Flow cytometric analysis of the basophil cell activating impact of potential drug delivery nanoparticle-candidate.

INTRODUCTION: Carbon nanotubes ­ as artificial nano-size ranged materials ­have increasing role in the modern biomedical, diagnostic and therapeutic applications.There is a promising option for their use as more potential drug carriers. Despite the favourable properties, their impact (accumulation, elimination, etc.) on biological systems is largely unknown. The main limiting factor of medical use of nanomaterials in most cases is the potential hypersensitive side effect. It can develop in different route, but the activation of basophil granulocytes may play a central role in this process. OBJECTIVE: Our aim was to test the direct activation ability of different, surface modified nanotubes on basophil granulocytes in vitro. In parallel we tested the effectiveness of BasoTest planned to use for this study. MATERIALS AND METHODS: Using the blood samples of allergic and healthy volunteers we examined the basophil degranulation in the presence of nanotubes and the expression level changes of cell-surface CD63 on FACS Calibur instrument. Our results were compared to positive(fMLP, Mite, Grass) and negative control samples. RESULTS: The test we have chosen proved to be sufficiently sensitive and specific for further study. Significant basophil activation was observed in the presence of carbon nanotubes in healthy persons and allergic patients, as well. The activating effect of nanotubes was more prevailed in allergic population. CONCLUSION: Our experiments have proven the fact that nanotubes may play a role in the development of hypersensitive allergic reactions through their basophil granulocyte activator effect.

Brain glucose sensing and counterregulatory response to hypoglycaemia.

An important obstacle to achieve optimal glycaemic control in diabetics on intensive insulin therapy is the frequent occurrence of insulin induced hypoglycaemic events. In healthy subjects and in diabetics without autonomic neuropathy hypoglycaemia activates the sympathetic nervous system, resulting in epinephrine and glucagon release. Both hormones increase hepatic glucose production and this counterregulatory response is of key importance of glucose homeostasis. Recent research shed light on the fact that antecedent hypoglycaemic episodes play pivotal role in hypoglycaemia associated autonomic failure (HAAF). In this condition the sympatho-adrenal response to decreased blood glucose level is blunted. The existence of HAAF clearly indicates that the nervous system contributes to glucose homeostasis in a substantial manner. This review outlines the mechanisms of both peripheral and central neuronal glucose sensing and of neural pathways involved in the counterregulatory response.

[New perspective in the treatment of chronic myeloid leukemia? gene silencing therapy]. / Új perspektíva a krónikus mieloid leukémia kezelésében? géncsendesítéses terápia.

Chronic myeloid leukemia is an incurable white blood cell disease with slow progression which affects myeloid stem cells. In the course of chromosome 22 shortening a fusion oncogene arises whose product, a Bcr-Abl oncoprotein, is a continuously expressed tyrosine kinase protein. Beside the opportunity of chemotherapy, stem cell therapy and interferon-a therapy, the application of tyrosine kinase inhibitors also became widespread in the treatment of the disease. Patients bearing the T315I point mutation, however, show resistance against all tyrosine kinase inhibitors, which can be managed by dose escalation or the combination of therapies. The discovery of RNA interference or gene silencing put the therapeutic opportunity of CML in new light. The in vitro application of anti-bcr-abl siRNA showed promising results in the causal treatment of the disease, feasible for identification of new genes associated to the disease, but we do not have sufficient evidence for the safety and efficacy of this method in human therapy.

Reduced physical fitness in children and adolescents with type 1 diabetes.

AIMS: To evaluate motor performance and cardiorespiratory function in youths with type 1 diabetes in comparison with age-matched control groups and to analyze the influence of physical activity level, anthropometric and physical fitness parameters on long-term metabolic control. METHODS: 106 youths with diabetes and 130 healthy youths aged 8-18 were assessed by the Eurofit test regarding motor performances, cardiorespiratory fitness (VO2max), skinfold thickness, and body mass index. Physical activity level was assessed through the use of questionnaires. Predictors of physical fitness and metabolic control were determined with regression analysis. RESULTS: There were no differences either in body composition or in physical activity level, but younger girls with diabetes had impaired results in speed of upper limb movement, abdominal muscle strength, upper body strength, running speed, and VO2max ; older girls with diabetes had poor results in speed of upper limb movement, abdominal muscle strength, upper body strength and VO2max . Younger boys with diabetes had impaired results in speed of upper limb movement, flexibility, static strength of hand, and abdominal muscle strength; and older boys with diabetes had poor results in speed of upper limb movement, flexibility, abdominal muscle strength, upper body strength, and VO2max compared with control groups. Older age, female gender, lower physical activity level, and higher HbA1c were significant independent predictors of poorer VO2max. Better VO2max proved to be the single predictor of favorable HbA1c . CONCLUSIONS: Youths with diabetes have reduced fitness parameters. Efforts should be carried out to improve physical fitness as part of treatment and care of children and adolescents with type 1 diabetes.

