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Biomedical subjects

V Pistoia

Publications and source records attributed to V Pistoia.

At least 73 records · Page 4Linked to original sources
PubMed1995

Characterization of a distal 5'-flanking region (-2010/-630) of human GM-CSF.

The 5'-flanking region of the human GM-CSF gene was subcloned from the phagic clone lambda J1-16 to detect cis-elements involved in GM-CSF constitutive expression. We determined and sequenced an uncharacterized promoter region of 1381 bp (-2010 to -630), named pPF2000. Putative binding sites of several transcriptional factors were found. Progressive deletion mutants of the PF2000 were analyzed by measuring the linked CAT activities, in constitutive (5637) and inducible (PEU) GM-CSF-producing cells. A positive distal sequence (268 bp), between -2010 and -1742, responsible for the high constitutive expression of GM-CSF in 5637 carcinoma cell line was found.

Base Sequence↗
PubMed1995

Differential effects of N-(4-hydroxyphenyl)retinamide and retinoic acid on neuroblastoma cells: apoptosis versus differentiation.

Retinoids exert various important biological effects in the control of normal growth, differentiation, and fetal development. While retinoic acid (RA) has entered clinical trials as a differentiation-promoting agent, it is only recently that the synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) has been shown to be of potential clinical interest in cancer chemoprevention and treatment. Since thus far no data exist on the effects of HPR on neural crest cell-derived tumors, we have examined its in vitro effects on neuroblastoma (NB) cell lines and found that at relevant pharmacological concentrations it induces a dose-dependent growth inhibition. The antiproliferative effects of HPR were, in six of six cell lines tested, drastically more potent that those induced by an equimolar dose of RA. Time course growth analysis showed that HPR at 3 x 10(-6) M induces a very rapid (24-72 h) fall in thymidine uptake (> 90%), whereas at 3 x 10(-7) M it exhibits cytostatic effects. In contrast to RA, HPR did not show morphological changes typical of NB cell maturation nor did it induce the expression of any cytoskeletal protein associated with neuronal differentiation. DNA flow cytofluorimetric analysis revealed that HPR did not induce an arrest in a specific phase of the cell cycle while triggering apoptosis. This phenomenon was evidenced both by the visualization of "DNA ladders" on gel electrophoresis and by a quantitative assay for evaluating programmed cell death based upon the labeling of DNA breaks with tritiated thymidine. With the latter method, apoptotic cells were detectable as early as 3-6 h after treatment of NB cells with 10(-5) M HPR, while more than 50% of cells were apoptotic by 24-72 h following exposure to 3 x 10(-6) M HPR. In contrast, RA induced a low rate of apoptosis in NB cells only after 3-5 days. Time lapse photomicroscopy showed that NB cells treated with HPR underwent a death process highly reminiscent of apoptosis, with progressive condensation of the cytoplasm around the nucleus and intense cell shrinkage. The cells then rounded up and detached from the plate. Furthermore, propidium iodide staining of the DNA showed that a high proportion of cells treated with HPR displayed a small and brightly staining nucleus; chromatin appeared aggregated into dense masses in the nuclear periphery, a typical feature of apoptotic cells. In conclusion, our study demonstrates that contrary to the differentiation-promoting activity of RA, HPR dramatically suppresses NB cell growth by inducing programmed cell death.(ABSTRACT TRUNCATED AT 400 WORDS)

Apoptosis↗
PubMed1995

Toxoplasma gondii-specific CD4+ T cell clones from healthy, latently infected humans display a Th0 profile of cytokine secretion.

