Biomedical subjects

L Frati

Publications and source records attributed to L Frati.

At least 217 records · Page 12Linked to original sources

PubMed1989

Steroid sensitivity of thymocyte subpopulations during intrathymic differentiation. Effects of 17 beta-estradiol and dexamethasone on subsets expressing T cell antigen receptor or IL-2 receptor.

We have studied the effects of the steroid hormones, 17 beta-estradiol and dexamethasone, on the relative proportion of thymocyte expression of CD4 (L3T4), CD8 (Ly-2), TCR and IL-2R, used to identify different stages of thymocyte differentiation. After short-term in vivo steroid treatment, a significant decrease in the number and proportion of the CD4+/CD8+, double positive subpopulation was observed in parallel with a proportional increase in the percentage of the CD4+/CD8- single positive, of the CD4-/CD8-, double negative and, to a lesser extent, of the CD8+/CD4- subsets. Either steroid treatment increased the proportion of cells expressing either the epsilon-chain of the CD3 complex and/or the beta-chain of the TCR (beta-TCR) (TCR+/CD3+) and the 55 kDa protein of the IL-2R (IL-2R+), related to the increase of CD4+ SP thymocytes and of DN cells, respectively. Furthermore, the increased proportion of CD3+ cells could also be partially related to the increase of both the CD4+ and DN subsets. A decrease of the effect on either DN/IL-2R+ cells or CD4+ SP cells was selectively observed after long-term treatments with 17 beta-estradiol or DEX, respectively. It is concluded that after short-term administration, different steroid hormones mediate a similar selective depletion of DP, TCR-/CD3-, IL2R- cells presumably in an intermediate stage of differentiation. However, either steroid effects evolve differently in long-term treatment schedules, resulting in different effects on early (DN/IL2R+) and late (SP/IL2R-) steps of thymocyte differentiation.

Animals↗

PubMed1989

Altered restriction pattern of the putative DNA binding domain of estrogen receptor or related genes in primary human meningiomas.

We analyzed by restriction mapping the genomic organization of the estrogen receptor gene in several primary human brain tumors in order to investigate the possible relationships between the development of these tumors and gonadal steroid hormones. In 5 out of 23 meningiomas the Eco RI restriction of genomic DNAs revealed 6 invariant normal fragments of 6.5, 4.8, 3.8, 3.1, 2.7 and 1.7 kb, plus 2 additional variant fragments of either 8.6 or 2.5 kb. More detailed analysis showed that these variant bands hybridized with a probe specific for the middle region of the estrogen receptor cDNA, coding for the DNA-binding domain of the receptor. This abnormal restriction pattern was found only in these meningiomas and not in other brain tumors or DNAs obtained from peripheral blood lymphocytes.

Blotting, Southern↗

PubMed1989

Molecular cytochemistry of CD3 and CD4 antigens in human lymphocytes as studied by label-fracture and by fracture-label.

Label-fracture and fracture-label membrane immunocytochemistry are used to analyze the surface distribution, dynamics and partition on fracture of CD3 and CD4 antigens of human T lymphocytes. Redistribution of the antigens, induced by treatment at 37 degrees C with specific monoclonal antibodies, results in patching and capping of the labeling as observed in label-fractured specimens. Examination of platinum/carbon replicas of freeze-fractured plasma membranes of antibody-treated cells does not reveal recognizable domains of intramembrane particles. However, in cells where the aggregation of intramembrane particles is induced by incubation with glycerol, colloidal gold-labeled CD3 and CD4 molecules are seen confined to particulate domains of the membrane. Therefore, the lack of visible aggregation of intramembrane particles in patched or capped regions of the membrane implies that migration of CD3 and CD4 antigens with concentration in domains of the membrane is achieved contemporaneously with export of other non-capped integral membrane proteins from the same regions, in a process of diffusional equilibrium. Examination of fracture-labeled specimens shows that CD4 molecules partition on fracture with the inner protoplasmic face of the plasma membrane. This partition illustrates the transmembrane attitude of the antigen molecule and is a probable consequence of interaction of the protein with other components of the membrane or with the cytoskeleton.