[New trends in the laboratory diagnostics of proteinuria and albuminuria]. / Uj irányzatok a proteinuria és albuminuria laboratóriumi diagnosztikájában.

According to current clinical trials, albumin excretion is an early indicator of cardiovascular damage. While proteinuria is considered as a marker of kidney function, albuminuria indicates cardiovascular risk first of all. Sensitivity of the previous laboratory tests does not meet the clinical requirements, and the error of urine collection makes the results misleading. For that reason recent guidelines suggest to calculate albumin/creatinine (ACR) and protein/creatinine (PCR) measured from the first morning urine. For the clinical diagnosis of albuminuria the sensitive immunoturbidimetric assays are suggested. Albumin dipsticks are not recommended for the measurement of albuminuria. Wide-range urinary protein reagents are also available with high sensitivity, while serum reagents are not applicable (Biuret). The traceability of calibrator to a reference material is a critical requirement. Proteinuria and albuminuria of a patient should be monitored in the same laboratory, using a fixed method and cut-off value. Albumin/creatinine value should be reported together with gender-dependent reference range.

[Challenge and limitations in determination of serum creatinine]. / A szérumkreatinin-meghatározás kihívásai és korlátai.

Since 2006 clinical guidelines have recommended that the estimated glomerular filtration rate should be calculated from serum creatinine for the early detection of chronic kidney disease. These brought into the limelight the limitations of the Jaffe method and for comparability of test results the different routine creatinine methods need to be harmonized. The disadvantage of the kinetic Jaffe creatinine determination is its low specificity. This was improved by the enzymatic, compensated-Jaffe and high resolution liquid chromatographic assays introduced in the last decade. Creatinine values determined by the new methods are more accurate, but give lower creatinine values, therefore the glomerular filtration rate would be overestimated, if the new creatinine results were applied in the previous formulae (4-variables Modification of Diet in Renal Disease-186, Cockcroft-Gault, Quadratic). Because of the new creatinine methods estimation of the glomerular filtration rate and classification in chronic kidney disease staging became uncertain. Therefore the 4-variables Modification of Diet in Renal Disease-186 formula has been adjusted in 2006 and for the new creatinine methods (traceable to the isotope-dilution mass spectrometry) only the new Modification of Diet in Renal Disease-175 formula is advised. Authors compare the diagnostic value and limitations of the creatinine methods.

On-line haemodiafiltration versus haemodialysis: stable haematocrit with less erythropoietin and improvement of other relevant blood parameters.

BACKGROUND: Controlled randomised studies to prove improved cardiovascular stability and improved anaemia management during on-line haemodiafiltration (oHDF) are scarce. METHODS: 70 patients were treated with both haemodialysis (HD) and oHDF in a cross-over design during 2 x 24 weeks at a dialysis dose of eKt/V> or =1.2. Patients randomised into group A started on HD and switched over to oHDF, whereas patients in group B began with oHDF and were treated with HD afterwards. Intradialytic morbid events (IME), such as symptomatic hypotension or muscle cramps, were noted in case of appearance. Blood parameters reflecting anaemic status, phosphate status, lipid metabolism, oxidative stress, and accumulation of advanced glycation end products were recorded either monthly or at the end of each study phase. RESULTS: The mean incidence of IME was 0.15 IME per treatment, and there was no statistical difference between oHDF and HD. A higher haematocrit (oHDF 31.5% vs. HD 30.5%, p < 0.01) at a lower erythropoietin dose (oHDF 4,913 vs. HD 5,492 IU/week, p = 0.02) was found during oHDF, when the sequence of HD and oHDF had not been taken into account. For the study groups, the results were less distinct: in group A, a higher haematocrit (HD 30.4% vs. oHDF 32.0%, p < 0.01) at a comparable erythropoietin dose (HD 5,421 vs. oHDF 5,187 IU/week, ns) was observed during oHDF, whereas in group B an identical haematocrit (oHDF 30.8% vs. HD 30.7%, ns) was achieved at a reduced erythropoietin dose (oHDF 4,622 vs. HD 5,568 IU/week, p < 0.01). During oHDF, lower levels of free and protein-bound pentosidine and of serum phosphate were found. CONCLUSION: In contrast to other studies, no benefit regarding cardiovascular stability for oHDF was found, but oHDF could well offer a potential benefit regarding anaemia correction, inflammation, oxidative stress, lipid profiles, and calcium-phosphate product.

