Search PubMed⌕ Search

Biomedical subjects

F Grignani

Publications and source records attributed to F Grignani.

At least 91 records · Page 5Linked to original sources
PubMed1992

C-MYB activation and the pathogenesis of ovarian cancer.

We have detected the expression of the MYB proto-oncogene in ovarian cancer. This oncogene was thought to be expressed in a tissue-specific manner in cells of hematopoietic lineage. Total RNA from three established cell lines and four human primary ovary cancers was examined by Northern and Southern blot, RNAse protection, in situ hybridization and cytogenetic analysis. A 3.8 kb RNA transcript was present in one human primary cell culture which is the same size as that found in the immature myeloid HL60 cell line. No expression was detected in normal ovary tissue. Southern blot analysis of DNA from five ovarian tumors indicated that this gene is not rearranged. Chromosomal analysis of three samples show many abnormalities in two cases and a normal karyotype in another one. The presence of MYB transcript in ovarian cancer suggests that MYB may play a specific role in the pathogenesis of this disease.

Blotting, Northern↗
PubMed1992

Alternative splicing of PML transcripts predicts coexpression of several carboxy-terminally different protein isoforms.

The acute promyelocytic leukaemia (APL)-specific chromosome 15;17 translocation leads to the fusion of a newly identified putative transcription factor, PML, and the retinoic acid receptor alpha. We have characterized the structure of the PML genomic locus and preliminarily characterized its expression pattern. The PML locus spans a minimum of 35 kb and is subdivided into nine exons. The putative PML DNA binding site is encoded by exons 2 and 3. We isolated a large number of alternatively spliced PML transcripts that encode numerous PML isoforms. Two groups of isoforms were identified that differed either in their C-terminal region or in the length of their central region, but retained the putative DNA-binding and dimerization domains. RNAase protection experiments revealed that the different PML isoforms are equally expressed in established cell lines of different histological origin.

Amino Acid Sequence↗
PubMed1992

IgG subclass deficiency and sinopulmonary bacterial infections in patients with alcoholic liver disease.

Abnormalities in IgG subclass distribution were sought in serum samples and bronchoalveolar lavage fluid from 15 patients with alcoholic liver disease to explain their increased susceptibility to bacterial respiratory infections. Serum IgG4 deficiency alone or in association with low IgG2 levels was revealed in approximately 30% of patients with alcoholic liver disease. This fact prompted us to further investigate the immunoglobulin concentrations in broncho-alveolar lavage fluid, paying special attention to the distribution of IgA and IgG subclasses. IgA levels were found to be normal or slightly elevated. However, there were substantial defects in total IgG and IgG1 concentrations, often associated with reduced IgG2 and IgG4 levels, in approximately 70% of patients with alcoholic liver disease, which proved to be closely correlated with the number and type (pneumonia) of bacterial respiratory infections. A prospective study of intravenous immunoglobulin substitutive therapy involving two patients with recurrent pneumonia and very low serum IgG2 values demonstrated a reduction in the number of respiratory infectious episodes as well as an increase in both serum and, to a lesser extent, bronchoalveolar lavage fluid IgG1 and IgG2 levels. We identified immune defects that may represent an important pathogenetic mechanism that, when considered together with the alcohol-related suppression of alveolar macrophage and ciliary functions and the inhibition of leukocyte migration into the lungs, should help clarify the complex relationships between alcohol and immune defense.

Adult↗
PubMed1991

A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry.

Corticosteroids, calcium ionophores and anti-CD3 monoclonal antibodies kill mouse thymocytes incubated in vitro. Cell death is preceded by extensive DNA fragmentation into oligonucleosomal subunits. This type of cell death (apoptosis), which physiologically occurs in the intrathymic process of immune cell selection, is usually evaluated by either electrophoretic or colorimetric methods which measure DNA fragmentation in the nuclear extracts. These techniques are unable to determine the percentage of apoptotic nuclei or recognize the apoptotic cells in a heterogeneous cell population. We have developed a flow cytometric method for measuring the percentage of apoptotic nuclei after propidium iodide staining in hypotonic buffer and have compared it with the classical colorimetric and electrophoretic techniques using dexamethasone (DEX)-treated mouse thymocytes. Apoptotic nuclei appeared as a broad hypodiploid DNA peak which was easily discriminable from the narrow peak of thymocytes with normal (diploid) DNA content in the red fluorescence channels. When the DEX-induced apoptosis was inhibited by either low-temperature (4 degrees C) incubation or cycloheximide treatment, no hypodiploid DNA peak appeared. Similarly, thymocyte death induced by sodium azide, a substance with cell-killing activity through non-apoptotic mechanisms, did not result in any variation in the normal DNA peak. The flow cytometric data showed an excellent correlation with the results obtained with both electrophoretic and colorimetric methods. This new rapid, simple and reproducible method should prove useful for assessing apoptosis of specific cell populations in heterogeneous tissues such as bone marrow, thymus and lymph nodes.

