Radiosensitising Effects of Metformin Added to Concomitant Chemoradiotherapy with Cisplatin in Cervical Cancer.

AIMS: The role of metformin on the radiosensitising effect of cisplatin is not clear. Here we investigated the radiosensitising effect of metformin alone and combined with cisplatin in HeLa cells, as well as the implications of the adenosine monophosphate-activated protein kinase (AMPK) pathway on the radiosensitising effect. MATERIALS AND METHODS: HeLa cells were treated with ionising radiation, metformin, cisplatin, A769662 (AMPK activator) and dorsomorphin (AMPK inhibitor) or in combination. A cell proliferation assay, Western blot and flow cytometry were carried out. RESULTS: Metformin potentiated cisplatin cytotoxicity when administered 4 h before ionising radiation. Although the radiosensitising effects of metformin and cisplatin alone were observed, which is more apparent at high ionising radiation doses, the metformin-cisplatin combination did not increase the radiosensitivity of cisplatin at any ionising radiation dose. Dorsomorphin alone significantly decreased cell proliferation and potentiated the radiosensitising effects of cisplatin with ionising radiation. Administration of A769662 24 h prior to cisplatin treatment resulted in an increased AMPK level that yielded resistance to cisplatin, but this effect was not observed in HeLa cells concomitantly treated with A769662 and cisplatin. CONCLUSIONS: Modulation of AMPK may have a role in cervical cancer treatment. Increased AMPK levels result in higher sensitivity to ionising radiation but causes resistance to cisplatin. Dorsomorphin is proven to be a potent radiosensitising agent. The use of metformin alone may be an option as a radiosensitiser during high-dose ionising radiation (e.g. intracavitary brachytherapy).

Vitamin D replacement ameliorates serum lipoprotein functions, adipokine profile and subclinical atherosclerosis in pre-menopausal women.

BACKGROUND AND AIMS: Low vitamin D (vitD) has been linked to increased cardiovascular (CV) risk, but the effects of vitD supplementation are not clarified. We evaluated the impact of vitD normalization on HDL cholesterol efflux capacity (CEC), which inversely correlates with CV risk, the proatherogenic serum cholesterol loading capacity (CLC), adipokine profile and subclinical atherosclerosis. METHODS AND RESULTS: Healthy premenopausal women with vitD deficiency (n = 31) underwent supplementation. Subclinical atherosclerosis was evaluated by flow-mediated dilation (FMD), pulse wave velocity (PWV) and augmentation index (AIx), measured with standard techniques. HDL CEC and serum CLC were measured by a radioisotopic and fluorimetric assay, respectively. Malondialdehyde (MDA) in HDL was quantified by the TBARS assay. Pre-ß HDL was assessed by 2D-electrophoresis. Serum adipokines were measured by ELISA. VitD replacement restored normal levels of serum 25-hydroxyvitamin D (25OHD) and significantly improved FMD (+4%; p < 0.001), PWV (-4.1%: p < 0.001) and AIx (-16.1%; p < 0.001). Total CEC was significantly improved (+19.5%; p = 0.003), with a specific increase in the ABCA1-mediated CEC (+70.8%; p < 0.001). HDL-MDA slightly but significantly decreased (-9.6%; p = 0.027), while no difference was detected in pre-ß HDL. No change was observed in aqueous diffusion nor in the ABCG1-mediated CEC. Serum CLC was significantly reduced (-13.3%; p = 0.026). Levels of adiponectin were increased (+50.6%; p < 0.0001) and resistin levels were decreased (-24.3%; p < 0.0001). After vitD replacement, an inverse relationship was found linking the ABCA1-mediated CEC with pre-ß HDL (r2 = 0.346; p < 0.001) and resistin (r2 = 0.220; p = 0.009). CONCLUSION: Our data support vitD supplementation for CV risk prevention.

Analysis of BAFF and TRAIL expression levels in multiple sclerosis patients: evaluation of expression under immunomodulatory therapy.