Antibodies, Monoclonal↗

PubMed1989

Granzyme A expression by normal rat natural killer (NK) cells in vivo and by interleukin 2-activated NK cells in vitro.

Granzyme A (TSP-1) is a serine esterase expressed in cytotoxic T and natural killer (NK) cell lines. Its presence in in vivo primed T cells is disputed at present. Here we show that normal rat NK cells contain granzyme A in vivo and that its expression is augmented by their short-term in vitro treatment with IL 2. Granzyme A expression in a NK cell population with LAK activity has also been examined.

Animals↗

PubMed1989

Effects of protein kinase C (PK-C) activators and inhibitors on human large granular lymphocytes (LGL): role of PK-C on natural killer (NK) activity.

The role of protein kinase C (PK-C) in the early metabolic events involved in human natural killer (NK) cell activation has been studied through the action of PK-C-specific activators and inhibitors. Highly purified human large granular lymphocytes (LGL) were treated for 1 hr with the diacylglycerol analog 1-oleoyl-2-acetyl glycerol (OAG) (10(-4)-10(-5) g/ml) or with 12-O-tetradecanoylphorbol-13-acetate (TPA) (10(-8)-10(-10) g/ml), both specific activators of PK-C. Both these agents consistently increased NK activity against K562 target cells. Suboptimal doses of either OAG or TPA also synergized with Ca2+ ionophores to augment spontaneous cytotoxic activity. Pretreatment of LGL with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrocloride (H7) (5-40 microM), a potent PK-C inhibitor, greatly reduced NK activity in a time- and dose-dependent fashion. By contrast, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (HA 1004), a potent cAMP- and cGMP-dependent PK inhibitor with almost no effect on PK-C, marginally reduced NK activity. Moreover, almost complete NK activity inhibition was observed when H7 (10 microM), but not HA 1004 (50 microM), was present in the NK assay. Finally, 48 hr stimulation of LGL with TPA (10(-6) g/ml), a treatment able to inactivate most of the PK-C cellular pool, almost completely abrogated NK activity. This functional evidence was supported by phosphorylation of several endogenous substrates which occurs within 5 min in TPA-treated LGL. Two proteins of 70 and 56 kDa have been identified as major PK-C substrates, together with other phosphorylated proteins with MW ranging from 177 to 43 kDa. H7, but not HA 1004, almost completely inhibited the TPA-induced phosphorylation of all of these proteins in the NK cells. These data strongly suggest that selective activation of PK-C plays an essential role in the mechanisms of NK cell activation.

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

PubMed1989

Laminin synthesis by NK cells and modulation of its expression by TPA (12-O-tetradecanoylphorbol-13-acetate).

Natural killer (NK) cells have been suggested to play a major role in resistance against metastatic spread of tumors. This study was aimed at understanding whether laminin (LM), a component of the extracellular matrix involved in the mechanism of tumor invasion and cell interaction, is expressed by NK cells. The results indicate that NK cells can synthesize and display on the cell surface LM and that TPA can modulate its expression. Our findings suggest that the presence of LM on NK cells could be relevant in the control of tumor invasion by NK cells.

Animals↗

PubMed1989

Epidermal growth factor binding and steroid receptor content in human benign prostatic hyperplasia.

The receptor for epidermal growth factor (EGF-R) was characterized on membrane fractions from human benign prostatic hyperplasia (BPH). Specific binding of [125I]EGF reached equilibrium after 40 min at 25 degrees C and was stable for up to 120 min. Saturation analysis of EGF-R, performed by incubating the membranes with 0.0156-15 nM [125I]EGF in the presence and in the absence of 100-fold excess of cold EGF for 60 min, revealed the presence of two classes of binding sites with high and low affinities (Kd = 0.35 +/- 0.23 and 9.60 +/- 2.87 nM respectively). Competition experiments revealed that FSH, insulin and calcitonin did not compete with [125I]EGF. The simultaneous determination of EGF-R and that of estradiol (ER), progesterone (PR) and androgen receptors (AR) was performed using the same buffer to homogenate the tissues and to obtain cellular membranes. The steroid receptors (SR) were determined by means of the dextran-coated charcoal method. There was a significant negative correlation between nuclear SR and binding capacity of EGF-R. The presence of specific and high affinity binding sites for EGF and the modulation of the level of these sites by steroid receptors suggest a possible role of EGF in prostatic hyperplasia.