Animals↗
PubMed1991

Molecular heterogeneity of the 1.0-kb T beta transcript in natural killer and gamma/delta lymphocytes.

The T cell receptor (TcR) beta chain is encoded by a 1.3-kb mRNA which contains variable (V), diversity (D), joining (J) and constant (C) regions. A shorter 1.0-kb transcript with C but no V sequences is found in thymocytes, alpha/beta and gamma/delta T lymphocytes and natural killer cells. The origin, clonal variability and function of this transcript is unknown. We isolated cDNA clones representative of the 1.0-kb mRNA from cDNA libraries of either polyclonal or clonal natural killer and gamma/delta lymphocytes. Ten different types of cDNA clones belonging to three separate groups were identified: (a) "J-C clones" consisting of one of five J beta regions and the corresponding C beta 1 or C beta 2 regions; (b) "D-J-C clones" composed of D beta 1/or D beta 2/J beta rearranged sequences and C beta 1 or C beta 2 sequences; (c) "5'C clones" made up of C beta 1 or C beta 2 regions preceded by the corresponding genomic 5' flanking region. The presence of recombination signal sequences in J-C and D-J-C clones suggests that the first derive from mRNA transcribed from promoters located in the 5' J region and the second from mRNA transcribed from promoters situated in the 5' D region. Nucleotide sequence analysis demonstrated that only three of the ten types of clones isolated had the potential to code a short cytoplasmic T beta protein with a variable D-J beta N terminus. These findings have implications for the mechanisms of T beta germ-line transcription and the function of the T beta transcripts.

Amino Acid Sequence↗
PubMed1991

Interleukin-6 is constitutively produced by human CTL clones and is required to maintain their cytolytic function.

Maturation of cytolytic T lymphocytes from nonlytic precursors requires cytokines in addition to IL2. Interleukin-6 is the principal cytokine that cooperates with IL2 in the induction of CTL differentiation from murine and human thymocyte precursors. However, a cytotoxic differentiation factor (CDF) role of IL6 for mature T cells is challenged by data indicating that IL2 alone is sufficient for CTL generation. The aim of this study was to identify a model system in which IL6 acted as a CDF for human peripheral T cells. We noted that IL6 was endogenously produced by CTL clones in the course of their expansion with APC, lectin, and IL2. The majority of several hundred T-cell clones, both CD4+ and CD8+, produced IL6 in response to relatively high doses of IL2. Other experiments that compared the cytolytic function of CTL clones cultured in the presence of IL6 with that of the same clones cultured in the absence of IL6 demonstrated that IL6 contributes to the cytolytic ability of the majority of human CTL clones. Our data suggest that IL6 acts in an autocrine fashion to support CTL differentiation in human T-cell clones.

CD4 Antigens↗
PubMed1991

Inhibition of human breast cancer cell (MCF-7) growth in vitro by the somatostatin analog SMS 201-995: effects on cell cycle parameters and apoptotic cell death.

Somatostatin (SS) and SS analogs have been shown to exert an antiproliferative effect on several transplantable tumors in animals and to reduce the growth of pancreatic, pituitary, and mammary tumor cells in vitro. We evaluated the effects that the SS analog SMS 201-995 exerts on growth, cell-cycle parameters, and suicidal cell death (apoptosis) of human breast cancer cells (MCF-7) in vitro. SMS 201-995 significantly reduced the MCF-7 cell growth induced by serum, estradiol, insulin, and insulin-like growth Factor-I in both short term and long term experiments. The effect was maximal when 10 nM estradiol was used as mitogen in long term cultures. SMS 201-995 treatment produced a slight but transient accumulation of cells in the G2/M phase but did not cause any noteworthy reduction in the percentage of proliferating cells. There was, instead, a time-related increase in the number of cells with the flow-cytometric characteristics of apoptosis in the cultures treated with the SS analog, which correlated well with its growth-inhibiting activity. It would, therefore, seem that SMS 201-995 exerts its inhibitory effect on MCF-7 cell growth in vitro mainly by enhancing the rate of programmed (or suicidal) cell death in the culture.

Breast Neoplasms↗
PubMed1991

Structure and origin of the acute promyelocytic leukemia myl/RAR alpha cDNA and characterization of its retinoid-binding and transactivation properties.