OBJECTIVES: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and B cell-activating factor (BAFF), the members of tumor necrosis factor superfamily, play essential roles in immune homeostasis and may have potential contributions to the autoimmune process in multiple sclerosis (MS). MATERIAL AND METHODS: Thirty-five relapsing remitting MS (RRMS) patients and 19 healthy individuals were enrolled in the study. The expression of TRAIL on peripheral blood lymphocytes was analyzed by flow cytometry. The serum levels of soluble TRAIL (sTRAIL) and soluble BAFF (sBAFF) were determined by ELISA. Further, we evaluated the effect of IFN-ß on sTRAIL, sBAFF levels and on TRAIL surface expression in these patients on the third and sixth months following the treatment. RESULTS AND CONCLUSION: These preliminary results signify that MS patients are heterogenous in TRAIL expression. Additionally, during the IFN-ß treatment, the soluble form of TRAIL increases concomitantly as its surface expression decreases on lymphocytes. The basal sBAFF levels of patients were significantly higher than the control group and no significant change was observed. Thus, the changes in TRAIL expression may be a potential parameter indicating the response to IFN-ß1 therapy at individual level.

Coexistence of different tissue tumourigenesis in an N-methyl-N-nitrosourea-induced mammary carcinoma model: a histopathological report in Sprague-Dawley rats.

N-methyl-N-nitrosourea (MNU), a highly potent carginogen, is widely used to generate mammary tumours in murine species. In a model of MNU-induced mammary carcinogenesis using immature female Sprague-Dawley rats, large mammary tumours (largest dimension > or =0.5 cm) were obtained within a very short period of time. In addition, in the rats bearing MNU-induced mammary carcinomas, there were a number of tumours whose origins were not from mammary tissue but from several different tissues and from mammary non-epithelial tissue. The tumours were of mesenchymal or epithelial origin and they were located in the inguinal region. These tumours were diagnosed as fibroadenoma, combined tubular adenoma and fibroadenoma, hyperkeratotic papilloma, keratinous cyst and malignant peripheral nerve sheath tumour (MPNST) with smooth muscle differentiation. The occurrence of these other tumours in addition to the development of the mammary carcinomas may be attributed to a direct local effect of the intraperitoneal administration of MNU during the sexual development of the immature rats. In the MNU-induced mammary tumour model, coexistence of tumourigenesis in various non-mammary tissues should be considered an important factor that may interfere with experimental procedures and results and also the quality of life of the tumour-bearing animals.

Enhanced expression of the local haematopoietic bone marrow renin-angiotensin system in polycythemia rubra vera.

Local bone marrow (BM) renin-angiotensin system (RAS) affects physiological and pathological haematopoiesis, including erythropoiesis. In this study, quantitative expression of the messenger RNAs of the major RAS components--angiotensin-converting enzyme (CD143), renin and angiotensinogen--were measured in BM samples by quantitative real-time polymerase chain reaction, to evaluate the activity of local BM RAS in polycythemia rubra vera (PV) in comparison with normal erythropoiesis. The presence of CD143 was also investigated in the same BM samples by flow cytometry. Increased local synthesis of the major RAS components has been identified by demonstrating corresponding mRNAs in the BM of the patients with PV. Our findings indicate up-regulation of local BM RAS, together with down-regulation of the cell surface angiotensin-converting enzyme receptors, in the autonomous neoplastic clonal erythropoiesis of PV.

Tamoxifen-induced tissue factor pathway inhibitor reduction: a clue for an acquired thrombophilic state?

BACKGROUND: Current understanding of hemostatic systems enables us to better explore the enigmatic pathobiology of tamoxifen (TAM)-induced thrombotic diathesis. We have therefore aimed to assess the hemostatic changes in breast cancer patients receiving TAM on an adjuvant basis. PATIENTS AND METHODS: The study population consisted of 43 female patients with hormone receptor-positive breast cancer who received TAM 20 mg/day as part of their adjuvant treatment. Mean age was 52+/-12 years (range 25-74). Twenty-one patients (49%) were premenopausal. Plasma samples were collected prior to and following 6 months of TAM therapy and were assayed for total tissue factor pathway inhibitor (TFPI), free TFPI, lipid-bound TFPI, thrombomodulin, D dimer, activated protein C resistance (APC res), factors VIIa, II, V, VII and X, and global fibrinolytic capacity (GFC). RESULTS: Median total TFPI decreased significantly from 48.5 ng/ml to 36.2 ng/ml (P=0.001), free TFPI from 10 to 7.6 ng/ml (P=0.001) and lipid-bound TFPI from 39.1 to 28.7 ng/ml (P=0.001). There were significant decreases in the levels of factor II (P=0.03), factor V (P=0.001), factor VII (P=0.06), thrombomodulin (P=0.01) and D dimer (P=0.001). However, APC res times were significantly prolonged (P=0.04). The remaining parameters that we have studied were not significantly affected. CONCLUSION: Our findings suggest that TAM tends to activate the coagulation pathway by counteracting major molecules involved in coagulation inhibition, namely TFPI and TM. As reflected by unchanged GFC, the drug appears to impair the expected compensatory activation of the fibrinolytic system, which removes fibrin polymers resulting from coagulation activation.

