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Biomedical subjects

L Frati

Publications and source records attributed to L Frati.

At least 109 records · Page 6Linked to original sources
PubMed1997

Modulation of cytokine gene expression by thymic lympho-stromal cell to cell interaction: effect of retinoic acid.

We have examined the expression of a panel of cytokines in thymic epithelial cells and CD4-CD8- (DN) thymocytes following cell to cell lymphostromal interaction, in an experimental model which enhances in vitro thymocyte maturation. Since retinoic acid (RA) has been previously shown to be an inhibitor of thymocyte maturation process in this model, we wanted to analyse cytokine expression in DN thymocytes and thymic epithelial cells following the RA-induced impairment of in vitro thymocyte maturation. Cell to cell lymphostromal interaction results in increased IL2 and decreased IL7 expression in thymocytes while the expression of IL1 beta and IL7 increased and decreased, respectively, in thymic epithelial cells. Addition of RA to lympho-stromal cell co-culture results in the enhancement of IL4 and IL7 expression in thymocytes while in thymic epithelial cells IL1 alpha decreased and IL6 and IL7 increased. These data indicate that discrete patterns of cytokine expression are present in thymocyte precursors and in thymic epithelial cells during in vitro T-cell development. They furthermore suggest that specific cytokine modulation might contribute to the RA-induced impairment of thymocyte differentiation.

Animals↗
PubMed1997

Microsatellite instability is correlated with lymph node-positive breast cancer.

We analyzed 81 cases of primary breast carcinoma and 7 cases of fibroadenoma for microsatellite instability at eight loci. Twenty-seven cases (33.3%) manifested aberrant microsatellite alleles: 7 (8.6%) at one locus and 20 (24.7%) at two or more loci [tumors with replication error-positive (RER+) phenotype]. No evidence of microsatellite instability was observed in fibroadenomas. We investigated correlations between RER+ phenotype and clinicopathological characteristics of the carcinomas. The RER+ phenotype was statistically associated with large tumor diameter; of 19 RER+ tumors with measured size, 16 were > 2 cm, compared to 28 of 58 tumors with no evidence of microsatellite instability or with shifts in allele sizes limited to one locus (P </= 0.005, chi2 test). Consistently, there was also a strong statistical association between RER+ phenotype and lymph node metastasis; 14 of 19 RER+ tumors with known lymph node status were N+, compared to 15 of 59 tumors with no evidence of microsatellite instability or with allele shifts limited to one locus (P </= 0.0002, chi2 test). Correlations with age of patients, proliferative activity, histotype (ductal versus lobular), and grade of differentiation were not statistically significant, although the RER+ phenotype was more frequent in lobular and high-grade ductal carcinomas, in carcinomas with high proliferative activity, and in carcinomas from patients </=50 years. Data concerning cancer(s) in first and/or second degree relatives were available for 66 cases, including 33 positive and 33 negative for family history of cancer. No correlations were detected between RER+ phenotype and family history of cancer. In conclusion, our results indicate that in breast cancer, microsatellite instability is associated with clinicopathological parameters that are considered predictors of recurrent disease and aggressive behavior.

Adult↗
PubMed1996

Expression of Egr-1 correlates with the transformed phenotype and the type of viral latency in EBV genome positive lymphoid cell lines.

In this paper we have investigated the role of Egr-1 in B cell growth regulation by examining the gene expression in a panel of B cell lines, including both EBV genome negative and EBV carrying cell lines. Egr-1 expression correlates with the cellular phenotype and the specific pattern of viral latency established within the individual cell lines. Thus, constitutive activation of Egr-1 gene is invariably associated with unrestricted expression of viral latent genes in all group III EBV genome carrying cell lines. In contrast, Egr-1 expression is abrogated in group I Burkitt tumor cells, irrespective of the EBV genome carrying status. Activated viral gene expression associated with phenotypic conversion of group I cell lines in to group II or III restores the Egr-1 gene expression. Several forms of EGR-1 protein are found within the different groups of cell lines, and the binding activity to DNA consensus sequences was investigated. Finally, time course analysis of Egr-1 expression during the early steps of EBV infection in vitro demonstrated that Egr-1 is upregulated within minutes from the initial interaction with the B lymphocyte.