Biomarkers↗

PubMed1989

Localization of Epstein-Barr virus envelope glycoproteins on the inner nuclear membrane of virus-producing cells.

Epstein-Barr virus-producing cells were used as a model to analyze, with a fracture-immunolabel technique, the distribution, behavior on fracture, and extent of glycosylation of viral transmembrane glycoproteins at the inner nuclear membrane. Surface and fracture immunolabeling with two monoclonal antibodies directed against the carbohydrate or polypeptide portions of the major viral envelope glycoproteins gp350/220 showed the following. (i) The glycoproteins present on the inner and outer nuclear membranes were labeled only with the monoclonal antibody directed against the polypeptide chain, whereas over the surface of virus-producing cells and on mature virions the labeling was dense and uniformly distributed with both monoclonal antibodies. (ii) The glycoproteins were nonuniformly distributed only over the inner nuclear membranes; at the sites of viral budding, the glycoproteins showed a preferential partition with the protoplasmic face. Since fully glycosylated glycoproteins were not present on the nuclear membranes, our observations support the proposed model of herpesvirus maturation. The peculiar distribution and partition on fracture of the envelope glycoproteins on the inner nuclear membrane are similar to those of Sindbis virus envelope glycoproteins on the plasma membrane of infected cells. Therefore, our results suggest that inner nuclear membranes may behave like plasma membranes during viral assembly.

Antigens, Viral↗

PubMed1989

Capping of HLA antigens in human lymphocytes as followed by immunogold label-fracture.

We used immunogold label-fracture to follow the migration of HLA I class and HLA II class antigens during capping as induced by specific monoclonal antibodies. Capping is achieved through a process of clustering and "consolidation" of clusters into larger patches and, finally, a single cap. All receptors appear to cluster from the very start, with no "stray" molecules joining already formed patches. Characterization of exoplasmic and protoplasmic fracture-faces of capping cells fails to reveal any corresponding accumulation of intramembrane particles and/or subtler rugosities. Our results are consistent with the concepts that view the migration of capping molecules as contemporaneous with the efflux of noncapping integral membrane proteins.

Antibodies, Monoclonal↗

PubMed1989

Tumor-promoting phorbol ester and ras oncogene expression inhibit the glucocorticoid-dependent transcription from the mouse mammary tumor virus long terminal repeat.

Oncogene activation has been suggested to play some role in determining the hormone independency of tumors. In order to study the role of protein kinase C in mediating the inhibition of the glucocorticoid-dependent transcription from the Mouse Mammary Tumor Virus (MMTV)-Long Terminal Repeat induced by overexpressed activated ras oncogene, we studied the effects of protein kinase C activators [the tumor promoting phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA)] and inhibitors [1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7)] on the dexamethasone (DEX)-mediated activation of a MMTV-Long Terminal Repeat-chloramphenicol acetyltransferase (pMMTV-CAT) chimeric reporter gene transiently transfected into NIH-3T3 cells and in Ha-ras-transformed fibroblasts (T24-NIH-3T3). TPA (30 ng/ml) together with DEX (0.1 microM) treatment of NIH-3T3 cells resulted in a significant decrease of CAT activity from pMMTV-CAT, compared to DEX treatment alone. The addition of H-7 (40 microM) was able to overcome the TPA-induced inhibition of DEX-dependent transcription from pMMTV-CAT. DEX-dependent expression of pMMTV-CAT was significantly reduced in T24-NIH-3T3 with respect to wild-type NIH-3T3 cells. Treatment of T24-NIH-3T3 cells with either H-7 or TPA significantly enhanced or decreased, respectively, the DEX-dependent expression of pMMTV-CAT. TPA and/or H-7 did not affect CAT activity from either pMMTV-CAT in the absence of DEX or from CAT gene under the control of the SV40 promoter. Similar glucocorticoid receptor sites and binding affinities were observed in T24-NIH-3T3 or TPA-treated NIH-3T3 cells compared to wild-type untreated cells. Our data suggest that activation of PKC is involved in the reduced transcriptional regulatory activity of glucocorticoid hormone induced by overexpressed Ha-ras oncogene in NIH-3T3 fibroblasts.

