Altered endothelial gene expression associated with hereditary haemorrhagic telangiectasia.

BACKGROUND: Mutations in endoglin (ENG) and activin receptor-like kinase 1 (ALK-1 or ACVRL1) genes are the underlying basis of hereditary haemorrhagic telangiectasia (HHT) types 1 and 2, respectively. Both genes belong to the transforming growth factor-beta (TGF-beta) receptors superfamily and are expressed in endothelial cells. The current model for HHT is that ENG or ALK-1 haplo-insufficiency affects angiogenesis and predisposes to vascular dysplasia and arteriovenous malformations. MATERIALS AND METHODS: Using microarray technology, we compared human umbilical vein endothelial cells (HUVEC) from newborns with ENG or ALK-1 mutations to control cells to search for gene profiles associated with early stages of the disease. Real-time polymerase chain reaction and Western blot analysis were used to validate a subset of the modulated genes and functionally related genes. RESULTS: Our results indicate that HHT endothelial cells in vitro display several gene expression disturbances, including genes associated with the activation phase of angiogenesis, with cell guidance and intercellular connections, and also with the TGF-beta pathway. Hierarchical clustering using modulated genes enables discrimination between affected and non-affected samples. CONCLUSION: HHT HUVECs display gene modulations which can suggest that ENG and ALK-1 haplo-insufficiency induces compensatory regulatory mechanisms at the expression levels.

Increased shedding of angiotensin-converting enzyme by a mutation identified in the stalk region.

Angiotensin-converting enzyme (ACE), an enzyme that plays a major role in vasoactive peptide metabolism, is a type 1 ectoprotein, which is released from the plasma membrane by a proteolytic cleavage occurring in the stalk sequence adjacent to the membrane anchor. In this study, we have discovered the molecular mechanism underlying the marked increase of plasma ACE levels observed in three unrelated individuals. We have identified a Pro(1199) --> Leu mutation in the juxtamembrane stalk region. In vitro analysis revealed that the shedding of [Leu(1199)]ACE was enhanced compared with wild-type ACE. The solubilization process of [Leu(1199)]ACE was stimulated by phorbol esters and inhibited by compound 3, an inhibitor of ACE-secretase. The results of Western blot analysis were consistent with a cleavage at the major described site (Arg(1203)/Ser(1204)). Two-dimensional structural analysis of ACE showed that the mutated residue was critical for the positioning of a specific loop containing the cleavage site. We therefore propose that a local conformational modification caused by the Pro(1199) --> Leu mutation leads to more accessibility at the stalk region for ACE secretase and is responsible for the enhancement of the cleavage-secretion process. Our results show that different molecular mechanisms are responsible for the common genetic variation of plasma ACE and for its more rare familial elevation.

Characterization of an upstream enhancer region in the promoter of the human endothelial nitric-oxide synthase gene.

The endothelial nitric-oxide synthase gene is constitutively expressed in endothelial cells. Several transcriptionally active regulatory elements have been identified in the proximal promoter, including a GATA-2 and an Sp-1 binding site. Because they cannot account for the constitutive expression of endothelial nitric-oxide synthase gene in a restricted number of cells, we have searched for other cell-specific regulatory elements. By DNase I hypersensitivity mapping and deletion studies we have identified a 269-base pair activator element located 4.9 kilobases upstream from the transcription start site that acts as an enhancer. DNase I footprinting and linker-scanning experiments showed that several regions within the 269-base pair enhancer are important for transcription factor binding and for full enhancer activity. The endothelial specificity of this activation seems partly due to interaction between this enhancer in its native configuration and the promoter in endothelial cells. EMSA experiments suggested the implication of MZF-like, AP-2, Sp-1-related, and Ets-related factors. Among Ets factors, Erg was the only one able to bind to cognate sites in the enhancer, as found by EMSA and supershift experiments, and to activate the transcriptional activity of the enhancer in cotransfection experiments. Therefore, multiple protein complexes involving Erg, other Ets-related factors, AP-2, Sp-1-related factor, and MZF-like factors are important for the function of this enhancer in endothelial cells.

Induction of angiotensin I-converting enzyme transcription by a protein kinase C-dependent mechanism in human endothelial cells.