B-Lymphocytes↗
PubMed1996

Infection of human T lymphoid cells by human herpesvirus 6 is blocked by two unrelated protein tyrosine kinase inhibitors, biochanin A and herbimycin.

Human herpesvirus 6 is a T lymphotropic herpesvirus that causes exanthem subitum in infants and is considered a potential cofactor in AIDS etiopathogenesis and progression owing to its in vivo and in vitro interactions with human immunodeficiency virus. We report that no differences in phosphorylation on tyrosine residues of cellular proteins were detectable at early times following HHV-6 infection in comparison to uninfected cells. On the contrary, several cellular proteins appeared phosphorylated on tyrosine at 24-48 hr postinfection. In addition, when tyrosine phosphorylation induced by HHV-6 infection was inhibited by the tyrosine kinase inhibitor biochanin A, the infection of HSB-2 cells was also coordinately reduced, as judged by inhibition of cytopathic effect and by inhibition of early and late viral antigen expression. Similar results were obtained with a second unrelated tyrosine kinase inhibitor, herbimycin. The inhibitors seem to act at a late stage of the viral infectious cycle, since neither viral binding nor internalization were affected. Thus, our results indicate that HHV-6 infection leads to the phosphorylation of protein tyrosine kinases, which may play a role in the course of viral infection, probably by participating in the cytopathic effect induced by the virus.

Benzoquinones↗
PubMed1996

Inactivation of the IL-6 gene prevents development of multicentric Castleman's disease in C/EBP beta-deficient mice.

Castleman's disease is a lymphoproliferative disorder thought to be related to deregulated production of IL-6. We have previously shown that mice lacking the trans-acting factor C/EBP beta, a transcriptional regulator of IL-6 and a mediator of IL-6 intracellular signaling, develop a pathology nearly identical to multicentric Castleman's disease, together with increasingly high levels of circulating IL-6. We describe here how the simultaneous inactivation of both IL-6 and C/EBP beta genes prevents the development of pathological traits of Castleman's disease observed in C/EBP beta-deficient mice. Histological and phenotypic analysis of lymph nodes and spleen of double mutant mice did not show either the lymphoadenopathy and splenomegaly or the abnormal expansion of myeloid, B and plasma cell compartments observed in C/EBP beta-/- mice, while B cell development, although delayed, was normal. Our data demonstrate that IL-6 is essential for the development of multicentric Castleman's disease in C/EBP beta-/- mice.

Animals↗
PubMed1996

Expression of trKB neurotrophin receptor during T cell development. Role of brain derived neurotrophic factor in immature thymocyte survival.

The relationships between the nervous and the immune systems raise the question of whether neurotrophic factors, in addition to the regulation of neural cell ontogeny, may influence lymphocyte development. We report in this work that the pattern of neurotrophin receptor expression depends on the developmental stage of T cells. The presence of nerve growth factor receptor trkA could not be detected in any of the thymocyte subsets, whereas brain-derived neurotrophic factor (BDNF) receptor trkB was expressed in all thymocyte subpopulations. Interestingly, both trkB mRNA and protein expression inversely correlated with the maturation stage and the differentiation potential of thymocytes, being more expressed in CD4-8- immature thymocytes and progressively declining in CD8+ and CD4+ single-positive and CD4+8+ more mature thymocytes. The developmentally regulated expression of trkB is further shown by the inhibition or enhancement of trkB expression induced by signals that either trigger or impair the transition from immature to more differentiated stages of the thymocyte developmental pathway. Signals generated following the interaction of BDNF with trkB receptor resulted in the stimulation of trkB autophosphorylation and in the up-regulation of the expression of the c-fos gene in CD4-8- cells and enhanced thymocyte survival. Finally, BDNF is expressed in thymic stroma and is further up-regulated by signals generated by the thymocyte/stromal cell interaction. These data suggest that BDNF may be a novel survival factor for thymocyte precursors and support the presence of developmentally regulated feedback mechanisms based on autocrine/paracrine neurotrophin/receptor interactions that may be involved in the thymocyte differentiation process.

Animals↗
PubMed1996

Prostaglandin E2 inhibits the interleukin-2 promoter activity through down-regulation of the Oct-dependent transcription of the octamer motif.

