IL-2 And IL-15 Induced NKG2D, CD158a and CD158b Expression on T, NKT- like and NK Cell Lymphocyte Subsets from Regional Lymph Nodes of Melanoma Patients.

Regional lymph nodes (LN)s represent important immunological barriers in spreading of malignant tumors. However, they are the most frequent early metastatic site in melanoma. Immunomodulatory agents including cytokines have been included in therapy of melanoma and have shown severe side effects and toxicity. In this sense, there is a growing need for bringing these agents to further in vitro testing that may enlighten aspects of their regional application. Therefore, the aim of this study was to investigate the effect of interleukin (IL)-2 and IL-15, the two cytokines with similar immune-enhancing effects, on the expression of activating NKG2D, inhibitory CD158a and CD158b receptors on CD8+ T, NKT-like and NK cell lymphocyte subsets from regional LNs of melanoma patients. In this study, we showed significant effects of IL-2 and IL-15 cytokine treatments on the expression of activating NKG2D and on inhibitory CD158a and CD158b receptors on lymphocytes, CD8+ T, NKT-like and NK cell lymphocyte subsets originating from regional LNs of melanoma patients. Furthermore, IL-2 and IL-15 by inducing the expression of NKG2D activating receptor on innate and on adaptive lymphocyte subsets and by augmenting NK cell antitumor cytotoxicity that correlated with the cytokine-induced NKG2D expression, increased antitumor potential of immune cells in regional LNs of melanoma patients irrespective of LN involvement. These findings indicate the importance of immune cell population from regional LNs of melanoma patients in the development of immune intervention strategies that may if applied locally increase antitumor potential to the level that controls tumor progressions.

Prognostic importance of steroid receptor status for disease free and overall survival after surgical resection of isolated liver metastasis in breast cancer patients.

PURPOSE: Breast cancer (BC) is the most common malignancy among women, while isolated operable liver metastases (LMs) from BC are very rare and occur in only 1-5% of the patients. Besides, positive steroid receptor (SR) status for oestrogen and/or progesterone is known as a factor which improves disease free survival (DFS) and overall survival (OS). The primary aim of this study was to examine the impact of SR status on DFS and OS after liver metastasectomy in female patients with primary BC. METHODS: We analyzed 32 medical records of female patients diagnosed and treated for primary BC with LMS as the first and only site of disease progression, at the Institute of Oncology and Radiology of Serbia (IORS), during 2006- 2009. All of them underwent primary BC surgery as well as LMs resection. RESULTS: Patients with metachronous BC and LMs and positive SR status in both BC and LM (BC+/LM+) had a median time from BC to LM occurrence (TTLM) of 36 months, compared to BC+/LM- and BC-/LM- subgroups, whose medians for TTLM were 30.5 and 14.5 months, respectively (p<0.01). For all patients, positive SR status showed high correlation with longer DFS and OS after LM resection (medians according survival analysis for DFS/OS in subgroups BC-/LM-, BC+/LM- and BC+-LM+ were 10/19, 25/45, 50/not reached months respectively; p<0.01 for DFS/ OS). Cox regression analysis confirmed that the subgroup of patients with BC-/LM- had 10.8 and 18.8 higher risk of events for DFS (disease relapse or death) and event for OS (death only), respectively, compared to BC+/LM+ subgroup of patients. CONCLUSION: Positive SR status in BC and LM has a high impact not only on time from BC to LM occurrence, but also on longer DFS and OS after LM resection.

Decreased Interferon γ Production in CD3+ and CD3- CD56+ Lymphocyte Subsets in Metastatic Regional Lymph Nodes of Melanoma Patients.

As lymphogenic dissemination is very common in melanoma, regional lymph nodes (LN)s represent first immunological barriers to tumor invasion and play a complex role in antitumor immune defense. In this sense, their most prominent role is the presentation of tumor-derived antigens to naïve T cells and generation of cell-mediated adaptive immune response. Since tumor micro-environment affects immune cell function in this study we have evaluated the ability of T cells and NK cells in metastatic (involved) and non-metastatic regional LNs to produce interferon γ (IFNγ), a pleiotropic cytokine that regulates adaptive antitumor immune response. Our results show reduced IFNγ production in both T and NK lymphocyte subsets and decreased prevalence of T cells in metastatic regional LNs of melanoma patients. The decrease of IFNγ production in T cells was more pronounced with increased number of involved regional LNs indicating tumor-induced functional impairment of both T and NK cell lymphocyte subsets in involved regional LNs. Therefore, shown low IFNγ production in metastatic LNs may represent an obstacle in adaptive cell-mediated antitumor immune response and hence may enable tumor progression.