Angiotensin I-converting enzyme (ACE) has been implicated in various cardiovascular diseases; however, little is known about the ACE gene regulation in endothelial cells. We have investigated the effect of the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) on ACE activity and gene expression in human umbilical vein endothelial cells (HUVEC). Our results showed a 3- and 5-fold increase in ACE activity in the medium and in the cells, respectively, after 24-h stimulation by PMA. We also observed an increase in the cellular ACE mRNA content starting after 6 h and reaching a 10-fold increase at 24 h in response to 100 ng/ml PMA as measured by ribonuclease protection assay. This effect was mediated by an increased transcription of the ACE gene as demonstrated by nuclear run-on experiments and nearly abolished by the specific PKC inhibitor GF 109203X. Our results indicate that PMA-activated PKC strongly increases ACE mRNA level and ACE gene transcription in HUVEC, an effect associated with an increased ACE secretion. A role for early growth response factor-1 (Egr-1) as a factor regulating ACE gene expression is suggested by both the presence of an Egr-1-responsive element in the proximal portion of the ACE promoter and the kinetics of the Egr-1 mRNA increase in HUVEC treated with PMA.

Human TcR gamma delta+ lymphocyte response on primary exposure to Plasmodium falciparum.

In 29 patients experiencing their first P. falciparum malarial attack, blood levels of TcR gamma delta+ lymphocytes were studied from the onset of infection to up to 6-9 months later. Blood TcR gamma delta+ lymphocytes, revealed using the TcR delta 1 monoclonal antibody (MoAb), were increased both in absolute and relative numbers. Alterations lasted for up to 3-4 months following the attack. A Ti gamma A/BB3 reactive V gamma 9 subset was preferentially amplified. In vitro, TcR gamma delta+ lymphocytes from both malaria-sensitized and unprimed donors responded to P. falciparum schizont extract (PFSE). PFSE-stimulated polyclonal T cell lines consisted principally in TcR gamma delta+ cells with a Ti gamma A+/BB3+ phenotype. Several TcR gamma delta+ T cell clones obtained from patients recovering from acute malarial attack were maintained in the presence of PFSE and autologous irradiated PBL. They belong to the V gamma 9 subset. In long-term cultures, TcR gamma delta+ clones progressively lost their capacity to react to PFSE antigen while they were able to proliferate and to exert cytotoxic activity in response to autologous TcR alpha beta+, PFSE-specific T lymphocyte clones. This suggests that regulatory interactions occur between activated TcR gamma delta+ and TcR alpha beta+ cells generated by P. falciparum. Sequential variations in blood TcR gamma delta+ and TcR alpha beta+ lymphocyte levels after primary exposure to P. falciparum suggest that such regulatory interactions may occur in vivo.

Responses of T cells from sensitized donors to recombinant and synthetic peptides corresponding to sequences of the Plasmodium falciparum SERP antigen.

In the present work, we intend to determine the capacity of human lymphocytes to recognize subfragments of the serine-stretch protein SERP, a blood-stage antigen from Plasmodium falciparum. Individuals sensitized by a previous P. falciparum infection were studied. Some recombinant proteins (RP) including RP7 and RP10 (amino acids 631-684 and 631-892 of SERP, respectively), were recognized in proliferation assays by lymphocytes from 28 sensitized individuals and not by lymphocytes from control, non-sensitized, donors. Synthetic peptides covering predefined zones of particular interest were tested and appeared to induce proliferative responses of lymphocytes from sensitized donors, allowing identification of putative T cell epitopes.

Interactions of CD4+ and CD8+ human T lymphocytes from malaria-unprimed donors with Plasmodium falciparum schizont stage.

During Plasmodium falciparum malaria, a wide spectrum of parasite-encoded blood-stage proteins is presented to the immune system of the host. To explore their multiple interactions with T cells from donors who have had no previous exposure to the parasite, whole schizont extract was used in vitro. Both CD4+ and CD8+ lymphocytes from all individuals tested were stimulated to proliferate. The responses were dependent on the presence of accessory cells and were only partially replaced by recombinant interleukin-1. Responses were inhibited by monoclonal antibodies to CD3, the alpha beta-chain T-cell receptor, or CD4 molecules but not to CD2. P. falciparum schizont extract-specific T-cell clones were generated and maintained by using sole stimulation by P. falciparum extract with autologous accessory cells or recombinant interleukin-2. Monoclonal antibodies to CD3 (or the alpha beta-chain T-cell receptor) blocked cloned T-cell responses to the schizont extract, and although the responses of the majority of the CD4+ or CD8+ T-cell clones were restricted by autologous accessory cells and inhibited by monoclonal antibodies to either CD4 or CD8, other clones responded to P. falciparum in the absence of accessory cells and were not regulated by the same monoclonal antibodies. The last category of clones consisted of autoreactive T cells. These data suggest that at the first contact with P. falciparum, requirements are met for significant T-cell stimulation.

Plasmodium falciparum exoprotein stimulation of human T-lymphocytes unsensitized to malaria.

