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L Frati

Publications and source records attributed to L Frati.

At least 181 records · Page 10Linked to original sources
PubMed1992

Patching and capping of LFA-1 molecules on human lymphocytes.

The distribution and dynamics of LFA-1 molecules over the surface of human lymphocytes were analysed using immunogold label-fracture and fracture-flip methods. Patching and capping were induced by incubation at 37 degrees C with antibodies directed against the alpha and beta chains respectively of the heterodimeric LFA-1 molecule, and were followed by immunofluorescence. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) to link LFA-1 molecules to the cytoskeleton increased the percentage of capped cells, implying a faster and more efficient process of capping. At all times of clustering or upon phorbol ester treatment, the concentration of LFA-1 in patches and then in caps was not accompanied by a parallel concentration of membrane particles on the freeze-fractured plasma membranes. Our results support the role of the cytoskeleton in regulating the capping phenomenon and in controlling the structural organization of the plasma membranes.

Antibodies↗
PubMed1992

Surface distribution and internalization of erbB-2 proteins.

We report the localization over the cell surface and the early steps of antibody-induced internalization of the product of the erbB-2 proto-oncogene, structurally related to the epidermal growth factor receptor (EGFR). We show that erbB-2/p 185 is mostly excluded from endocytic pits on the cell surface. Incubation at 37 degrees C with an anti-erbB-2/p185 monoclonal antibody induces the rapid entry of the protein into the cell. Similar internalization is shown by a chimeric molecule EGFR/erbB-2 in response to EGF. Both the timing and the pathway of internalization followed by the erbB-2/p185 appear totally similar to those described for the EGFR. At variance with the normal erbB-2/p185, two mutant activated erbB-2 proteins are frequently localized within endocytic pits of the cell surface, indicating that mutations in the transmembrane regions may determine constitutive internalization of the protein.

3T3 Cells↗
PubMed1992

Infection by human herpesvirus 6 (HHV-6) of human lymphoid T cells occurs through an endocytic pathway.

We have analyzed by immunoelectron microscopy the early events of binding and internalization of human herpesvirus 6 (HHV-6, strain GS) on a susceptible T-lymphoblastoid cell line, HSB-2. The virions bound to the cell surface at 4 degrees C were tightly associated with the plasma membrane. Gold immunolabeling of the viral envelope proteins was strong and specific. Warming at 37 degrees C for different times showed viral internalization through smooth surfaced pits and vesicles. Fusion events of the virions with the cell plasma membrane were never observed. Gold immunolabeling performed in parallel experiments before or after viral internalization showed: (1) absence of viral envelope proteins on the cell plasma membranes at all times of internalization, again excluding fusion events; (2) entry of the virions with their envelopes. Treatment of the cells with chloroquine, a drug known to affect the endocytic pathway, led to an almost complete inhibition of viral infectivity, suggesting that the endocytosed virions are responsible for a successful infection. Comparable results were obtained using a second strain of HHV-6 (BA92), with biologic and molecular characteristics similar to the prototype strain Z29. The chloroquine inhibition was effective on two different T cell lines (HSB-2 and J-Jhan), as well as on phytohemagglutinin-stimulated peripheral blood mononuclear cells.

Cell Line↗
PubMed1992

TNF impairs in vivo and in vitro natural killer (NK) susceptibility of B16 melanoma cells.

Tumour necrosis factor alpha (TNF) is a multipotent cytokine which affects many biological properties of both normal and neoplastic cells. Here we show that treatment with TNF reduces B16-A melanoma cell susceptibility to normal and in vivo- and in vitro-activated NK cell-mediated killing. This resistance is associated with an enhancement of B16-A metastatic potential in normal syngeneic mice, but not in anti-asialo GM1-treated animals, further supporting the NK dependence of TNF-induced enhancement of metastatic ability. A significant increase of MHC class I expression on B16-A murine melanoma cells is observed after TNF treatment. In all these effects TNF interacts positively with interferon gamma (IFN gamma). Taken together, these results indicate that TNF treatment negatively affects the susceptibility of B16-A murine melanoma to NK effectors in vivo and in vitro. This decreased susceptibility may be related, at least in part, to enhanced expression of MHC class I antigens on tumour cells.

Animals↗
PubMed1992

Charge and pH effect on the early events of Epstein-Barr virus fusion with lymphoblastoid cells (Raji).