Animals↗

PubMed1989

The irresistible fascination of medical theories about opposites.

Theurgical medicine was a part of the eternal fight between good and evil and health was reconciliation with the gods. This duality characterized ancient medicine, i.e. Greek medicine after the Homeric age or Chinese traditional medicine. In the passage to medicine of observation due to the School of Cos duel between good and evil becomes substrate of new medicine and the balances between*opposites represented by elements and qualities were the fundaments of the humoralism. Fascination of opposites continues for centuries up to now, both in western and far eastern medicine: yin/yang, antibody/antigen, cAMP/cGMP, oncogene/antioncogene are examples of this attractive theory. Although fundaments of biological and medical observations are the basis of theories of opposites, the trend is to overcome reality and today represents, following idealism in the 19th century, an inconscious ancestral reminiscence of theurgical philosophy and medicine.

History, Ancient↗

PubMed1989

Overnight incubation of mouse spleen cells in recombinant IL-2 generates cytotoxic cells with NK characteristics from precursors enriched with or devoid of LGL.

Interleukin-2 (IL-2) augments natural killer (NK) activity as well as generating effector cells named lymphokine activated killer cells (LAK) which are capable of lysing a wide spectrum of target cells. A large body of evidence has been accumulated to evaluate the relationship between NK and LAK cells and conflicting results have been reported. Our study was addressed to further analyse this relationship and in particular to investigate whether in a short incubation IL-2 is merely capable of augmenting the activity of pre-existing killer cells, or whether it can also promote the differentiation of precursor cells. Eighteen-hour culture of mouse spleen cells in human recombinant IL-2 induced a DNA-synthesis-independent generation of cytotoxic cells bearing an NK phenotype (aGM-1+, Thy1.2+/-, CD8-, CD4-). These were generated from precursor cells also bearing an NK phenotype, recovered either from low density Percoll fractions enriched in lytic cells with LGL morphology as well as from high density fractions devoid of LGL and cytotoxic activity.

Animals↗

PubMed1988

MicroELISA detection of thymosin alpha 1 released in thymic organ cultures.

Using a polyclonal specific rabbit anti-thymosin alpha 1 a highly sensitive enzyme-linked immunosorbent assay (ELISA) has been developed to measure thymosin alpha 1. Production of thymosin alpha 1 was detected in both thymic organ cultures and in mouse serum. The method is rapid (5 h), reproducible and easy to perform.

Animals↗

PubMed1988

Proliferative effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and calcium ionophores on human large granular lymphocytes (LGL).

The proliferative responses of natural killer (NK) cells to 12-O-tetradecanoylphorbol-13-acetate (TPA), which directly activates protein kinase c(PKC), and to the Ca2+ ionophores A23817 and ionomycin, known to enhance the intracellular calcium, have been investigated. Highly purified large granular lymphocytes (LGL) were cultured for 12-30 hr in the presence of TPA, ionomycin, or A23817. TPA alone (1-20 ng/ml) triggered rapid LGL proliferation, whereas the calcium ionophores were ineffective. The addition of either calcium ionophore to suboptimal doses or TPA (0.1-0.5 ng/ml) resulted in a synergistic effect on LGL proliferation. Under these conditions high levels of IL-2 activity were released by the LGL. Phenotypic analysis revealed the rapid loss of the Fc gamma receptors (CD16) on LGL and the induction of the expression of IL-2 (CD25) and transferrin receptors and of HLA-DR, but not of CD3. Removal of extracellular Ca2+ by addition of EGTA at the beginning of the culture greatly depressed LGL proliferation and IL-2 production, and blocked phenotypic changes, such as the expression of Tac antigen. Finally, progression to the proliferative phase of LGL, activated by TPA alone or with ionomycin, was completely abrogated by a hyperimmune anti-IL-2 antiserum.

Antigens, Surface↗

PubMed1988

Nonrandom distribution of epidermal growth factor receptors on the plasma membrane of human A431 cells.

