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Biomedical subjects

G Fossati

Publications and source records attributed to G Fossati.

At least 91 records · Page 5Linked to original sources
PubMed1987

Production of monoclonal antibodies to calcitonin and development of a two-site enzyme immunoassay.

A procedure is described for the production of calcitonin-specific hybridomas which involves primary and secondary immunization of Balb/c mice in the hind footpads with free, synthetic human calcitonin and cell fusion of lymphocytes from popliteal lymphonodes with a P3 x 63 myeloma line. This protocol offers the following advantages: (a) it is short and easy to perform, (b) it requires small amounts of unconjugated antigen, and (c) it gives a high yield of antigen-specific IgG-secreting hybridomas. Routine screening was carried out by ELISA on solid phase calcitonin and binding of the monoclonals to free antigen was studied with calcitonin linked to biotin through a 13 carbon atom spacer. Monoclonal couples capable of simultaneously binding calcitonin in solution were found by pairing in all possible combinations 25 purified antibodies, in their unlabelled and biotinylated form, in a checkerboard matrix experimental system. Of the over 30 positive pairs identified, four were used in a one-step enzyme immunoassay for calcitonin determination on microtiter plates in a concn range between 0.1 and 5 ng/ml. With the detecting monoclonal directly conjugated to peroxidase with a heterobifunctional crosslinker, the range of the assay with one monoclonal pair was between 25 and 1000 pg/ml with an 18 hr incubation at 4 degrees C.

Animals↗
PubMed1986

Control of human melanoma growth in nude mice by autologous allo-activated peripheral blood lymphocytes.

Human peripheral blood lymphocytes (PBL) were activated in vitro by means of a pool of allogeneic PBL from normal donors and then evaluated for in vivo activity against human melanoma cells xenografted in splenectomized and irradiated athymic (nude) mice. The subcutaneous (s.c.) growth of human melanoma cells was inhibited by intravenous (i.v.) injection, 2 hr later, of such allo-activated, autologous and allogeneic PBL in 7/8 and in 6/9 mice respectively. Unstimulated PBL were ineffective. When allo-activated patients' lymphocytes were administered 3 days after s.c. implantation of autologous melanoma cells, inhibition of tumor growth was observed in 1/6 mice. A significant delay in tumor appearance was noted in the remaining animals. Unstimulated as well as allo-activated, lymphokine-releasing helper-enriched human PBL had no effect on melanoma xenografts, indicating that the tumor inhibition by tumor-cytotoxic allo-activated PBL was not due to recruitment of murine immuno-competent cells by human lymphokines. These results indicate that allo-stimulated, tumor-cytotoxic human PBL given i.v. to nude mice can circulate and inhibit the growth of autologous or allogeneic human melanoma cells implanted s.c.

Animals↗
PubMed1986

Phenotypic profile of clones from early cultures of human metastatic melanomas and its modulation by recombinant interferon gamma.

Fifty-six tumor clones isolated by cloning in soft agar from early cultures (before the 10th in vitro passage) of two different human metastatic melanomas (Me9229 and Me28) were characterized by FACS analysis for surface expression of class-I and class-II HLA antigens and of melanoma-associated antigens (MAA) with a panel of 15 monoclonal antibodies (MAbs). A marked phenotypic heterogeneity involving MAA and/or HLA markers was observed among the clones derived from both tumors. The differences among the tumor clones and between them and the uncloned melanoma were qualitative and quantitative for each antigen considered. Clones derived from Me9229 expressed the same HLA profile as the parental culture (class I+, class II-) while strong heterogeneity was observed for MAA expression. Clones from Me28 presented a marked heterogeneity for class-I and class-II HLA antigens but were more homogeneous for MAA. The phenotype of the clones was repeatedly checked over the first month in culture and found to remain generally unchanged and not linked to the cell cycle. However, major changes in antigenic expression of the clones could be observed upon treatment with recombinant interferon-gamma (rIFN-gamma): class-I and -II HLA antigens could be induced or augmented while a moderate inhibition was seen on MAA expression. Furthermore, an apparent hierarchy in expression and/or induction of class-II antigens by rIFN-gamma was observed among the tumor clones. DR antigens were more frequently expressed (Me28 clones) and upon treatment with rIFN-gamma reached higher levels than DP and DQ products. Taken together these results indicate that antigenic heterogeneity for MAA and HLA antigens can be detected in cells isolated from early cultures of human metastatic melanomas and suggest that the original uncloned tumor might be considered as a complex mixed population made up of a number of neoplastic cells each expressing a distinct phenotype which can be modulated by lymphokines such as IFN-gamma.