No effect of transfusion transmitted virus viremia on the distribution and activation of peripheral lymphocytes in hemodialyzed patients.

AIM: We aimed to examine the distribution and activation of peripheral T cells in TTV positive (n = 32) and negative (n = 17) hemodialyzed patients. The control group (n = 20) consisted of healthy blood donors. METHOD: TTV-DNA was detected by seminested PCR. CD3, CD4, CD8, CD19, CD56, CD3/HLA-DR, CD3/CD69 and the Th1/Th2 ratio of T cells were analyzed by flow cytometry. Circulating IFN-gamma, IL-2, IL-4, IL-6, IL-10, IL-13, TNF-alpha, TGF-beta levels were measured by ELISA in the sera. RESULTS: There was no difference between the CD3, CD4, CD8 and CD19 values of HD subjects. In addition, the expression of both activation markers, HLA-DR and CD69, was significantly elevated in the TTV-positive and -negative HD groups compared to the controls, but not showing any difference from each other. The measurements of intracellular cytokines showed the enhanced occurrence of INF-gamma + CD4 T cells, and decreased appearance of IL-4 + CD4 lymphocytes in the HD groups without any significant difference between the TTV virus positive and negative patients. In addition, HD also elevated the expression of IL-10 in CD4 and CD8 (Th2) cells. There were only two significant changes in the levels of circulating cytokines: (a) IL-2 increased; (b) IL-13 decreased in both groups of HD patients compared to the controls, independently of TTV positivity or negativity. CONCLUSIONS: We assume that transfusion-transmitted virus does not cause any specific change in the distribution and activation of lymphocytes in the peripheral blood of hemodialyzed patients. Hemodialysis itself, however, results in a significant activation of peripheral T cells with the domination of increased production of Th1 type cytokines, IFN-gamma, IL-2, in contrast to the decreased synthesis of Th2 type cytokines, IL-4 and IL-13. Furthermore, the increased expression of IL-10 in the CD4 and CD8 cells of HD patients can be the sign of a contraregulatory Th2 activation as an answer on the Th1 effect.

[Does the TT virus affect T-cell subgroups in patients undergoing hemodialysis?]. / Hatással van-e a TT-vírus a hemodializált betegek T-sejt alcsoportjaira?

BACKGROUND: Recently many publications have appeared about the new DNA virus, called transfusion transmitted virus (TT virus), first described in 1997. These are mainly about the virus epidemiology, gene sequences and the distribution of different genotypes. In spite of the fact that the prevalence of this type of infection can reach 40 percent rate in polytransfused patients, such as in hemodialysis patients, the real pathogenetic effect of the virus has not yet been known. AIMS: The aim of the authors was to examine the activation and distribution of mononuclear cells in peripheral blood and to analyse the possible changes in Th1/Th2 immune regulatory mechanism through the soluble and intracellular cytokine profile beside the biochemical parameters of hepatic lesions in TT virus positive (n = 32) and negative (n = 17) hemodialysed patients. Healthy blood donors were the control group (n = 20). METHOD: Semi-nested PCR was used to detect the DNA of TT virus. For the surface antigen (CD3, CD4, CD8, CD19, CD56, CD3/HLA-DR, CD3/CD69) and intracellular cytokine analysis the authors applied flow cytometric method. RESULTS: The authors did not find any differences in the liver specific biochemical parameters between TT virus positive and negative hemodialysed and the healthy control group. The number of total T, T helper and total B cells were decreased. The percentage of CD8+, CD3+/HLA-DR+, CD3+/CD69+ and CD56+ cells were increased significantly in both hemodialysed population independently the presence or absence of TT virus. The soluble and intracellular cytokines showed significant growth of the Th1/Th2 cells ratio in hemodialysed patients, which has not been modified by the virus. CONCLUSIONS: From these results the authors assume that the TT virus does not cause any significant changes in the immune regulation, although it could play some role in the pathogenesis of hepatitis by local reaction.