Prostaglandins, mainly those of the E series (PGE), are modulators of immune responses. Indeed PGE2 inhibits T cell activation and the transcription of the interleukin-2 (IL-2) gene, the major T cell growth factor. We observed that PGE2 inhibits IL-2 promoter transcription activity by interfering with signals activating the (-96 to -66 bp) octamer motif. This motif binds Oct-1 and Oct-2 as well as the phorbol ester and calcium ionophore-inducible jun and fos AP-1 factors. The PGE2-dependent down-modulation is observed in the presence of either the endogenous transacting factor Oct-1 or the exogenously expressed Oct-2. PGE2 does not regulate octamer function by influencing the jun and fos mRNA or Oct-1 protein levels or their DNA-binding abilities. Functional dissection of the octamer motif, through mutations of either the AP-1 or the octamer sites, revealed that the AP-1 site is dispensable for PGE2-dependent inhibition which instead may occur through the interference with the Oct-mediated transactivation of the octamer element. Our data suggest that the Oct-octamer interaction is a novel target of the PGE2-induced down-regulation of the IL-2 promoter.

DNA-Binding Proteins↗
PubMed1996

Bcl-2/bax mRNA expression ratio as prognostic factor in low-grade urinary bladder cancer.

Apoptotic cell death represents an important mechanism for the precise regulation of cell numbers, and a defence mechanism against tumoral cell. bcl-2 and bax genes are known to be involved in the control of apoptotic pathways; in particular, the ratio between bcl-2 and bax represents a cell rheostat that is able to predict a cell's response toward life or death to an apoptotic stimulus. In the present study we investigated the role of bcl-2 and bax gene expression in a panel of 37 low-grade tumours of the urinary bladder, and correlated the expression of these genes to the prognosis of patients in a follow-up of more than one year. We found that levels of bax expression higher than bcl-2 in bladder tumours well correlates to a better outcome for patients. Early relapses are much more frequently observed in those patients whose tumours express more bcl-2 than bax mRNA. We conclude that the bcl-2/bax expression ratio may be considered as a marker for disease progression in low grade bladder tumours, independently of clinical staging and histological grading.

Adult↗
PubMed1996

Cloning of a novel human RNA polymerase II subunit downregulated by doxorubicin: new potential mechanisms of drug related toxicity.

Using the differential display PCR method, we have isolated an mRNA downregulated in doxorubicin resistant human cell lines. The full length cDNA clone was identified as the human homologue of yeast RPB11 subunit of RNA polymerase II. Northern blot analysis of normal tissues detected a particularly high expression of RPB11 mRNA in heart and skeletal muscle. Reduction of this mRNA expression was observed in all the cell lines tested after drug treatment and was paralleled by a similar decrease of the protein levels. These findings suggest that doxorubicin may exert in vivo specific inhibitory effects on a major component of the transcription machinery.

Amino Acid Sequence↗
PubMed1996

Human immunodeficiency virus type 1 tat protein modulates fibronectin expression in thymic epithelial cells and impairs in vitro thymocyte development.

Infection of both lymphoid and stromal components of the thymus by human immunodeficiency virus type 1 (HIV-1) suggests that impairment of lymphocyte differentiation from early T cells progenitors in the thymus may contribute to the HIV-induced T cell depletion. Cross-talk between immature thymocyte and thymic epithelium through cell-to-cell adhesion mediated by fibronectin/receptor interaction plays a central role in driving T cell development. HIV-1 tat protein, like fibronectin, contains an RGD sequence involved in the interaction with fibronectin receptor. We demonstrated that gene transfer-mediated tat expression in thymic stroma is able to influence the in vitro maturation of T cell progenitors as tat-expressing epithelial cells have a decreased ability to drive the generation of CD4+8+ thymocytes from CD4-8- precursors. Furthermore, tat-expressing cells produce more fibronectin and display upregulation of VLA-5 cell surface receptor levels compared to control cells, while alpha v expression was unchanged. Cellular distribution of fibronectin is also influenced by tat. Fibronectin is distributed in the whole cell surface and along cell processes in control cells whereas it is mainly concentrated in the intracytoplasmic area in tat-expressing cells. Therefore, expression of tat in thymic epithelial cells impairs thymocyte maturation and modulates fibronectin expression: this suggests a crucial role of this viral protein in regulating the T lymphocyte differentiation program through modulation of intrathymic lympho-stromal interactions.

