol for patients with AIDS.

In vitro human immunodeficiency virus-1 infection of purified hematopoietic progenitors in single-cell culture.

Uni- or multi-lineage suppression of hematopoiesis is observed in the majority of acquired immunodeficiency syndrome (AIDS) patients. The mechanism(s) underlying these abnormalities is not understood: particularly, the human immunodeficiency virus (HIV) infection of hematopoietic progenitor and stem cells (HPCs/HSCs) is highly controversial. We report that CD34+ HPCs from adult peripheral blood (PB) are in part CD4+ and susceptible to in vitro HIV infection. Primitive CD34+ HPCs were approximately 80% purified from PB. Double labeling for CD34 and CD4 membrane antigens was shown for 5% to 20% of the purified cells, thus suggesting their potential susceptibility to HIV-1 infection. The enriched HPC population, challenged with purified or unpurified HIV-1 strains, was cloned in unicellular methylcellulose culture. The single colonies generated by erythroid burst-forming units (BFU-E), granulocyte-macrophage colony-forming units (CFU-GM), and granulocyte-erythroid-macrophage-megakaryocyte colony-forming units (CFU-GEMM) were analyzed for the presence of HIV, ie, for gag DNA, tat mRNA, and p24 protein by PCR, reverse transcription PCR (RT-PCR), and enzyme-linked immunosorbent assay, respectively. In the first series of experiments incubation of HPCs with HIV-1 at multiplicities of infection (MOI) ranging from 0.01 to 10 TCID50/cell consistently yielded an 11% to 17% infection efficiency of BFU-E-generated colonies, thus indicating the sensitivity of HPCs to in vitro HIV infection. An extensive series of experiments was then performed on HPCs challenged with HIV at 0.1 MOI level. In the initial studies proviral gag sequences were detected in 9.2% of 121 analyzed CFU-GM colonies. In further experiments tat mRNA was monitored in 17% and 23% of BFU-E and CFU-GM colonies, respectively, but never in CFU-GEMM clones. Finally, 12% of CFU-GM clones and rare erythroid bursts were shown to be positive for the p24 viral protein. In control studies, purified HPCs grown in liquid suspension culture were induced to terminal unilineage erythroid, monocytic, or granulocytic differentiation: monocytes were consistently HIV-infected, whereas mature-terminal erythroblasts and granulocytes were not. Our observations indicate that a minority of primitive HPCs, but not of the multipotent type, is susceptible to in vitro HIV infection. These observations may reflect on the in vivo hematopoietic impairment in AIDS patients; more important, they provide an experimental model for studies on HIV hematopoietic infection and in vitro tests for anti-HIV HSC gene therapy.

Efficient transfer of selectable and membrane reporter genes in hematopoietic progenitor and stem cells purified from human peripheral blood.

We have utilized highly purified hematopoietic progenitor and stem cells (HPCs, HSCs) from normal peripheral blood to develop methodology for: (a) efficient transfer into HPCs of a non-hematopoietic membrane reporter, i.e., the nerve growth factor receptor complementary DNA; and (b) effective gene transduction of putative HSCs, i.e., cells initiating Dexter-type long-term culture (LTC-ICs). Purified HPCs induced into cycling by growth factors (interleukin 3, interleukin 6, c-kit ligand) were transduced with the N2 retroviral vector containing the neomycin resistance (neor) gene. More than 80% of transduced HPCs were resistant to the toxic G418 level. Thereafter, the HPCs were effectively transduced with the LNSN retroviral vector containing a nerve growth factor receptor complementary DNA; the nerve growth factor receptor was detected on > or = 18% of the transduced HPCs. These experiments provide a new tool from which (a) to monitor expression of a transduced membrane report on hematopoietic cells, particularly at the level of HPCs/HSCs, and (b) to characterize the transduced cells by double- and triple-labeling membrane antigen analysis. Purified HPCs/HSCs grown in Dexter-type LTC were transduced at 1 week by exposure to supernatant N2 retroviral particles in the absence of exogenous hematopoietic growth factors. The procedure, devoid of toxic effects, allowed an efficient neor transduction into LTC-ICs. Thus, we consistently detected neomycin-resistant mRNA in the clonal progeny of HPCs produced in LTC at 5-8 weeks in both the nonadherent and adherent fractions; this timing of expression coincides with that of HPC production by LTC-ICs, thereby indicating the effective transduction of the LTC-ICs. These experiments represent a first step toward development of preclinical models for gene transfer into human peripheral blood HSCs by complex retroviral vectors.

PCR analysis of HIV-1 sequences and differential immunological features in seronegative and seropositive haemophiliacs.