Antibodies, Monoclonal↗
PubMed1986

Infusion of autologous alloactivated lymphocytes in melanoma patients: toxicity and immunologic monitoring.

Previous work has shown that infusion of autologous helper-enriched, alloactivated lymphocytes in melanoma patients may induce, in addition to other mild signs of toxicity, a transient but sharp elevation of blood pressure. To avoid such a disturbing symptom, the in vitro protocol of peripheral blood lymphocyte activation has been modified. In the present study we show that such a modification has led to a lower toxicity of autologous lymphocyte infusion in 4 melanoma patients; in particular, hypertension was no longer observed. In addition, an immunologic monitoring was carried out in these patients. In 1 of 4 patients the treatment enhanced the in vitro cytotoxic activity of peripheral blood lymphocytes against autologous tumor cells. Other parameters such as NK activity and T4/T8 ratio did not show significant trends. The possible implications of these findings for clinical trials of adoptive immunotherapy with lymphocytes are discussed.

Adolescent↗
PubMed1986

Heterogeneity of clones from a human metastatic melanoma detected by autologous cytotoxic T lymphocyte clones.

The possibility that a single human tumor may be composed of an heterogeneous population of cells with respect to susceptibility to lysis by autologous CTL clones was investigated by testing six cytolytic clones derived by micromanipulation against the autologous metastatic melanoma, Me28, and against 31 clones derived from Me28 by cloning in soft agar. Highly significant differences in the lysis of many tumor clones were observed by three of the CTL effectors in comparison with the cytotoxicity achieved on Me28. These results indicate that cloned cellular reagents can detect heterogeneity among cells isolated from the same melanoma, and suggest that the target determinants recognized on the autologous tumor might be differentially expressed on different neoplastic cells.

Analysis of Variance↗
PubMed1986

Systemic administration of autologous, alloactivated helper-enriched lymphocytes to patients with metastatic melanoma of the lung. A phase I study.

A phase I study was carried out to test the feasibility and toxicity of infusing large numbers of autologous, alloactivated helper lymphocytes into patients with metastatic melanoma. Patient peripheral blood lymphocytes (Pt-PBL) obtained by lymphopheresis and expressing the helper phenotype BT5/9 were separated and stimulated for 48 or 72 h with a pool of PBL from four to six healthy donors. Patients were then infused with such activated lymphocytes over a 2-3 h period. A total of 4 phereses and infusions (2/week for 2 weeks) were carried out for each cycle in each patient. Of the five patients treated, two received a second round of infusions. Infusion of autologous PBL stimulated in vitro for 48 h caused chills, fever, headache, and increased blood pressure. All symptoms disappeared in 2-3 h and were easily controlled by appropriate therapy. When lymphocytes were given after 72 h of allostimulation, no or very mild toxicity was observed. Serum chemistry, coagulation, autoimmunity, and urine analysis showed no gross abnormalities during therapy or follow-up of the patients. Immunological parameters (OKT4/OKT8 ratio, NK activity and cytotoxic T cell activity to autologous melanoma) were evaluated before starting the therapy, during its course and during the 3 to 6 months follow-up. The OKT4/OKT8 ratio increased significantly but transiently soon after the first course of infusions in one of the two patients tested. NK activity increased after 75-100 days in the three patients tested and in one of them it was high even after 180 days. No correlation between NK activity and prognosis was apparent. Cytotoxicity to autologous tumor was assessed in two patients, only of one of whom exhibited an increased activity from 75 to 180 days, which was associated with a prognosis better than that of the negative patient. Five patients were treated: two had progressive disease, two had stable disease for 5 and 6 months, respectively. In the first of these patients, a new cycle of lymphocyte infusions was carried out which caused a measurable reduction of lung tumor nodules whose growth, however, resumed 4 months later. This patient died 14 months after the onset of therapy. The fifth patient had a partial regression of pulmonary and intracranial metastases after therapy, but eventually died 3 months later. These results indicate that infusion of a high numbers of autologous, allostimulated helper PBL is a feasible and safe procedure, which could therefore be used in future studies of adoptive immunotherapy of cancer.