The localization of epidermal growth factor (EGF) receptors over the plasma membranes of human epidermoid carcinoma A431 cells was analyzed at the electron microscopic level using surface replica techniques and conventional thin sections, in combination with immunocytochemistry. Immunolabeling was performed using two distinct monoclonal antibodies directed against the extracellular portion of the receptor, followed by protein A-colloidal gold conjugates. Unexpectedly, with the first monoclonal antibody used, the distribution of the receptors in both unfixed and glutaraldehyde-fixed cells was clearly regionalized, showing a preferential localization of the immunolabeling at the cell periphery as well as over the areas rich in microvilli and in coated and uncoated pits. A similar pattern of distribution was observed also with the other monoclonal antibody, but only when the cells were fixed with glutaraldehyde before immunolabeling. Treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate modifies this distribution, inducing a more disperse pattern. Our observations suggest that a minor group of EGF receptors, which may represent the high-affinity receptors, presents a regional distribution, similar to that described for typical recycling receptors.

Carcinoma, Squamous Cell↗

PubMed1988

Wheat germ agglutinin fracture-label of Golgi apparatus membranes in proliferating cells.

The thin section fracture-label technique has been recently used to analyze the distribution and compartmentalization of fully glycosylated components on intracellular membranes. Labelling with the lectin wheat germ agglutinin over the freeze-fractured membranes of Golgi apparatus in various secretory and non-secretory cells as well as in human peripheral lymphocytes was always very weak or absent even over the trans-most cisternae. In order to investigate if the labelling density may reflect the cellular activity in membrane biogenesis, we used in this study wheat germ agglutinin fracture-label of rapidly proliferating cells and mitogen-activated lymphocytes. Labelling over the fractured cisternae of the medial and trans portions of the Golgi apparatus was intense. Treatment with cycloheximide of proliferating cells induced a drastic reduction of the labelling over the Golgi cisternae.

Cell Division↗

PubMed1988

Treatment of severe psoriasis with somatostatin: four years of experience.

Over a period of 4 years, 20 patients suffering from severe forms of psoriasis (erythrodermic, sub-erythrodermic, resistant generalized forms and/or forms associated with acute arthropathy) were treated with 96 h of continuous i.v. infusion of somatostatin (Stilamin, Serono) diluted in D5W at 250 micrograms/h. In addition to the usual blood chemistry parameters, circadian levels of growth hormone (GH) and epidermal growth factor (EGF) were measured before, during, and after therapy. Approximately 2-3 weeks after termination of therapy, erythrodermic and suberythrodermic symptoms had disappeared. In some patients, a few lesions of psoriasis vulgaris remained, although they were much less severe. Remission of acute arthropathy was impressive. Blood chemistry parameters were unchanged after therapy. Circadian levels of GH and EGF, normal before therapy, were significantly decreased after therapy. The infusion was well-tolerated. Infusion rates of greater than 250 micrograms/h caused only some complaints of abdominal pain, nausea, and vomiting. During the 4 years, erythrodermic symptoms reappeared only in seven patients, three of whom were also arthropathic. After 6-8 months, they underwent a second course of somatostatin therapy with good results. The other patients are still able to control their disease with tar-based products alone or with low-dose 8-methoxypsoralen + UVA (PUVA) or UV therapy. The arthropathic patients control their symptoms with periodic low-dose nonsteroidal antiinflammatory drug therapy.

Epidermal Growth Factor↗

PubMed1987

TPA induction of Epstein-Barr virus early antigens in Raji cells is blocked by selective protein kinase-C inhibitors.

We have shown that modulation of intracellular calcium in EBV latently infected cells could induce the expression of viral antigens, and suggested that a protein kinase-C (PKC) may play a major role in the EBV genome activation. We now report further investigations on the role of PKC using 2 selective enzymatic inhibitors [1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine] (H-7) and Staurosporine. We show that these inhibitors can abrogate the inductive effect of TPA or the combination of TPA plus n-butyrate. The inhibitors have no effect on induction by calcium ionophores or by viral superinfection. In this context the effect of verapamil (a specific calcium channel blocker) and of several calmodulin antagonists was investigated. No inhibitory effect of these agents could be demonstrated on any of the induction systems examined. These observations strengthen the idea that in some instances cellular PKC plays a role in the expression of viral antigens; however, alternative regulatory mechanisms cannot be excluded.

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