Human Toxoplasma gondii (Tg)-specific T cell clones were raised by infecting peripheral blood mononuclear cells (MNC) from two healthy, latently infected individuals with Tg trophozoites. All of the clones had a CD4+ immunophenotype and produced simultaneously interleukin (IL)-2, interferon (IFN)-gamma, IL-4 and IL-5 upon mitogen or antigen stimulation. Tg-specific T cell clones were classified as T helper of type 0 (Th0) since most of them released roughly comparable amounts of IFN-gamma and IL-4. In some clones, a trend to an increased production of IFN-gamma following antigen-specific as compared to non-specific stimulation was observed. The Th0 phenotype was also expressed by T cell clones that had been raised from bulk cultures performed in the presence of IL-4 or IFN-gamma. All of the Tg-specific T cell clones were cytolytic in a non-specific assay which involves the triggering of the CD3-T cell receptor (TcR) complex. Some clones specifically lysed an autologous lymphoblastoid cell line (LCL) that had been infected with Tg trophozoites. Finally, most of the Tg-specific T cell clones produced IL-10, irrespective of whether they had been raised from bulk cultures incubated in the presence or absence of IL-4 or IFN-gamma. Taken together, these findings suggest that Tg-specific Th0 helper cell clones from healthy, latently infected individuals, beside activating toxoplasmacidal mechanisms through IFN-gamma release, might limit the magnitude of the immune response of the parasite by killing Tg-infected antigen-presenting cells and by releasing IL-10.

Animals↗
PubMed1995

Relationships between B cell cytokine production in secondary lymphoid follicles and apoptosis of germinal center B lymphocytes.

In vivo or in vitro activated human B lymphocytes can produce a wide spectrum of cytokines which are involved in the regulation of hematopoiesis and of the inflammatory and immune responses. Three major B cell subsets have been identified in peripheral lymphoid organs: the germinal center (GC), the mantle zone (MZ) and the marginal zone B lymphocytes. GC and MZ B cells can be isolated as CD39- surface (s)IgD- or CD39+ sIgD+ cells, respectively. Therefore, it is now possible to investigate the cytokine producing potential of purified GC and MZ B lymphocytes. In this article, the optimal conditions for the assessment of cytokine production by human B cells are first discussed; thereafter, the spectrum of B lymphocyte-derived cytokines is described together with their possible physiological meaning. Next, data concerning the cytokines released in vitro by either GC or MZ B cells are presented. Some cytokines, such as granulocyte colony stimulating factor (G-CSF) or granulocyte-macrophage CSF (GM-CSF), are produced only by GC or MZ B lymphocytes, respectively, whereas other cytokines, such as tumor necrosis factor-alpha (TNF-alpha), interleukin 6 (IL-6) or IL-10 are synthesized by both B cell subsets. Finally, the relationships between B cell-derived cytokines and apoptosis of GC B lymphocytes are discussed, and a hypothetical model of the cytokine networks in secondary lymphoid follicles is presented. It is expected that these notions will help to clarify the pathophysiology of lymphoproliferative and autoimmune diseases.

Apoptosis↗
PubMed1995

Production of inflammatory cytokines by Epstein-Barr virus (EBV)-infected lymphoblastoid cell lines spontaneously originated from the peripheral blood of patients with human immunodeficiency virus (HIV) infection.

In this study we have raised spontaneous Epstein-Barr virus (EBV)-positive lymphoblastoid cell lines (LCL) from the peripheral blood of human immunodeficiency virus (HIV)-infected individuals and of control patients with primary EBV infections. These LCLs were also raised in the presence of the viral inhibitor phosphonoformate (PFA); under these conditions, the in vitro infection of bystander B lymphocytes with EBV released in culture by in vivo infected B cells is inhibited. Thus, the latter LCLs are likely to represent the progeny of B cells latently infected by EBV in vivo. The LCLs raised in the presence or absence of PFA had the same phenotypic features, type of EBV latency, and growth pattern irrespective of whether they had been raised from HIV-seropositive individuals or patients with primary EBV infections or had been generated by infecting normal B cells in vitro. Studies on the production of inflammatory cytokines were conducted by Northern blotting or by determining the cytokine concentrations in the cell supernatants by immunoassays or bioassays. Three of eight LCLs from HIV-seropositive patients released TNF alpha and 5/5 released TNF beta, IL6 was present in the supernatants of 1/8 LCLs, and IL1 alpha and IL1 beta were not detected in any culture supernatant. No differences were noticed in the patterns of cytokine secretion among the LCLs from HIV-seropositive patients and in those raised from patients with primary EBV infections or obtained by infecting normal B cells in vitro with EBV. It is tempting to speculate that abnormally expanded EBV-harboring B cells in HIV-seropositive patients may participate in the pathogenesis of certain clinical manifestations by releasing inflammatory cytokines; some of these cytokines might also contribute to the in vivo spreading of HIV infection. However, the spontaneous LCLs from HIV-seropositive individuals do not display abnormal features compared to latently EBV-infected LCLs from other sources despite the high frequency of EBV-driven lymphoproliferative disorders observed in AIDS patients.

