Biomedical subjects
Publications and source records attributed to W H Fridman.
Cancer immunotherapy has been carried out since the early fifties and first involved nonspecific system (BCG, Corynebacterium parvum, levamisole...). More recently, the production of cytokines as interferons or interleukin 2, the introduction of monoclonal antibodies have allowed a new development to cancer immunotherapy. Nevertheless, these new approaches have to be considered as a step in the biological therapy of cancer.
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Thyroid dysfunction and abnormalities in corticosteroid regulation were observed after clinical use of cytokines (IL2, IFN alpha), molecules with immunostimulating properties. Interleukin 2 administration induces hypothyroidism often associated with anti-thyroglobulin and anti-thyroid microsomal antibody. All these features recall Hashimoto's thyroiditis. Frequency of thyroid dysfunction during immunotherapy varies according to authors between 20 and 40 percent of all patients treated. A correlation between thyroid dysfunction and response to IL2 therapy was reported. Hypothyroidism and hyperthyroidism with development of anti-TSH receptor antibody are observed after IFN alpha administration. An auto-immune aetiology of these thyroid side effects was suggested because fine needle aspirates of the thyroid of these patients, when available, revealed a mixed cellular infiltrate with lymphocytes and histiocytes and immunocytochemical staining showed strong HLA-DR expression of all thyrocytes. Involvement of lymphokine activated killer (LAK) or direct effect of cytokines on thyroid are also possible. After intravenous injection of IL2, a marked increase in ACTH levels occurred, peaking at 4 hour with a comparable peak in cortisol level at 5 hour. Expression of IL2-Receptor in pituitary cells suggests a direct action of IL2 on anterior pituitary. Nevertheless, indirect action of IL2 via induction of other cytokines or possible secretion of ACTH by activated lymphocytes, cannot be ruled out. Testosterone serum levels decreased significantly after IL2 or IFN alpha administration. It is noteworthy that these endocrine effects induced by cytokines may modulate the clinical efficacy of these substances underlying the bi-directional communication between the immune and endocrine system.
Soluble forms of low affinity receptors for the Fc portion of IgG circulate in body fluids and regulate immune functions. We describe the transfection, production and purification techniques which allow the preparation, at a laboratory scale, of milligram amounts of glycosylated recombinant mouse and human soluble Fc gamma receptors. These recombinant products bind IgG and are biologically active on immune responses, like their normal counterparts.
Interleukin 6 (IL-6) is a multifunctional cytokine which has recently been shown to act in vitro as a growth factor for cervical carcinoma cell lines. This prompted us to measure IL-6 gene expression using a new quantitative polymerase chain reaction assay in 13 invasive cervical cancers, 5 cases of cervical intraepithelial neoplasia, and 2 normal cervix. A significant increase in the expression of the IL-6 gene in invasive cervical carcinoma as compared to cervical intraepithelial neoplasia and normal cervix was demonstrated (P < 0.05). Unlike IL-6, the expression of other cytokine genes such as gamma-interferon was not correlated with any particular cervical histological lesion. Immunohistochemical analysis identified IL-6 protein only on stroma cells which, based on morphological criteria, most likely belong to the macrophage lineage. This was reinforced by the correlation observed between IL-6 gene expression and macrophage tumor infiltration (P < 0.007). No IL-6 immunostaining of cervical tumor cells was shown. Therefore this study confirms, in vivo, that IL-6 may play a role in the pathogenesis of carcinoma of the uterine cervix since its increased expression is associated with advanced neoplastic cervical lesions. In contrast to in vitro studies, the stromal origin of IL-6 suggests that this cytokine may modulate tumor cell proliferation by a paracrine rather than an autocrine mechanism.
The engagement of the B cell antigen receptor is the first step of antigenic stimulation of B lymphocytes. This step is followed by a series of biochemical events, including the activation of protein-tyrosine kinases, phosphoinositide turnover, and multiple patterns of calcium mobilization, which lead to the regulation of gene transcription and cellular responses. The B cell antigen receptor complex is composed of membrane immunoglobulins (as antigen recognition subunits) and associated chains (Ig-alpha and Ig-beta) that couple the receptor to cytoplasmic protein kinases. To investigate independently the relative signaling capacity of Ig-alpha and Ig-beta, chimeric proteins containing their cytoplasmic domains were expressed in a B cell line. We found that Ig-alpha and Ig-beta activate two distinct intracellular signaling pathways. The engagement of Ig-alpha chimeras induces a complete release of calcium from intracellular stores, followed by transmembrane calcium influx and late cell activation signals, detected by lymphokine secretion. In contrast, Ig-beta chimeras do not induce lymphokine secretion or calcium influx, but induce short oscillatory release of calcium, dependent on the activity of the Ca-ATPase pump of the endoplasmic reticulum. These results provide a structural basis for the diversity of B cell responses.