Acute promyelocytic leukemia (APL) is characterized by the 15;17 chromosomal translocation. Cloning experiments have established that the chromosome 17 breakpoint maps to the RAR alpha and the 15 to the myl locus. The resulting chimeric gene is transcribed as a myl/RAR alpha fusion mRNA. By isolating both normal myl and APL myl/RAR alpha cDNAs, we showed that the myl/RAR alpha mRNA encodes for a putative fusion protein with a molecular weight of about 103 kDa, which is made up of 530 amino acids derived from the myl N-terminus and 402 amino acids originating from the RAR alpha C-terminus. The protein includes the RAR alpha DNA and retinoid-binding regions but lacks the A portion of the N-terminal region (A/B region) which is thought to contain one of the RAR alpha transactivation domains. The myl/RAR alpha protein acted as a retinoid-inducible transcription factor with both ligand-independent repressor and ligand-dependent activator functions in transactivation experiments of a retinoic acid-responsive gene. Myl/RAR alpha exerted this dual function three times more effectively than RAR alpha and had about 10-fold greater affinity for RA than RAR alpha. Comparison of myl/RAR alpha genomic and cDNA sequences from the same case demonstrated that both chromosome 15 and 17 breakpoints occurred within introns and the myl and RAR alpha sequences are spliced in the same polyadenylated RNA.

Amino Acid Sequence↗
PubMed1991

T-lymphocyte activation pathways in alcoholic liver disease.

Immune system derangement in cirrhotic patients with evidence of malnutrition is a well-recognized characteristic of chronic alcohol abuse. However, in vitro studies on cellular immune function performed with lectin mitogens have produced conflicting results. The recent development of more accurate immunological techniques for studying lymphocyte transformation, that use monoclonal antibodies directed against surface structures (CD3 and CD2) involved in antigen recognition, as well in adhesion functions, prompted us to study discrete in vitro T-cell hypo-responsiveness in a series of alcoholic liver disease (ALD) patients with no evidence of malnutrition or hepatic cirrhosis. The results indicated that the CD2 pathway is markedly defective in ALD T lymphocytes, accompanied by reduced interleukin-2 (IL-2) receptor expression upon in vitro activation. This defect cannot be reversed by the addition of recombinant IL-2 (rIL-2) or rIL-1. Faulty intracellular signal transduction by protein kinase C (PKC) and defective intracellular Ca2+ mobilization may be responsible for the CD2 pathway impairment. The addition of small amounts of phorbol 12-myristate, 13-acetate, but not Ca2+ ionophore A23187, is able to overcome the defect, thereby suggesting a direct PKC involvement. The hypothesis of a direct ethanol effect on transmembrane signal transduction systems is suggested by the demonstration of an expansion of circulating virgin (naive) T cells (CD3+/UCHL1-low) that binds tyrosine phosphatase (CD45RA antigen) on their surface.

Adult↗
PubMed1991

Analysis of T cells bearing different isotypic forms of the gamma/delta T cell receptor in patients with systemic autoimmune diseases.

The expression of gamma/delta T cell receptor (TCR) on peripheral blood CD3+ cells circulating in 74 patients with different systemic autoimmune diseases was evaluated. There was a significant increase in the gamma/delta T cell number only in patients with primary Sjögren's syndrome (SS) and in untreated patients with systemic lupus erythematosus (SLE). Unlike healthy subjects, a subgroup of patients with SLE and SS displayed a marked increase in gamma/delta T cells. Immunosuppressive treatment of patients with active SLE led to a normalization of the gamma/delta T cell number. Analysis of surface phenotype showed that when patient gamma/delta T cells were expanded in the peripheral blood, they were not activated but bore "memory" markers. In addition, they preferentially expressed the disulfide linked form of the TCR, except in progressive systemic sclerosis where the nondisulfide form was displayed. Serial determinations in single patients demonstrated that the gamma/delta T cell increase is a persistent immunological feature in these patient subgroups.

Adolescent↗
PubMed1991

GM-CSF: clinical trials in non-Hodgkin's lymphoma patients with chemotherapy induced leucopenia.

Fourteen patients (M/F, 6/8; age, 48/23-64 yrs) with relapsing or primary resistant intermediate-high grade non-Hodgkin lymphomas were treated with ARA-C (2 g/m2 x 4 on days 1 and 2), DDP (100 mg/m2 96 hr infusion) and VP-16 (150 mg/m2 on days 1, 2 and 3). GM-CSF or placebo was administered from the 5th day until neutrophil count reached greater than or equal to 1000/microliters on 2 consecutive days. Three PR and 6 CR were documented. Two CR pts are still in CR at 19 and 23.5 months. With the exception of one case of cerebral haemorrhage, life-threatening liver toxicity, exfoliative colitis, capillary leak syndrome and anaphylactoid reaction, the protocol regimen provoked only modest haematological and extra-haematological toxicities.

Adult↗
PubMed1990

Down-regulation of LFA-1 adhesion receptors by C-myc oncogene in human B lymphoblastoid cells.

