CD69 on CD56+ NK cells and response to chemoimmunotherapy in metastatic melanoma.

BACKGROUND: The few chemoimmunotherapy trials that together with dacarbazine (DTIC) and interferon-alpha 2a (IFNalpha), include retinoic acid (RA), did not include detailed immunological evaluation of functional and phenotypic natural killer (NK) cell characteristics, and have shown contradictory clinical results. MATERIALS AND METHODS: Malignant melanoma (MM) patients undergoing phase II-randomized chemoimmunotherapy trials were treated with DTIC, IFNalpha (Hoffmann-La Roche) (group A, n = 31), and with DTIC, IFNalpha and 13-cis-RA (Isotretinoin, Hoffmann-La Roche, Basel, Switzerland) (group B, n = 29). Patients and 42 healthy controls were evaluated by FACS flow analyses for CD3/CD56/CD69 positive cells, NK cytotoxicity in fresh peripheral blood lymphocytes (PBL) and for interferon regulatory factor-1 mRNA expression by reverse transcriptase polymerase chain reaction in treated PBL. RESULTS: The addition of RA to a DTIC-IFN regime did not bring any therapeutical benefit in terms of response or survival. Immunological follow-up on days 1, 6 and 27 of each therapy cycle shows a significant increase in NK cell activity in both groups, only on day 6 of the first cycle, while CD69+CD56+ expression increased significantly on day 6 of each therapy cycle, in both groups. Evaluation of the dynamics of expression of IRF-1 of in vitro treated PBL, shows its strong and prompt up-regulation by IFNalpha and synergistic effect of IFNalpha and RA combination. CONCLUSION: The dynamics of the increase in CD69 early activation antigen expression on CD56+ NK cells is systematic and serial with the increase being significantly higher on day six of the first cycle in group B patients with clinical response, compared to those without, indicating possible predictive value of CD69 expression for clinical response to chemoimmunotherapy.

TNF-alpha induced changes in cell membrane antigen expression on K-562 cells associated with increased lactate dehydrogenase (LDH) release.

TNF-alpha is a pleiotropic cytokine, which induces death of sensitive cells, whose effect depend on cell membrane receptor expression, cell cycle phases, as well as on intracellular ratio of pro- apoptotic and anti-apoptotic molecule expression. Since determination of LDH release from cultured cells in vitro, reflects early membrane alterations, we estimated and compared LDH release from cultured cells with changes in cell membrane antigen expression on K-562 cells after TNF-alpha treatment by flow cytometry. The significant increase in LDH release activity and cytotoxicity values was associated with decrease in membrane molecule expression for CD45 and CD30 as well as for low expressed CD45RA and CD38 after TNF-alpha treatment. However, percentage of decrease of all examined molecules is not uniform, and appears to depend on the respective level of pre treatment values expression and molecule type. These results indicated the complexity of events on cell membrane, including association between increasing LDH release and decrease of antigen expression of membrane molecules following TNF-alpha mediated processes.

The comparison of spontaneous LDH release activity from cultured PBMC with sera LDH activity in non-Hodgkin's lymphoma patients.

Based on the fact that lactate dehydrogenase (LDH) enzyme is a very sensitive indicator of the cellular metabolic state, aerobic or anaerobic direction of glycolysis, activation status, and malignant transformation, in this study we compared values of the spontaneous LDH release from circulating PBMC with sera LDH activity in 53 different subtypes of non-Hodgkin's lymphoma (NHL) patients. Results shows that serum LDH was significantly (p < 0.05) elevated in comparison to the range values only in the advance clinical stage (III and IV) in all investigated subtypes of NHL according to The Working and REAL classification. On the other hand, the spontaneous LDH release from cultures PBMC is significantly (p < 0.01) elevated in early and advanced stage in all investigated forms of NHL in comparison to healthy controls. Based on consideration that an increase in spontaneous LDH release appears before elevated sera LDH activity, we conclude that determination of spontaneous LDH release by microassay from cultured cells together with other findings may help in the diagnosis of NHL patients, especially in patients with early stage of disease.

TNF-alpha induces changes in LDH isotype profile following triggering of apoptosis in PBL of non-Hodgkin's lymphomas.