Expression of adhesion molecules LFA-1, ICAM-1, CD44, and L-selectin in childhood non-Hodgkin lymphomas.

BACKGROUND: The aim of the present study was to analyse the expression of adhesion molecules in childhood non-Hodgkin lymphomas and to correlate the findings with clinical features and prognosis. PROCEDURE: Samples were obtained from pleural and peritoneal fluids, bone marrow aspirates, and tissue biopsies from 21 patients (median age: 8 years). There were 9 T-cell and 12 B-cell lymphomas. The expression of CD18, CD44s, CD54, CD62L were investigated with flow cytometry by using monoclonal antibodies. RESULTS: Absence of CD18, which was independent from immunophenotype, was found in 67% of patients. Positive CD44s and CD62L expression were observed in 48 and 63% of the cases, respectively. In all of the cases with T-cell lymphoma, CD54 was negative, whereas 8 of 12 cases with B-cell lymphoma expressed this molecule (P = 0.005). There was no correlation between location of disease and the expression of adhesion molecules, except CD54 that was negative in all mediasten lymphoma (P = 0.004). CD62L (+) patients had more frequently stage IV disease than CD62L (-) ones (P = 0.01). Two-year overall survival was 83 and 29% in CD18 (+) and CD18 (-) cases; 55 and 36% in CD44s (+) and CD44s (-) cases; 46 and 42% in CD54 (+) and CD54 (-) cases; 42 and 50% in CD62L (+) and CD62L (-) cases. CONCLUSIONS: The expression of LFA-1 on lymphoblasts is lost in the majority of childhood non-Hodgkin lymphomas. ICAM-1 is not detected on neoplastic cells of patients with T-cell lymphoblastic lymphoma. L-selectin positivity correlates with disseminated disease. There is no significant relationship between the expression of adhesion molecules and the survival rates, although CD18(+) cases had better overall survival rate than CD18(-) cases.

Correlation of flow cytometric parameters and transferrin receptors with gallium-67 scintigraphic images in lymphoma patients.

The purpose of this study was to determine the correlation of flow cytometric parameters and transferrin receptors with gallium-67 scintigraphic imaging results in Hodgkin's and non-Hodgkin's lymphoma patients. DNA content and cell cycle analyses were performed using flow cytometry and transferrin receptor analysis was carried out by the immunohistochemistry technique in 24 patients aged between 16 and 62 years. All patients underwent gallium-67 scintigraphy, and tumour to background ratios were calculated. The findings were correlated with computed tomography and/or magnetic resonance imaging. A strong relationship was observed between flow cytometry and transferrin receptor expression with gallium-67 tumour scintigraphy [P = 0.005, r = 0.054 and P = 0.038, r = 0.54 (Spearman test), respectively]. The results of this study show that there is a close correlation between each of these modalities and, as they reflect the biological activity of the tumour, together they have a major role in treatment and follow-up.

Gastric lymphomas in Turkey. Analysis of prognostic factors with special emphasis on flow cytometric DNA content.

BACKGROUND: In contrast to DNA ploidy, to the authors' knowledge the prognostic significance of S-phase fraction (SPF) in gastric lymphomas has not been determined. In the current study, the prognostic significance of various parameters including SPF and DNA aneuploidy were analyzed and some distinct epidemiologic and biologic features of gastric lymphomas in Turkey were found. METHODS: A series of 78 gastric lymphoma patients followed at Hacettepe University is reported. DNA flow cytometry was performed for 34 patients. The influence of various parameters on survival was investigated with the log rank test. The Cox proportional hazards model was fitted to identify independent prognostic factors. RESULTS: The median age of the patients was 50 years. There was no correlation between patient age and tumor grade. DNA content analysis revealed 4 of the 34 cases to be aneuploid with DNA index values < 1.0. The mean SPF was 33.5%. In the univariate analysis, surgical resection of the tumor, modified Ann Arbor stage, performance status, response to first-line chemotherapy, lactate dehydrogenase (LDH) level, and SPF were important prognostic factors for disease free survival (DFS). The same parameters, excluding LDH level, were important for determining overall survival (OS). In the multivariate analysis, surgical resection of the tumor, disease stage, performance status, and age were found to be important prognostic factors for OS. CONCLUSIONS: To the authors' knowledge the current study is the first to demonstrate the prognostic significance of SPF in gastric lymphomas. The distinguishing features of Turkish gastric lymphoma patients are 1) DNA indices of aneuploid cases that all are < 1.0, which is a unique feature; 2) a lower percentage of aneuploid cases; 3) a higher SPF; 4) a younger age distribution; and 5) lack of an age-grade correlation. The authors conclude that gastric lymphomas in Turkey have distinct biologic and epidemiologic characteristics.

