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Biomedical subjects

L Frati

Publications and source records attributed to L Frati.

At least 199 records · Page 11Linked to original sources
PubMed1990

T-cell restricted and unrestricted expression of transfected human interleukin-2 gene: phorbol ester- and calcium-inducible versus constitutive expression.

Interleukin-2 (IL-2) gene expression is tightly controlled and generally limited to antigenic stimulation of T cells. To study the cell-specific expression of the IL-2 gene, we transfected the intact human IL-2 gene, including 2.0 kb of 5' and 0.3 kb of 3' flanking sequences, into mouse NIH-3T3 fibroblasts and BFS lymphoma T cells and into human epithelial HeLa cells. Stable transformants (NIH-3T3,HeLa and BFS cells) carried an intact transfected IL-2 gene and constitutively expressed cytoplasmic human IL-2 mRNA which was not detected in vector-transfected cells. Constitutive expression of IL-2 mRNA in human IL-2 gene-transfected NIH-3T3 and HeLa cells was associated to the secretion of bioactive IL-2 protein, while no IL-2 production was observed in untransfected or vector-transfected cells. Cytoplasmic IL-2 mRNA observed in transfectants was larger (1.4 kb) than endogenous IL-2 mRNA of human T cells, although smaller than RNA containing unspliced intact introns. No alternative promoters or polyadenylation signals were used by these cells, but some intronic sequences were present in the 1.4 kb mRNA. Phorbol ester and calcium ionophore did not modulate the expression of the transfected IL-2 gene in NIH-3T3 and HeLa cells, while these agents increased its expression in transfected BFS lymphoma T cells. We conclude that when transfected into lymphoid and non-lymphoid cells the intact human IL-2 gene is constitutively expressed, while its phorbol ester/calcium-mediated inducible expression is restricted to T cells. This suggests that the constitutive and inducible expression of the IL-2 gene can be dissociated and are presumably subjected to separate regulatory pathways.

Animals↗
PubMed1990

CD69 is expressed on platelets and mediates platelet activation and aggregation.

CD69, a surface dimer so far considered an early activation antigen restricted to lymphocytes, was found constitutively expressed on human platelets. Biochemical analysis revealed that platelet CD69 appears on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a broad 55-65-kD band, in which three 55-, 60-, and 65-kD components were detectable when nonreduced, and as two 28- and 32-kD bands when reduced, corresponding to the two disulfide-linked chains of the dimer. It therefore closely resembles lymphoid CD69, although the resolution of the three bands under nonreducing conditions is not usually seen in lymphoid cells. Moreover, as CD69 expressed on activated lymphocytes and CD3bright thymocytes, both chains are constitutively phosphorylated. CD69 stimulation by anti-Leu-23 monoclonal antibodies induced platelet aggregation in a dose-dependent fashion. This effect was associated with Ca2+ influx and platelet degranulation, as revealed by adenosine triphosphate release. In addition, CD69 stimulation in platelets induced production of thromboxane B2 and PGE2, suggesting activation of arachidonic acid metabolism by cycloxygenase. As observed for CD69-mediated T cell activation, platelet activation through CD69 requires molecular crosslinking. These results suggest that CD69 may function as an activating molecule on platelets, as on lymphocytes, and point toward a more general role of this surface dimer in signal transduction.

Adenosine Triphosphate↗
PubMed1990

Transcriptional regulation of the interleukin 2 gene by glucocorticoid hormones. Role of steroid receptor and antigen-responsive 5'-flanking sequences.

Dexamethasone treatment of the Jurkat T77 cell clone inhibited the enhancing effect of 12-O-tetradecanoylporbol-13-acetate (TPA) and the calcium ionophore A23187 on the interleukin 2 (IL2) mRNA levels and gene transcription from intact nuclei. Dexamethasone treatment of Jurkat T77 cells inhibited the TPA/A23187-dependent activation of the transcription from the transfected pIL2CAT, containing 600 base pairs of the genomic sequences upstream of the coding region of IL2 gene, including the TPA/calcium responsive cis-regulatory elements and promoter sequences, driving the expression of the chloramphenicol acetyltransferase (CAT) gene. Transfection of either Jurkat T77 cell clone or glucocorticoid-resistant Jurkat cells with a human glucocorticoid receptor cDNA under the transcriptional control of the Rous sarcoma virus long terminal repeat (LTR) (pRShGR alpha) significantly increased or induced, respectively, the dexamethasone-mediated inhibition of the TPA/A23187-dependent expression of pIL2-CAT as well as the enhancing effect on the expression of the cotransfected CAT gene under the control of the mouse mammary tumor virus LTR, as a marker of glucocorticoid receptor action. This suggests a role for the glucocorticoid receptor in mediating the dexamethasone action on IL2 gene expression. To study the cis-regulatory sequence specificity of the dexamethasone-induced interference with the TPA/A23187-mediated T cell activating signals, we studied the effect of the hormone on the regulatory elements contained in the Rous sarcoma virus and human T lymphotropic virus 1 long terminal repeats and the SV40 promoter, which are known to be transcriptionally enhanced by those activating agents. Dexamethasone was unable to interfere with the TPA/A23187-mediated enhancement of these cis-regulatory elements, suggesting that the hormone effect is specific for IL-2 gene sequences. Our data suggest that the dexamethasone-mediated transcriptional inhibition of the IL2 gene is mediated by an interference with the protein kinase C and calcium-mediated trans-activation of the antigen-responsive and T cell-specific elements lying in the 5'-flanking region of the gene.