Based on the possibility of tumor necrosis factor (TNF)-alpha to perform multiple and opposite biologic effects, we simultaneously investigated in vitro its effects on intracellular lactate dehydrogenase (LDH)-H and LDH-M isoenzyme activity and morphological characteristics following induction of apoptosis in peripheral blood mononuclear cells (PBMC) of non-Hodgkin's lymphoma patients (NHL) prior to and after the end of applied chemotherapy. TNF-alpha showed a significant increase ( p<0.05) of LDH-H and LDH-M activity in sonified PBMC of healthy controls after 18 h cultures accompanied with an increase of apoptotic index (AI) from 2.3 to 16.2%. Contrary to this, in PBMC of NHL patients prior to therapy TNF-alpha induced a significant decrease ( p<0.05) of LDH-H isotype activity. In patients after administration of chemotherapy, TNF-alpha in a dose of 100 U/ml induced a significant increase ( p<0.05) of LDH-M isotype activity, but not of LDH-H. In the PBMC of NHL patients prior to chemotherapy, TNF-alpha in vitro induced an increase of AI from 2.8 up to 6.8%, while in PBMC of NHL patients after applied chemotherapy AI changed from 7.2 to 14.4%. However, there was no significant difference in the increase of apoptosis in PBMC of NHL patients with high-grade malignancy and high rate response among patients who received first-line therapy, high-dose therapy, or third-line therapy regimens after in vitro TNF-alpha treatment. These results indicated different susceptibilities of PBMC of NHL to TNF-alpha when effects were analyzed by determination of intracellular LDH isotype profile and induction of apoptosis prior to and after administration of therapy in comparison to effects on healthy controls PBMC.

[Accomplishments and perspectives in tumor diagnostics and treatment]. / Dostignuca i perspektive u dijagnostici i lecenju tumora.

In contemporary medicine cancer has an exceptionally important role, even though it is not a new disease. Tumors are found in all animals and plants, and today are more frequent than before, when they were found predominantly in advanced ages. The frequency occurrence depends of many factors, and it is more frequent in countries with higher degree of civilization, what is the consequence of irradiation of other diseases as well as more pronounced industrialization, leading to in proper nourishment, many sources of intoxication with carcinogenic factors arising from environmental pollution, and even by the use of some diagnostic and therapeutic procedures. Cancer is the result of disturbance in cell growth and differentiation. In the appearance and spreading of tumors many factors are involved. In the Department of Experimental and Clinical Oncology of the Institute for Oncology and Radiology, we in the first place investigated the role of immunity, hormones and central nervous system, predominantly in experimental conditions, based on the investigations on animals, the in vitro investigations is tissue cultures and some disturbances which are found in blood of patients with tumors. As in the processes of carcinogenesis the immune system has in important role, in the immunological investigations we primarily investigated parameters of the status of the immune system in patients with malignant processes. The number and function of particular cell subsets of the immune system was investigated, namely T and B lymphocytes and their subclasses, NK cells and monocytes-macrophages. In the majority of analyzed patients with breast and lung cancer, lymphoproliferative diseases and other malignancies, it was shown that the majority of them had a decreased number of immune cells that correlated with the clinical advances of disease and applied cytostatic therapy. However, it was shown that the function of these cells, primarily NK, but also of T lymphocytes, was markedly decreased even before changes in the cell number, indicating that a functional impairment is present even before the cell number decrease and proportional to the advancement of the disease. As the results of exploring of investigation of the immunological status indicate the concrete defects in the function of the separate components of the immune system, these findings make it possible to direct immunotherapy for the correction of existing defects. The activity and pathways of mechanisms of NK cells and the possibility of their modulation were also investigated. Also, the possibility of the application of mAbs in the precise diagnostics of some malignancies was explored. The role in carcinogenesis was investigated in tumors whose appearance, growth and spreading is hormone-dependent. One of the hormone-dependent tumors is breast cancer. It was shown that they are significantly dependent on estrogen and growth factor presence, steroid-receptor content, and that these characteristics can change during the disease and do not have to be identical in their metastasis. Numerous investigations that we performed were in the in vitro conditions, i.e. in cell cultures. The obtained data show how some tumor cells react to applied agents, cytostatical and biological. However their effect in vivo is very often different, as in the in vivo conditions many other factors are involved, suggesting a need for further investigations of these factors. The role of the central nervous system neurotransmiters in carcinogenesis in experimental animals exposed to chemical cancerogen (5-methylcholantrene) with simultaneous treatment of the monoamine system was investigated. It was shown that monoamines expressed their influence on carcinogenesis by regulating the brain homeostasis, as well as by direct influence on the intracellular processes during cell development and differentiation. The obtained results will direct our further investigations toward obtaining mAbs for receptors for TNFalpha and IFNgamma, transfection of suppressor gene into tumor cell cultures and genes for IL-2 and TNFalpha. At the same time we will work on isolation of malignant melanoma tumor antigen and construction of a vaccine using some epitopes and adjuvantes. We will try to introduce appropriate immunotherapy in the advancedehZAD.

