Ana Damjanović

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

BackgroundDipeptidyl 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.MethodsThe 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.ResultsData 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.ConclusionThis 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

BackgroundThe 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.MethodsThe 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.ResultsELISA 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).ConclusionAutoantibodies 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.

Good tolerance to goat's milk in patients with recurrent aphthous ulcers with increased immunoreactivity to cow's milk proteins.

BACKGROUND Recurrent aphthous ulcers (RAU) represent a very common, but poorly understood mucosal disorder. The connection between immunity to cows milk proteins (CMP) and oral diseases was noted earlier. The goal of this study was to determine the prevalence of the increased levels of serum antibodies to goats milk proteins (GMP), by enzyme-linked immunosorbent assay (ELISA) test, in subjects who have RAU and proven increased immunity to CMP. METHODS Fifty subjects with RAU (36 with proven increased immunity to CMP and 14 without this increased immunity) were included in this research. Levels of serum IgA, IgG, and IgE antibodies to the same quantity of the examined antigens were determined by ELISA. The statistical analysis of data was performed by Wilcoxon rank-sum test and Mann-Whitney test. RESULTS The levels of serum antifresh cows milk IgA, IgG, and IgE antibodies were significantly higher than the levels of serum antifresh goats milk, in subjects with RAU with proven increased immunoreactivity to CMP (P = 0.0003; P < 0.0001; P < 0.0001). CONCLUSIONS These results indicate that patients with RAU with increased immunity to CMP could consider the use of goats milk as the alternative protein source.

Immunoreactivity to food antigens in patients with chronic urticaria.

The goal of study was better understanding of complex immune mechanisms that can help to evaluate patients with chronic urticaria (CU), especially those with unknown etiology. The study involved 55 patients with CU. Control group consisted of up to 90 healthy persons. The presence and intensity of serum IgG, IgA, IgM and IgE antibodies to common food antigens: cow’s milk proteins (CMP), gliadin and phytohemagglutinin were determined by ELISA. Determination of subpopulations of immunocompetent cells was performed by flow cytometry. Significantly enhanced IgE, but also IgA immunity to CMP was found in patients with CU in comparison to healthy controls: (p < 0.000004) and (p < 0.002), respectively. Notably, in 40 out of 55 CU patients, the increased levels of some type of immunoglobulin reactivity to CMP were found. Regarding gliadin, only the levels of serum IgE anti-gliadin antibodies were significantly enhanced in patients with CU (p < 0.04). Significantly enhanced percentage of CD89+ cells accompanied with significantly lower percentage of lymphocytes and significantly higher mean fluorescence intensity of CD26 expression on lymphocytes were found in patients with CU in comparison to healthy controls (p < 0.04), (p < 0.02) and (p < 0.003), respectively. Results of this study may help in better understanding the complex immune disturbances in patients with CU.

Peripheral White Blood Cell Subsets in Metastatic Colorectal Cancer Patients Treated with Cetuximab: The Potential Clinical Relevance

It was demonstrated that cetuximab-induced tumor regression is based on the effects exerted by immune cells included mainly in the innate immune response. Therefore, the focus of this study was to explore the alterations in the percentages of CD16+, and/or CD56+ lymphocytes, which are comprised of NK cells, and minority of CD56+CD3+ cells, in patients with metastatic colorectal cancer before or 2 months after the treatment with cetuximab-based regimens associated with the response to therapy. The changes in the percentages of lymphocytes and granulocytes in these patients were evaluated as well. We enrolled 50 patients with wild-type KRAS metastatic colorectal cancer. Disease progression was observed in 11/50 patients (non-responders), while other patients achieved partial response or stable disease (responders). Control groups included up to 72 healthy individuals. A significant decrease in the percentages of CD56+ and CD16+CD56+ lymphocytes together with a significant decrease in the percentage of lymphocytes and an increase in the ratio of granulocyte to lymphocyte percentages were observed in patients with metastatic colorectal cancer before therapy, compared with those in the healthy individuals. In contrast to those in the responders, the percentage of CD16+ lymphocytes in the overall white blood cell pool was shown to be significantly decreased in the non-responders, together with a significantly decreased percentage of lymphocytes, a significantly increased percentage of granulocytes, and an increased ratio of granulocyte to lymphocyte percentages before treatment compared with those in the healthy controls. Two months after the initiation of the treatment, significantly decreased percentages of CD16+, CD56+, and CD16+CD56+ lymphocytes were observed in patients, compared with those determined in the healthy controls. The same changes in the amounts of circulating immune cells were also observed in the responder subgroup, but the percentages of CD16+, CD56+, and CD16+CD56+ lymphocytes 2 months after treatment in the non-responder group did not differ significantly in comparison with healthy individuals. Considerable alterations of immune cell percentages observed in patients with metastatic colorectal cancer with disease progression indicate that the assessment of peripheral white blood cell architecture before treatment initiation may be clinically relevant.