Antibodies, Monoclonal↗
PubMed1986

Classes I and II HLA and melanoma-associated antigen expression and modulation on melanoma cells isolated from primary and metastatic lesions.

Human melanoma cells freshly isolated from 20 patients with primary and 73 patients with metastatic melanomas were analyzed by indirect immunofluorescence staining with monoclonal antibodies (MoAb) to class I (HLA-A, -B, and -C) and class II (HLA-DR and -DQ) antigens and to melanoma associated antigen (MAA). The latter included the GD3-MAA and the high molecular weight MAA. HLA class I antigens were present in 91 and 93% of primary and metastatic tumors, respectively. GD3-MAA was detected in 100% of primary and 80% of metastatic tumors. Whereas the high molecular weight MAA was expressed in 75% of tumors. Sixty % of primary and 50% of metastatic melanomas were stained by anti-HLA-DR MoAb, whereas 38 and 21% of cases, respectively, were stained by anti-HLA-DQ MoAb. Marked phenotypic heterogeneity was evident among primary and metastatic tumors, including different metastases from the same patient. Moreover, in vitro culture of melanoma cells isolated from metastases was associated with an increase from 50 to 75% of tumors stained by anti-HLA-DR MoAb but not of tumors positive for HLA class I antigens and MAA. In vitro incubation with partially purified or recombinant human gamma-interferon enhanced the expression of HLA-DR antigens on all short-term cultured melanoma cells tested but induced and/or augmented the expression of HLA-DQ antigens only in 5 of the 8 cases examined. The average increase in antigenic expression was higher for HLA-DQ than for HLA-DR antigens. Flow cytometric measurement of DNA content of melanoma cells treated with gamma-interferon revealed that the increase of HLA-DR and -DQ expression induced by gamma-interferon was independent from the cell cycle of the tumor cells.

Antibodies, Monoclonal↗
PubMed1986

Cancer procoagulant in human tumor cells: evidence from melanoma patients.

It has repeatedly been proposed that fibrin plays a role in tumor growth and metastasis. Among tumor cell products or activities which may promote clot formation, cancer procoagulant (CP), a direct activator of coagulation factor X, has been suggested to be selectively associated with the malignant phenotype. We report here the enzymatic and immunological identification of this cysteine proteinase procoagulant in extracts and cells from human melanoma. CP activity was independent of both the intrinsic and extrinsic pathways of blood coagulation, using factor IX and factor VII deficient plasmas, and was inhibited by the cysteine proteinase inhibitors iodoacetamide and HgCl2. CP activity was detectable in extracts and cell suspensions from all 32 patients studied and was higher in extracts from metastases (14.8 +/- 3.9 units/mg protein) than from the primary tumors (3.7 +/- 1.0 units/mg protein). CP activity was not affected by an anti-apoprotein III antibody or by concanavalin A, a known inhibitor of thromboplastin. In contrast, no CP activity or antigen was detected in extracts from six benign melanocytic lesions. The procoagulant activity was dependent on factor VII and was inhibited by anti-apoprotein III antibody and by concanavalin A, properties that suggest that the procoagulant was tissue thromboplastin. These data indicate that CP can be expressed by human tumor cells and that, among melanotic lesions, its presence is associated with the malignant phenotype and its activity is particularly high in metastatic cells.

Adolescent↗
PubMed1985

Clonal analysis of cytotoxic T-lymphocyte response to autologous human metastatic melanoma.