[The possibilities of modulation of NK cell activity]. / Mogucnosti modulacije NK celijske aktivnosti.

The immune system has an important role in tumor appearance and spreading. One of the most efficient subpopulations of cytotoxic cells in the destruction of tumors are NK cells. NK cells are activated and increase their cytotoxic potential and modulate their cytokine production after treatment with IFNgamma, IL-12, TNFalpha and IL-2. The investigation of the activity of NK cells was performed on peripheral blood lymphocytes (PBL) of 16 healthy controls and of 40 patients with metastatic breast carcinoma. Modulation of NK cells was performed with IL-2, IL-7, IL-12, TNFalpha, monoclonal antibodies (mAb) for TNFalpha and TNFalpha receptors type I and II, as well as with sera of healthy controls and patients with breast cancer in different clinical stages. Modulating effect of the applied factors after in vitro treatment of PBL was evaluated by the cytotoxic assay using 51chromium. Our results indicate that IL-2 significantly increased the activity of NK cells of controls and breast cancer patients. The sera of patients with advanced breast cancer significantly reduced NK cell activity. IL-7, IL-12 and mAb for TNFalpha do not significantly change the activity of NK cells. The presence of anti-TNFalpha mAb did not change the inhibitory effect of the sera of breast cancer patients with advanced disease on the activity of NK cells of controls and patients with breast cancer. Blocking of TNFalpha Rcs with mAbs decrease the reactivity of NK cells for IL-2. The treatment of breast cancer patients with advanced clinical stage of breast cancer with IL-2, as an additional therapy, could be advantageous, as NK cells after this treatment increase their cytotoxic activity against tumor cells and can improve therapeutical results.

[Correlation between functional capability and phenotypic characteristics of peripheral blood lymphocytes in patients with malignant melanoma]. / Odnos funkcionalinih sposobnosti i fenotipskih karakteristika limfocita periferne krvi bolsnika sa malignim melanomima.

Malignant melanoma is well known for its poor response to chemotherapy, radiotherapy and its susceptibility to immunotherapy. Considering that dacarbazine (DTIC) and interferon alpha (IFNalpha) are among the most frequently used agents in the treatment of melanoma, the aim of this study was to evaluate the kinetics of immunological changes during adjuvant treatment of melanoma patients with DTIC or with IFNalpha monotherapy, as well as with their combination in metastatic disease. Pre-therapy values of immunological parameters showed significantly decreased NK cell activity, altered in vitro production of TNFalpha, IL-2 and proliferative response of peripheral blood lymphocytes (PBL), while percentage of PBL subpopulations was unchanged. During therapy, NK cell activity was significantly increased after the 1st cycle of combined chemoimmunotherapy (DTIC + IFNalpha), followed by a significant decrease after the 2nd cycle of therapy. Furthermore, in this group, there was a significant increase in CD4+ T helper cell percentage after the 1st cycle of therapy. Serial monitoring of activation antigens also showed a significant increase in the expression of CD38 on CD8+ cytotoxic T cells, after the 1st and 2nd cycle in combined chemoimmunotherapy group and, after 30 days, in the group of patients treated with IFNalpha, only. The increase in the expression of HLA-DR activation antigen on CD3+ and CD8+ T cells had a gradual increase and significant rise after the 2nd cycle of combine chemoimmunotherapy, only. The dynamic of immunological changes, mostly observed in combined chemoimmunotherapy, and rarely in IFNalpha monotherapy gives valuable insight into induced immunomodulation, suggesting early, but transient favourable changes, that could be prolonged by timely introduction of other immunotherapeutic agents.

[Lactate dehydrogenase (LDH) in peripheral blood lymphocytes (PBL) of patients with solid tumors]. / Laktat dehidrogenaza za u limfocitima periferne krvi bolesnika sa solidnim tumorima.