Dipeptidyl peptidase IV: serum activity and expression on lymphocytes in different hematological malignancies

Abstract The aim of this research was to determine the serum dipeptidyl peptidase IV (DPPIV) activity as well as the percentages of CD26 + lymphocytes and CD26 + overall white blood cells in patients with hematological malignancies: non-Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL), leukemia, plasmacytoma and multiple myeloma, and in healthy individuals. Data from our study showed significantly decreased serum DPPIV activity and a significant decrease in the percentage of: CD26 + lymphocytes, CD26 + overall white blood cells and lymphocytes in patients with NHL in comparison to healthy controls. Patients with leukemia had a statistically significant lower activity of DPPIV in serum and significant decrease in the percentage of CD26 + lymphocytes in relation to healthy controls. Furthermore, significantly decreased DPPIV serum activity associated with a significantly reduced percentage of CD26 + overall white blood cells and percentage of lymphocytes was found in patients with multiple myeloma when compared to the healthy control group. The obtained results indicate that immune disturbances that can occur in hematological malignancies might be related to the decreased expression and activity of CD26/DPPIV that we observed.

Identification of cytotoxic metabolites from Mahonia aquifolium using 1 H NMR-based metabolomics approach

Herein, we propose a 1H NMR-based metabolomics method to reveal cytotoxic metabolites from Mahonia aquifolium stem-bark. Primary and secondary metabolites in the Mahonia aquifolium extracts were identified by thorough analysis of 1H and 2D NMR spectra, without prior isolation. An OPLS multivariate analysis method was used to correlate the chemical composition of the plant extracts with the results of cytotoxic activity against Human cervical adenocarcinoma cell line. Protoberberine alkaloids berberinе and palmatine, along with bisbenzylisoquinoline alkaloid berbamine were identified as the most influential in the OPLS model, with the highest cytotoxic activity.

LC/DAD determination of biogenic amines in serum of patients with diabetes mellitus, chronic urticaria or Hashimoto's thyroiditis

Biogenic amines are integral part of nearly every cell. In present study, we used method of acidic extraction of histamine (His), of polyamines putrescine (Put), spermidine (Spd) and catecholamines epinephrine (Epi) and norepinephrine (NE) from human serum; precolumn derivatization with dansyl chloride, and LC/DAD analysis of the biogenic amines, in aim to monitor differences of their levels in patients with diabetes mellitus , chronic urticaria , and Hashimotos thyroiditis, compared to healthy subjects, and to observe them as possible markers for immune mediated diseases. Method of retention times was used for determination of serum biogenic amines. We found statistically significant differences in putrescine and histamine levels in diabetes mellitus patients; putrescine, histamine, spermidine and epinephrine levels in chronic urticaria patients compared to healthy controls, and putrescine and spermidine levels in Hashimotos thyroiditis patients, compared to controls. Norepinephrine was found only in serum of patients with chronic urticaria . The values of recovery, evaluated in controls, varied between 85.7% and 106.7%. The statistically significant changes in putrescine, histamine, spermidine and epinephrine levels in patients compared to healthy people reflects the existence of biochemical disturbances in mentioned immune-mediated diseases.

Metformin decreased human melanoma cell survival but did not affect viability of normal resting PBMC

Introduction: Epidemiological studies indicate that diabetes mellitus type 2 (T2D), could be associated with increased risk for some forms of cancer. While the antitumour action of high metformin concentrations in experiments in vitro is well documented there are no data about the action of the much lower, maximally achievable plasma metformin concentrations (present in diabetic patient’s blood during the therapy). Therefore, the first aim of this work was to determine the antiproliferative action of low plasma metformin concentrations on human melanoma Fem-x cell line and on normal human peripheral blood mononuclear cells, (PBMC). Second aim was to evaluate the contribution of externally, in vitro added metformin on the decrease in the survival of target Fem-x cells induced by PBMC. Materials and methods: Up to five patients with hyperglycemia and seven healthy volunteers were included in the study. Metformin hydrochloride was the product of Harman Finochem. Human melanoma Fem-x cell line and (PBMC) non-stimulated, or stimu-