Fusion of Epstein-Barr virus (EBV) with Raji cells was measured after exposure of the virus to neutral or low pH, enzymatic modification of the viral spike glycoproteins, or chemical modification of the target membrane. The relief of octadecylrhodamine (R18) fluorescence self-quenching was used to monitor fusion. Fusion of EBV with Raji cells at pH 5.9 was significantly enhanced compared to that at neutral pH. Treatment of Raji cells with agents known to modify the surface net charge (trinitrobenzene sulfonic acid) totally prevented fusion at a neutral pH. Desialylation of EBV significantly reduced the extent of fusion with Raji cells. Our results demonstrate that EBV is rapidly internalized and then fuses with lymphoblastoid cells in the endocytic vesicles.

Cells, Cultured↗
PubMed1992

[Does an efficacious adjuvant treatment exist in resected colonic carcinoma?].

The prognosis of colon cancer after curative resection is mainly related to the onset of metastases, and especially of liver metastases. In order to prevent metastatic recurrences, the value of adjuvant medical therapy is widely admitted. The aim of the present review was to analyse the conclusions of the main recent randomized trials assessing the comparative value of different adjuvant protocols. The results obtained using either classic systemic infusion or intraportal infusion, which is mainly used with the intent of preventing liver metastases, are reported. At term of this review, we conclude that: adjuvant chemotherapy using combined drugs (5-Fluorouracil + Methyl CCNU, 5-Fluorouracil + Oncovin) did not prove to be more active than 5-FU alone. the beneficial action of a combined 5-FU + Levamisole regimen has been clearly demonstrated for patients with a Dukes C tumor. intraportal adjuvant therapy has been shown to be effective for patients with Dukes B tumors in only one limited trial but this remains to be confirmed. On the basis of the present data, new adjuvant programs using combined chemotherapeutic and immunotherapeutic compounds, and combined systemic and regional infusion, can be envisaged.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1991

Regional heterogeneity and complementation in the expression of the tumor-associated glycoprotein 72 epitopes in colorectal cancer.

We analyzed the immunohistochemical expression of three epitopes of the tumor-associated glycoprotein 72 (TAG-72) in whole cross-sections of primary colorectal carcinomas and in regional lymph node metastases using monoclonal antibodies (MAbs) B72.3, CC-49, and CC-83, which recognize distinct carbohydrate antigenic determinants. B72.3, CC-49, and CC-83 reacted with 13 of 27 (48%), 25 of 27 (92%), and 21 of 27 (77%) carcinomas, respectively. The immunoreactivity with lymph node metastases followed a similar pattern; MAb CC-49 was again the most reactive of the three antibodies, since it labeled 13 of 15 metastatic lesions. Positive reactions of the MAbs with the primary tumors were not always predictive of the immunorecognition of their metastases. Distinct areas within whole cross-sections of TAG-72-positive primary carcinomas demonstrated marked differences in the expression of the three epitopes. CC-49 tended to react with the highest number of areas and with the highest percentages of carcinoma cells within each area. In no instances did B72.3 demonstrate reactivity superior to that of either CC-49 or CC-83. Tumors negative for the CC-49 epitope in any area also did not express the other two TAG-72 epitopes. However, the comparison of the immunostaining obtained with each MAb in TAG-72-positive primary lesions revealed areas where CC-83 was clearly more reactive than CC-49. Moreover, one lymph node metastasis, negative for CC-49, was recognized by CC-83. Thus, the combined use of MAbs CC-49 and CC-83 resulted in additive immunostaining of primary and metastatic colorectal carcinoma cells. The study provides evidence of intratumoral heterogeneity in the glycosylation pattern of the TAG-72 antigen in colorectal cancer and emphasizes the advantages of cocktails of anti-tumor-associated antigen MAbs in the immunodetection of colorectal tumor cells.

Antibodies, Monoclonal↗
PubMed1991

Immunohistochemical evidence of immune responses to tumor-associated antigens in lymph nodes of colon carcinoma patients.

The authors investigated by immunohistochemical study the drainage of three tumor-associated antigens in unaffected regional lymph nodes of colon cancer patients. The study was conducted using monoclonal antibodies (MoAb) directed against different epitopes of the tumor-associated glycoprotein, TAG-72 (CC-49, CC-83, B72.3), of the carcinoembryonic antigen (CEA) (COL-4, COL-12), and of the colon-associated antigen, CAA (anti-CAA). The authors detected immunohistochemical reactions of MoAb CC-49 and anti-CAA with antigen-presenting cells (APC), such as peritumoral and sinus macrophages and lymphatic endothelial cells and with specific areas of germinal centers in lymph nodes draining 11 of 24 colorectal carcinomas studied. The corresponding primary tumors expressed the TAG-72 and CAA antigens. No immunostaining was detectable in lymph nodes using the anti-CEA MoAb, even when the primary tumors strongly expressed the specific epitopes. In germinal centers of regional lymph nodes, the immunostaining was often distributed at the periphery with a characteristic crescentic or circular pattern, which strongly suggested the exposure of the specific epitopes defined by MoAb CC-49 and anti-CAA on follicular dendritic cells. This would indicate that these epitopes are selectively recognized and presented to germinal center B-cells. This phenomenon may have clinical and diagnostic implications.