FcR capable of triggering cell activation share with BCR and TCR a conserved intracytoplasmic tyrosine-containing activation motif (TAM). Besides cell activation, these receptors trigger other biologic responses, such as endocytosis of soluble ligands. Murine mast cells express two types of FcR that, when aggregated by antibodies and multivalent Ag, trigger the release of inflammatory mediators and cytokines. These are high affinity receptors for IgE (Fc epsilon RI) and low affinity receptors for IgG (Fc gamma RIII). They comprise each an IgE- or IgG-binding alpha-subunit and two TAM-containing subunits that associate with both receptors: a beta-subunit and a homodimeric gamma-subunit that can associate also with the other subunits of the TCR. Herein, we focused on biologic activities triggered in mast cells via the TAM of the gamma-subunits. Using rat basophilic leukemia (RBL) cells stably transfected with cDNA-encoding murine Fc gamma RIII alpha, we found that murine Fc gamma RIII trigger the phagocytosis of antibody-coated erythrocytes. Using RBL transfectants expressing Fc gamma RIII with a deletion of the intracytoplasmic domain of Fc gamma RIII alpha or chimeric receptors having the extracellular and transmembrane domains of Fc gamma RII and the intracytoplasmic domain of Fc gamma RIII alpha, we showed that intracytoplasmic sequences of Fc gamma RIII alpha are neither necessary nor sufficient for Fc gamma RIII to trigger phagocytosis. Using RBL transfectants expressing chimeric receptors having the extracellular and transmembrane domains of Fc gamma RII and the TAM-containing intracytoplasmic domain of murine Fc gamma RIII gamma, we demonstrated that intracytoplasmic sequences of Fc gamma RIII gamma are sufficient to trigger phagocytosis. Using RBL transfectants expressing the same Fc gamma RII-III gamma chimeras, in the TAM of which one, the other, or both tyrosine residues were mutated, we established that tyrosines in the TAM sequence are required for phagocytosis. Our results endow TAM gamma with previously unknown triggering capacities and Fc gamma RIII with new biologic properties.
Langerhans cells (LC) express Fc gamma RII on their cell surface. In this paper, we demonstrate that these cells also release soluble Fc gamma RII (sFc gamma RII) molecules. LC express transcripts encoding a membrane-associated receptor and a transmembrane-deleted Fc gamma RIIA. The latter form was identified in LC culture supernatants using specific antibodies. CHO cells, transfected with LC-derived cDNA encoding the transmembrane-deleted Fc gamma RIIA, secrete sFc gamma RIIA that include the intracellular domain and exhibit the same backbone as the protein identified in LC supernatants. Secreted sFc gamma RIIA exhibits the same pattern of binding to human and mouse IgG subclasses as do membrane Fc gamma RII and inhibits the binding of immune complexes to Fc gamma RII+ cells. In addition, CHO cells expressing the membrane-associated Fc gamma RIIA release truncated and unstable Fc gamma RIIA molecules that lack the intracellular domain. Thus, sFc gamma RII can result from shedding of membrane molecules and/or from secretion of soluble receptors lacking the transmembrane domain.
The frequency of peripheral blood cells expressing the perforin gene or the granzyme B gene was evaluated by in situ hybridization in nine patients suffering from metastatic melanoma and treated with recombinant interleukin-2 (rIL-2). A spontaneous expression of both genes was detected in five to seven patients. rIL-2 administration increased the frequency of positive cells in all patients (P < 0.03 for each gene), the highest frequency being reached in the patients who already expressed these genes prior to rIL-2 treatment (P < 0.02). Expressions of the granzyme B gene and of the perforin gene were strongly correlated before IL-2 treatment and they were similarly affected by rIL-2 administration. In contrast, their modification under treatment did not correlate with that of CD56+ cell counts, of natural killer activity and of sCD8 release. This indicates that perforin and granzyme B gene expressions are markers of cytotoxic cell activation independent of those previously described, and that they should be further evaluated in patients with malignancies to delineate their potential value in predicting clinical outcome.
Transforming growth factor-beta 1 (TGF beta 1) is a pleiotropic cytokine which inhibits growth of many cell types and positively or negatively regulates the production of Ig isotypes. By using mouse resting B cells stimulated by lipopolysaccharide (LPS), we investigated whether the effect of TGF beta 1 on Ig production is related to its effect on cell growth. We show that low doses of TGF beta 1 stimulate IgG3 and IgG2b production whereas higher doses inhibit IgM, IgG3, IgG1 and IgG2b secretion and cell proliferation. TGF beta 1 titration curves and kinetics experiments suggested that the inhibitory effect on Ig secretion and B-cell growth are closely related. We defined the phase at which TGF beta 1 exerts its anti-proliferative effect on mouse B cells. TGF beta 1 does not modify the increase in expression of class II antigens which occurs before transition from G0 to G1. However, it partially inhibits the induction of expression of low-affinity Fc gamma RII and cell enlargement which both begin during the early G1 phase, and it totally blocks induction of the expression of transferrin receptors, a marker of the late G1 phase. Thus, TGF beta 1 blocks LPS-stimulated mouse B cells in the early G1 phase, and this results in inhibition of Ig production.
