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Biomedical subjects

W H Fridman

Publications and source records attributed to W H Fridman.

At least 55 records · Page 3Linked to original sources
PubMed1999

Modulation of interleukin-18 expression in human colon carcinoma: consequences for tumor immune surveillance.

The production in colon cancer of interferon-gamma (IFN-gamma), a type-1 T-helper (TH1) cytokine, is considered as a marker of good prognosis. We asked whether interleukin-18 (IL-18), which strongly induces IFN-gamma and regulates Fas ligand (Fas-L)-dependent cytotoxicity, may play a role in colon homeostasis, and if its expression was modulated in colon adenocarcinomas. We analyzed 14 specimens of colon adenocarcinomas, 6 of normal colon mucosa of the series, and 6 colon-tumor cell lines. The expression of IL-18, of ICE protease, involved in the processing of this cytokine, and of the downstream effectors of IL-18, IFN-gamma and Fas-L was analyzed by RT-PCR. We further performed IL-18 immunostaining of normal and tumor specimens. The results were correlated with tumor dissemination and clinical outcome. We report the synthesis of IL-18 in human normal colon, mainly by epithelial cells of the mucosa. Out of the 6 tumor cell lines, 4 expressed IL-18 transcripts, but neither ICE mRNA nor secreted forms of IL-18 were detected. We observed decreased or abolished synthesis of IL-18 in colon adenocarcinomas, as compared with normal mucosa. Thus, half of the colon-cancer tissues (7/14 cases) expressed neither IFN-gamma nor Fas-L. This feature was correlated with the existence of distant metastases (Fischer's exact test, p = 0.02) and an unfavorable outcome. These findings suggest that production of IL-18 in human colon may play a role in homeostasis and in tumor immune surveillance, by enhancing IFN-gamma production and Fas-L-dependent cytotoxicity of immune cells.

Caspase 1↗
PubMed1999

Vav and SLP-76 recruitment by cross-linking of FcgammaRIIa1 in promyelocytic HL-60 cells.

The signaling events induced upon cross-linking of the human FcgammaRIIa1 (CD32) which contains an immune receptor tyrosine-based activation motif (ITAM) in its intracellular region, were investigated in the promyelocytic HL-60 cells. It is shown here that the FcgammaRIIa1 engagement recruits the Ras pathway in these cells, as evidenced by the tyrosine-phosphorylation of the Shc adaptator protein and of the extracellular signal-regulated kinase 2 (ERK2). However, p95vav, a molecule able to interact with Rac-1 and to regulate cytoskeletal reorganization, was also found to be phosphorylated. In addition, co-immunoprecipitation experiments demonstrated that Vav is associated with SLP-76 upon FcgammaRIIa1 activation. A strong phosphorylation of p120cbl was also observed. The phosphorylation of molecules such as p95vav, SLP-76 and p120cbl suggests that FcgammaRIIa1 triggering also activates signaling pathways other than the Ras pathway.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Regulation of production of soluble Fc gamma receptors type III in normal and pathological conditions.

CD16 (Fc gamma R type III), a low affinity IgG Fc receptor, is found in two forms, a transmembrane Fc gamma RIIIa expressed by NK cells and monocytes and a phosphatidylinositol-linked Fc gamma RIIIb present on neutrophils. Exposure of neutrophils to inflammatory signals induces a rapid loss of CD16 expression and release of a soluble form of CD16 (sCD16). Soluble CD16 circulates in plasma, levels being reduced in sera from patients with multiple myeloma. In the present manuscript the authors summarize work that aimed to better understand: (i) the role of proteinases in sCD16 production and CD16 membrane shedding; and (ii) the regulation of sCD16 levels in multiple myeloma patients and the possible biological consequences of its decrease in this disease. Soluble CD16 was purified from human serum. Its N-terminal sequencing demonstrated that it originates from neutrophil CD16 and its C-terminal sequencing showed that the cleavage site was between Val 196 and Ser 197, close to the membrane anchor. Analysis of the effect of protease inhibitors revealed that the cleavage leading to sCD16 production by PMA-activated neutrophils was metalloproteinase-dependent. In addition, membrane and sCD16 were sensitive to serine proteinases released by azurophil granules or added under purified form. The reduction of sCD16 levels that occurs in patients with multiple myeloma was associated with a slight decrease in circulating neutrophils, but not with a significant defect in sCD16 production by neutrophils, as detected in vitro. Moreover, addition of a recombinant sCD16 to plasmocytoma lines did not significantly modify their proliferation and Ig secretion.