Antibodies, Monoclonal↗
PubMed1991

GTP-binding proteins transduce signals generated via human FC gamma receptor IIIA (CD16).

This study demonstrates that GTP-binding proteins regulate Fc gamma RIII-mediated signal transduction and inositol phosphate (IPn) generation in human NK cells. In addition the cross-linking of CD16 by mAb, guanosine 5'-o-3-thiophosphate induced 1,4,5 inositol trisphosphate (IP3) release in permeabilized NK cells and their membranes. By contrast, guanosine 5'-o-2-thiophosphate, almost completely inhibited IP3 generation induced by cross-linking with anti-CD16 mAb. Pretreatment of NK cells with 10 to 100 ng/ml Vibrio cholerae toxin (Ctx) almost completely inhibited the generation of IP3 and of other Ipn as well as Fc gamma RIII-operated cell functions such as antibody-dependent cell-mediated cytotoxicity against antibody-coated P815 mastocytoma cells. Isolated B subunit of Ctx was inactive. Bordetella pertussis toxin (0.1 to 1 microgram/ml) only marginally affected IP3 release and antibody-dependent cell-mediated cytotoxicity. Ctx increased cAMP levels in NK cells. However, inhibition of IP3 release preceded the rise of cAMP. Moreover, cAMP analogues (8-chlor-cAMP, 8-bromo-cAMP, dibutiryl-cAMP), as well as intracellular cAMP-enhancing agents (PGE1, PGE2, and forskolin) did not mimicked the effects of Ctx on IP3 generation, suggesting that the adenylate cyclase pathway is not responsible for the early effects of Ctx on Fc gamma RIII-mediated signalling. Overall these results demonstrate that signal transduction via Fc gamma RIII is mediated by Ctx-sensitive cellular membrane GTP-binding protein.

Adenylate Cyclase Toxin↗
PubMed1991

Upregulation of epidermal growth factor receptor induced by alpha-interferon in human epidermoid cancer cells.

Unregulated or increased expression of epidermal growth factor receptor (EGF-R) is a common event in neoplastic transformation; modulation of such a receptor by physiological agents could be, therefore, of clinical interest. We have studied the binding ability, the availability at cell surface, and the synthesis of EGF-R in the A431 and KB human epidermoid cancer cell lines after treatment with recombinant alpha-interferon (IFN-alpha). After 48 h of treatment, IFN-alpha induces, in both cell lines, growth inhibition and enhances class I major histocompatibility HLA complex expression, which is a common marker of IFN action. [125I]EGF total binding assessed after 48 h of treatment with IFN-alpha shows a dose-dependent upregulation of EGF-R binding capacity. Saturation plots of the binding data show that IFN-alpha treatment does not dramatically alter the affinity of the EGF-R and indicate that IFN-alpha only increases the number of low affinity receptors. We show that this effect is due to a specific increase in the synthesis of the receptor protein, as assessed by immunoprecipitation of [35S]methionine-labeled cell extracts. Electron microscopy analysis has confirmed an increase of EGF-R proteins at cell surface without major changes in the morphology of the cells. Taken together, these results indicate that IFN-alpha consistently induces both the binding capacity and the synthesis of EGF-R in human epidermoid cancer cells and suggest the use of such a mechanism for new anticancer therapies.

Binding Sites↗
PubMed1991

Enhancement of natural-killer-cell susceptibility of human breast-cancer cells by estradiol and v-Ha-ras oncogene.