Animals↗
PubMed1990

Epstein-Barr virus internalization and infectivity are blocked by selective protein kinase C inhibitors.

Selective protein kinase C inhibitors can either block or significantly reduce Epstein-Barr virus infectivity: inhibition of transformation and decreased 3H-thymidine (3H-TdR) incorporation in human B lymphocytes infected with B95-8 EBV, as well as a significant reduction in the induction of early antigens in Raji cells superinfected by P3HRI EBV was achieved by pre-treating the cells with the inhibitors. The inhibitors do not act by blocking binding of the virus to its cellular receptor CR 2, but rather are effective in the viral internalization process. Our results suggest that protein kinase C may be involved in the process of viral entry into cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

Post-transcriptional control of c-myc proto-oncogene expression by glucocorticoid hormones in human T lymphoblastic leukemic cells.

We have studied the regulation of the human c-myc proto-oncogene by glucocorticoid hormones in T lymphoblastic leukemic cells. A significant decrease (50%) of the steady state levels of c-myc mRNA was observed as early as 3 h after dexamethasone treatment of CEM-1.3 human lymphoma cells, reaching less than 5% values, with respect to untreated cells, 24 h after hormone administration. Nuclear run-on experiments showed no modifications of the transcriptional rate from the first exon. However, a slight decrease (15%) of the transcript elongation from the first exon/first intron boundary was observed in the dexamethasone-treated cells. Using actinomycin D to block gene transcription, we have observed a significant increase in the rate of c-myc RNA specific decay after dexamethasone treatment. Furthermore, cycloheximide was able to overcome completely the dexamethasone-induced down-regulation of the c-myc gene expression. Our data suggest that dexamethasone is able to inhibit human c-myc gene expression primarily at the post-transcriptional level, through the synthesis of hormone-induced regulatory protein(s) controlling c-myc transcript stability.

Blotting, Northern↗
PubMed1990

Effects of natural beta-interferon and recombinant alpha-2B-interferon on proliferation, glucocorticoid receptor content, and antigen expression in cultured HL-60 cells.

In the current study, we investigated the effects of natural beta-interferon (beta-IFN) and recombinant alpha-2b-interferon (alpha-IFN) on the growth of the HL-60 cell line. Cells cultured in a medium that contains various concentrations (from 10 to 1000 IU/ml) of interferons showed a growth inhibition, which reaches the maximum after a 6-day treatment, at the highest dose used. Furthermore, we studied the effect of both beta-IFN and alpha-IFN on the level of glucocorticoid receptors. This was enhanced more than 30% with respect to control in HL-60 cells exposed for 24 hours to concentrations of beta-IFN that ranged from 100 to 1000 IU/ml. The increase of the receptor amount was seen even if cells were treated for 5 days, and was not accompanied by a modification of antigen expression of HL-60 cells. alpha-IFN did not modify the glucocorticoid receptor level substantially in our experimental conditions. Our data indicate that both beta-IFN and alpha-IFN regulate HL-60 cell proliferation. Additional studies are required to clarify if modifications of the receptor level induced by beta-IFN could be related to the modulation of hormone-sensitivity in this model.

Antigens↗
PubMed1990

Human keratinocyte growth factor activity on proliferation and differentiation of human keratinocytes: differentiation response distinguishes KGF from EGF family.

Human keratinocyte growth factor (KGF) is an epithelial cell specific mitogen which is secreted by normal stromal fibroblasts. In the present studies, we demonstrate that KGF is as potent as EGF in stimulating proliferation of primary or secondary human keratinocytes in tissue culture. Exposure of KGF- or EGF-stimulated keratinocytes to 1.0 mM calcium, an inducer of differentiation, led to cessation of cell growth. However, immunologic analysis of early and late markers of terminal differentiation, K1 and filaggrin, respectively, revealed striking differences in keratinocytes propagated in the presence of these growth factors. With KGF, the differentiation response was associated with expression of both markers whereas their appearance was retarded or blocked by EGF. TGF alpha, which also interacts with the EGF receptor, gave a similar response to that observed with EGF. These findings functionally distinguish KGF from the EGF family and support the role of KGF in the normal proliferation and differentiation of human epithelial cells.

