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E Fecarotta

Publications and source records attributed to E Fecarotta.

13 recordsLinked to original sources
PubMed2000

Evidence of alloreactive T lymphocytes in fetal liver: implications for fetal hematopoietic stem cell transplantation.

The use of hematopoietic stem cells for in utero transplantation to create permanent hematochimerism represents a new concept in fetal therapy, although this approach has provided heterogeneous results. In this paper we have undertaken molecular, phenotypic and functional studies aimed at identifying the presence of fully competent T lymphocytes in samples of fetal livers and cord blood. We found mature VDJ TCR beta chain transcripts in fetal liver cells taken from 7 to 16 weeks of gestation and a similar pattern was detected in cord blood cells sampled from 13.5 to 20.5 weeks of gestation. A Vbeta8 gene sequence comparable to that detected in adult PBMC was found in fetal liver samples at 9 or 17 weeks gestation. PreTalpha message was detected in all samples and its expression decreased in fetal blood samples with increasing gestational age while Calpha message appeared at 9.4 weeks and its expression increased during gestational age. T cell clones obtained from fetal liver cells showed a mature TCR alphabeta+, CD8+ phenotype and displayed strong alloreactivity against allo-MHC class I molecules. The presence of alloreactive T lymphocytes may explain the failure to engraft in fetuses older than 13 to 16 weeks and may provide insights into fetal liver transplantation. Bone Marrow Transplantation (2000) 25, 135-141.

CD8 Antigens↗
PubMed1996

Alternative splicing of the estrogen receptor primary transcript normally occurs in estrogen receptor positive tissues and cell lines.

Several laboratories have described estrogen receptor mRNA variants created by skipping internal exons. Some of the putative proteins encoded for by these variants have been functionally characterized by transfection analyses. The variant lacking exon 5 would lead, if translated, to a truncated receptor which shows dominant positive transactivation activity in the absence of hormone. It has been postulated that the variant could account for anti-estrogen resistant tumor growth and for expression of the progesterone receptor in estrogen negative tumors. In order to understand the possible role this and other variants may have in the tumorigenesis of mammary tissue we have carried out a thorough analysis of variants expressed in a tumor cell line (MCF-7), in a tumor sample and in a sample of normal breast tissue derived from mammary reduction surgery. We performed rt-PCR analyses followed by hybridization with exon specific oligonucleotide probes. By these means we have detected nine different variants co-expressed in MCF-7 cells and at least the major variants were equally expressed in normal and neoplastic breast tissue. The same is true for the variant lacking exon 5 which, however, resulted to be a variant of low expression in the three samples analyzed. Variant formation appeared to be restricted to the estrogen receptor messenger since several other members of the superfamily of nuclear receptors did not show variant formation. We also have analyzed the effect of the most abundantly expressed variant, the exon 4 lacking variant, on normal estrogen receptor function, on the growth and on the response to estradiol and to tamoxifen of MCF-7 cells. Although over-expressed at high levels this variant has, if any, only marginal effects on the expression of endogenous estrogen regulated genes and on growth and response to the hormone and its antagonist. Although the lack of function of this variant cannot be extrapolated to other variants, their involvement in tumor formation appears rather unlikely since they are also expressed in normal tissue and the single variant is expressed in addition to many others, some of which might have opposing effects. Variant formation is, however, specific for the estrogen receptor and apparently regulated with tissue specificity as our expression analysis in normal mouse tissues shows. Therefore the variants probably have a physiological significance yet to be discovered.

Adenocarcinoma↗
PubMed1996

Prednisone increases apoptosis in in vitro activated human peripheral blood T lymphocytes.

