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G Parmiani

Publications and source records attributed to G Parmiani.

At least 109 records · Page 6Linked to original sources
PubMed1994

Expression of interleukin 1 alpha, interleukin 6, and tumor necrosis factor alpha genes in human melanoma clones is associated with that of mutated N-RAS oncogene.

To assess whether RAS oncogenes may affect the expression of cytokines in tumor cells, the presence of interleukins (IL) 1 alpha, 1 beta, 4, 6, 7, and 8, tumor necrosis factor (TNF) alpha and interferon gamma mRNA has been analyzed by reverse transcriptase-polymerase chain reaction in 19 melanoma clones derived from the metastatic cell line 665/2 and previously characterized for RAS mutation and expression. Five of these clones and the parental cell line showed a mutation at codon 61 of N-RAS that resulted in Gln-->Arg substitution (N-RAS/61+), while in the remaining 14, only the wild-type allele for N-RAS was present (N-RAS/61-). With the exception of interferon gamma and IL-4, all the cytokines tested were expressed by the parental 665/2 cell line, whereas IL-1 alpha, IL-6, and TNF-alpha were coordinately transcribed only in the subset of the clones bearing the mutated N-RAS gene. The other cytokine genes studied (IL-1 beta, IL-4, IL-7, and IL-8) displayed a variable degree of expression, and such an heterogeneity was not correlated to the N-RAS phenotype of the clones. The association between N-RAS oncogene and IL-1 alpha, IL-6, and TNF-alpha expression was also found in a 665/2 subline (665/2/5) in which loss of mutated N-RAS genes simultaneously occurred with the loss of IL-1 alpha, IL-6, and TNF-alpha expression. Direct evidence that N-RAS oncogene could influence the pattern of cytokine expression was provided by the coordinate induction of IL-1 alpha, IL-6, and TNF-alpha messenger RNA achieved in N-RAS/61+ transfectants of the N-RAS wild-type melanoma clone 2/21. Furthermore, IL-1 alpha, IL-6, and TNF-alpha could be detected by enzyme-linked immunosorbent assay in the culture medium obtained from N-RAS/61+ melanoma clones as well as from positive transfectants, indicating that lymphokine mRNA expression triggered by the activated N-RAS oncogene lead to a secreted protein. In an N-RAS/61+ melanoma clone, by adding specific antibodies against each cytokine, it was found that soluble IL-1 alpha exerted a positive control on IL-6 mRNA and a negative one on its own expression. In addition, IL-1 alpha and IL-6 were negatively regulated by soluble IL-6 and TNF-alpha.

Base Sequence↗
PubMed1994

Frequency of cytotoxic T lymphocyte precursors (CTLp) interacting with autologous tumor via the T-cell receptor: limiting dilution analysis of specific CTLp in peripheral blood and tumor-invaded lymph nodes of melanoma patients.

The frequencies of cytotoxic T-lymphocyte precursors (CTLp) that lyse autologous tumor by a T-cell receptor (TCR)-dependent mechanism (specific CTLp) were evaluated by limiting dilution analysis (LDA) using lymphocytes from peripheral blood (PBL) and from surgically resected, tumor-invaded lymph nodes (LNL) in 9 melanoma patients. The frequency of specific CTLp was determined in PBLs and/or LNIs of all patients by a modified LDA assay, enabling us to measure lytic activity on the autologous tumor that could be significantly inhibited by an anti-CD3 monoclonal antibody (MAb). This assay allowed us to detect frequencies of specific CTLp ranging from 1/720 to 1/32,037 in peripheral blood and from 1/328 to 1/22,061 in tumor-invaded lymph nodes. These frequencies indicated that lymphoid populations from PBLs or LNLs of melanoma patients may contain as low as 30 to as much as 3,000 specific CTLp/10(6) lymphocytes. In addition, comparison of wells containing specific CTLp with those showing no inhibition by anti-CD3 MAb indicated that specific CTLp represent between 3 and 88% of all precursors with lytic activity on the tumor. In 6 of 9 patients, no marked differences between PBLs and LNIs in specific CTLp frequencies were found. A 10-fold increase of specific CTLp, in comparison to PBL and LNL, was found only in lymphocytes isolated from a subcutaneous metastasis of one patient. Our results indicate that CTLp interacting with autologous tumor by a TCR-dependent mechanism exist in PBL and LNL of most melanoma patients, although a wide variation in their absolute number is evident among different patients.

