In vitro increased natural killer cell activity of metastatic melanoma patients with interferon-α alone as opposed to its combination with 13-cis retinoic acid is associated with modulation of NKG2D and CD161 activating receptor expression.

PURPOSE: Considering tumor-induced suppression of natural killer (NK) cell activity the aim of this study was to investigate the in vitro effect of a standard immunotherapeutic cytokine, interferon (IFN)α, and a less investigated agent, 13-cis retinoic acid (RA) on the functional and receptor characteristics of CD16-defined NK cells and their functionally diverse dim and bright subsets in patients with metastatic melanoma (MM). METHODS: Peripheral blood lymphocytes (PBL) of patients with clinical stage IV MM were stimulated in vitro for 18 h in RPMI 1640 culture medium (CM) alone, CM supplemented with IFN-α (250 U7sol;ml), RA (10-6M) and their combination. NK cell activity was determined using standard 4 h radioactive cytotoxicity assay, while the expression of activating (NKG2D, CD1617rpar; and inhibitory (CD158a, CD158b) NK cell receptors on CD3-CD16+ NK cells and their functional bright and dim subsets were analyzed by flow cytometry. RESULTS: NK cell cytotoxic activity was increased after in vitro treatment with IFN-α alone and in combination with RA, while only IFN-α induced increase in NKG2D and CD161 activating NK cell receptor expression. Contrary to this, RA treatment increased the expression of inhibitory KIR CD158b. IFN-α-obtained increase in CD161 expression was due to its induction on both NK cell subsets, while for NKG2D only on CD16bright subset. CONCLUSION: The favorable enhancement of NK cell activity of MM patients obtained with IFN-α is associated with upregulation of activating NKG2D and CD161 receptors, while the lack of RA-associated upregulation is probably due to the shown increased expression of inhibitory KIR receptor CD158b after in vitro treatment with this agent.

Association of decreased NK cell activity and IFNγ expression with pSTAT dysregulation in breast cancer patients.

PURPOSE: Impaired IFNγ production in peripheral blood lymphocytes (PBL) and their subsets reflects immunosuppression and inadequate antitumor immune response in cancer patients. Decreased function of natural killer (NK) cells has not been investigated in breast cancer with respect to altered pSTAT signaling pathways. METHODS: PBL of breast cancer patients and healthy controls were analyzed for IFNγ and pSTAT1 expression and NK cell activity using flow cytometry and (51)Cr-release assay, respectively. The level of pSTAT1, 3 and 5 was investigated by Western blotting. RESULTS: Our results indicated that PBL and CD3(-) CD16(+) NK cells of patients had significantly lower level of IFNγ. The patients had a significantly decreased NK cell cytotoxicity compared to controls, with the decrease being dependent on the stage of disease. Positive correlation between IFNγ level in PBL and NK cytotoxicity in controls and patients was also shown. The PBL of patients, compared to controls, expressed lower level of pSTAT1, 3 and 5. The patients' T and NK cell subsets had lower pSTAT1 level. CONCLUSION: This study indicates that pSTAT1 in PBL of breast cancer patients could be a biomarker of decreased NK cell cytotoxicity and IFNγlevel that are associated with progression of this disease.

Novel aspects of in vitro IL-2 or IFN-α enhanced NK cytotoxicity of healthy individuals based on NKG2D and CD161 NK cell receptor induction.