DNA content and proliferative activity in children with neuroblastoma.

Many studies have reported that neuroblastoma patients with aneuploid DNA content and a low cellular proliferative activity have better outcome; other studies have reported contradictory results. Formalin-fixed, paraffin-embedded archival pretreatment specimens of 56 neuroblastomas were studied. Thick sections from paraffin blocks were deparaffinized, and rehydrated. Following enzymatic digestion and filtration, cellular suspensions were analyzed by flow cytometry. Six tumors were aneuploid (13.3%) and 39 samples were diploid (86.7%). S-phase fraction (SPF) ranged from 1 to 78% with a median of 31%. DNA ploidy and proliferative activity results showed no correlation with the prognostic variables. There was no significant difference between the 5-year overall and event-free survival rates of the aneuploid and the diploid neuroblastomas or between the neuroblastomas with a high and low proliferative activity. The results revealed the prognostic significance of neither DNA ploidy nor the cellular proliferative activity in neuroblastoma in contrast to other studies.

Expression of complement regulatory proteins CR1, DAF, MCP and CD59 in haematological malignancies.

Host cells are protected from the lytic effect of the complement system by complement regulatory proteins. This study was designed to investigate the expression of complement regulatory proteins on leukemic blasts which may be susceptible to the lytic effects of the complement system in the circulation. The surface expressions of complement regulatory proteins, complement receptor 1 (CR1, CD35), decay accelerating factor (DAF, CD55), and homologous restriction factor 20 (HRF20, CD59), on peripheral blood and bone marrow blasts were evaluated by using flow cytometry in 16 acute myeloblastic leukemia (AML), 16 acute lymphoblastic leukemia (ALL), 4 chronic lymphocytic leukemia (CLL), 3 chronic myelocytic leukemia (CML) patients and control granulocytes and lymphocytes obtained from 15 healthy volunteers. mRNA expression was investigated by Northern blot analysis. mRNA abundances were calculated after normalization according to 28s rRNA. Surface expressions of CRI and DAF were marginally (p = 0.08 and p = 0.08, respectively) lower in AML, and DAF expression was significantly lower (p=0.0008) in ALL patients in comparison to their normal counterparts. Except from a slight increase that is detected for CD59 in CML patients (p=0.06), there was no significant difference between the surface expressions of CD59 in any of the groups studied. Densitometric analysis of autoradiographs obtained from Northern blots revealed that in AML patients, CR1 mRNA expression were 5.5-fold lower than controls (p=0.06), while DAF mRNA expression was significantly higher (p=0.0046). Furthermore, the mRNA expression of CRI in ALL patients was found significantly lower than in the control group (p = 0.0419). None of the values obtained from the other groups were significantly different from each other. These results suggest that leukemic blasts are protected from the lytic attack of the complement system at all levels, since all of the complement membrane regulatory proteins were expressed in all leukemia types (although at lower amounts in some cases), and it is also possible to use CRI and DAF as differentiation markers in acute leukemias.

Influence of perioperative whole blood transfusions on lymphocyte subpopulations in patients with stage II breast cancer.

Preliminary reports have suggested an adverse relationship between blood transfusion and survival after surgery in patients with solid tumour. One might postulate that from these studies, perioperative blood transfusions alter host immune defences. We therefore examined the influence of homologous whole blood transfusion on circulating lymphocyte subpopulations in transfused patients compared with non-transfused patients. Fifty-one women with Stage II breast cancer who underwent surgical procedures were studied. Patients were classified into two groups on the basis of whether or not they had received blood transfusion. The lymphocyte subpopulations were analyzed by flow cytometry before cancer surgery and three weeks after the operation. CD3+, CD4+, CD8+, and CD20+ cells as the lymphocyte subsets were quantitated using appropriate monoclonal antibodies. No significant differences between pre- and postoperative lymphocyte subset levels were seen in non-transfused patients. However, there was a statistically significant increase in the CD8+ cell count; decreasing CD4+ cell count and decreased CD3+ cells levels were observed in the transfused group (P < 0.05). Although these early results of the study suggest that the blood transfusions could be associated with alterations in lymphocyte populations, additional studies are needed to elucidate the possible mechanism of the transfusion-induced immunological modulations.

