Use of cryopreserved lymphocytes for assessment of the immunological effects of interferon therapy in renal cell carcinoma patients.

Experiments were designed to assess whether cryopreserved PBL could be used to monitor the immunological effects of IFN-alpha therapy in renal cell carcinoma (RCC) patients. It was found that programmed freezing and thawing of peripheral blood lymphocytes (PBL) from normal blood donors did not substantially change lymphocyte subset proportions and that cryopreserved PBL were able to proliferate in response to IL-2. It was also possible to activate the cytolytic activity of frozen PBL, and the frozen leukocytes did not lose their ability to secrete IFN-gamma after PHA activation. We have used these findings to investigate the immunological effects of IFN-alpha therapy in RCC patients. Cryopreservation of PBL samples collected from various patients over a period of 9-14 months enabled us to compare the in vitro reactivity of PBL from individual RCC patients repeatedly and under standard conditions. It was found that IL-2 induced proliferative responses of PBL from IFN-alpha non-responders, collected prior to IFN-alpha therapy, were significantly decreased as compared to those from normal blood donors. The proliferative responses of PBL from IFN-alpha responders, collected prior to IFN-alpha therapy, did not substantially differ from normal controls. Culture of PBL from IFN-alpha responders for 3 days in IFN-alpha-containing medium increased their lytic activity towards RCC targets, whereas no such increase was observed with non-RCC targets or using PBL from IFN-alpha non-responders or PBL from normal-blood donors. Enzyme-linked immunospot (ELISPOT) assays performed with cryopreserved lymphocytes from IFN-alpha non-responding RCC patients, collected prior to IFN-alpha therapy, revealed a substantially decreased ability to secrete IFN-gamma, as compared to IFN-gamma secretion of PBL from IFN-alpha responders or normal blood donors.

IFN-alpha therapy of renal-cell carcinoma - defect of lymphocyte sensitivity to mitogenic and activating cytokine signals in patients not-responding to therapy.

The present prospective study was designed to assess whether the renal cell carcinoma (RCC) patients treated with recombinant interferon alpha (IFN alpha), whose tumours respond (responders) and do not respond (non-responders) to IFN alpha therapy, differ with regard to in vitro sensitivity of peripheral blood lymphocytes (PBL) to interleukin 2 (IL-2), IFN alpha, and IFN gamma signals prior to therapy. Twenty-one patients with advanced RCC after nephrectomy, 15 responders and 6 non-responders, were entered into a protocol. The protocol involved isolation and freezing of PBL samples followed by IFN alpha treatment of patients, assessment of proliferative and activating PBL responses, and evaluation of the therapeutic results. Freezing of PBL samples allowed us to compare the in vitro reactivity of PBL from individual RCC patients, repeatedly and under standard conditions. Substantial differences in proliferative responses to the mitogenic IL-2 signal of PBL derived from IFN alpha responders and nonresponders were found. Whereas the IL-2-induced proliferative responses of PBL from normal blood donors and IFN alpha responders were comparable, the proliferative responses of PBL from IFN alpha non-responders were significantly decreased, suggesting an immune dysfunction in non-responders. Cultivation of PBL from RCC patients in medium supplemented with IFN alpha increased the lytic activity of PBL from IFN alpha responders directed against RCC targets; no such increase could be observed with non-RCC targets, with PBL from IFN alpha non-responders, or with PBL from normal blood donors. Detection of phytohaemagglutinin (PHA)-stimulated IFN gamma secretion by PBL at the single cell level using enzyme-linked immunospot (ELISpot) assay revealed that the ability to produce IFN gamma was substantially decreased in IFN gamma non-reponders, as compared to IFN alpha responders and to normal blood donors.

Subcutaneous interleukin-2 in combination with vinblastine for metastatic renal cancer: cytolytic activity of peripheral blood lymphocytes.