The effects of Plasmodium falciparum proteins released in asexual blood stage culture supernatants on human T-lymphocytes from malaria non-immune donors were examined. Supernatants from several plasmodial strains stimulated both CD+4 and CD+8 T-lymphocytes to proliferate and secrete interferon gamma in vitro. Active moieties were predominantly released during the final stages of the parasite cycle. They were enriched by gel filtration and were further purified by anion-exchange and Superose 12 column fast protein liquid chromatography. Three active fractions of apparent 250, 70 and 18 kilodaltons were identified. The parasitic origin of the predominant 70-kilodaltons protein(s) was shown by biosynthesis experiments with radioactive amino acid precursors and was also demonstrated by in vitro translation of parasitic mRNA species. Interestingly, antibodies to the 70-kilodalton exoprotein(s) also reacted to a schizont protein of similar molecular weight.

Immunological evaluation of cell-mediated and humoral immunity in Thai patients with cerebral and non-cerebral Plasmodium falciparum malaria: I. Cutaneous delayed hypersensitivity, blood leukocytes and in vitro lymphocyte responses.

In Thai patients with acute P. falciparum malaria including cerebral cases, cell mediated immune functions were studied in vivo and in vitro. Initial cutaneous delayed reactions to phytohaemagglutinin and soluble protein antigens were negative in most cerebral malaria patients. No major alteration of the number of circulating T and B cells was observed. In lymphocytes cultures, proliferatives responses to lectins or protein antigens were generally found within normal ranges. This study shows a direct role of P. falciparum on the impairment of cell mediated immunity.

Human lymphocyte responses to Plasmodium falciparum merozoite antigens. A functional assay of protective immunity?

Merozoites obtained from cutaneous cultures of Plasmodium falciparum were used as antigen for an in vitro lymphocyte assay. Antigen specific proliferative responses were observed with lymphocytes from individuals with long-standing immunity to P. falciparum. Donors whose last P. falciparum challenge occurred within the year preceding the assay exhibited lymphocyte responses significantly higher than those from donors whose infection was more remote. This suggests that a lymphocyte dependent assay may relate to the protective status of the donor.

Major histocompatibility complex restriction of tetanus toxoid-specific human T lymphocyte clones.

Peripheral blood mononuclear cells (PBMC) from an HLA DRw6/7 individual were stimulated with tetanus toxoid (TT). T cell blasts were cloned by the limiting dilution technique in the presence of TT and irradiated autologous PBMC (iPBMC). Twelve were propagated under interleukin 2 and restimulated weekly with TT and iPBMC. All proliferated specifically in response to TT or either the alpha or beta chain of the toxin molecule. HLA restriction of specific proliferative responses was analyzed using a panel of HLA-typed unrelated donors and selected families, and blocking experiments with anti-HLA class I and class II monoclonal antibodies (mAb). Three types of restriction were observed: (a) autologous restriction; the inhibition observed using anti-HLA DR mAb as well as family studies performed previously with a similarly restricted clone obtained from the same donor suggest an HLA DRw6-linked restriction; (b) an HLA DR7 restriction was found for 2 clones, specific for alpha or beta chain; the identical pattern of inhibition obtained with two different mAb belonging to the same cluster suggests that these clones may be restricted by the same (or a very close) epitope of the HLA DR7 molecule; (c) an unusual restriction pattern was found for one clone; PBMC from more than 80% of donors could present TT whatever their degree of HLA compatibility with the autologous donor. Family studies were unable to disclose any restriction with known class II (or class I) antigens. While no inhibition was observed with anti-DR or -DC reagents, a mAb that recognizes class I antigen when associated with beta 2-microglobulin did inhibit the proliferation of this clone.

Impaired cell-mediated immunity in Plasmodium falciparum-infected patients with high-parasitemia and cerebral malaria.

Several cell-mediated functions were studied in vivo and in vitro in 63 Thai patients with acute falciparum malaria, including 21 cases with cerebral manifestations and 10 cases with initial parasitemia over 10%. Initial delayed cutaneous reactions to phytohemagglutinin and soluble protein antigens were negative in most cerebral malaria cases. In other patients, skin reactions were impaired or abolished as a direct function of parasitemia. No major alteration in the numbers of blood T and B lymphocytes was found. In lymphocyte cultures, proliferative responses to lectins were generally found within normal ranges; in contrast, proliferative responses to candidin were suppressed in parallel with delayed cutaneous responses to the same antigen. From these data, it can be concluded that the alteration of specific cell-mediated responses are predominantly detectable in acute cases with major parasite invasion, i.e., high parasitemia and/or cerebral manifestations. A direct role of Plasmodium falciparum was further suggested by the rapid restoration of cell-mediated functions observed in several cases under successful antimalarial therapy. These results do not support any evidence in favor of a preexisting cellular immune deficiency in relation with the occurrence of cerebral or high-parasitemia acute malaria in these patients.