Natural killer (NK) cells are putative components of the cellular immune response to transformed cells. Since both estradiol treatment and ras-oncogene overexpression enhance tumorigenicity of hormone-dependent breast-cancer cells, we studied the effects of estrogen and of the activated v-Ha-ras oncogene on NK susceptibility of MCF-7 human breast-cancer cells. MCF-7 cells were sensitive to cytolysis mediated by resting and IL2-activated peripheral-blood non-adherent lymphocytes. Lysis appeared to be mediated by NK cells, since it was abrogated by treatment of effector cells with alpha-CD16 monoclonal antibody (MAb) plus complement (c'). Estradiol treatment of MCF-7 cells was able to significantly increase their sensitivity to the lysis by IL2-activated and unactivated peripheral-blood lymphocytes, as early as 24 hr throughout 10 days of hormone treatment. Hormone-insensitive, estrogen-receptor-negative breast-cancer cells (BT20) did not change their NK susceptibility after estradiol treatment. Increased NK susceptibility was also observed in v-Ha-ras-transfected and oncogene product overexpressing MCF-7 cells (MCF-7-ras) with respect to cells transfected with the selectable gene marker gpt alone (MCF-7-gpt). Overexpression of v-Ha-ras appeared to be able to bypass the need for estrogen to increase NK susceptibility, since estradiol-treated MCF-7-ras cells were not lysed more than untreated MCF-7-ras cells. The enhancement of NK susceptibility observed after both estradiol treatment and v-Ha-ras overexpression suggests that the hormone-mediated and the ras-oncogene-mediated signalling systems share events involved in the control of tumor-cell/host-effector-cell interactions.

Breast Neoplasms↗
PubMed1991

Human natural killer cells express VLA-4 and VLA-5, which mediate their adhesion to fibronectin.

Very late Ag (VLA)-3, VLA-4, and VLA-5, belonging to the beta-1 subfamily of integrins, have been recently identified as receptors for different binding regions of fibronectin (FN). We have detected VLA-4 and VLA-5, but not VLA-3, on fresh CD3-, CD16+, CD56+ human NK cells by flow cytometry and immunochemical analyses using mAb directed against beta-1, alpha-3, alpha-4, and alpha-5 subunits. Binding assays, performed on FN-coated plates, showed that NK cells specifically adhere to FN and their binding capacity is increased by MgCl2 but not by CaCl2. Using as inhibitory probes a polyclonal antibody against the beta-1 chain of the human FN receptor, the synthetic peptide GRGDSP, which is able to inhibit cellular adhesion mediated by VLA-5, the CS1 fragment, which contains the principal adhesion site in the IIICS domain recognized by VLA-4, and functional mAb directed against alpha-4 or alpha-5 subunits, we show that both VLA-4 and VLA-5 mediate the adhesion of human NK cells to FN. The expression of these integrin receptors may be relevant for NK interaction with extracellular matrix components and other cell types.

Amino Acid Sequence↗
PubMed1991

In vivo modulation of the distribution of thymocyte subsets: effects of estrogen on the expression of different T cell receptor V beta gene families in CD4-, CD8- thymocytes.

Estrogen treatment of mice has been shown to deplete CD4+, CD8+ double-positive (DP) thymocytes and to alter the relative proportion of CD4+ and CD8+ single-positive (SP) thymocytes. In this work, we have studied the effect of the steroid hormone 17 beta-estradiol (E2) on the different subsets of CD4-/CD8- double-negative (DN) thymocytes by analyzing the expression of CD5, CD3-epsilon and of several V beta gene family products of the T cell antigen receptor (TCR). After in vivo administration of E2 a significant decrease in the number and proportion of dull CD5+, CD3-, beta-TCR- DN thymocytes was observed. In contrast E2 treatment significantly increased the proportion of bright CD5+, CD3+, beta-TCR+ DN cells. The E2-induced increase in DN/TCR+ cells was observed for subsets expressing V beta 6, V beta 8, and V beta 11, but not V beta 3 gene products of the TCR. Thus, estrogen administration results in a selective inbalance of the DN thymocyte subsets by depleting an immature, dull CD5+, CD3-, TCR beta- DN subset, while enriching a mature, bright CD5+, CD3+, TCR beta+ DN subset of cells. In addition to TCR beta+ DN thymocytes, an increased proportion of CD4+ and CD8+ SP thymocytes expressing V beta 8, V beta 6, and V beta 11, but not V beta 3, TCR proteins was also observed after E2 administration. An involvement of intrathymic cytokine production in mediating the hormone action is suggested by the ability of estrogen to increase the levels of IL-1 alpha mRNA of intact thymus. Our data suggest that estrogen exerts its effects on a broad range of immature cells, including dull CD5+, CD3-, beta-TCR- DN and DP thymocytes.

Animals↗
PubMed1991

Retinoic acid-induced down-regulation of the interleukin-2 promoter via cis-regulatory sequences containing an octamer motif.