Serum LDH level is a prognostic factor in different malignancies as its increase reflects tumor mass and response to therapy. Serum LDH is the consequence of the disruption of the cell membrane of a large fraction of dividing malignant cells whose metabolic hallmark is anaerobic glycolysis that leads to increased LDH enzyme activity. Moreover, as we have previously shown that spontaneous LDH release from cells represents a measure of cell membrane damage, and this parameter is used for the estimation of cell destruction in cytotoxic assays, the aim of this study was to evaluate the characteristics of LDH activity of PBMC of patients with different solid tumors (non-Hodgkin's lymphomas--NHL, n=47; Hodgkin's disease--HD, n=45; ovarian cancer--OvCa, n=6; breast cancer--BrCa, n=34; thyroid cancer--TyCa, n=3; cancer of PVU--CaPVU, n=4 and head & neck--H&N, n=6) in all clinical stages of NHL and HD and in advanced clinical stages of disease for BrCa, OvCa, CaPVU and H&N. Spontaneous LDH release from PBMC was determined by the spectrophotometric method from supernatants of 8 x 10(6)/ml PBMC cultured for 2 h in RPMI 1640 without phenol red using and LDH substrate mixture. The total LDH activity was determined after lysis of PBMC by ultrasound. The obtained results indicate that PBMC in all the investigated malignancies, compared to control PBMC, demonstrate a significant increase (p<0.01) in spontaneous LDH release act, which correlates with advanced clinical stage in all malignancies except in Hodgkin's disease, in which the spontaneous LDH release was increased in all clinical stages. Contrary to this, the total LDH activity was not increased in PBMC in all investigated tumors. However, the "percent of spontaneous LDH release" was always increased, regardless of the total LDH activity, indicating that spontaneous LDH release is the consequence of PBMC membrane damage present in advanced stages of different solid tumors.

Therapeutic implications of the kinetics of immunomodulation during single or combined treatment of melanoma patients with dacarbazine and interferon-alpha.

The therapy of metastatic melanoma has not given satisfactory results. Single chemo- or immunotherapeutic agents in the adjuvant setting or combined chemoimmunotherapy for metastatic disease have generally been evaluated only in terms of clinical benefit. Considering that dacarbazine (DTIC) and interferon-alpha (IFN-alpha) are among the most frequently used agents in the treatment of melanoma, the aim of this study was to evaluate the kinetics of immunological changes during adjuvant treatment of melanoma patients with DTIC or with IFN-alpha monotherapy, as well as by their combination in metastatic disease. The evaluated immunological parameters showed significant early increase in the activity of NK (natural killer) cells, CD4/CD8 ratio, CD4+ T cell number in patients treated with combined chemoimmunotherapy and an increase in expression of the early activation antigen CD38 on CD8+ cytotoxic T cells, both, in patients treated with combined chemoimmunotherapy and with IFN-alpha alone, while, no significant change in any one parameter was detected in the group of patients receiving DTIC. The kinetics of the observed immunological changes, restricted to combined chemoimmunotherapy, indicate that the engagement of antitumor immune response appears early but is short-lived and that this favorable effect should be augmented and prolonged by the timely introduction of additional immunomodulating agents.

Association of NK cell dysfunction with changes in LDH characteristics of peripheral blood lymphocytes (PBL) in breast cancer patients.

The cytotoxic activity of NK (natural killer) cells is very important in immunological surveillance against the appearance and especially the spread of malignant disease. The aim of this study was to investigate the function of this subpopulation of cells in breast cancer patients in different clinical stages of disease prior to therapy. NK cell activity was determined in breast cancer patients and healthy controls by three different methods: standard 51-chromium-release assay and by the original colorimetric uncorrected and corrected lactate dehydrogenase (LDH) release assay. A discrepancy was shown between the assays, as the uncorrected LDH assay showed, not only, much higher values, but no stage-dependent depression in NK cell activity compared to the chromium-release assay. Further analyses of separately cultured peripheral blood lymphocytes (PBL) revealed that this difference arose from an increasing, clinical stage-dependent, spontaneous LDH release from PBL of breast cancer patients. Furthermore, a stage-dependent increase in intracellular LDH activity of PBL was found, although without difference in LDH-H and LDH-M isotype ratio, compared to controls. Increased spontaneous LDH release and intracellular LDH activity was more evident in young patients, under 40 years. Correction of the original LDH-release assay for the spontaneous LDH release activity from PBL present in the assay, gave values of NK cell activity comparable to those determined by the chromium assay and indicated that breast cancer patients have a significant depression in NK cell activity which correlates with the stage-dependent increase in spontaneous LDH release. Moreover, as both assays measure the secretory, perforin-mediated, NK cell cytotoxic pathway against tumor cells, it can be concluded that the appearance of spontaneous LDH release is an indicator of cell membrane damage which not only allows the loss of LDH, but also of the components of the secretory killing pathway, resulting in NK cell dysfunction with the progression of disease. The novel findings obtained in this work reveal the association of PBL membrane damage with clinical stage of breast cancer that can, aside from reflecting NK cell depression, underlie the defect in other PBL subsets and subsequently facilitate progression of the malignant process.