Eight patients with progressive metastatic renal cell carcinoma were selected for one course of subcutaneous recombinant interleukin-2 (IL-2) plus vinblastine (VBL) treatment lasting for seven weeks. Seven of the eight patients were evaluable for response, eight for toxicity. Peripheral blood lymphocytes (PBL) from the evaluable patients were isolated and frozen prior to, during, and after the treatment courses; kinetics of their cytolytic activity was assessed and compared under standard conditions in 51Cr microcytotoxicity assay with natural killer (NK)-sensitive and NK-resistant human tumor targets. Among the evaluable patients treated, there was 1 partial responder (10+ months, regressions occurred in lung, retroperitoneal lymph nodes and adrenal metastases) and 3 patients achieved a stable disease (10+, 10+, 5+ months). Systemic toxicity was mild to moderate with treatment-limiting adverse effects in one patient (severe thrombocytopenia, grade IV). In the IL-2-treated patients, the cytolytic activity of PBL directed against NK-sensitive targets rapidly decreased during the first week of IL-2 treatment, approaching the negative values on day 10. Then the cytolytic activity was slowly increasing and reached its maximum within another two weeks. Afterwards, the cytolytic activity of PBL was again decreasing and the approximate values of the initial cytolysis were reached after 6-8 weeks. In contrast, with NK-resistant targets such characteristic kinetics of PBL cytolytic activity was not observed. The kinetics of PBL-mediated cytolysis was similar in IL-2-responders and non-responders, so that no correlation of in vivo and in vitro effects of subcutaneous IL-2 and VBL treatment could be established.

Activity of PDGF (A) gene in the human cell line derived from a renal carcinoma metastasis.

PDGF-like activity was investigated in conditioned media of cell cultures derived from 4 human renal carcinomas. Transient production of PDGF-like factor was found only in the cell line derived from a subcutaneously growing metastasis. Further analysis of this cell line showed an increase of PDGF (A) gene activity in one cellular clone.

[Hydatidiform changes in the chorion]. / Hydatiformní zmeny choria.

Revision of 630 cases of mola enabled a description of morphology in complete hydatid mole, partial hydatid mole, hydropic degeneration and their relation to the origin of trophoblastic disease. A survey covers pathogenesis of molar syndrome, cytogenetic findings and genetic methods for discrimination of complete and partial hydatid mole. To express grade of certainty in diagnostic of the lesions is recommended.

[Detection of hCG serum levels in choriocarcinoma using monoclonal antibodies]. / Sérové hladiny hCG u choriokarcinomu sledované pomocí monoklonálních protilátek.

hCG values and those of its alpha- and beta-subunits are assessed in the serum of all patients treated in the Centre of trophoblastic disease. Commercial RIA kits with conventional antibodies are used. There was an opportunity to work with kits of Serono Co. with monoclonal antibodies, the so-called hCG MAIA clone kit which assesses hCG and its beta-subunit. In this IRMA-MAIA system 240 sera of patients with choriocarcinoma were processed. Comparison of hCG values found in the IRMA-MAIA test with values assessed with kits from Kosice revealed the following: 33% of the values in MAIA were higher, 57% were in agreement. 10% of the specimens were MAIA negative, while the Kosice values were low (50-100 i.u./l) and a negative finding was assumed. The authors investigated low (or false positive) hCG values and values obtained during the so-called residual reaction. According to the obtained results in this system of three highly sensitive monoclonal antibodies no cross reaction with LH occurs.

Analysis of epidermal growth factor and epidermal growth factor receptor expression in human renal carcinoma cell cultures.

In cells derived from two human renal carcinomas only the precursor form of epidermal growth factor (EGF) was found. The binding assay revealed a high level of EGF receptor expression in both cell types tested. However, these receptors are not involved in the growth activity of the cells under in vitro conditions used. The source of DNA synthesis-stimulating activity found in conditioned media of the cells tested is discussed with respect to possible participation of TGF beta.