Glucocorticoid hormones (GCH) regulate, through the apoptotic process, the negative selection of immature T cells in the thymus. Because apoptosis seems to occur also in the maintenance of peripheral tolerance, we have investigated whether GCH may induce apoptosis in human mature lymphocytes. Peripheral blood lymphocytes (PBL) or peripheral CD4(+) and CD8(+) T cell subsets were cultured in the presence of phytohaemaglutinin (PHA) or PHA and prednisone (PDN) at 10(-3)-10(-12)M concentrations for 72, 96 and 120h. Cell cycle and membrane antigen expression were evaluated by flow cytometry and DNA degradation was detected by agarose gel electrophoresis. PDN blocks PBL growth in the G1 phase of cell cycle and inhibits both IL-2 receptor (IL-2R) expression and IL-2 secretion. Apoptosis is clearly increased by PDN in PHA-activated human PBL, and the apoptotic effect of PDN is stronger on CD8(+) than on CD4(+) T lymphocytes. All these effects are dose- and time-dependent. The addition of exogenous IL-2 did not rescue lymphocytes from PDN-increased apoptosis. These results show that PDN increases apoptosis in mature activated human peripheral blood lymphocytes, suggesting a possible role of GCH in the maintenance of immune tolerance at post-thymic level.

Adult↗
PubMed1995

Coexpression of multiple estrogen receptor variant messenger RNAs in normal and neoplastic breast tissues and in MCF-7 cells.

Mammary cancers often develop into a hormone-independent and antagonist-resistant growth phase. The molecular mechanisms of this transition are not clear. Recently, it has been proposed that estrogen receptor variants derived from alternative splicing might lead to dominant positive transcription factors acting on estrogen response elements, even in the absence of the hormone. We show here the comprehensive analysis of expression of estrogen receptor variants lacking internal exons in the estrogen receptor-positive mammary carcinoma cell line MCF-7, in a tumor sample, and in healthy breast tissue taken from reduction surgery. Variants are identified by reverse transcription PCR and hybridization to exon-specific oligonucleotide probes. In MCF-7 cells we detected 10 variants including 5 that have not been described before. Skipping one, two, or three exons occurs. The major variants detected in the cell line are also present in normal and neoplastic tissues. Quantitative variations allow no conclusions of a potential involvement of the variants in neoplastic processes. Rather, the variants appear to be present normally and thus might have a physiological role. Given the expression of the variants in normal tissue, and given the expression of potentially dominant positive variants in conjunction with potentially dominant negative ones, we suggest that these variants do not account for hormone antagonist resistance.

Amino Acid Sequence↗
PubMed1994

Estradiol inhibits growth of hormone-nonresponsive PC3 human prostate cancer cells.

Significant inhibition of proliferative activity in PC3 human prostate cancer cells by estradiol is reported, accompanied by experimental evidence for a specific estrogen receptor (ER). Radioligand-binding assays revealed the presence of high affinity sites of estrogen binding in the nuclear compartment of PC3 cells. In addition, using a reverse transcriptase-polymerase chain reaction system, we obtained evidence of either normal or a variant ER mRNA; the latter, which lacks the entire exon 4, is coexpressed with normal ER mRNA and has been recently characterized in our laboratories. The likelihood that the inhibitory effect exerted by estradiol could be mediated by an increase of transforming growth factor beta (TGF beta) production was also investigated. Use of monoclonal antibodies against TGF beta 1 produced a 3-fold increase of growth rate in PC3 cells; this clearly speaks for high levels of endogenous TGF beta 1. This effect was almost completely abolished after addition of 100 nM estradiol. However, we failed to demonstrate any increase of TGF beta 1 mRNA after estradiol administration using Northern blot analysis. Further studies are needed to ascertain whether the estradiol-induced growth inhibition of PC3 cells is either mediated by other TGF beta species or exerted via alternative mechanism(s).

Blotting, Northern↗
PubMed1993

Estrogen receptor variant messenger RNA lacking exon 4 in estrogen-responsive human breast cancer cell lines.

We have identified a messenger RNA coding for a variant estrogen receptor jointly expressed with the normal mRNA in the estrogen receptor-positive, -responsive mammary carcinoma cell lines MCF-7 and ZR 75-1 by means of reverse transcription polymerase chain reaction. This variant mRNA was not observed in estrogen receptor-negative, -unresponsive MDA-MB 231 cells. Partial sequence analysis of the variant complementary DNA revealed identity to sequences of the estrogen receptor exons 3, 5, and 6, but the absence of the entire exon 4. We suggest that this variant receptor messenger is created by alternative splicing. The variant protein is expected to lack most of the hinge domain and part of the hormone binding domain, and it might have a cellular distribution and estrogen-binding affinity different from that of the normal receptor protein.

Amino Acid Sequence↗
PubMed1992

Evidence for the presence of androgen receptors in the synovial tissue of rheumatoid arthritis patients and healthy controls.