Amino Acid Sequence↗
PubMed1999

Control of tumor development by intratumoral cytokines.

The local immune reactions may influence the clinical outcome of human tumors. In carcinoma of the cervix, high gene expression of IL6 with tumor invasiveness whereas lack of gene expression of IFNbeta is correlated with poor prognosis. In colorectal cancer, lack of expression of IFNbeta is associated with the presence of distant metastasis and poor survival. The production of IL17 and IL18, inducers of IL6 and IFNbeta respectively is regulated in these tumors and may control the levels of the effector cytokines, i.e. IL6 and IFNbeta. The mechanisms by which these cytokines act are linked to the recruitment of effector cells such as macrophages.

Animals↗
PubMed1999

Negative regulation of c-kit-mediated cell proliferation by Fc gamma RIIB.

Fc gamma RIIB are single-chain low-affinity receptors for IgG that bear an immunoreceptor tyrosine-based inhibition motif in their intracytoplasmic domain and that negatively regulate immunoreceptor tyrosine-based activation motif-dependent cell activation. They are widely expressed by cells of hematopoietic origin. We investigated here whether Fc gamma RIIB could also negatively regulate protein tyrosine kinase receptor (RTK)-dependent cell proliferation. As an experimental model, we used growth factor-dependent mast cells that constitutively express Fc gamma RIIB and c-kit, an RTK prototype. We found that anti-c-kit Abs mimicked the effect of stem cell factor and induced thymidine incorporation in Fc gamma RIIB-/-, but not in wild-type (wt) mast cells unless Fc gamma RIIB were blocked or anti-c-kit F(ab')2 were used. When coaggregated with c-kit by intact Abs in wt mast cells, Fc gamma RIIB inhibited thymidine incorporation, as well as cell proliferation, and inhibition was correlated with an arrest of cells in G1 during the cell cycle. The coaggregation of c-kit with Fc gamma RIIB did not affect ligand-induced c-kit phosphorylation and induced the tyrosyl-phosphorylation of Fc gamma RIIB, which selectively recruited the Src homology 2 domain-bearing inositol 5-phosphatase SHIP. Our results indicate that IgG Abs to growth factors or growth factor receptors may control RTK-dependent proliferation of a variety of cells that express Fc gamma RIIB.

Animals↗
PubMed1999

Differential roles of N- and C-terminal immunoreceptor tyrosine-based inhibition motifs during inhibition of cell activation by killer cell inhibitory receptors.

Killer cell inhibitory receptors (KIRs) inhibit NK and T cell cytotoxicity when recognizing MHC class I molecules on target cells. They possess two tandem intracytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs) that, when phosphorylated, each bind to the two Src homology 2 domain-bearing protein tyrosine phosphatases SHP-1 and SHP-2 in vitro. Using chimeric receptors having an intact intracytoplasmic KIR domain bearing both ITIMs (N + C-KIR), a deleted domain containing the N-terminal ITIM only (N-KIR), or a deleted domain containing the C-terminal ITIM only (C-KIR), we examined the respective contributions of the two ITIMs in the inhibition of cell activation in two experimental models (a rat mast cell and a mouse B cell line) that have been widely used to analyze KIR functions. We found that the two KIR ITIMs play distinct roles. When coaggregated with immunoreceptor tyrosine-based activation motif-bearing receptors such as high-affinity IgE receptors or B cell receptors, the N + C-KIR and the N-KIR chimeras, but not the C-KIR chimera, inhibited mast cell and B cell activation, became tyrosyl-phosphorylated, and recruited phosphatases in vivo. The N + C-KIR chimera recruited SHP-1 as expected, but also SHP-2. Surprisingly, the N-KIR chimera failed to recruit SHP-1; however, it did recruit SHP-2. Consequently, the N-terminal ITIM is sufficient to recruit SHP-2 and to inhibit cell activation, whereas the N-terminal and the C-terminal ITIMs are both necessary to recruit SHP-1. The two KIR ITIMs, therefore, are neither mandatory for inhibition nor redundant. Rather than simply amplifying inhibitory signals, they differentially contribute to the recruitment of distinct phosphatases that may cooperate to inhibit cell activation.