Metformin effects on malignant cells and healthy PBMC; the influence of metformin on the phenotype of breast cancer cells.

The aim of research was to determine the effects of maximally therapeutically achievable concentrations of metformin on malignant cells and healthy peripheral blood mononuclear cells (PBMC). Eight patients with T2D or hyperglycemia and nine healthy volunteers were included in the study. For determination of the influence of metformin on the phenotype of breast carcinoma, 1,410 patients with surgically removed tumors were included. From this group 37 breast cancer patients had DM type 2 or hyperglycemia and were pretreated with metformin alone or sometimes in combination with other antidiabetic drugs. Our results proved that metformin at low concentrations induced mild decrease in survival of malignant cells and PBMC stimulated for proliferation, but it didn't affect survival of resting PBMC. The effects of plasma of hyperglycemic patients who were under metformin therapy on autologous PBMC-induced decrease in survival of MDA-MB-361 cells, was noticeable in some patients. Metformin pretreatment for 24 h of HER2+ MDA-MB-361 cells, which were subsequently treated for 48 h with Herceptin, induced additional decline in cell survival. The analysis of influence of metformin on phenotype of breast cancer cells revealed significantly lower number of diabetic cancer patients treated with metformin with overexpressed HER2+ tumors (p < 0.013), while the number of patients with ER+PR+ tumors was not significantly changed (p < 0.832). In conclusion, therapeutically used concentrations of metformin exhibit mild cytotoxic action on malignant and dividing normal cells pointing to its preferred role in malignant and autoimmune diseases. The use of metformin was associated with pronounced decrease in HER2 overexpressing tumors.

In-vitro activation of natural killer cells from regional lymph nodes of melanoma patients with interleukin-2 and interleukin-15.

Regional lymph nodes (LNs) represent the first barrier in lymphogenic tumor dissemination in melanoma. Natural killer (NK) cells, the effector cell subpopulation of the innate immune system, are in the first line of antitumor immune defense. Therefore, the aim of this study was to investigate the effect of interleukin (IL)-2 and IL-15, two cytokines with similar immune-enhancing effects, on antitumor cytotoxic function and immunophenotype of NK cells from regional LNs of melanoma patients. Mononuclear cells purified from regional LNs of 50 melanoma patients in clinical stage II-IV were treated in vitro for 72 h and 7 days with 200 IU/ml rhIL-2 and 25 ng/ml IL-15 at 37°C in 5% CO2. Both cytokines significantly augmented NK cell cytotoxic activity, transcription of the cytotoxic molecule perforin, and the level of functionally mature perforin in both nonmetastatic and metastatic regional LNs. IL-2 treatment increased the percentage of CD3CD56 NK cells by increasing the CD56 NK cell subset in both nonmetastatic and metastatic LNs, whereas IL-15 treatment did not affect the percentage of NK cells and their subsets. Both cytokines increased on NK cells from nonmetastatic and metastatic LNs the expression of CD69 early activation antigen, the NKG2D activating receptor, as well as CD16 and inhibitory killer-cell immunoglobulin-like receptor CD158b, both inherent to the mature and the cytotoxic NK cell phenotype. In conclusion, our data may indicate the therapeutic potential of the NK cell population from regional LNs either as immunotherapeutic targets or as adoptively transferred after activation with IL-2 or IL-15.

Distribution of several activating and inhibitory receptors on CD3(-)CD56(+) NK cells in regional lymph nodes of melanoma patients.

BACKGROUND: Natural killer (NK) cells, as the main effector subpopulation of the innate immune system, play an important role in the control of the rise and spread of malignant tumors. Regional lymph nodes (LN) represent the first immunologic barrier to tumor metastasis. Since there are scarce data on NK cells from regional LN of cancer patients, the aim of this study was to investigate the expression of several activating and inhibitory receptors on the entire NK cell population as well as their CD3(-)CD56(dim) and CD3(-)CD56(bright) functional NK subsets from regional LN of melanoma patients. MATERIALS AND METHODS: Mononuclear cells were isolated from 50 regional LN of melanoma patients. The expression of several receptors on NK cells and their functional subsets was analyzed by flow cytometry. RESULTS: We show increased percentages of CD3(-)CD56(+) NK cells in involved LN compared with uninvolved LN, mostly in favor of the CD56(dim) NK cell subset. NK cells in involved LN express similar levels of activating receptor NKG2D, while the level of another activating receptor, CD16, is increased compared with uninvolved LN. Regarding the expression of inhibitory NK cell receptors, we show increased CD158b, but similar low CD158a, inhibitory killer Ig-like cell receptor expression in involved LN compared with uninvolved LN. Furthermore, NK cells in involved compared with uninvolved LN displayed increased CD69 early activation antigen expression. CONCLUSIONS: Our results indicate that with tumor infiltration into regional LN of melanoma patients, NK cells, mostly of the CD56(dim) subset, are recruited into draining LN. The invading NK cells show counterbalance of the increased expression of CD16 activating receptor and increased CD158b inhibitory killer Ig-like cell receptor.