The function of the c-myc gene and its role in tumorigenesis are poorly understood. In order to elucidate the role of c-myc oncogene activation in B cell malignancy, the phenotypic changes caused by the expression of c-myc oncogenes in human B lymphoblastoid cells immortalized by Epstein-Barr virus were analyzed. C-myc oncogenes caused the down-regulation of lymphocyte function-associated antigen-1 (LFA-1) adhesion molecules (alpha L/beta 2 integrin) and loss of homotypic B cell adhesion in vitro. Down-regulation of LFA-1 occurred by (i) posttranscriptional modulation of LFA-1 alpha L-chain RNA soon after acute c-myc induction, and (ii) transcriptional modulation in cells that chronically express c-myc oncogenes. Analogous reductions in LFA-1 expression were detectable in Burkitt lymphoma cells carrying activated c-myc oncogenes. Since LFA-1 is involved in B cell adhesion to cytotoxic T cells, natural killer cells, and vascular endothelium, these results imply functions for c-myc in normal B cell development and lymphomagenesis.

B-Lymphocytes↗
PubMed1990

The C gamma 1-encoded disulphide-linked and the C gamma 2-encoded nondisulphide-linked forms of the gamma/delta heterodimer use different gamma and delta variable regions.

We identified two types of gamma/delta T-cell clones: (1) BB3+ clones, which express the disulphide-linked form of the gamma/delta heterodimer (type 1 receptor) and carry rearrangements of the C gamma 1 gene; and (2) delta TCS1+ clones, which express the nondisulphide-linked form of the gamma/delta heterodimer (type 2 receptor) and carry rearrangements of the C gamma 2 gene. To determine the joining (J) and variable (V) region usage of the two receptor types, T gamma and T delta gene rearrangements and expression were analyzed in each gamma/delta T-cell clone. The results showed that the J and V regions used by the two receptor types are distinct: clones that express the type 1 receptor carry potentially functional rearrangements in J gamma P-V gamma 9 for gamma and in J delta 1-V delta 2 for delta; and clones that express the type 2 receptor had potentially functional rearrangements in J gamma 2-V gamma 2/V gamma 4 for gamma and in J delta 1-V delta 1 for delta. Therefore, two clearly different gamma/delta receptors (V gamma 9-J gamma P-C gamma 1/V delta 2-J delta 1-C delta and V gamma 2/V gamma 4-J gamma 2-C gamma 2/V delta 1-J delta 1-C delta) predominate in peripheral blood.

Adult↗
PubMed1990

Cytolytic function of clonable T cells after human bone marrow transplantation.

We evaluated T-cell mediated lymphokine activated killer (LAK) function during the late (greater than 5 months) reconstitution phase after T cell-depleted allogeneic bone marrow transplantation (BMT) for hematologic malignancy. Since LAK cells are sustained by interleukin-2 (IL-2), we also investigated the ability of post-BMT T cells to produce IL-2. These functions were investigated at the clonal level. More than 200 T-cell clones from six long-term BMT recipients were generated and compared with 60 T-cell clones derived from two normal controls. Almost all the CD8+ clonal cultures from BMT recipients expressed cytolytic activity in a lectin-dependent cellular cytoxicity assay. Interestingly, a higher proportion of BMT recipient-derived cytolytic clones were able to mediate LAK activity in comparison with control clones (28% versus 4%, P less than .05). However, T-cell clones from BMT recipients, as opposed to control clones, were largely incapable of producing IL-2. Given the high proportions of post-BMT circulating CD8+ T cells, it appears that, in long-term BMT recipients, the precursors of nonspecific LAK effectors are present at above normal levels. However, their function may be defective in vivo due to poor IL-2 production.

Bone Marrow Transplantation↗
PubMed1990

Negative autoregulation of c-myc gene expression is inactivated in transformed cells.

Negative feedback regulation of c-myc gene expression has been observed in some, but not all, cell types. In order to demonstrate conclusively the existence of this mechanism and gain insight into the cause of its inactivation, we have directly examined its function in B cells and then investigated its activity in a number of cell types. We demonstrate the existence of negative c-myc autoregulation by showing the rapid, dose dependent and reversible suppression of endogenous c-myc expression in EBV-immortalized B lymphoblastoid cells transfected with a c-myc gene expressed under the control of a heavy metal inducible promoter. Autoregulation occurs at the level of transcriptional initiation and is mediated by at least one stable intermediate or cofactor molecule. The c-myc autoregulatory mechanism was found operative in all (11 of 11) non-tumorigenic cells tested, including normal and immortalized lymphocytes and fibroblasts. However, this mechanism was found to be inactive in all (10 of 10) tumor cell lines derived from a variety of tissues including those carrying normal and oncogenically activated c-myc genes. These data establish the existence of an important regulatory circuit modulating c-myc expression in normal cells and suggest that its inactivation may represent a general regulatory disturbance of transformed cells.

Animals↗