Anti-cytokines belong to a new family of biological response modifiers which interfere with the biological functions of cytokines. An imbalance between cytokines and natural anti-cytokines may represent an important factor to explain the pathogenesis of some human diseases. Indeed, dysregulation of anti-cytokine serum levels was observed in cancers and auto-immune and infectious diseases. It was sometimes associated with disease activity and in some circumstances they may help to predict clinical response to treatment. Control of disease activity in septic shock and auto-immune diseases was demonstrated after administration of anti-cytokines such as soluble TNF receptors and IL1ra inhibitors. Therefore these agents are promising tools for diagnosis and immunotherapy.
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Interleukin 6 and C-reactive protein (CRP) were determined prior to IL-2 therapy in sera from metastatic melanoma patients. Patients with elevated serum IL-6 (> 20 pg ml-1) and/or CRP (> 10 mg l-1) levels were associated with resistance to IL-2 therapy. A correlation between high serum IL-6 levels and a shorter median survival was also observed.
GM-CSF induces proliferation and activation of Langerhans' cells in vitro. The density of Langerhans' cells in human tumours is correlated to the in situ density of GM-CSF, and intradermal injection of GM-CSF induces local accumulation of Langerhans' cells. Therefore, we investigated the presence of GM-CSF in the sera of children with Langerhans' cell histiocytosis (LCH). We detected GM-CSF in the sera of all children with disseminated and active LCH, but not in the sera of patients with localized (i.e. bone) LCH. These results suggest that GM-CSF level is related to extent and the activity of LCH.
Twenty renal cell carcinomas were cultured in the presence of 200 IU/ml of recombinant interleukin-2; tumour-infiltrating lymphocytes (TIL) developed in 70% of cases; the phenotypic profile was heterogeneous in 11 TIL tested on day 30: 4 were mostly CD8 positive, 4 mostly CD4 and 3 showed CD4-CD8-CD56 mixed phenotypes. The cytotoxic activity was also heterogeneous, and no TIL developed a tumour-specific cytotoxic activity. The phenotypic profile and cytotoxic activity were also tested after thawing, and this study demonstrated that TIL can be frozen at the time of the nephrectomy, then thawed and cultured for the purposes of therapeutic trials when metastases appear. The differences between TIL derived from renal cell carcinoma and TI1 derived from melanoma are discussed.
Neuroblastoma is the most frequent tumour of the childhood under the age of 5. The staging and the follow up are achieved by MIBG scintigraphy, considered as the method of reference, but sometimes difficult to interpret . The availability of monoclonal antibodies against the ganglioside GD2, expressed on the cell membrane of neuroblastoma and neuro-endocrine cancers offers novel tools that deserve to be carefully explored. We investigated four mouse monoclonal antibodies (3 IgG3: BW704, 7A4, 60C3, and the IgG1 variant of BW704: MAK704), on nude mice xenografted with a human neuroblastoma (REM). Sixty one nude mice were included. The three former MAbs provided tumour imaging, the best results being obtained with BW704, followed by 7A4 and 60C3. MAK704 was disappointing. A control antiphosphorylcholine antibody (P51-1) did not give any tumour image in the three tested mice. Scintigraphy ratios tumour/liver and tumour/muscle reached 20 and 100 with BW704, respectively, on the 10th day. Good imaging quality was already obtained from the 24th h. The tumour uptake, calculated from radioactivity countings of resected samples, reached 22 +/- 3% of injected dose per gramme. These results let us hope that these antibodies could also provide highly contrasted images in humans and could open the way for therapeutic applications.
This study analyzed clinical response induced by TIL in patients with renal cell carcinoma previously treated by interleukin-2. Six patients (4 men, 2 women, mean age 44.3 years) with measurable metastatic localizations have been treated by TIL reinjection (0.02 to 7.6 x 10(10) cells). TIL phenotype was a combination of CD4 and CD8 in 3 patients, predominantly CD4 in two patients and predominantly CD8 in one patient. Previous treatment by interleukin-2 induced one partial response, 2 stabilizations of the disease and 3 tumoral progressions. TIL led to an amelioration for 4 patients: 2 were in complete response, 2 were stabilized and 2 had tumoral progression and decreased. This study shows that CD4 TIL may improve an initially response induced by IL-2 therapy.
Membrane and soluble forms of Fc gamma receptors play important role in immune reactions. Upon interaction with immune complexes, Fc gamma R transduce activation or inhibition signals via their intracytoplasmic portions. Soluble Fc gamma R contain the Fc gamma R ectodomain. They bind to the Fc portion of IgG and inhibit Fc dependent immune reactions. In addition, they bind directly to various cell types and regulate immune reactions.