Impaired perforin-dependent NK cell cytotoxicity and proliferative activity of peripheral blood T cells is associated with metastatic melanoma.

AIMS AND BACKGROUND: Patients with metastatic melanoma often have defects in the percentage and function of peripheral blood NK cells, which are involved in the non-specific innate antitumor immune response, and T cells, which participate in the specific acquired antitumor immune response. The aim of this study was to investigate in more detail not only the percentage but also the activation status and function of NK and T cells in patients with metastatic melanoma prior to therapy. METHODS: The percentage of peripheral blood CD56+ NK cells, CD3+ T cells and their CD4+ and CD8+ subsets, as well as the expression of the activation antigens CD69, CD38 and HLA-DR were analyzed by flow cytometry. The functional capacity of NK cells was evaluated by the 51-chromium release cytotoxicity assay, while the proliferative activity of T cells was estimated by the lymphocyte transformation test to mitogen phytohemagglutinin. RESULTS: The results obtained in this study have revealed a new aspect of NK and T cell dysfunction that is not, as commonly reported, associated with a decrease in their percentage. Moreover, a significant number of the investigated patients had a higher percentage of NK cells that did not lead to improved NK cell cytotoxicity as a result of the detected defect in the NK cell perforin-mediated cytotoxic mechanism of tumor cell lysis. The impaired proliferative response of T cells was associated with a decreased expression of the activation antigen HLA-DR. CONCLUSION: The novel finding in this study of melanoma patients with metastatic disease is the impaired perforin-dependent NK cell cytotoxic mechanism, which was recently shown to be primarily responsible for preventing metastasis. Another interesting finding was the generally hyporeactive status of T cells, possibly resulting from persistent antigenic stimulation. The observed dysfunction of NK and T cells in patients with metastatic melanoma prior to therapy point to the need to supplement chemotherapy with appropriate immunotherapeutic agents in order to overcome the immunosuppression associated with advanced malignancy.

Content of epidermal growth factor receptor in metastatic breast cancer: its role in endocrine sensitivity prediction.

Epidermal growth factor receptor (EGF-R) is known as an indicator of endocrine independence of breast cancer. However, a small proportion of EGF-R expressing tumors was found to respond to endocrine treatments. On the other side, a cut-off point of EGF-R positivity is not yet defined. In the aim to find out whether there exists a cut-off value that sharply discriminate the endocrine sensitive and endocrine insensitive breast cancers, the quantitative EGF-R content was analyzed in a group of 42 female patients with metastatic disease, being routinely treated with chemo-, chemo-endocrine, or endocrine therapy alone. Steroid receptors (SR) and EGF-R were determined by biochemical methods in tissue samples of an unselected group of patients. Patients with metastatic disease, either at diagnosis, or developed after the treatment of operable or locally advanced breast cancer, were included in the present analysis. According to the treatments used, and their therapeutic response, all patients were divided in endocrine sensitive or resistant, and chemo-sensitive or resistant. The SR and EGF-R status and content was analyzed in relation to the sensitivity to both systemic treatments. The EGF-R content was significantly lower in responders to endocrine treatments, compared to non-responders, while there was no difference in EGF-R level, in relation to the sensitivity to chemotherapy. In addition, the EGF-R content was significantly higher in chemo-sensitive tumors, than in endocrine sensitive. On the contrary, ER content was significantly higher in endocrine sensitive, than in endocrine resistant, and in chemo sensitive patients, as well. Similar differences were found in PR content, but they were less pronounced. While the individual ER contents in endocrine sensitive and endocrine resistant tumors overlapped, the EGF-R ranges were different: no one endocrine sensitive tumor exceeded the EGF-R content of 26 fmol/mg, thus suggesting the EGF-R cut-off point of endocrine sensitivity. The clinical use of EGF-R, with the cut-off point of 26 fmol/mg, in addition to clinical criteria of endocrine sensitivity and SRs, would significantly improve the correct endocrine sensitivity prediction (from 52 to 78%). In conclusion, in a group of metastatic breast cancer patients, treated routinely by systemic therapies it was found, that the use of higher cut-off point for EGF-R positivity can improve the prediction of endocrine sensitivity. The prognostic relevance of this cut-off value remains to be analyzed.