As IL-2 and IFN-α modulate NK cell activity it was of interest to investigate the expression of newly defined NK cell receptors and augmented NK cell activity in healthy individuals after cytokine in vitro treatment. Peripheral blood lymphocytes (PBL) obtained from 31 healthy volunteers treated for 18 h with 200 IU/ml IL-2 and 250 IU/ml IFN-α were evaluated for NK cell cytotoxicity. Expression of NKG2D, CD161, CD158a, CD158b receptors was analyzed on CD3⁻CD16+ NK cells, cytotoxic CD16(bright) and regulatory CD16(dim) subsets by FACS flow. The found induced significant in vitro enhancement of NK cell activity by both cytokines is supported by specific cytokine induction in PBL of pSTAT1 and pSTAT5, determined by Western blotting, as well as induction of IRF-1 transcription. Both cytokines induce significant up-regulation of NKG2D expression while only IFN-α induced significant up-regulation of CD161, with no alteration in KIR expression by either cytokine on CD3⁻CD16+ NK cells. Investigated cytokines did not induce change in NK cell bright and dim subset distribution. Moreover, we find that, not only cytokine receptor induction on the CD3⁻CD16+ NK cells, but also simultaneous increase in their percentage and/or density on CD16(bright) and CD16(dim) subsets, represent good indicators of receptor cytokine-susceptibility. As the role of NK cells has been shown in the loss of tolerance, infection and cancer, the data obtained in this study may be of help in NK cell profiling, by giving referent values of cytokine-induced novel NK cell receptor expression either in evaluation of these diseases or in immunomonitoring during cytokine immunotherapy.

[Immunoglobulins G, A, M and circulatory immune complexes in patients with laryngeal carcinoma].

INTRODUCTION: Malignant tumors of the head and neck represent 5% of all malignancies, of which the most common are tumors of the larynx and oral cavity. In the blood serum of patients with malignant tumors increased levels of immunoglobulins and circulating immune complexes have been detected, with a diverse relevance to the clinical course and prognosis of the disease. AIM: Due to contradictory findings of the correlation between the humoral immune response and the clinical course of the disease, we examined 42 male patients with laryngeal carcinoma. All patients underwent surgery, of which 15 patients were treated postoperatively with radiotherapy. The changes in the levels of immunoglobulins and circulating immune complexes in the blood serum were recorded and evaluated. RESULTS: Analysis of the results showed an immunological disorder of an abnormal level of circulating immune complexes in the blood serum that normalized after the surgical removal of the tumor. The levels of immunoglobulins G and A were abnormal during the whole postoperative period of examination. CONCLUSION: The normalizing of the levels of circulating immune complexes in the blood serum, after surgical removal of the tumor, shows a strong association between the two, and this could consequently mean that it could be used as a prognostic tool, particularly in correlation with other immunological parameters.

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.

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.

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.

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.

[Mechanisms in onset of postoperative immunodepression and its importance in the surgical treatment of cancer patients]. / Mehanizmi nastanka postoperativne imunodepresije i njen znacaj za hirursko lecenje onkoloskih bolesnika.

Postoperative immunodepression is inevitable transient path of operative trauma. Changes in the immunologic status are related to the duration of the anesthesia and surgery procedure. Mortality and morbidity and recurrence rate may be related with immunodepression.

[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.

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.

[Steroid receptors and NK cell activity in patients with breast cancer]. / Receptori za steroidne hormone i aktivnost celija "prirodne ubice" kod bolenica s karcinomom dojke.

Taking into account the important role of oestrogen and NK cells in the process of cancerogenesis, the content of receptors for steroid hormones and NK cell activity was investigated in 31 patients with breast cancer. The analysis of steroid receptors showed the usual distribution with regard to clinical stage. The values of NK cell ativity in peripheral blood of these patients were decreased x = 38.27 +/- 2.84%) compared to controls (X = 57.76 +/- 2.21%), especially in advanced stages, as was previously shown fot this malignancy. General analysis of values for these two parameters did not show a correlation of NK activity and content of steroid receptors. However, the presence of positive progesterone receptors, which reflects the active effect of oestrogen, was associated with very low NK activity (n = 12 PR+, NK x = 29.47 +/- 3.18%), indicating possible negative regulation of immunological parameters by estrogen.

[Immunological activity of NK cells]. / Imunska funkcija celija "prirodnih ubica".

The article reviews the general characteristics of NK cell which represent a population of lymphocytes engaged through unique mechanisms in immunological defense of the organism. Data concerning morphological characteristics, mechanism of action, perforin, surface antigens and regulation, are given. The new results in human studies related to NK cells are given in detail. The possibility of NK cell activation in vivo and in vitro as applicable methods in adoptive immunotherapy, is described.