Adult↗
PubMed1995

Antiphospholipid antibodies in paediatric systemic lupus erythematosus, juvenile chronic arthritis and overlap syndromes: SLE patients with both lupus anticoagulant and high-titre anticardiolipin antibodies are at risk for clinical manifestations related to the antiphospholipid syndrome.

Antiphospholipid antibodies (APA) are often associated with severe clinical manifestations, especially in the setting of systemic lupus erythematosus (SLE). Here we have investigated the prevalence of anticardiolipin antibodies (aCL) and lupus anticoagulant (LA) in paediatric patients affected with SLE, JCA and overlap syndromes (OS) and correlated the presence of aCL and LA with clinical features. aCL were assayed by enzyme-limited immunoassay; LA was determined by activated partial thromboplastin time and the kaolin clotting time test. aCL and LA assays were performed in parallel on at least two occasions over a 7-30-month follow-up. Fifteen out of nineteen (79%) SLE patients had aCL and 8/19 (42%) had LA. Six SLE patients displayed manifestations that were APA-related: deep venous thromboses, autoimmune haemolytic anaemia, pulmonary hypertension, neurological alterations. Five out of six symptomatic patients had both LA and high-titre aCL. In contrast, JCA and OS patients had usually low-titre aCL, no detectable LA and no APA-related manifestations. aCL persisted at high titre over time in SLE patients, but was only transiently detected in JCA and OS patients. This study shows that the simultaneous positivity for LA and high-titre aCL allows the identification of paediatric SLE patients who are at risk not only for thrombosis, but also for other APA-related clinical features.

Adolescent↗
PubMed1994

Spontaneous production of granulocyte colony-stimulating factor in vitro by human B-lineage lymphocytes is a distinctive marker of germinal center cells.

The ability of human B lymphocytes to produce granulocyte (G)-CSF in vitro was investigated. Highly purified tonsillar B cells were fractionated into large and small cells by a Percoll density gradient, cultured, and tested for G-CSF gene expression. Large B cells spontaneous produced G-CSF mRNA and protein, whereas small B cells did not, even after incubation with various stimuli. Immunophenotypic analyses showed that large B lymphocytes contained approximately 60 to 70% of cells with the characteristic surface markers of germinal center (GC) B cells (CD38+, CD10+, and surface IgG+). The remaining cells expressed CD39, CD23, and surface IgD and were presumably in vivo-activated follicular mantle zone B cells. Fractionation of the large B lymphocytes into CD39+, surface IgD+, and CD39-, surface IgD- cells showed that the latter, but not the former, cell type produced G-CSF spontaneously in culture. Stimulation of purified (CD39-, surface IgD-) GC B cells with a CD40 mAb alone or in combination with IL-4 increased G-CSF production. Because these stimuli rescued a large fraction of GC cells (up to 50%) from spontaneous apoptosis in vitro, the finding may suggest that prevention of apoptotic death resulted in an increased G-CSF production or that CD40 mab and/or IL-4 increased G-CSF gene expression in G-CSF-producing GC B cells. Malignant B cells purified from the invaded lymph nodes of three patients with follicular center cell lymphoma and three Burkitt lymphoma cell lines, which had an immunophenotype identical with that of normal GC B cells, spontaneously produced G-CSF in vitro, thus confirming the GC origin of the cytokine. Incubation of normal purified GC B cells with rG-CSF resulted in the rescue of GC B cells from apoptosis, suggesting that G-CSF may be used by GC B cells in an autocrine manner. This autocrine loop of production and response to G-CSF by GC B cells may be activated by stimuli such as those delivered via the surface CD40 molecule, that participate in the rescue of GC B cells from apoptosis.