We have compared the immunological features of two matched groups of seronegative and seropositive haemophilia A individuals. Both groups were exposed from 1981 to 1985 to comparable amounts and batches of FVIII concentrates not subjected to virus inactivation procedures, and had therefore a 100% probability of receiving HIV-contaminated material. The presence of proviral HIV-1 sequences was evaluated by PCR in the DNA from peripheral blood lymphocytes and/or monocytes. After hybridization with specific probes, DNA from all seropositive haemophiliacs revealed HIV sequences; no HIV sequences were observed from the DNA of seronegative patients, even after two rounds of amplification, thus suggesting that these patients were not affected by a latent HIV infection. Seronegative/PCR- and seropositive/PCR+ patients showed a normal and reduced number of CD4+ lymphocytes, and a slight and marked increase of CD8+ cells respectively. Activated T cells expressing the HLA-DR antigen were elevated in both groups. Interestingly, a significant reduction of circulating CD56+/CD3- NK lymphocytes was observed only in seropositive haemophiliacs, whereas NK lymphocytes with CD56+/CD3+ phenotype were within normal levels in both groups. In seropositive patients no correlation was found between the number of CD4+ and CD56+/CD3- lymphocytes. The marked reduction of CD56+/CD3- lymphocytes observed in seropositive haemophiliacs in addition to the CD4+ cell depletion may represent a key pathogenetic factor which facilitates the onset and/or the progression of HIV-1 infection in haemophiliacs, and is related to the capacity of HIV to infect NK cells.

Astrocyte cultures from human embryonic brain: characterization and modulation of surface molecules by inflammatory cytokines.

Astrocyte-enriched cultures were established upon passaging of primary cultures from the myelencephalon and mesencephalon of 7-9-week-old human embryos. Immunocytochemical analysis showed that third-fourth passage cultures were composed of a highly enriched population of proliferating, epithelioid cells, up to 90% of which expressed glial fibrillary acidic protein (GFAP); no macrophages and very few fibroblasts (less than 2%) were present. GFAP expression and proliferation declined upon further culturing in serum-containing medium but could be transiently reinduced by growing the cells in a serum-free chemically defined medium. Large numbers of GFAP+ astrocytes were obtained from each embryo and could be stored frozen and recultured. Using flow cytometric analysis, human astrocyte cultures were examined for basal and cytokine [interferon-gamma (IFN-gamma), interleukin-1 beta (IL-1 beta), and tumor necrosis factor-alpha (TNF-alpha)]-induced expression of molecules that may be involved in astrocyte-T-lymphocyte interactions. Cultured human astrocytes spontaneously expressed major histocompatibility complex (MHC) class I antigens and variable levels of MHC class II; MHC class I levels were increased upon IFN-gamma and TNF-alpha treatment, whereas MHC class II antigens were induced on most of the astrocytes by IFN-gamma. Among the molecules involved in antigen-independent interactions between T lymphocytes and target cells, lymphocyte function-associated molecule-3 (LFA-3) was spontaneously expressed by most cultured human astrocytes, whereas intercellular adhesion molecule-1 (ICAM-1) was present at variable levels in non-stimulated astrocytes and was greatly induced by IFN-gamma, TNF-alpha, and IL-1 beta. In this study we also show that the above cytokines upregulate astroglial expression of adhesion molecules of the integrin family (VLA-1, VLA-2, and VLA-6) that may be involved in astrocyte-extracellular matrix interaction and play a role in the astrocyte reactive changes occurring at sites of brain injury and inflammation. The human astrocyte cultures developed here represent a useful in vitro model to further investigate mechanisms involved in bidirectional communication between central glia and cells of the immune system.

Transforming growth factor beta (TGF-beta) inhibits expression of fibrinogen and factor VII in a hepatoma cell line.

We have investigated the effect(s) of transforming growth factor (TGF)-beta 1 and interleukin (IL)-6 on the expression of fibrinogen and blood coagulation factors VII, IX, X mRNAs in a hepatoma cell line (Hep 3B). The results indicate that TGF-beta induces a decrease of the basal level of fibrinogen and factor VII mRNAs, but does not affect factor X expression. Furthermore, TGF-beta efficiently antagonizes the IL-6 induction of fibrinogen mRNA at late (12-48 h) but not early (6 h) times: this effect is apparently mediated by posttranscriptional mechanism(s). These findings, together with previously reported data on the inhibitory effect of TGF-beta on acute phase genes (e.g., ApoA1 and albumin), suggest a role for TGF-beta in the regulation of liver genes expression. The early stimulatory and late inhibitory effect exerted by IL-6 and TGF-beta respectively on fibrinogen mRNA level may play a role in the regulatory mechanism(s) of clot formation in a variety of conditions.

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.

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.

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.

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.