Adult↗
PubMed1994

Cytotoxic T lymphocytes recognize tumor antigens of a murine colonic carcinoma by using different T-cell receptors.

To see whether different antigens expressed by the same tumor are recognized by distinct T-cell receptors (TCR), we used cytotoxic T-lymphocyte (CTL) lines known to lyse in vitro the syngeneic BALB/c adenocarcinoma C-26. Four of these CD3+ CD8+ lines showed 4 different patterns of lysis on a panel of MHC-class-I-compatible targets. The activity was H-2d-restricted and could be blocked by anti-CD3 and anti-TCR-alpha/beta monoclonal antibodies (MAbs). The CTL lines were also effective, although to a different extent, in adoptive immunotherapy of mice bearing lung metastases. Phenotypic analysis revealed in all the lines a high frequency of cells positive for CD45, asialo GM1 (ASGM1), lymphocyte-function-associated antigen-1 (LFA-1), intercellular adhesion molecule-1 (ICAM-1) and CD44, but negligible expression of L-selectin (LAM-1) and very late antigen-4 (VLA-4); 2 lines expressed the vitronectin-receptor (VN-R). Analysis of TCR V beta-chains used by the 4 lines showed selective presence of V beta 6, V beta 8.2, V beta 8.3 and of V beta 13 chains. MAbs directed to these V beta chains blocked their lytic activity in vitro. V alpha-chain transcripts of the lines were identified by polymerase chain reaction (PCR) as V alpha ITT11 and V alpha 52 in 2 lines, while one could not be identified. Analysis of V beta s in mixed lymphocyte-tumor-cell cultures (MLTC) of T cells deriving from tumor-infiltrating lymphocytes (TIL) or from spleen of C-26 tumor-bearing or immune animals indicated that the TCR of the CTL lines were representatives of the TCR repertoire recognizing C-26 tumor, since their V beta s were shown to be selectively expanded in MLTC.

Amino Acid Sequence↗
PubMed1994

Multiple sub-sets of CD4+ and CD8+ cytotoxic T-cell clones directed to autologous human melanoma identified by cytokine profiles.

CD4+ and CD8+ cytotoxic T-cell (CTL) clones, selected for T-cell-receptor (TcR)-dependent lysis of the autologous tumor and isolated from peripheral-blood lymphocytes (PBL) or tumor-infiltrating lymphocytes (TIL) of 3 melanoma patients, were characterized for the pattern of 13 different cytokines released by antibody- or tumor-mediated triggering. Induction or enhancement of cytokine release by anti-CD3 monoclonal antibody (MAb) led to the identification of 2 major sub-sets of CD8+ CTL clones on the basis of production of IL-4. Within the 2 groups of IL-4-producing or non-producing clones, further sub-sets could be identified on the basis of differential production of IL-1 beta, IL-2, IL-6, IL-8, IL-10, TNF-alpha, TNF beta and IFN-gamma. A similar analysis performed on a panel of CD4+ CTL clones indicated multiple patterns consistent with at least 4 major sub-sets, but further complexity was evident in each sub-set on the basis of differential production of IL-1, IL2, IL-6, IL-10 and G-CSF. The cytokine profile of CD4+ and CD8+ clones, as determined after anti-CD3 stimulation, was different from the pattern seen after co-culture with autologous tumor, since many clones released cytokines such as IL-4, IL-10, IFN-alpha and -gamma, TNF-alpha and GM-CSF after activation with only 1 of the 2 stimuli. These results indicate that CD4+ and CD8+ CTL clones reacting to human melanoma belong to a highly complex repertoire of functional subsets characterized by distinct cytokine profiles. In addition, the cytokine pattern of each T-cell sub-set can be modulated by changing the activation signals delivered to the T cell.