Different alterations in lactate dehydrogenase activity and profile of peripheral blood mononuclear cells in Hodgkin's and non-Hodgkin's lymphomas.

The enzyme lactate dehydrogenase (LDH) activity of peripheral blood mononuclear cells (PBMC), LDH isotype H and M pattern and PBMC spontaneous LDH release activity were examined in 55 non-Hodgkin's lymphoma (NHL) patients, 46 Hodgkin's disease (HD) patients and 47 controls. The intracellular LDH and M isotype activity of PBMC, their spontaneous LDH release activity significantly increase (p < 0.01) in NHL with progressing histological grade of malignancy. Contrary to this, all classical HD patients have a significant elevation (p < 0.05) of each of these parameters. Furthermore, unlike HD, in NHL clinical stage is associated with significant (p < 0.05) increase in the level of spontaneous LDH release activity in each histological form. It is also shown that spontaneous LDH release activity of PBMC for HD and NHL patients demonstrates significant positive correlation (p < 0.005) with serum LDH level, although elevation of spontaneous LDH release precedes serum LDH increase in both diseases. The results obtained regarding alterations in intracellular, isotype and spontaneously released LDH activity of circulating PBMC show that these parameters are dependent, in NHL patients, on the grade of malignancy and tumor burden, while they are persistently present in HD patients.

Expression of epidermal growth factor receptor in breast cancer, from early stages to advanced disease.

Epidermal growth factor receptor was determined in 106 newly diagnosed breast cancer patients, using the biochemical method. The group consisted of 58 patients in stage I-II, and 48 patients in stage III-IV. Although a significant inverse correlation was found between EGF-R status, and ER or PR status, quantitative content of EGF-R did not correlate either with quantitative ER, or PR levels. The ER/PR content was similar in all clinical stages, suggesting their stability during the clinical course of the disease. EGF-R content was significantly higher in stage IV, compared to stage I, while intermediate clinical stages and all substages did not differ according to the EGF-R content. EGF-R was confirmed as a weak prognostic factor within clinical stages. However, in a whole group, the overall survival was significantly better in patients whose tumors EGF-R content was lower than 26 fmol/mg, compared to those with higher ERF-R content. EGF-R content was highly predictive for the response to systemic endocrine treatment, in metastatic breast cancer patients. In locally advanced breast cancer a trend towards higher levels of EGF-R was found in inflammatory breast cancers, compared to non-inflammatory ones. Slightly higher levels were found in responders to local non-endocrine primary treatments (radiotherapy with or without chemotherapy), compared to non-responders, suggesting the possible predictive role of EGF-R for the response to such treatments. Our results emphasized the usefulness of quantitative receptor determination suggesting the relative stability of EGF-R content during the clinical course of breast cancer, its independence from ER, its significant predictive and weak prognostic values, and a possible correlation with the aggressiveness of the disease, and response to non-endocrine treatments.

A comparison of the NK cell cytotoxicity with effects of TNF-alpha against K-562 cells, determined by LDH release assay.

Effects of r h TNF-alpha as a single cytotoxic mediator against K-562 cells was examined by LDH release and compared with NK cell cytotoxicity. The mean values of the percentage of LDH release (x = 6.25 +/- 3.68%, for ten individual experiments) from K-562 cells cultured for 2 h with r h TNF-alpha 100 U/ml of culture medium did not give significant difference in comparison with mean values of percentage LDH release (x = 6.43 +/- 2.97%, for 37 individual experiments) from K-562 cells which were cultured without r h TNF-alpha (Student's t-test, P > 0.05). The results also showed, that in the presence of increasing concentrations of r h TNF-alpha there was no significant increase of LDH release through the cell membrane in these short term incubations. However, significant difference in LDH release from K-562 cells was found after 6 h between cultures treated for 30 min with or without r h TNF-alpha (Mann-Whitney test, P < 0.05). Since TNF-alpha alone shows a lower degree of K-562 cell membrane damage than NK effectors, this suggested that TNF-alpha is neither an only nor a major mediator of cell destruction, based on determination of LDH release.