Animals↗
PubMed1999

Immunoglobulin variable regions usage by B-lymphocytes infiltrating a human breast medullary carcinoma.

Breast medullary carcinoma are heavily infiltrated by B-lymphocytes and associated with a good prognosis despite their high histological grade. We investigated the Ig repertoire of B-lymphocytes infiltrating one such tumour. A single cell suspension was obtained from a tumor specimen by enzymatic digestion. VH, Vkappa, and Vlambda regions were amplified by RT-PCR using mixtures of primers optimized to maximize the diversity of the PCR products. They were then cloned and sequenced. Analysis of 9 VH, 5 Vkappa, and 10 Vlambda sequences using the Kabat database indicated that several VH and VL region subgroups (I, II and III) are expressed by B-lymphocytes infiltrating this tumor. The analysis of CDR3 regions also showed a variability, although some VH and VL clones exhibited identical or nearly identical sequences. Thus, the B-cell infiltration observed in this breast medullary carcinoma does not reflect a monoclonal proliferation and represents an oligoclonal or a polyclonal B-cell proliferation.

Amino Acid Sequence↗
PubMed1999

Generation of phagocytic MAK and MAC-DC for therapeutic use: characterization and in vitro functional properties.

Phagocytic cells with macrophage or dendritic cell phenotype, able to capture and ingest tumor cells, were derived in large numbers from peripheral blood mononuclear cells using two different activation procedures. Peripheral blood mononuclear cells were stimulated in nonadherent conditions in the presence of human AB serum with either granulocyte-macrophage colony-stimulating factor and dihydroxy-vitamin D3 for 7 days and with interferon-gamma for the last 18 hours to obtain activated macrophages (MAK) or with granulocyte-macrophage colony-stimulating factor and interleukin-13 for 7 days (with fresh interleukin-13 added on day 4) to obtain macrophage-dendritic cells (MAC-DC). A strong ability of MAC-DC to phagocytose yeasts was observed, in contrast to a low-intermediate phagocytosis capacity by MAK. Both CD14+ FCgammaR+ (FcgammaRI/CD64, FcgammaRII/CD32, FcgammaRIII/CD16) MAK and CD1a+/CD86+, CD14- MAC-DC were able to phagocytose whole tumor cells. However, only MAK phagocytosis was enhanced by FcgammaR engagement. MAK but not MAC-DC could lyse tumor cell in antibody-dependent cell cytotoxicity assays, via FcgammaRI. Thus, MAK as well as MAC-DC may represent valuable tools for different in vivo therapy strategies that do or do not include the use of monoclonal antibodies.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1999

Ras and p53 intracellular targeting with recombinant single-chain Fv (scFv) fragments: a novel approach for cancer therapy?

Intracellular expression of recombinant antibodies allows one to interfere with the functions of oncogenic molecules expressed in various cell compartments and has therefore a vast clinical potential in cancer therapy. We inhibited the functions of oncogenic Ras mutant forms by intracellular expression of a neutralizing single-chain antibody (scFv). In vitro studies indicated that the scFv is expressed in the cytosol of Xenopus laevis oocytes and of tumor cells, blocks ras-mediated activation processes, and induces tumor cell death. In vivo studies performed using scFv cDNA inserted into an adenoviral vector showed that the scFv dramatically affects tumor growth. Second, intracellular expression of scFvs directed against p53 indicated that these antibody fragments can be successfully targeted to cell nucleus, bind p53, and partially restore the transcriptional activity of p53 mutants in human tumor cells. Thus, intracellular scFvs directed against oncogenic molecules may represent a new class of antitumor agents.

Animals↗
PubMed1999

Soluble CD16 in plasma cell dyscrasias.