Antibodies, Monoclonal↗
PubMed1994

Expression of cytokine/growth factors and their receptors in human melanoma and melanocytes.

Human tumors can constitutively express cytokines and growth factors, but the extent of this expression has not been investigated. Using 44 different probes to cytokines, growth factors, and their receptors, we tested 21 melanoma and 5 melanocyte cultures for RNA transcript expression by reverse transcriptase-polymerase chain reaction. With 30 amplification cycles, expression of the cytokines interleukin (IL)-1 beta, IL-6, leukemia inhibitory factor (LIF), IL-7, gro alpha, IL-8 and the p35 chain of IL-12 was detected in more than 60% of melanomas. Concomitant receptors for IL-6 and IL-7 were also detected. IL-1 alpha, IL-5, Rantes, IL-10, interferon (IFN)-beta, tumor-necrosis factor (TNF)-alpha, G-colony-stimulating factor (CSF) and GM-CSF were expressed at lower levels. Melanocytes showed greatly reduced cytokine RNA transcripts, and only gro alpha was consistently detected. No expression of IL-2, IL-3, IL-4, IL-9, the p40 chain of IL-12, IFN-alpha or IFN-gamma RNA transcripts was detected in melanomas or melanocytes. The growth factors expressed by melanomas and, after further signal amplification, by melanocytes were transforming growth factor (TGF)-alpha, epidermal growth factor (EGF), TGF-beta, endothelial-cell growth factor (ECGF), basic-fibroblast growth factor (bFGF), nerve growth factor (NGF) and steel. The receptors EGFR, FGFR, NGFRp70 and c-kit were also expressed by melanomas and melanocytes. These results point to new possible autocrine and paracrine pathways in melanoma biology.

Base Sequence↗
PubMed1994

Loco-regional immunotherapy with recombinant interleukin-2 and adherent lymphokine-activated killer cells (A-LAK) in recurrent glioblastoma patients.

Nine patients with recurrent glioblastoma were given autologous adherent lymphokine-activated killer (A-LAK) cells and interleukin-2 (IL-2) administered directly into the tumor cavity through an Ommaya tube placed during surgery/biopsy. The immunotherapy was well tolerated and the response rate was 33% (one complete response, two partial responses, four with stable disease and two with progressive disease). However, survival 18 months from initial diagnosis did not differ from that reported in the literature for patients treated conventionally. Serial determinations of IL-2 in the tumor cavity during the course of treatment revealed that IL-2 concentrations were sufficient to maintain lymphocyte activation. Since steroid medication was discontinued during treatment and A-LAK cells have greater antitumor activity than standard LAK cells, other factors are discussed that might explain the limited results.

Adult↗
PubMed1994

Interleukin-2 gene-transduced human melanoma cells efficiently stimulate MHC-unrestricted and MHC-restricted autologous lymphocytes.