The difference in NK-cell activity between patients with non-Hodgkin's lymphomas and Hodgkin's disease.

Natural killer (NK) cells play an important role in immune surveillance against malignant diseases. Considering the lymphoid origin of malignant lymphomas, as well as scarce data concerning NK-cell function in these neoplasms, we evaluated NK-cell activity in 49 patients with non-Hodgkin's lymphomas (NHL) and 47 patients with Hodgkin's disease (HD), prior to therapy. Using the recommended International Working Formulation and the Ann Arbor staging system for classification of lymphomas we found, by the LDH release cytotoxicity assay, that decreased NK-cell activity (P < 0.05) in NHL patients was essentially related to unfavourable histology (13 indolent lymphomas, 25 intermediate and 11 very aggressive lymphomas were included), but that within these categories clinical stage of the disease also contributed to the degree of NK-cell dysfunction. In contrast, in HD, NK-cell activity was persistently decreased (P< 0.05), compared to controls, irrespective of histological type and clinical stage. It is of interest also that the most profound NK-cell dysfunction that is present and persistent from the onset of HD, and which appears in very aggressive NHL was associated with the phenomenon of increased spontaneous lactate acid dehydrogenase (LDH) release activity from the separated PBMC of these patients. The difference in the level of NK-cell impairment between patients with various histological grades of malignancy in NHL and HD suggests different initial participation of innate immune reactions in these diseases.

[The effect of sera of breast cancer patients on NK cell activity]. / Dejstvo seruma bolesnica s karcinomom dojke na aktivnost celija "en-ka".

INTRODUCTION: The immune system may influence tumorogenesis through natural killer (NK) and specific T cytotoxic cells. It is considered that NK cells, which constitute 15% of peripheral blood lymphocytes, have a main role in defense, due to their ability to destroy tumor cells in situ or in the blood stream in first contact without major histocompatibility restriction. Determination of NK cell activity in malignancies indicates that the evaluation of their activity is more reliable than their number, as well as that IL-2 and interferons increase, while prostaglandines and some hormones decrease their activity. With regard to this, investigations of the effect of sera of patients with malignancies on NK cell activity are being performed [10] in order to see whether the impairment of this activity, which is common in advanced disease, may be due to the presence of different factors in their sera or is the consequence of a defect in NK cells, themselves. This work analysis the influence of sera of healthy persons and patients with different stages of breast cancer on the activity of NK cells of controls and patients with malignancies. MATERIALS AND METHODS: NK cell activity was evaluated in 58 patients with stage I-III, 11 patients with stage IV of breast cancer and 18 healthy controls. The sera of 30 patients with stages I-III (CaSa), 11 patients with stage IV of breast cancer without or with metastases (CaSb, CaSm, respectively) and 35 healthy controls (ZS/HS) was used for analysis of their effect on NK cell activity. Foetal calf sera (FCS) were used as controls. Isolation and cultivation of peripheral blood lymphocytes: Lymphocytes from 30 ml of blood were isolated by sedimentation on lymphoprep gradient (Nycomed, Norway) and resuspended in RPMI 1640 culture medium without sera in concentration of 4 x 10(6) cells/ml of medium Lymphocytes of each person were treated in a short term culture for 18 h, simultaneously with 10% of inactivated FCS, 10% inactivated healthy sera (HS) and 10% inactivated sera of patients with different stages of breast cancer (CaSa, CaSb, CaSm). NK cell assay: NK cell activity was evaluated as previosly reported by the radioactive 51-Cr release assay. Namely, 100 microl of lymphocytes as effector cells (E) in concentration of 4 x 106 cells/ml of medium and three 1:1 dilutions there of were mixed with 100 ul of target tumor cell line K 562 (T) previosly labeled with 51-chromium (As = 100 microCl) concentration of 5 x 10(5) cells/ml of medium in 96 round bottom plates and incubated for 4 h in an incubator at 37 degrees C with 5% CO2 and humidity. After that the plates were centrifuged and 100 microl of supernatants were used for determination of 51-Cr release in a gamma-counter and expressed in counts per minute (cpm). The percent of specific NK cytotoxicity was determined by the following formula: ((cpm in experimental release - cpm in spontaneous release/ (cpm in maximal release - cpm in spontaneous release)) x 100 where the spontaneous and maximal release were obtained by incubation of tumor cells in culture medium i.e. with 5% Triton x 100 solution, respectively. The results were interpreted by the Mann-Whitney statistical method. RESULTS NK cell activity determined in 18 healthy controls was x = 57.75 +/- 2.21% (E:C=80:1, in patients with breast cancer in clinical stages I-III n=58) it was x = 36.64 +/- 1.92% (E:C=80:1) which is significantly (p < 0.01) below that found in controls, while in patients in stage IV (n=11) it was x = 18.31 +/- 1.38% (E:C=80:1) which is significantly (p < 0.01) below the stages I-III (Table 1). Short term in vitro treatment of PBL in culture medium with FCS gave a significant increase in NK cell activity of both groups of patients: in stages I-III and stage IV (Table 1). Treatment of PBL of patients with stages I-III with sera CaSa, CaSb and CaSm gave a progressively greater inhibition of their NK cell activity compared with FCS (Figure 1). The same type of treatment of PBL of patients with stage IV of breast cancer with HS and CaSb also showed a significant inhibition of their NK cell activity compared to treatment with FCS (Figure 2). Treatment of PBL of healthy controls with CaSa, HS and CaSm demonstrated the same type of progressive inhibition of NK activity (Figure 3). DISCUSSION: In this work it has been shown that patients with breast cancer in clinical stages I-II, and especially stage IV, have a significantly decreased NK cell activity compared to controls. However, the treatment of both controls' and patients' PBL with sera of patients with different stages of this disease changed NK cell activity. The sera of the first three stages of breast cancer increased this activity, while sera of advanced disease caused a progressive decrease compared to control treatments with FCS. Some of these results were partially obtained in different investigations indicating the presence of stimulative factors in sera of patients with locoregional disease, and inhibitory factors in sera of advanced disease, as well as a slight, probably homeostatic, inhibitory effect of sera of healthy controls 2 These results indicate that the changes in NK cell activity are governed more by the factors in the present sera than by the changes in the activity of NK cells, themselves. The nature of these factors in sera should be further investigated.