Adenosine Triphosphatases↗
PubMed1994

Expression and genomic configuration of GM-CSF, IL-3, M-CSF receptor (C-FMS), early growth response gene-1 (EGR-1) and M-CSF genes in primary myelodysplastic syndromes.

Peripheral blood mononuclear cells from seventeen patients with primary myelodysplastic syndromes (MDS) in advanced stage were enriched for blasts and tested for (1) karyotype, (2) genomic configuration and (3) expression of IL-3, GM-CSF, FMS and EGR-1 genes which are all located on the long arm of chromosome 5. The expression of the M-CSF gene, that has been recently reassigned to the short arm of chromosome 1 (lp), was also investigated. Aims of the study were to (1) assess the potential role of the expression of these genes in the maintenance and expansion of the neoplastic clones and (2) search for constitutional losses or rearrangements of one allele followed by a deletion of the second allele of the same genes in the leukemic cells. The latter issue was investigated by comparing, in 8 cases, constitutive DNA from skin fibroblasts with leukemic DNA. Eleven of the 17 patients had abnormal karyotypes. The M-CSF gene was expressed in 6 cases and the FMS and the EGR-1 genes were expressed in 2 of the latter cases. An autocrine mechanism of growth could be hypothesized only for the 2 patients whose cells expressed both the M-CSF and FMS genes. No germline changes or rearrangements were observed in any of the genes studied. Thus, deregulation of genes encoding for certain hemopoietic growth factors or receptors does not seem to represent a major mechanism of MDS progression.

Aged↗
PubMed1993

Production of hematopoietic growth factors by human B lymphocytes: mechanisms and possible implications.

In this study we have investigated the ability of human B lymphocytes to produce granulocyte-macrophage colony stimulating factor (GM-CSF) and, in preliminary experiments, granulocyte CSF (G-CSF). The sources of human B cells were surgically removed tonsils from normal individuals and peripheral blood from patients with B cell chronic lymphocytic leukemia (B-CLL). Tonsil B lymphocytes were purified by E rosetting and complement-mediated cytotoxicity with selected monoclonal antibodies and subsequently fractionated by a Percoll density gradient into in vivo activated and resting cells. The latter cell fractions were subsequently cultured with or without stimuli. GM-CSF was detected by a bioassay, G-CSF by an enzyme-linked immunoassay. In vivo and in vitro activated B cells produced GM-CSF, whereas in vivo activated, but not in vitro activated, B lymphocytes produced G-CSF. These results were confirmed by Northern blot experiments with cDNA probes specific for GM-CSF and G-CSF genes. Many B cell suspensions from B-CLL patients produced GM-CSF or G-CSF only following Staphylococcus Aureus Cowan I (SAC) stimulation; in some cases, a spontaneous production or no production at all of the two cytokines was detected. The possible implications of these results for B cell physiology and for the pathogenesis of immunologically mediated diseases will be discussed.

B-Lymphocytes↗
PubMed1993

Clonal analysis of peripheral blood lymphocytes from three patients with advanced neuroblastoma receiving recombinant interleukin-2 and interferon alpha.

In this study we have investigated, at the population and the clonal levels, the immunophenotypes and the non-specific cytotoxic functions of peripheral blood lymphocytes from three stage IV neuroblastoma patients receiving treatment with recombinant interleukin-2 (IL-2) and interferon alpha (IFN alpha). Both IL-2 alone and the combination of IL-2 and IFN alpha caused an in vivo expansion of CD56+, CD3- NK cells most of which expressed the p75 molecule, i.e. the beta chain of the IL-2 receptor. Peripheral blood mononuclear cells (PBMC), drawn after treatment, displayed an increased NK activity, but no lymphokine-activated killer (LAK) activity. However, the subsequent in vitro culture of PBMC with high-dose IL-2 induced the generation of a potent LAK activity, which was mediated by an expanded population of CD3+, CD8+ T cells. Finally lymphocytes that had been isolated after cytokine therapy were cloned, in the presence of low-dose phytohemagglutin, immediately or following culture with IL-2. Clones derived from LAK cells expanded in vitro had predominantly a CD3+, CD8+ immunophenotype, whereas those raised from freshly separated lymphocytes were either CD3+, CD4+ or CD3+, CD8+ in equal proportions. Most of the above clones were poorly or not at all cytolytic against NK-sensitive or NK-resistant targets. In contrast, the few NK clones obtained (CD3-, CD56+) lysed all targets with high efficiency.