Two human melanoma lines were transduced by a retroviral vector with the gene of the human interleukin-2 (IL-2) and characterized for their immunological properties in comparison with the parental lines. Transduction resulted in the production of biologically active IL-2 in the average amounts of 2,282 and 2,336 pg/ml per 10(5) cells per 24 hr over 3 and 2 months by the Me14932/IL-2 and the Me1B6/IL-2 lines, respectively. Melanoma-transduced cells lost their tumorigenicity in nude mice. No major changes in the phenotype were observed in IL-2 gene-transduced lines. In fact, more than 90% of cells expressed class I and II(DR) HLA, adhesion molecules, integrins, and melanoma-associated antigens. Irradiation with 100-400 Gy, while inhibiting tumor cell growth in vitro, allowed the release of IL-2 by the transduced cells for at least 5 weeks. The two melanoma lines also maintained susceptibility to lysis by lymphokine-activated killer (LAK) cells and by a HLA-A2-restricted melanoma-specific cytotoxic T lymphocyte (CTL) clone recognizing the melanoma antigen (Melan-A). In a limiting dilution assay, transduced, but not parental melanoma lines unless added with an amount of IL-2 comparable to that released by the transduced cells, were able to expand both nonspecific and melanoma-specific CTL precursors from autologous peripheral blood lymphocytes (PBL). In mixed lymphocytes-tumor cultures, IL-2 gene-transduced melanoma cells stimulated the expansion of major histocompatibility complex (MHC)-unrestricted effectors from autologous PBL, and of CD3+ CD8+ MHC-restricted CTL from tumor-invaded lymph nodes. These results indicate that IL-2 gene transduction does not alter significantly the expression of the immunologically relevant molecules of human melanoma lines while increasing their ability to stimulate both specific and nonspecific lymphocyte responses. These lines will be of value in the vaccination of melanoma patients.

Animals↗
PubMed1994

N-RAS mutations and susceptibility to lymphokine-activated killer (LAK) cells in human melanoma.

RAS oncogene expression has been reported to affect several biological features of rodent tumours cells, including lysability by activated natural killer cells. In order to examine whether expression of mutated RAS genes in human melanoma cells alters their susceptibility to lysis by LAK cells, seven melanoma lines were assessed for the presence of Ki- and N-RAS genes bearing all possible mutations at codons 12, 13 and 61. A panel of 21 clones deriving from the metastatic lesion Me665/2, which had a Gln-->Arg substitution at codon 61 of N-RAS (N-RAS/61+), were also examined. Melanoma cells and clones were used as targets of allogeneic LAK in a 4-h 51Cr-release assay. LAK showed a higher lysis on melanoma lines and clones harbouring a mutated RAS compared with counterparts bearing no RAS mutations. In addition, LAK-mediated lysis drastically decreased on Me665/2 sublines progressively selected by exposure to LAK. This loss was paralleled by a reduction or even disappearance of N-RAS/61+ mRNA signal in Me665/2 sublines. To evaluate whether N-RAS could directly modulate LAK susceptibility to lysis, N-RAS/61+ gene was transfected in two N-RAS wild type (N-RAS/61-) 665/2 melanoma clones by a cosmid vector. In contrast to the high lysability of melanoma cells constitutively expressing the mutationally active N-RAS oncogene, N-RAS/61+ transfectants did not show a consistent high lysability by LAK, compared with some control pSV2neo transfectants.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1994

Successful adoptive immunotherapy with lymphokine-activated killer cells in the treatment of medulloblastoma disseminated via cerebrospinal fluid: case report.

We report the case of a girl who developed cerebellar medulloblastoma at the age of 12 years and in whom, 4 years after surgical removal and radiotherapy, neoplastic dissemination via the cerebrospinal fluid took place. After only partially effective systemic and intrathecal chemotherapy, an intrathecal administration of lymphokine-activated killer cells and recombinant interleukin-2 allowed complete clinical recovery persisting after a follow-up of 30 months.

Adult↗
PubMed1993

Lymphotoxin gene expression by melanocytes and melanoma cell lines and persistence of unspliced mRNA.

Human melanoma cell lines express many different cytokines [1], including lymphotoxin (LT), the production of which has been considered to be restricted to cells of the lymphocytic lineage in response to cell activation. LT expression by melanomas is constitutive and characterized by the presence of two mRNAs. In the present paper we report an analysis of the origin of the two LT-specific transcripts in four human melanoma cell lines at different stages of progression and in four melanocytic cell lines. Reverse transcription-polymerase chain reaction (RT-PCR) performed with primers lying in the first and fourth exons and hybridization with intron probes showed a spliced and a full-unspliced LT mRNA. This pattern was also displayed by one of four melanocyte cell lines. Western blot analysis indicated that LT RNA is properly translated to a 23-25 kDa protein and immunocytochemistry showed its localization within the cytoplasm and on the cell membrane.