Peripheral blood lymphocytes (PBL) from a melanoma (Me) patient, previously shown to be unable to react against the autologous tumor (Me 28) after mixed lymphocyte-tumor culture (MLTC), were cultured in vitro with the autologous tumor in MLTC and/or with IL-2-containing supernatants. T-cell clones were then obtained by limiting dilution and by micromanipulation. Eleven clones, selected for autologous tumor (Auto-Tu) cytotoxicity, were tested for specificity on a panel of 17 cell cultures of normal and neoplastic origin, revealing a complex spectrum of lytic activities. Three groups of clones could be identified depending on the patterns of cytotoxicity. One clone (B11.12) lysed Me28 and expressed a borderline reactivity against one allogeneic Me. A second group of clones (A4, A4.18, H10, E12, C9) lysed the Auto-Tu and allogeneic Me. The last group of clones (A4.2, A4.3, A4.4, A7, B7) expressed a broader pattern of reactivity with significant cytotoxicity against targets of different histologic origin. Furthermore, the second and third groups of clones lysed K562 while B11.12 did not. The Auto-Tu-restricted reactivity of clone B11.12, confirmed by a further test on 13 allogeneic Me and on autologous IL-2 cultured lymphocytes, suggests the recognition of antigenic structures preferentially expressed on Me28. Blocking studies, performed with monoclonal antibodies (MAb), revealed that an anti-HLA class I MAb (w6/32), but not two anti-DR MAbs (L243, DI.12), could inhibit the cytotoxic activity of clones B11.12 on Me28. A significant blocking effect by w6/32 on Me28 was achieved also with clones A4.4 and H10 but not with clones A4.2, A4.3 and A7. The phenotype of all clones was T3+, T4-, T8+, HNK-I-, suggesting that the anti-tumor effectors were of the T-cell lineage. Taken together, these data indicate that it is possible to isolate anti-tumor CTL-clones after MLTC from a PBL population of a metastatic melanoma patient. Furthermore, we present evidence suggesting a role of class-I antigens in the interaction of some cloned effectors with the autologous tumor target.

Antibodies, Monoclonal↗
PubMed1985

Autologous cellular immune response to primary and metastatic human melanomas and its regulation by DR antigens expressed on tumor cells.

Evidence for heterogeneity of several biological features of human malignant melanoma (Me) like morphology, cytogenetics, oncogenes activation, antigenic expression, metastatizing capacity and procoagulant activity are briefly reviewed in an attempt to distinguish findings related to primary vs. metastatic lesions. In our own studies monoclonal antibodies were used to study expression of MHC class I, class II products and of Me-associated antigens (MAA) on primary and metastatic Me cells. High expression of class I antigens was found in a high percentage of both primary and metastatic tumors, whereas DR and MAA showed a significant variation (from 3 to 90% of cells) in expression both in primary and in metastatic Me. When autologous cell-mediated immune responses were evaluated, it was found that Me cells from primary tumors but not those from lymph node metastases were able to stimulate autologous lymphocytes to proliferate and become cytotoxic for autologous Me. Clonal analysis of cytotoxic lymphocytes was then carried out in order to see whether the lack of lymphocytes reactivity to metastatic cells was due to the absence or to a low frequency of cytotoxic cells in the unstimulated PBL. CTL clones cytotoxic for autologous Me (Auto-Me) cells were indeed isolated. Three classes of CTL clones were identified: 1) one which is cytotoxic for Auto-Me; 2) a second one which lyse Auto-Me and allogeneic Me; and 3) a third one which is cytotoxic for Auto-Me and allogeneic normal and neoplastic cells. Metastatic Me cells, however, had the ability to suppress the stimulation of autologous PBL by alloantigens or IL-2. This effect was dose-dependent and was not due to absorption of IL-2 by Me cells. Since it has been reported that Me cells express class II MHC antigens, we investigated whether there was any correlation between autologous immune responses and DR expression on Me cells. Autologous lymphocytes stimulation was found to occur only with DR+ Me cells from primary lesions, whereas metastatic cells, either DR+ or DR-, did not stimulate autologous PBL. Moreover, the suppressive effect of metastatic Me cells was associated with their expression of DR antigens. The modulation of DR antigens on Me cells by Interferon-gamma correlated positively with their suppressive capacity. Thus, it appears that primary Me can behave differently from the metastatic one in their interactions with the immune system of autologous host. These findings suggest that DR antigens on Me cells may have an important role in the regulation of autologous immune responses.