Antigens, CD↗
PubMed1993

Transforming growth factor beta-1 (TGF-beta 1) released by an Epstein-Barr virus (EBV) positive spontaneous lymphoblastoid cell line from a patient with Kostmann's congenital neutropenia inhibits the growth of normal committed haemopoietic progenitors in vitro.

This study reports the characterization of a spontaneous lymphoblastoid cell line (LCL) raised from the peripheral blood of a patient with Kostmann's congenital neutropenia. The LCL was composed of EBV-infected polyclonal B cells and displayed surface markers and pattern of growth in vitro typical of normal LCLs. The supernatant of the LCL contained a colony inhibiting activity (CIA) that decreased the cloning efficiency of normal committed haemopoietic progenitors and was identified as immunoreactive transforming growth factor beta 1 (TGF-beta 1) by neutralization experiments with a specific antiserum. Control studies with a panel of LCLs spontaneously derived from the peripheral blood of patients seropositive for Epstein-Barr virus (EBV) infections showed that 5/30 LCLs produced a CIA. This CIA was not identifiable as TGF-beta 1 but rather was due to the combined effects of tumour necrosis factor alpha (TNF alpha), tumour necrosis factor beta (TNF beta) and interferon alpha (IFN alpha), that were present in the LCL supernatants. The hypothesis that the B cells latently infected by EBV in vivo and possibly expanded as a consequence of the infection may have contributed to the inhibition of the patient granulopoiesis by releasing TGF-beta 1 will be discussed.

B-Lymphocytes↗
PubMed1993

ICF syndrome with variable expression in sibs.

We describe a new familial case of ICF syndrome (immunodeficiency, centromeric instability, facial anomalies) in a woman of 29 years and in her brother of 30 years. The proband showed mental retardation, facial anomalies, recurrent respiratory infections, combined deficit of IgM and IgE immunoglobulin classes, and paracentromeric heterochromatin instability of chromosomes 1, 9, and 16. The brother had minor signs of the syndrome and had an apparently normal phenotype. Their parents were healthy and non-consanguineous. Chromosome anomalies consisted of homologous and non-homologous associations, chromatid and isochromatid breaks, deletions of whole arms, interchanges in the paracentromeric region, and multibranched configurations of chromosomes 1, 9, and 16. CD bands and fluorescence in situ hybridisation with alphoid DNA sequence probes specific for the centromeres of chromosomes 1 and 16 showed that the centromere was not directly implicated in the formation of multibranched configurations. These cases indicate the autosomal recessive mode of inheritance and the variable expressivity of the ICF syndrome.

Adult↗
PubMed1993

Clinical diversity of Raynaud's phenomenon in childhood: report of six cases.

Six pediatric patients with Raynaud's phenomenon are reported. Three patients had clinical and immunological evidence for a connective tissue disease; one had primary Raynaud's phenomenon associated with antinuclear antibodies and two had Raynaud's phenomenon secondary to exposure to lead and to a vibrating instrument, respectively. The diagnostic and therapeutic implications of these observations will be discussed.

Adolescent↗
PubMed1993

H and L ferritin gene expression in U937 cells induced to macrophage differentiation.

Ferritin is an ubiquitous protein that has been shown to regulate cell differentiation in several experimental systems. In this study we have investigated the expression of ferritin genes encoding the heavy (H) and light (L) chains in t'B U937 cell line, induced to differentiate to macrophage-like cells by 12-O-tetradecanoylphorbol-13-acetate (TPA), retinoic acid (RA) or 1-beta-D-arabinofuranosylcytosine (Ara-C). An increase in the level of H ferritin mRNA was detected in U937 cells that had been incubated with Ara-C. Treatment of U937 cells with Actinomycin D suggested that the H ferritin mRNA increase was mediated by post-transcriptional mechanisms. The L ferritin mRNA level increased only following stimulation of U937 cells with RA. Immunophenotypic and cytochemical analyses showed that Ara-C was the strongest inducer of the macrophagic differentiation of U937 cells. These results suggest that the increase of H ferritin mRNA expression may represent a sensitive marker of myeloid cells differentiating along the monocyte-macrophage lineage.