Retinoic acid (RA) is known to influence the proliferation and differentiation of a wide variety of transformed and developing cells. We found that RA and the specific RA receptor (RAR) ligand Ch55 inhibited the phorbol ester and calcium ionophore-induced expression of the T-cell growth factor interleukin-2 (IL-2) gene. Expression of transiently transfected chloramphenicol acetyltransferase vectors containing the 5'-flanking region of the IL-2 gene was also inhibited by RA. RA-induced down-regulation of the IL-2 enhancer is mediated by RAR, since overexpression of transfected RARs increased RA sensitivity of the IL-2 promoter. Functional analysis of chloramphenicol acetyltransferase vectors containing either internal deletion mutants of the region from -317 to +47 bp of the IL-2 enhancer or multimerized cis-regulatory elements showed that the RA-responsive element in the IL-2 promoter mapped to sequences containing an octamer motif. RAR also inhibited the transcriptional activity of the octamer motif of the immunoglobulin heavy chain enhancer. In spite of the transcriptional inhibition of the IL-2 octamer motif, RA did not decrease the in vitro DNA-binding capability of octamer-1 protein. These results identify a regulatory pathway within the IL-2 promoter which involves the octamer motif and RAR.

Base Sequence↗
PubMed1991

Partition of epidermal growth factor receptors on freeze-fractured plasma membranes of A431 cells is affected by the ligand.

The fracture immunolabel technique, which permits assessment of the partition of transmembrane proteins with the inner or outer leaflets of the freeze-fractured membrane, was used to analyze the behavior on fracture of epidermal growth factor (EGF) receptors over the plasma membranes of A431 cells. The receptors partition mainly with the outer leaflet of the freeze-fractured plasma membranes, whereas they become associated with the inner leaflet when they are occupied by the ligand. This modified partition is even more evident after receptor clustering induced by incubation with EGF at 37 degrees C. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) decreases the number of receptors over both inner and outer leaflets. An effect similar to that induced by the ligand is obtained when receptor aggregation is achieved using anti-receptor monoclonal antibodies (MAb). The modified partition therefore indicates receptor activation and appears to be a consequence of receptor cross-linking rather than to reflect a conformational change of the receptor molecule. Parallel immunolabeling with anti-phosphotyrosine antibodies of freeze-fractured EGF-treated A431 cells reveals that the receptors, when activated, are associated only with the inner leaflet of the plasma membrane.

Cell Line↗
PubMed1991

Inhibition of NK cell generation by Corynebacterium parvum.

Treatment of mice with Corynebacterium parvum (Cp) resulted in a substantial decrease of splenic NK activity associated with a reduced number of LGL. Cp also inhibited in vitro augmentation of NK cytotoxicity by IFN or IL-2 as well as generation of LAK activity. Localization experiments by using radiolabelled LGL indicated that the lower number of LGL in the spleen was not attributable to a Cp-induced alteration of LGL homing. Finally Cp was found to affect the ability of bone marrow cells to reconstitute NK activity in lethally irradiated mice, indicating that it can interfere with development of NK cells from bone marrow progenitors.

Animals↗
PubMed1990

Complementary reactivities of anti-carcinoembryonic antigen and antitumor-associated glycoprotein 72 monoclonal antibodies in lung carcinomas.

Monoclonal antibodies (MAbs) COL-4 and COL-12, to the carcinoembryonic antigen (CEA), and B72.3, CC-49, CC-83, to the tumor-associated glycoprotein 72 (TAG-72), were used to study the expression of distinct epitopes of the two molecules in 71 cases of lung carcinoma of differing histotype. These MAbs reacted with the majority of adenocarcinomas by immunoperoxidase on tissue sections, but demonstrated a more restricted reactivity with squamous carcinomas. MAb CC-49 detected the highest percentages of adenocarcinoma cells while the B72.3 epitope was expressed more in squamous carcinoma cells. No significant reactivity with any of these MAbs was observed in small cell carcinomas. The expression of the CEA and TAG-72 epitopes in non-small cell lung cancers was highly heterogeneous: a distinct epitopes in non-small cell lung cancers was highly heterogeneous: a distinct epitope could be expressed by the majority of cells, whereas another of the same antigenic molecule was either poorly or not expressed. In adenocarcinomas, mixtures of anti-CEA, anti-TAG-72, and anti-(TAG-72 plus CEA) MAbs resulted in additive reactivity with an increase of the immunopositive tumors and of the percentages of immunostained cells. This was particularly evident for the anti-(TAG-72 plus CEA) mixture. In squamous cell carcinomas the increase was modest and was mainly related to anti-TAG-72 reactivity. These studies suggest variability in the antigenic structure of tumor-associated antigens expressed by carcinomas and indicate that anti-(TAG-72 plus CEA) mixtures may represent an immunological adjunct for clinical application in adenocarcinoma patients. On the other hand, TAG-72 should be considered a better target antigen, as compared to CEA, in the detection of squamous cell carcinomas.

Adenocarcinoma↗