OBJECTIVE: To study the presence of androgen receptors in the synovial tissue of male and female patients with rheumatoid arthritis (RA) and matched healthy controls. METHODS: Both site I (high affinity, low binding capacity) and site II (reduced affinity, higher binding capacity) androgen receptors were investigated in soluble and nuclear fractions of homogenized synovial samples, using the dextran-coated charcoal method. The finding of pure, high-affinity site I receptors in both fractions was considered indicative of androgen receptor positivity. In order to determine what type of synovial cell was positive for androgen receptors, cryosections of synovial tissues were immunostained with a specific monoclonal anti-androgen receptor antibody (MAb), using both immunofluorescence and immunoperoxidase techniques. Double immunostaining with this MAb and specific MAb directed toward different macrophage/granulocyte antigens was also performed. RESULTS: Remarkable differences were found between male and female controls: Most males were positive for androgen receptors, and most females were negative. The fetomolar content of androgen receptor in the nuclear fraction was fairly constant, but the soluble fraction showed a higher femtomolar concentration in female RA patients than in controls of either sex, as well as in male RA patients compared with female RA patients. The androgen receptor-positive cells in both RA and control synovial cryosections were found by immunostaining to be macrophage-like synoviocytes, and were also found to be HLA-DR positive. CONCLUSIONS: The immunosuppressive action exerted by androgens might, at least in part, be mediated through their interaction with macrophage-like synoviocytes functioning as antigen-processing and antigen-presenting cells in rheumatoid synovia.

Aged↗
PubMed1992

Soluble and nuclear type I and II androgen-binding sites in benign hyperplasia and cancer of the human prostate.

This paper presents an approach for the assessment of the androgen receptor (AR) status in benign prostatic hyperplasia (BPH) and prostate cancer (PCa) tissues. Evaluation of AR was carried out in both soluble and nuclear fractions by a standard competition method, using tritiated mibolerone as radioligand. Based on our experience with breast and endometrial cancer, this approach focused on both type I (high affinity, low capacity) and type II (reduced affinity, higher capacity) binding sites, aiming mainly at establishing a putative "functional" receptor mechanism, i.e., the presence of type I AR in both cytosol and nucleus. Ancillary studies were carried out to exclude a potential overestimation of the AR content by interference with other steroid receptors, namely, progesterone (PgR) or glucocorticoid (GcR) receptors. Results showed that the interaction by PgR or GcR upon AR measurement was not relevant. The distribution of AR, namely the percent of positivity either in a single or in both cell compartments, was not significantly different in BPH (N = 32) or PCa (N = 24) tissues. For type I binding, the percent of positivity in both soluble and nuclear fractions (i.e., the "functional" AR status) was very close to that observed for other endocrine-related tumors, like breast cancer. Concentrations of type I AR appeared significantly higher in PCa than in BPH tissues; this was true for both soluble and nuclear fractions. In contrast, no significant difference was found in type II AR concentrations in either cell fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1992

The prognosis of breast cancer patients in relation to the oestrogen receptor status of both primary disease and involved nodes.

Nodal involvement is accepted as the best single marker of prognosis in breast cancer. However, there is little information on the sub-division of node-positive patients according to the oestrogen receptor status of the nodal tissue. We have previously reported (Eur. J. Ca. 1987, 23, 31) that, in almost all cases, involved nodes are only oestrogen receptor positive (ER+) in patients whose primary tumours are uniformly ER+. This paper presents clinical follow-up on a larger group of patients with node positive breast cancer. For each patient, both soluble and nuclear receptor concentrations were determined in three separate parts of the primary tumour and in at least one involved node (we have previously defined tumours which contained ER in all six fractions of the primary as HS++, those lacking receptor in some fractions as HS+- and wholly receptor negative tumours as HS--). Median follow-up time was 71.5 months. As expected, patients whose tumours were HS++ had a significant (P less than 0.008) survival advantage. More importantly, patients with ER in both the soluble and nuclear fractions of their involved nodes survived significantly (P less than 0.003) longer than those with ER- nodes. Thus, full oestrogen receptor status of involved nodes will give sufficient prognostic information when adequate primary tissue is not available.

Biomarkers, Tumor↗