Antigens, Neoplasm↗
PubMed1985

Allostimulation of patients' lymphocytes generates both T and NK-like cells cytotoxic for autologous melanoma.

Killing of autologous melanoma (auto-Me) was obtained with pooled allostimulated peripheral blood lymphocytes (PBL) in 34/42 cases and found not to be due to a cross-reactivity between melanoma and allogeneic normal antigens. To see whether generation of tumour cytotoxic PBL by allostimulation was due to release of IL-2, PBL from 34 patients were divided into two aliquots and stimulated either by alloantigens or IL-2. Allostimulated PBL were cytotoxic for auto-Me in 30/34 cases (85%) whereas IL-2 generated tumour cytotoxic cells in 22/34 cases (64%). Lysis of K562, a target for monitoring NK-like activity, was obtained in 95-100% of cases with both stimuli. A similar frequency of OKT3+, OKT4+, OKT8+ and HNK1+ cells was found in PBL activated by allostimulation and IL-2, whereas a higher frequency of OKM1+ cells was evident in IL-2-stimulated PBL. Cold-target competition studies indicated that allostimulation generated at least two different types of effectors, one lytic to auto-Me but not to K562, and the other which lysed both targets. Allostimulated, FACS-separated T3- cells killed both auto-Me and K562 cells whereas T3+ cells lysed only auto-Me. It is concluded that allostimulation generated two subpopulations of auto-Me killer cells, one of the T lineage and the other NK-like, which both can destroy auto-Me targets.

Antibodies, Monoclonal↗
PubMed1984

The inhibition of lymphocyte stimulation by autologous human metastatic melanoma cells correlates with the expression of HLA-DR antigens on the tumor cells.

Previous studies indicated that peripheral blood lymphocytes from patients (Pt-PBL) with lymph node metastatic melanomas proliferated in vitro and developed into tumor-restricted cytotoxic lymphocytes in response to alloantigens or interleukin 2 (IL-2). However, Pt-PBL were not stimulated by irradiated autologous metastatic melanoma (Auto-Me) cells. In the present study we report that the lack of stimulatory activity of Auto-Me cells may be due to a suppressive effect exerted by Auto-Me cells on the responder lymphocytes. In fact, we found that in 62% of cases examined, the addition of 5-10% Auto-Me cells to Pt-PBL cultures strongly inhibited both proliferation and the generation of tumor cytotoxic lymphocytes induced by alloantigens or IL-2. The inhibition was dose-dependent and tumor-restricted, and was not due either to toxicity, medium depletion or IL-2 absorption by Auto-Me cells. Normal fibroblasts, K562 cells and autologous E-lymphocytes were not suppressive. Auto-Me cells were able to inhibit Pt-PBL responses only when added during the first 24 h of culture and not later. Phenotypic analysis of Auto-Me cells using monoclonal antibodies directed against HLA-A,B,C, HLA-DR and melanoma-associated antigens revealed that the expression of high levels of DR antigens on Auto-Me cells was associated with an elevated suppressive activity. Conversely, Auto-Me cells with low or undetectable levels of DR antigens were not inhibitory. Furthermore, the increased expression of DR antigens on Auto-Me cells obtained by in vitro treatment with human interferon gamma (IFN-gamma) also resulted in an increased suppressive activity. We conclude that HLA-DR+ metastatic melanoma cells can interfere with the generation of an anti-tumor immune response, thus potentially favoring the escape of the tumor from the host's control mechanism.

HLA-DR Antigens↗
PubMed1984

Primary but not metastatic human melanomas expressing DR antigens stimulate autologous lymphocytes.