Steroid receptors in pleural effusions of advanced breast cancer patients.

The steroid receptor content in breast carcinoma correlates with the responsiveness of malignant cells to endocrine manipulation. Although the steroid receptor status of the primary tumor is mostly used to select systemic therapy, it was suggested that steroid receptor content should be evaluated in metastatic lesions whenever possible. In this study the estrogen and progesterone receptor content was determined biochemically in 38 pleural effusions from advanced breast cancer patients. In 17/38 patients the steroid receptor status was assessed twice during the course of the disease - at diagnosis in the primary tumor/lymph nodes, and subsequently in metastatic pleural effusion fluid. A trend towards lower receptor values in pleural fluids was evident. There was no correlation between pleural steroid receptor content and pleural response to endocrine or chemo/endocrine therapy, indicating that the usefulness of effusional steroid receptors for therapy planning of advanced breast cancer could not be confirmed in this study.

Analysis of perforin expression in human peripheral blood lymphocytes, CD56+ natural killer cell subsets and its induction by interleukin-2.

In this study new evidence is obtained by the use of an anti human perforin monoclonal antibody (mAb), anti P1, concerning the number of perforin positive cells in human peripheral blood lymphocytes (PBL). It is shown that about 23% of PBL is perforin positive and that this percent increases by the treatment in RPMI 1640 medium alone to 33% and with 1000 U r hIL-2 to 46%. Assessment of the cytotoxicity potential of NK cells from PBL, freshly isolated and treated, against tumor cell line K562 by the standard NK cell 4-hr 51-chromium release assay, indicates a significant enhancement in their cytotoxicity. By FACStar sorting and analysis of the CD56+ NK cell population new evidence is obtained which shows that about 25-30% of this population represents the CD56bright+ subset, while 70-75% represents the CD56dim+ subset. As the two subsets were shown to differ functionally they were stained with anti P1 for the evaluation of perforin content and it was found that both of them are positive for perforin from 97-99%, suggesting that the functional difference is not due to perforin content. In this sense, as NK cells are constitutively positive for perforin, the increase in the cytotoxicity of NK cells induced by IL-2 is most likely due to the synthesis and expression of various adhesion molecules on NK cells which increase their cytotoxic potential, as well as, that the detected increase in the number of perforin positive cells by this lymphokine does not belong to NK, but to the T lymphocyte population. The data obtained in this study indicate the possibility of perforin detection in human lymphocytes by an anti human perforin mAb and the change in the number of perforin positive cells after stimulation with interleukin-2.