Serum activity of DPPIV and its expression on lymphocytes in patients with melanoma and in people with vitiligo.

BACKGROUND: Dipeptidyl peptidase IV, a multifunctional serine protease, is implicated in regulation of malignant transformation, promotion and further progression of cancer, exerting tumor-suppressing or even completely opposite - tumor-promoting activities. The aim of present research was to determine the serum DPPIV activity, as well as the percentages of CD26+ lymphocytes, CD26+ overall white blood cells and the mean fluorescence intensity of CD26 expression on lymphocytes in patients with melanoma, people with vitiligo and in healthy controls. METHODS: The activity of DPPIV in serum was determined by colorimetric test. Expression of DPPIV (as CD26) on immunocompetent peripheral white blood cells was done using flow cytometry analysis. RESULTS: Data from our study show for the first time statistically significant decrease: in the serum DPPIV activity, in the percentage of CD26+ overall white blood cells and in the percentage of lymphocytes in patients with melanoma in comparison to healthy control people. In addition, significantly lower serum DPPIV activity was found in the group of patients with melanoma in relation to people with vitiligo too. CONCLUSION: This study indicates the need for exploring the cause and the importance of the disturbances in the serum DPPIV activity and in the CD26 expression on immunocompetent cells in complex molecular mechanisms underlying the development and progression of melanoma.

Immunity to melanin and to tyrosinase in melanoma patients, and in people with vitiligo.

BACKGROUND: The aim of this study was to determine the presence and the intensity of humoral immunity to melanoma-associated antigens: tyrosinase and melanin, in patients with melanoma, in persons with vitiligo and in control healthy people. METHODS: The study involved 63 patients with melanoma and 19 persons with vitiligo. Control group consisted up to 41 healthy volunteers. Mushroom tyrosinase and synthetic melanin were used as the antigens. RESULTS: ELISA test showed significantly (p < 0.0000004 and p < 0.04) lower levels of IgM anti-tyrosinase autoantibodies, in melanoma and vitiligo patients respectively, compared to controls.Although there was no significant difference between the levels of IgA anti-melanin autoantibodies in melanoma or vitiligo patients in comparison with controls, the enhanced concentrations of anti-melanin IgA autoantibodies were preferentially found in melanoma patients with metastatic disease. Significantly high percentage in the Fc alphaRI (CD89) positive cells was determined in melanoma patients (p < 0.002 and p < 0.008) in comparison to that found in healthy people or in patients with vitiligo, in the already mentioned order, pointing that IgA dependent cellular cytotoxicity is not important for the immune action against melanoma, even more that it is included in some immune suppression.Levels of IgG autoantibodies to mentioned antigens in melanoma patients although low were not significantly lower from controls. These findings analyzed together with the statistically significant low percentage of FcgammaRIII, (CD16) positive immunocompetent cells (p < 0.0007 and p < 0.003), which was found in patients with melanoma compared with healthy or vitiligo people respectively, and statistically significant low percentage of (CD16 + CD56+) natural killer (NK) cells (p < 0.005) found in melanoma patients in comparison to healthy controls pointed to the low probability for anti-melanoma IgG mediated, antibody mediated cellular cytotoxicity, (ADCC) and NK cytotoxicity. Moreover the ratio of the percentages of granulocytes and percentage of lymphocytes was statistically higher in patients with melanoma in relation to healthy people as well as to people with vitiligo (p < 0.0007 and p < 0.05 respectively). CONCLUSION: Autoantibodies to tyrosinase and to melanin which are found even in healthy people, point that consummation of edible mushrooms that carry the antigen tyrosinase and melanin, could influence the humoral anti-melanoma immune response.Levels of different immunoglobulin classes of anti-melanin and anti-tyrosinase antibodies varied depending on the presence and the stage of studied diseases. Besides, the statistically enhanced ratio of the percentages of granulocytes and percentage of lymphocytes, together with statistically decreased percentage of NK cells is found in analyzed melanoma patients.