Soluble forms of Fc gammaR type III (sFc gammaRIII or sCD16) are present in many biological fluids. Their main ligand is IgG in the form of complexes. In plasma, sCD16 essentially derive from cleavage of membrane CD16 (or Fc gammaRIII) present on neutrophils and, to a lesser extent, on NK cells. Determination of sCD16 serum level during monoclonal gammopathies has demonstrated markedly reduced levels in multiple myeloma and in monoclonal gammopathy of undetermined significance (MGUS) rapidly evolving to multiple myeloma, compared to stable MGUS or controls, indicating a prognostic value for this biological parameter. The biology and functions of sCD16 are described, together with the biological significance of modifications of the sCD16 serum level in monoclonal gammopathies.

Biomarkers↗
PubMed1998

Highly resolved chemical imaging of living cells by using synchrotron infrared microspectrometry.

Using synchrotron radiation as an ultra-bright infrared source, we have been able to map the distributions of functional groups such as proteins, lipids, and nucleic acids inside a single living cell with a spatial resolution of a few microns. In particular, we have mapped the changes in the lipid and protein distributions in both the final stages of cell division and also during necrosis.

Animals↗
PubMed1998

Intracellular expression of an antibody fragment-neutralizing p21 ras promotes tumor regression.

Mutated ras genes are found in a large number of human tumors and, therefore, constitute one of the primary targets for cancer treatment. Microinjection of the neutralizing anti-Ras monoclonal antibody Y13-259 was previously reported to induce transient phenotypic reversion of ras-transformed rodent fibroblasts in vitro. We have prepared a single-chain Fv fragment (scFv) derived from Y13-259, and here, we show that intracellular expression of the scFv led to the specific inhibition of the Ras signaling pathway in Xenopus laevis oocytes and NIH3T3 fibroblasts. Moreover, neutralizing Ras with the scFv specifically promoted apoptosis in vitro in human cancer cells but not in untransformed cells. As a step toward cancer gene therapy, we finally demonstrated that intratumor transduction of HCT116 colon carcinoma cells with the anti-Ras scFv using an adenoviral vector elicited sustained tumor regression in nude mice.

Animals↗
PubMed1998

Prognostic value of intratumoral interferon gamma messenger RNA expression in invasive cervical carcinomas.

BACKGROUND: The production of the cytokine interferon gamma (IFN gamma) by activated peripheral blood mononuclear cells may be reduced in patients with invasive cervical carcinoma. This study was designed to assess the prognostic value of intratumoral IFN gamma messenger RNA (mRNA) levels in such patients. METHODS: Biopsy specimens of primary cervical lesions were obtained from 27 patients with invasive squamous cell carcinoma before they received any therapy. Two prognostic groups were considered: 1) a group of 14 patients who had no apparent disease recurrence and who were alive 2 years after diagnosis (good-prognosis group) and 2) a group of 13 patients who had disease recurrence or died during the 2-year follow-up (poor-prognosis group). A competitive reverse transcription-polymerase chain reaction assay was used to measure levels of IFN gamma and beta actin mRNA. The expression of human leukocyte antigen (HLA) class II proteins (which is stimulated by IFN gamma) in tumor cells was studied by immunostaining. RESULTS: Tumor specimens from all 14 patients in the good-prognosis group contained more than 10(3) IFN gamma mRNA copies per 5 x 10(5) beta actin mRNA copies, whereas tumor specimens from only six of the 13 patients in the poor-prognosis group contained this level of IFN gamma mRNA (two-sided P = .006). No clear relationship was observed between levels of IFN gamma mRNA and T-cell or natural killer cell infiltration in tumors; however, a statistically significant association was observed between HLA class II expression on tumor cells and IFN gamma mRNA levels (two-sided P = .01). CONCLUSIONS: A subgroup of poor-prognosis cervical carcinoma patients who have low levels of intratumoral IFN gamma mRNA was identified.

Carcinoma, Squamous Cell↗
PubMed1998

Fc epsilon receptor I-associated lyn-dependent phosphorylation of Fc gamma receptor IIB during negative regulation of mast cell activation.