Animals↗
PubMed1996

Involvement of an arachidonic-acid-dependent pathway in the interferon-beta-mediated expression of C202 gene in Ehrlich-ascites-tumor cells.

We have investigated the signal transduction mechanism of the expression of the C202 gene mediated by interferon beta (IFN-beta) in the murine Ehrlich's ascites tumor cell line. We have shown that treatment of cells with IFN-beta transiently enhances within minutes the release of free arachidonic acid through membrane phospholipase activity. Furthermore, prior treatment with either p-bromophenacyl bromide, an antagonist of both cytosolic and secretory phospholipase A2, or neomycin, which blocks phospholipase C activity, significantly decreased the activation of the murine IFN-beta-inducible gene, C202. Moreover, an increase of the expression of the C202 gene was observed after blocking of both the cyclooxygenase and lipoxygenase pathways. This suggests that further metabolism of arachidonic acid to epoxides via epoxygenase-catalysed pathways may be a mechanism by which second messengers for IFN-beta-mediated effects on C202 gene expression are generated. Taken together, these results indicate that lipids as second messengers may be important mediators in the IFN-beta-based activation of C202 gene expression.

Animals↗
PubMed1996

CD16-mediated p21ras activation is associated with Shc and p36 tyrosine phosphorylation and their binding with Grb2 in human natural killer cells.

The Src homology (SH) 2/SH3 domain-containing protein Grb2 and the oncoprotein Shc have been implicated in a highly conserved mechanism that regulates p21ras activation. We investigated the involvement of these adaptor proteins in the signaling pathway induced by CD16 or interleukin (IL) 2R triggering in human natural killer (NK) cells. Both p46 and p52 forms of Shc were rapidly and transiently tyrosine phosphorylated upon CD16 or IL-2 stimulation with different kinetics. Shc immunoprecipitates from lysates of CD16- or IL-2-stimulated NK cells contained Grb2 and an unidentified 145-kD tyrosine phosphoprotein. Grb2 immunoprecipitates from anti-CD16-stimulated NK cells contained not only Shc, but also a 36-kD tyrosine phosphoprotein (p36). The interaction between Grb2 and Shc or p36 occurred via the Grb2SH2 domain as indicated by in vitro binding assays using a bacteriologically synthesized glutathione S-transferase-Grb2SH2 fusion protein. We also present evidence that p21ras is activated by CD16 and IL-2R cross-linking. Accumulation of guanosine triphosphate-bound Ras was detected within 1 minute and occurred with kinetics similar to inductive protein tyrosine phosphorylation and Grb2 association of Shc and p36 adaptor proteins.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Effects of the lipidosterolic extract of Serenoa repens (Permixon) on human prostatic cell lines.

BACKGROUND: Permixon is a drug used in the treatment of benign prostatic hyperplasia. We studied its androgenic and antiandrogenic effects in the prostatic cell lines LNCaP and PC3, respectively responsive and unresponsive to androgen stimulation. METHODS: We performed FACScan analysis to investigate toxicity, 3H thymidine and 35S methionine incorporation to determine antiproliferative and metabolic effects, electron microscopy to study ultrastructural changes and cotransfection experiments to elucidate the role of wild type androgen receptor. RESULTS: In LNCaP cell line, Permixon induced a double proliferative/differentiative effect, not observed in PC3 cells. In PC3 cells cotransfected with wild-type androgen receptors and CAT reporter genes under the control of a androgen responsive element, the drug inhibited androgen-induced CAT transcription. CONCLUSIONS: Our data indicate a role of the androgen receptor in mediating the effects of Permixon in LNCaP cells. Cotransfection experiments in PC3 cells support a clear antiandrogenic action of the drug.

Androgen Antagonists↗
PubMed1996

Tyrosine kinase-dependent activation of human NK cell functions upon triggering through CD44 receptor.