Gene Expression↗
PubMed1993

T cell receptor (TCR) structure of autologous melanoma-reactive cytotoxic T lymphocyte (CTL) clones: tumor-infiltrating lymphocytes overexpress in vivo the TCR beta chain sequence used by an HLA-A2-restricted and melanocyte-lineage-specific CTL clone.

HLA-A2+ melanomas express common melanoma-associated antigens (Ags) recognized in vitro by autologous cytotoxic T lymphocytes (CTL). However, it is not known whether tumor Ags can drive in vivo a selective accumulation/expansion of Ag-specific, tumor-infiltrating T lymphocytes (TIL). Therefore, to evaluate this possibility, 39 CTL clones isolated from several independent mixed lymphocyte tumor cultures (MLTC) of TIL and peripheral blood lymphocytes (PBL) of an HLA-A2+ melanoma patient and selected for T cell receptor (TCR)-dependent, HLA-restricted tumor lysis, were used for analysis of TCR alpha and beta chain structure by the cDNA polymerase chain reaction (PCR) technique with variable gene-specific primers followed by sequencing. Despite absence of oligoclonality in fresh TIL and PBL, as well as in T cells of day 28 MLTC (day of cloning), sequence analysis of TCR alpha and beta chains of TIL clones revealed a dominance of a major category of melanoma-specific, HLA-A2-restricted T cells expressing a V alpha 8.2/J alpha AP511/C alpha and V beta 2.1/D beta 1/J beta 1.1/C beta 1 TCR. The same TCR was also found in 2 out of 14 PBL clones. The other PBL clones employed a V alpha 2.1 gene segment associated with either V beta 13.2, 14, or w22. Clones A81 (V alpha 2.1/J alpha IGRJ alpha 04/C alpha and V beta 14/D beta 1/J beta 1.2/C beta 1) and A21 (V alpha 8.2/J alpha AP511/C alpha and V beta 2.1/D beta 1/J beta 1.1/C beta 1), representative of the two most frequent TCR of PBL and TIL, respectively, expressed different lytic patterns, but both were HLA-A2 restricted and lysed only HLA-A2+ melanomas and normal melanocytes, thus indicating recognition of two distinct HLA-A2-associated and tissue-related Ags. Finally, by the inverse PCR technique, the specific TCR beta chain (V beta 2.1/D beta 1/J beta 1.1/C beta 1) expressed by the dominant TIL clone was found to represent 19 and 18.4% of all V beta 2 sequences expressed in the fresh tumor sample and in the purified TIL, respectively, but < 0.19% of V beta 2+ sequences expressed in PBL. These results are consistent with the hypothesis that a clonal expansion/accumulation of a melanocyte-lineage-specific and HLA-A2-restricted T cell clone occurred in vivo at the site of tumor growth.

Amino Acid Sequence↗
PubMed1993

Occurrence of resistance to retinoic acid in the acute promyelocytic leukemia cell line NB4 is associated with altered expression of the pml/RAR alpha protein.

The mechanism(s) by which acute promyelocytic leukemia (APL) cells acquire resistance to all-trans retinoic acid (ATRA) is poorly understood. We describe here an APL cell line, named NB4.306, that shows resistance to the anti-proliferative action of ATRA. This cell line is also operationally resistant to most ATRA-induced phenotypic modifications (CD11b, CD11c, CD13, and CD33). No significant differences in ATRA intracellular accumulation, efflux, or metabolism were found between NB4.306 and the parent NB4 cell line that could explain the observed resistance of the NB4.306 line. The NB4.306 cell line was found to be positive for the t15;17 translocation and showed the usual pml/RAR alpha fusion bands in both Southern and Northern blot assays, but expressed no detectable amount of the usual pml/RAR alpha protein, as assayed by Western blot analysis using an anti-RAR alpha antibody. These results were confirmed in 14 of 14 clones obtained from the NB4.306 cell line, while 30 of 30 clones obtained from the parental NB4 line expressed the usual 110-Kd fusion polypeptide. It is concluded that the occurrence of resistance to ATRA in the NB4.306 cell line is closely associated to the loss of expression of the intact pml/RAR alpha protein.