Biomarkers↗
PubMed1990

Efficacy and toxicity of 5-fluorouracil and folates in advanced colon cancer.

From February 1987 to December 1988, 34 patients with histologically confirmed advanced colorectal carcinoma were entered in a phase II trial with 5-fluorouracil (5-FU) and folinic acid, for evaluation of treatment effectiveness and toxicity. Our data confirmed that the association 5-FU and folates represents an effective and moderately tolerated palliative treatment, with diarrhea being the only dose-limiting toxicity.

Clinical Trials as Topic↗
PubMed1990

High-resolution surface views of human lymphocytes during capping of CD4 and HLA antigens as revealed by immunogold fracture-flip.

The surface ultrastructure of lymphocytes during capping of two transmembrane proteins is shown. As seen by fracture-flip the plasma membranes of human lymphocytes are covered by a high density of surface particles. Incubation in 30% glycerol leads to aggregation of these surface particles. Immunogold labelling shows that the transmembrane proteins bearing HLA class I and CD4 antigens are confined to the particle aggregates. These results indicate that surface particles revealed by fracture-flip represent surface protrusions of integral membrane proteins seen as intramembrane particles in freeze-fractured lymphocytes. During capping HLA or CD4 antigens aggregate into progressively larger patches and, finally, into single caps. As revealed by fracture-flip the patches/caps are seen as clearly differentiated raised platforms that are clearly and sharply demarcated relative to contiguous areas of the surface. In non-patched (non-capped) regions, the pattern of distribution and apparent density of surface particles remain unaltered. Immunogold labelling clearly demarcates patches and caps, and shows that virtually no antigen molecules remain dispersed over the non-patched (non-capped) regions. Estimates of the surface density of either HLA or CD4 antigens over the capped areas point to high planar concentrations of the transmembrane proteins that bear these antigens.

Antigens, Surface↗
PubMed1990

Characterization of Corynebacterium parvum-induced suppressor cells of mouse NK and ADCC activity.

Treatment of mice with Corynebacterium parvum (Cp) resulted in a substantial decrease in natural killer activity in the spleen at 10 days. The decrease in cytotoxicity was associated with the presence of splenic nonadherent (NA) suppressor cells, capable of inhibiting natural as well as antibody-dependent cellular cytotoxicity (ADCC). The nonadherent suppressor cells appeared to be null cells, lacking detectable expression of Thy 1, L3T4 (CD4), Lyt 2 (CD8), or asialo-GM1 and could be physically separated from cells with NK activity by centrifugation on Percoll discontinuous density gradients. Our results indicate that Cp can negatively modulate cytolytic functions of NK cells by inhibiting the effector phase of cytotoxicity.

Animals↗
PubMed1990

Augmentation of mouse natural killer (NK) activity by GM-1/P, a processed form of monosialoganglioside GM-1.

We describe the immunomodulatory activity of GM-1/P a processed form of GM-1 (monosialoganglioside) extracted from ox brain, purified and physically modified. We examined the effect of in vivo and in vitro treatment of GM-1/P on natural (NK) activity and its ability to induce the production of interleukin-2 (IL-2) in the mouse. In vivo treatment with GM-1/P (1 mg/Kg, i.v., day-1) resulted in a marked increase and in a change of distribution of NK activity, which was associated with lower density Percoll fractions. Marked increase was already observed at 18 hrs and then declined by day 4. In vitro treatment with GM-1/P (2 micrograms/ml) enhanced NK activity of B6 spleen cells, already after 6 hours of incubation, remaining at plateau levels within 18 hours. A role of IL-2 in this enhancement was suggested by the ability of an anti-IL-2 rabbit antiserum to abolish in vitro increased cytotoxicity. The presence of IL-2 in the supernatants of splenocytes from GM-1/P (1mg/Kg, i.v., ,day-1) treated mice stimulated with Con A or Con A plus TPA for 48 hrs was evaluated by proliferation of an IL-2 dependent CTLL cell line. GM-1/P by itself was unable to stimulate IL-2 production; however it markedly increased IL-2 production induced by Con A or Con A plus TPA.

Adjuvants, Immunologic↗
PubMed1990

Enhancement of lymphocyte proliferation and IL-2 receptor expression by a processed form (GM-1/P) of monosialoganglioside GM-1.