We recently showed evidence of CD44-mediated enhancement of natural killer (NK) cell cytotoxic activity and induction of intracellular Ca2+ flux. In this study, we evaluated whether CD44 plays a stimulatory role in NK cell functions, such as cytokine production and activation antigen expression. Our results indicate that ligation of the CD44 receptor results in the induction of expression of the CD69 surface activation antigen as well as in the enhancement of phorbol ester-induced TNF-alpha secretion. We report also evidence for the coupling of CD44 receptor to a protein tyrosine kinase(s) pathway. CD44 engagement rapidly stimulates the tyrosine phosphorylation of several cellular substrates. Pretreatment of NK cells with the tyrosine kinase inhibitor herbimycin A resulted in marked decrease of CD44-stimulated phosphorylation, indicating that it activates tyrosine kinase(s). Furthermore, the drug also prevents CD44-mediated TNF-alpha production and CD69 expression. These findings indicate that protein tyrosine phosphorylation is an early and critical event in CD44-mediated activation of NK cell functions.

Antigens, CD↗
PubMed1996

Human antibodies against the polymorphic epithelial mucin in ovarian cancer patients recognise a novel sequence in the tandem repeat region.

The humoral immune response to the polymorphic epithelial mucin (PEM) was studied by characterising the reactivity of human antibodies generated by EBV-immortalised B-cells from tumour-draining lymph nodes of ovarian cancer patients. All the human antibodies, selected in ELISA for their reactivity to the protein tandem core repeat sequence, reacted with PEM-expressing tumour cells. Aberrant glycosylation of the peptide core of the PEM molecule in cancer cells leads to the exposure of peptide epitopes that can be considered tumour specific. The epitope mapping of six human antibodies revealed that only one of them contained the PDTR sequence, shown to be the immunodominant epitope in the mouse. Four of the six human antibodies recognised a novel common immunogenic sequence (APPAH) in the tandem repeats. The binding of these human antibodies did not appear to be modulated by the length of the carbohydrate side chains, as shown by O-glycosylation inhibition studies. These results indicate that distinct sequences within the tandem repeat of PEM are target for a humoral immune response in humans. The presence of antibodies directed against different epitopes within the same antigenic region may modulate the antigen presentation process and the ongoing immune response. This data may help in clarifying the mechanisms of the immune response to PEM in cancer patients for the development of PEM-based immunotherapy.

Antibodies, Neoplasm↗
PubMed1996

Cellular receptors for sex steroids in human pituitary adenomas.

Cellular receptors for sex steroids (SSRs) were studied in an unselected series of 55 human pituitary tumors. Cytosolic receptors for estrogen (ERcs) and progesterone (PgRcs) were determined in all cases and cytosolic androgen receptors (ARcs) in 47 cases. Nuclear receptors (ERns, PgRns, ARns) were also studied in 33 cases. ERs and PgRs were determined by an ELISA and ARs by [3H]methyltrienolone binding. Where both cytosolic and nuclear receptors were studied (n = 33), ERs, PgRs and ARs were found in at least one subcellular fraction in 66.7, 60.6 and 81.8% of cases respectively, ERs and ARs being mainly recovered from the cytosol and PgRs from the nucleus. No linear correlation was found between pre-operative plasma steroid hormones and their specific cellular receptors. Nonetheless, the differential expression of SSRs according to sex and gonadal status at the time of surgery strongly supports their regulation by the steroid environment in vivo: PgRcs were more frequent in tumors found in women (41.4 vs 15.4%, P < 0.05), whereas a high expression of ERcs and ARcs (> 15 fmol/mg protein) was more common in tumors found in men (34.5 vs 10.3%, P < 0.05 and 54.5 vs 24.0% respectively). PgRs were positively correlated with ERns, indicating the possibility of estrogen priming of their expression, and negatively correlated with ARs in nuclear fractions. SSRs appeared to be widely distributed among pituitary tumors, although, compared with other hormone-secreting groups, prolactinomas displayed a higher ERc expression (34.8 +/- 11.3 vs 4.8 +/- 5.1 fmol/mg protein, P = 0.007) and gonadotroph cell adenomas lower ARc values (1.3 +/- 0.8 vs 38.2 +/- 10.6 fmol/mg protein, P = 0.048). Microadenomas were characterized by a higher PgR expression than macroadenomas, whereas hemorrhagic (macro)adenomas were characterized by a high ER expression (> 90%). The present results indicate that most pituitary tumors are targets for sex steroids, SSR expression being partially triggered by the steroid environment itself. Possible physiopathological and therapeutic implications of these findings are discussed.

Adenoma↗