Specific immune responses after booster immunization with tetanus toxoid in man: study of kinetics, family segregation, and linkage to HLA of in vitro lymphocyte proliferative responses and serum-antibody responses.

Kinetics and family transmission of antigen-specific in vitro cell-mediated responses were investigated in 68, and serum-antibody responses to tetanus toxoid (TT) in 73 individuals from a total of 12 families. Proliferative responses to highly purified TT monomer were studied in 6- to 7-day lymphocyte cultures. The effect of booster immunization was detectable 7 (D7) and 30 (D30), but not 120 days (D120) later. The sex of donors was not found to have any influence. A significant influence of the time interval since the last immunization was found for the responses at D7 and D30. Data were correspondingly adjusted for segregation and linkage analyses. Several transmission hypotheses for the data obtained at D7 and D30 were evaluated by likelihood ratio tests. Observations at D30 were compatible with the hypothesis of a control by a dominant genetic determinant for high responses closely linked to the major histocompatibility complex region. No such evidence could be found for D7. After booster immunization, mean antibody levels determined on D7, D30 (peak of response), and D120 were found to be higher than those prior to immunization (D0). The sex of the donors was found to have no influence on antibody responses. The time interval since the last immunization and the age of donors both had a slight influence, and data were correspondingly adjusted for segregation and linkage analyses, which showed no evidence of genetic control of the antibody responses or of linkage to HLA.

Human T cell clones specific for tetanus toxoid: characterization of antigen specificity and HLA restriction.

T cell blasts isolated from six-day cultures of peripheral blood mononuclear cells stimulated with tetanus toxoid (TT) were cloned in a two-layer agar system in the presence of autologous irradiated PBM (iPBM) and TT. Colonies were individually isolated and expanded in interleukin 2-containing medium. The antigen specificity of three T cell clones was attested by their capacity to proliferate under restimulation by TT and not by an unrelated antigen. The clones were specific for either the alpha or the beta chain of the toxin. T cells from these clones expressed Ia determinants and antigens of the helper/inducer T cell subset as defined by anti-T monoclonal antibodies. In the case of the alpha chain-specific clone MA 11 from the donor MA, allogeneic iPBM from HLA-compatible unrelated donors, including seven donors sharing one HLA DR specificity with MA, were found inefficient as antigen-presenting cells. A familial study, however, demonstrated that antigen presentation could be obtained using nonautologous cells. The presenting capability of cells from relatives of the donor MA segregated in association with the HLA-DRw6-bearing maternal haplotype present in MA. Results suggest that MA 11 cells recognized the antigen in the context of a surface determinant closely linked to HLA-DRw6.

Effect of isoprinosine on in vitro proliferative responses of human lymphocytes stimulated by antigen.

Isoprinosine, a synthetic purine derivative, did not significantly interfere with the thymidine uptake of triggered normal lymphocytes, nor exhibit a detectable mitogenic activity. Isoprinosine was able to significantly enhance specific proliferative responses of human lymphocytes from sensitized donors to soluble antigens. Isoprinosine did not alter the early interaction of human mononuclear cells with 125I-labelled antigen, nor the expression of their membrane receptors for immunoglobulin Fc fragments. The agent could not replace the accessory rôle of adherent cells in proliferative responses to antigens. The enhancing effect of isoprinosine on antigen specific responses of T-cells was observed upon adding the compound on any one of the seven days of culture. Isoprinosine partially restored the inhibited proliferation of lymphocytes cultured in the presence of deoxyadenosine and deoxycoformycin. Data suggest that metabolic changes involving purines may account for the effect of isoprinosine on the expression of T-cell responses.

Parasite-derived mitogenic activity for human T cells in Plasmodium falciparum continuous cultures.

Supernatants from Plasmodium falciparum continuous cultures exhibited mitogenic activity against human blood lymphocytes from unsensitized donors. This effect, which was not observed with supernatants from control cultures grown in the absence of the parasites, was dependent upon (i) the concentration of supernatant added to the lymphocyte cultures and (ii) the parasite concentration in the P. falciparum continuous cultures. T cells were the predominant target cells of this mitogenic activity. We observed similar response in lymphocytes from malaria-sensitized individuals to P. falciparum continuous culture material. We also detected a mitogenic activity in parasite-infected erythrocytes from P. falciparum continuous cultures. P. falciparum continuous cultures may provide practical quantities of parasite-derived substances which, presumably, are able to manipulate the immune effector mechanisms of an infected host.