P-glycoprotein expression by technetium-99m-MIBI scintigraphy in hematologic malignancy.

UNLABELLED: Our aim was to ascertain the relationship between the degree of 99mTc-MIBI uptake and the level of p-glycoprotein (Pgp) expression determined by flow cytometry and reverse transcription-polymerase chain reaction (RT-PCR) techniques in patients with hematologic malignancy. METHODS: A total of 21 samples (19 patients) were evaluated. Two patients had repeat studies after therapy. Thirteen samples were studied at the time of initial diagnosis and 8 during relapse after therapy. After MIBI imaging, either bone marrow aspiration or peripheral blood was obtained for flow cytometric and RT-PCR analyses. Flow cytometry was performed using two different antibodies. After the injection of 555 MBq MIBI, whole-body and pelvic spot images were acquired using a dual-head gamma camera. The uptake in the bone marrow was evaluated against the background (adjacent soft tissue) by both qualitative (scoring system) and quantitative (tm/bkg ratios) analyses. RESULTS: For flow cytometry, the limit for Pgp overexpression was set at >15% Pgp-positive mononuclear bone marrow or peripheral blood cells. There was an inverse correlation between the levels of Pgp and MIBI imaging using both the qualitative (scoring system) and quantitative (tm/bkg ratios) analyses (p = 0.022). Mean values were statistically different between Pgp+ and Pgp- groups for both qualitative and quantitative analyses (p = 0.009 and 0.024, respectively). For RT-PCR, there was statistical support toward a difference in the mean values between Pgp+ and Pgp- groups by qualitative analysis (p = 0.061); however, no statistical difference was found between these two groups by quantitative analysis (p = 0.179). CONCLUSION: Based on the strong correlation between the imaging and flow cytometry and a statistical support toward the correlation between the imaging and RT-PCR, MIBI imaging may be used for the in vivo detection of Pgp in patients with hematologic malignancy.

Immune alterations in lead-exposed workers.

Peripheral blood lymphocytes, serum immunoglobulins (IgG, IgA and IgM), C3 and C4 complement protein concentrations of 25 male lead-exposed workers from storage-battery plants were examined and compared to 25 healthy male controls. Lead exposure was assessed using blood lead levels measured by atomic absorption spectrophotometry and zinc protoporphyrin (ZPP) levels assayed by hematofluorometry. The absolute number and the percentage of functionally different subsets of peripheral blood mononuclear lymphocytes, i.e. T, T-suppressor, B and natural killer cells, were unchanged. However, T-helper lymphocytes were significantly lower in lead-exposed workers compared to healthy controls (P < 0.05). In addition, lead-exposed workers had a significant reduction in the IgG, IgM and C3, C4 complement levels (P < 0.05). These results suggest that human chronic exposure to lead may be detrimental to the immune system.

T-cell subsets, interleukin-2 receptor expression and production of interleukin-2 in minimal change nephrotic syndrome.

This study was designed to investigate T-lymphocyte subsets interleukin-2 receptor (IL-2R) expression and IL-2 production in minimal change nephrotic syndrome (MCNS). Peripheral blood T-lymphocytes and IL-2R expression were analysed using fluorescein isothiocyanate-labelled CD3, CD4, CD8 and CD25 monoclonal antibodies with flow cytometry. IL-2 production was determined by enzyme immunoassay. Ten children with MCNS in relapse and in remission were evaluated. Thirteen healthy children served as controls. The patients in relapse demonstrated a moderate decrease in the total absolute lymphocyte counts and CD8(+) T-lymphocytes compared with controls (P < 0.05) and had a greatly increased IL-2R expression in freshly isolated, unstimulated peripheral lymphocytes compared with patients in remission and controls. While this was not statistically significant, IL-2R expression on cultured lymphocytes stimulated with phytohaemagglutinin was significantly elevated in relapse compared with those in remission and controls (P < 0.05). IL-2 production did not correlate well with IL-2R expression and there was no significant difference between the groups. Our results suggest that T-cell subset changes and high IL-2R expression on peripheral lymphocytes may indicate the presence of stimulated T-cell populations in MCNS which could contribute to the immunopathogenesis.