Antigens, CD↗
PubMed1992

Production of granulocyte-macrophage colony-stimulating factor but not IL-3 by normal and neoplastic human B lymphocytes.

The ability of human B cells to produce granulocyte-macrophage (GM)-CSF and IL-3 was investigated. B cells, isolated from tonsils or from the peripheral blood of patients with B cell chronic lymphocytic leukemia using mAb and immune rosettes, were cultured with or without Staphylococcus aureus Cowan strain I. GM-CSF and IL-3 were measured in the culture supernatants using a bioassay based on the selective proliferative response of the MO7e megakaryoblastic cell line to IL-3 or GM-CSF. S. aureus Cowan I-stimulated normal B cells released measurable amounts of GM-CSF but not of IL-3 as determined in neutralization assays with specific mAb in the MO7e cell line test. Some of the unstimulated normal B suspensions also produced GM-CSF, albeit in lower quantities. When normal B cells were fractionated into small (resting) and large (activated) B cells by Percoll density gradients, spontaneous GM-CSF production was detected only in the large cell fractions, but small cells were induced to produce GM-CSF upon S. aureus Cowan I stimulation. On a per cell basis, tonsillar B cells were found capable of releasing more GM-CSF than activated peripheral blood monocytes. The amount of GM-CSF produced by B cells was always inferior to that released by stimulated peripheral blood T cells or NK cells. The purified B cell suspensions from all 14 B cell chronic lymphocytic leukemia patients studied released GM-CSF but not IL-3 in the culture supernatants, sometimes even in the absence of stimuli. Northern blot analysis on total or poly(A)+ RNA confirmed the presence of GM-CSF, but not of IL-3, mRNA transcripts in both normal and malignant B cells. The results of these studies support the notion that activated human B lymphocytes release sufficient GM-CSF to play a role in the control of both hematopoiesis and the inflammatory process.

B-Lymphocytes↗
PubMed1991

Emergence of a B-cell lymphoblastic lymphoma in a patient with B-cell chronic lymphocytic leukemia: evidence for the single-cell origin of the two tumors.

A patient is described who presented with a chronic lymphocytic leukemia (CLL) and later developed a lymphoblastic lymphoma. The cells from the CLL were typical mature B lymphocytes as could be assessed by morphologic, cytochemical, and surface marker analyses. The cells from the lymphoblastic lymphoma were immature B cells that expressed CD10, CD20, and HLA-DR markers, but not surface Ig or cytoplasmic mu chains, and were negative for terminal deoxynucleotidyl transferase (TdT). The cells of two continuous cell lines, obtained from the bone marrow and the peripheral blood of the patient, had the same phenotype as the lymphoblastic lymphoma cells, did not contain the Epstein-Barr virus genome, and displayed malignant features in vitro, including the capacity to form colonies in agar. The two cell lines also shared identical chromosomal abnormalities, a finding which suggests that they derived from the same malignant cell already present in vivo. Such chromosomal abnormalities were not seen in the karyotype of the peripheral blood cells at the onset of the disease. Analysis of the Ig heavy chain genes using a DJ-specific probe showed the very same monoclonal rearrangement in the cells from the B-CLL, the lymphoblastic lymphoma and the two cell lines, thus demonstrating their common clonal origin. By contrast, a monoclonal rearrangement of the lambda chain gene locus was found in the B-CLL cells only, a finding consistent with their exclusive capacity to express surface IgM lambda. This patient represents a rare case in whom a chronic lymphoproliferative disorder with mature malignant cells transforms into a lymphoblastic lymphoma characterized by cells frozen at a very early maturational stage. The possible mechanisms leading to such transformation within the same cell clone are discussed.

Adult↗