In this study we investigated the ability of GM-1/P, a calcium mediated processed form of monosialoganglioside GM-1, of in vivo augmenting mouse T and B-lymphocyte blastogenesis induced by mitogens. We have also determined its effect on IL-2 responsiveness by analyzing the induction of the expression of IL-2 receptor (IL-2r) on mouse spleen cells. Lymphocyte blastogenesis was evaluated by 3H-TdR incorporation of spleen cells from untreated or GM-1/P (1mg/Kg, i.v., day-1) treated mice cultured in the presence of T (PHA, ConA) B (LPS) cell specific mitogens. The stimulatory effects appeared to be due to a direct action on T and B lymphocytes, since proliferative response was not abolished by removal of macrophages. Splenocytes from GM-1/P treated mice showed increased proliferation in response to various concentrations of HrIL-2; moreover under these conditions an increased generation of LAK activity was found. A direct evidence for enhanced expression of IL-2r was obtained by immunofluorescence and FACS analysis using a monoclonal antibody (PC.61) directed against the p55 subunit of murine IL-2r. 29% PC.61+ cells were found in IL-2 cultures from treated spleen cells.

Animals↗
PubMed1990

In vivo effects of cytokines on development of natural killer cells and antitumor activity in lethally irradiated bone marrow transplanted recipients.

We have evaluated the effects of combinations of various cytokines on the reconstitution of natural killer (NK) cell activity and resistance to metastases from B16 melanoma, in lethally irradiated mice transplanted with syngeneic bone marrow. Treatment with some combinations of interleukin-2 (IL-2) and other cytokines (IL-2 + IL-1 + TNF alpha or IL-2 + IL-1 + LT) induced appreciably greater and more rapid augmentation of NK cell regeneration than IL-2 alone, as measured in vitro in the 4-h 51Cr release assay against YAC-1 or in vivo in an assay of lung clearance of 125IUdR-labeled tumor cells. The same treatments also induced significant augmentation of in vivo resistance against pulmonary metastases in C57BL/6 mice injected with B16 melanoma cells. These data indicate that stimulation of NK activity in tumor-bearing bone marrow transplanted recipients may be of value in the control of metastatic disease.

Animals↗
PubMed1989

Enhancement of c-erbA proto-oncogene expression by glucocorticoid hormones in S49.1 lymphoma cells.

The modifications of the mRNA levels of the c-myc and c-erbA proto-oncogenes during the dexamethasone-induced decrease of S49.1 cell proliferation have been studied. The levels of c-myc mRNA decreased significantly between 3 and 18 h after dexamethasone (1 microM) treatment. In contrast, a significant increase in the levels of a 2.6 kb c-erbA mRNA was observed between 6 and 18 h after hormone treatment. Cycloheximide treatment of S49.1 cells increased the levels of c-erbA RNA and overcome the enhancing effect of dexamethasone on the expression of this proto-oncogene, suggesting that ongoing protein synthesis is necessary to elicit this hormone effect. The associated decrease of cell proliferation and changes in c-myc and c-erbA mRNA levels after dexamethasone treatment suggest that such oncogenes might be involved in the dexamethasone-mediated control of lymphoid cell growth.

Animals↗
PubMed1989

Rat natural killer cells synthesize fibronectin. Possible involvement in the cytotoxic function.

The ability of NK cells to synthesize and secrete fibronectin (FN), an extracellular matrix glycoprotein which plays a key role in many biologic processes including cellular adhesion, morphology, cytoskeletal organization, cell migration, and invasiveness, was studied. By using affinity-purified polyclonal antibodies directed against human cellular or plasma FN, the presence of FN was evidentiated on Percoll-purified rat large granular lymphocyte or on a large granular lymphocyte tumor cell line (CRC) by flow cytometry and immunoelectron microscopy. Its expression increased after NK cell activation by poly I:C administration. Biochemical analysis by immunoprecipitation and SDS-PAGE indicated that FN was associated to cell surface and secreted in the supernatant in a molecular form similar to that of FN from L929 fibroblasts. In an attempt to understand the role of FN in the NK cell function, we found that an antibody against human plasma FN and its F(ab')2 fragment inhibited NK cytotoxicity against YAC-1 target at the effector cell level. Inhibition occurred at the postbinding level, because F(ab')2 anti-FN inhibited induction of phosphatidylinositol hydrolysis by YAC-1 target cells, whereas binding to target cells was not affected. The possible role of FN in the NK cytotoxic function is suggested.

Animals↗
PubMed1989

Genomic alterations in human breast cancer: a review.

We review and discuss data on the genetic alterations documented in human breast carcinomas at the molecular level. These alterations may result in: 1) deletion of genetic material (chromosome 11p, 13q, 3p, 1q, 17p); 2) amplification of genes or entire chromosomal segments (c-myc, c-erb-B2, locus DF3/PUM, loci on 11q13); 3) rearrangements (c-myc); 4) point mutations (c-ras). Presently available informations do not allow the development of cohesive pathogenetic models but indicate that the molecular basis of human breast cancer is heterogeneous.

Breast Neoplasms↗