Blotting, Southern↗
PubMed1993

Regression of an established tumor genetically modified to release granulocyte colony-stimulating factor requires granulocyte-T cell cooperation and T cell-produced interferon gamma.

Using the murine colon adenocarcinoma C-26 cell line, engineered to release granulocyte colony-stimulating factor (G-CSF) (C-26/G-CSF), were studied the mechanisms responsible for inhibition of tumor take in syngeneic animals and of regression of an established tumor in sublethally irradiated mice injected with these cells. Immunocytochemistry and in situ hybridization, performed to characterize tumor-infiltrating leukocytes and their cytokine expression, respectively, indicated that polymorphonuclear leukocytes (PMN) were the major cells responsible for inhibition of tumor take and that they expressed mRNA for interleukin 1 alpha (IL-1 alpha), IL-1 beta, and tumor necrosis factor alpha (TNF-alpha). Expression of interferon gamma (IFN-gamma) and of IL-4 was undetectable, consistent with the absence of T lymphocytes at the site of tumor injection. In mice injected with C-26/G-CSF cells after 600-rad irradiation, the tumors grew to approximately 1.5 cm in 30 d, regressing completely thereafter in 70-80% of mice. During the growing phase, tumors were infiltrated first by PMN (between days 15 and 20), then by macrophages, and last by T lymphocytes. Both CD4+ and CD8+ T cells were present but only CD8 depletion significantly abrogated tumor regression. Depletion of PMN by the RB6-8C5 antigranulocytes monoclonal antibody reduced the number of T cells infiltrating the tumor and prevented tumor regression. In situ hybridization performed at the beginning of tumor regression revealed the presence of mRNA for IL-1 alpha, IL-1 beta, and TNF-alpha, but also the presence of cells, with lymphoid morphology, expressing IFN-gamma. Tumors from mice treated with recombinant IFN-gamma (between days 20 and 35) were rejected faster, whereas mice treated with antibodies to IFN-gamma (from day 20) died of progressive tumor. Cyclosporin A treatment (started at day 20) also abrogated tumor regression. These results indicate that inhibition of tumor take and regression in this model occurs through different mechanisms that involve PMN and PMN-T cell interactions, respectively, as well as a combination of cytokines that, for tumor regression, require IFN-gamma. Thus, gene transfer of a single cytokine gene such as G-CSF into tumor cells appears to be sufficient to trigger the cascade of cell interactions and cytokine production necessary to destroy a cancer nodule.

Adenocarcinoma↗
PubMed1993

Interleukin-6 expression in human neutrophil and eosinophil peripheral blood granulocytes.

Human peripheral blood granulocytes were analyzed for expression of interleukin-6 (IL-6) using reverse-transcriptase polymerase chain reaction (RT-PCR) and in situ hybridization. Neutrophil granulocytes from healthy donors were shown to express variable levels of IL-6. This expression was rapidly down-regulated after the removal of the cells from the circulating blood. In vitro culture of neutrophils abolished IL-6 expression, which could be reactivated by addition of GM-CSF to the culture medium. Constitutive expression of IL-6 was instead demonstrated in eosinophil granulocytes purified from normal donors and from a hypereosinophilic patient. In situ hybridization of unstimulated granulocytes confirmed that IL-6 expression occurs both in eosinophils and in neutrophils from peripheral blood. These findings show that granulocytes can actively contribute to cytokine expression in the peripheral blood and suggest their role in the afferent limb of the immune response, since by IL-6 production they might modulate T- and B-lymphocyte functions, granulocyte self-priming, and endothelial interaction.

Cells, Cultured↗