Lymphocytes from melanoma patients were stimulated in mixed culture with autologous tumor cells (MLTC) in order to evaluate lymphocyte proliferation and subsequent cytotoxicity on autologous melanoma cells. It was found that melanoma cells from lymph node metastases were unable to induce autologous tumor-cytotoxic cells in 21 cases examined, in 15 of which MLTC also failed to induce lymphocyte proliferation. Patients' lymphocytes, however, were significantly stimulated by allogeneic irradiated lymphocytes and by interleukin 2. To investigate whether the lack of autologous stimulation was restricted to metastatic cells, the immune response of patients with only primary lesions of malignant melanoma was evaluated. It was found that primary melanoma cells were able to induce proliferation in 7 out of 9 (77%) patients, whereas positive cytotoxicity was obtained in 2 out of 4 patients tested. In order to see whether the presence of DR molecules was important for the stimulatory activity, melanoma cells were examined for the expression of DR antigens by indirect immunofluorescence with monoclonal antibodies. Positive autologous MLTC was found in all of six DR+ primary melanomas, whereas the two DR-tumors were unable to stimulate autologous lymphocytes. An anti-DR but not an anti-DC monoclonal antibody was able to block the proliferation of lymphocytes induced by an autologous primary melanoma. Neither MLTC nor cell-mediated killing was obtained with either DR+ or DR-metastatic melanoma. In 60% of the cases tested, however, DR+ metastatic melanoma cells were able to stimulate allogeneic lymphocytes of normal individuals. Increased expression of DR antigens was induced by in vitro treatment with human gamma-interferon in metastatic tumor cells; this caused an increase in the proliferation of allogeneic but not autologous lymphocytes. These findings indicate that primary but not metastatic DR+ melanoma cells are able to activate the proliferation and cytotoxicity of autologous peripheral blood lymphocytes, suggesting a potential role of DR antigens in regulating tumor-host relationships in melanoma patients.

Cytotoxicity, Immunologic↗
PubMed1984

Inhibition of human melanoma growth in nude mice by autologous, alloactivated peripheral blood lymphocytes.

Peripheral blood lymphocytes of melanoma patients were stimulated in vitro by a pool of allogeneic lymphocytes and shown to be cytotoxic against autologous melanoma cells. To evaluate the in vivo antitumor activity of the cytotoxic alloactivated autologous peripheral blood lymphocytes, tumor neutralization (Winn) assay was carried out by injecting such lymphocytes admixed with autologous melanoma cells in athymic BALB/c nude mice. In 3 of 6 cases, complete inhibition of tumor growth was obtained at lymphocytes to tumor cells ratio of 10:1 and in one case also of 5:1. In all cases the appearance of tumors was delayed and the growth rate was significantly reduced in a dose-dependent fashion as compared to control mice injected with tumor cells alone. We conclude that in vitro alloactivated peripheral blood lymphocytes can inhibit and/or impair the growth of autologous melanoma cells in nude mice.

Animals↗
PubMed1984

Human renal antigen defined by a murine monoclonal antibody.

Fusion of spleen cells from a mouse immunized with a surgical specimen of a human renal carcinoma with murine P3 myeloma cells resulted in the establishment of a hybridoma cell line that secreted a monoclonal antibody (MKi-1), of IgG1 subclass, which preferentially reacted on kidney crude membrane (CM) preparations. This monoclonal antibody was tested by solid-phase radioimmunometric assay and immunofluorescence (IF) on a panel of tumor cell lines and on CM preparations and cell suspensions from surgical specimens of normal and neoplastic tissues. In addition, cryosections of normal and cancer tissues of various histologic types were tested by IF. The expression of the MKi-1 antigen was limited to normal kidney epithelium, renal cancers, some areas in the pancreas, the apical region of some breast ducts, and a proportion (5-50%) of activated lymphocytes. Electron microscopic study by the immunoperoxidase technique on fixed sections from normal kidney showed that MKi-1 stained the brush border of almost all proximal tubules. The molecule recognized by MKi-1 was a single polypeptide chain with a molecular weight of 140,000.

Animals↗