Fc gamma RIIB are low-affinity receptors for IgG whose intracytoplasmic domain contains an immunoreceptor tyrosine-based inhibition motif (ITIM). Fc gamma RIIB inhibit cell activation triggered by receptors that signal via immunoreceptor tyrosine-based activation motifs. This inhibition requires ITIM tyrosyl phosphorylation and is correlated with the binding of SH2 domain-containing phosphatases that may mediate the inhibitory signal. In the present work, we investigated the mechanism of Fc gamma RIIB phosphorylation and its consequences in mast cells. We demonstrate that the phosphorylation of Fc gamma RIIB requires coaggregation with Fc epsilon RI and that, once phosphorylated, Fc gamma RIIB selectively recruit the inositol polyphosphate 5 phosphatase SHIP, in vivo. In vitro, however, the phosphorylated Fc gamma RIIB ITIM binds not only SHIP, but also the two protein tyrosine phosphatases, SHP-1 and SHP-2. We show that the coaggregation of Fc gamma RIIB with Fc epsilon RI does not prevent Fc epsilon RI-mediated activation of lyn and syk. Both kinases can phosphorylate Fc gamma RIIB in vitro. However, when coaggregated with Fc epsilon RI, Fc gamma RIIB was in vivo phosphorylated in syk-deficient mast cells, but not in lyn-deficient mast cells. When Fc epsilon RI are coaggregated with Fc gamma RIIB by immune complexes, Fc epsilon RI-associated lyn may thus phosphorylate Fc gamma RIIB. By this mechanism, Fc epsilon RI initiate ITIM-dependent inhibition of intracellular propagation of their own signals.

Animals↗
PubMed1998

A tyrosine-based signal present in Ig alpha mediates B cell receptor constitutive internalization.

B lymphocytes express Ag receptors (BCR) that are composed of ligand binding subunits, the membrane Igs, associated with Ig alpha/Ig beta heterodimers. One main BCR function is to bind and to internalize Ags. Peptides generated from these internalized Ags may be presented to T lymphocytes. Here, we have analyzed the involvement of BCR Ig alpha/Ig beta components in BCR constitutive endocytosis. The role of Ig alpha subunit in BCR constitutive endocytosis was first determined in the context of an IgM-based BCR. In contrast with BCR that contain wild-type Ig alpha, surface BCR lacking Ig alpha cytoplasmic domain were not constitutively internalized. The respective roles of Ig alpha and Ig beta subunits were then analyzed by expressing chimeric molecules containing the cytoplasmic domains of either subunits in a B cell line. Only the Ig alpha cytoplasmic domain contained an internalization signal that allowed constitutive endocytosis of Ig alpha chimeras via coated pits and accumulation in sorting-recycling endosomes. This internalization signal is contained in its immunoreceptor tyrosine-based activation motif. These results indicate that Ig alpha, through its immunoreceptor tyrosine-based activation motif, may account for the ability of IgM/IgD BCR to constitutively internalize monovalent Ags.

Amino Acid Sequence↗
PubMed1998

Identification of the cleavage site involved in production of plasma soluble Fc gamma receptor type III (CD16).

CD16 (FcgammaR type III) is a low-affinity IgG Fc receptor (R) that exists in two isoforms, a transmembrane FcgammaRIIIa expressed by NK cells and monocytes, and a phosphatidylinositol-linked FcgammaRIIIb expressed by neutrophils. A soluble form of CD16 (sCD16) circulates in plasma. The cleavage site and the nature of the enzyme(s) involved in production of sCD16 were investigated. Soluble CD16 was purified to apparent homogeneity from human serum by eight steps, including anion exchange and immunoaffinity chromatography. Serum sCD16 was sequenced at both ends, as well as a recombinant form of sCD16 used as control. N-terminal sequencing demonstrated that serum sCD16 originates from neutrophil FcgammaRIIIb and C-terminal sequencing suggested that the cleavage site is between Val 196 and Ser 197, close to the membrane anchor. Addition of a hydroxamate-based inhibitor of Zn2+ metalloproteinases (RU36156) led to a dramatic decrease of sCD16 production by phorbol 12-myristate 13-acetate-activated neutrophils, whereas inhibitors of serine proteinases had no significant effect, showing the metalloproteinase dependence of this cleavage process.

Amino Acid Sequence↗