Miodrag Vučić

The effect of curcumin and PI3K/Akt inhibitor on monosodiumglutamate-induced rat thymocytes toxicity

Monosodium glutamate (MSG), the sodium salt of glutamic acid, is widely used in modern nutrition as flavor enhancer. However, it has been shown that curcumin has ability to induce apoptosis in the cells of the immune system. In the present study, we evaluate the potential protective effects of curcumin in MSG-induced apoptosis and signaling pathway which may be involved. Rat thymocytes were treated with increased (1, 10, 50 mM) MSG concentrations and/or curcumin (3 μM). Cell apoptosis rate, reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP), Bcl-2, Bax protein expression and caspase-3 activity were determined after 24 hours of incubation. Treatment with MSG resulted with increased apoptosis, ROS production and caspase-3 activity, followed with decreased MMP and Bcl-2/Bax protein ratio. Inhibition of caspase-3 and caspase-9 activity reduced cell apoptosis, indicating the involvement of mitochondrial apoptotic pathway. Co-treatment with curcumin markedly reduced apoptosis and ROS production, together with increased MMP and Bcl2/Bax protein ratio. Inhibition of PI3K/Akt signaling pathway abolished protective effect of curcumin in MSG-induced toxicity in rat thymocytes. Obtained findings suggest that curcumin may attenuate the MSG-induced apoptosis through PI3K/Akt signaling pathway which could be useful in preventing the potential deficiencies in T cell-mediated immunity.

The significance of angiogenesis for predicting optimal therapeutic response in chronic myeloid leukaemia patients

In this study the correlation and the prognostic value of the morphometric parameters of angiogenesis for optimal therapeutic response to tyrosine kinase inhibitor (TKI) therapy in patients with chronic myeloid leukaemia (CML), i.e. complete cytogenetic response (CCgR) and major molecular response (MMoR), were investigated. Microvascular density (MVD) and a number of different size- and shape-related morphometric parameters of microvessels of bone marrow biopsy from 40 CML patients and 20 controls were examined. Microvessels of bone marrow were examined by using immunohistochemical staining for CD34 and quantified in the region of the most intense vascularisation by using image analysis. CML patients had significantly higher angiogenesis parameters when compared with controls. A statistically significant correlation was found between some parameters of angiogenesis and evaluated CCgR and MMoR. For achievement of CCgR, lower values of MVD, minor axis, area, circularity, and roundness and higher value of aspect ratio, while for achievement of MMoR only lower values of MVD have been identified as positive prognostic factors. Besides confirming increased angiogenesis in CML patients, this study also demonstrated prognostic significance of the degree of angiogenesis for the clinical outcome and identified angiogenic predictive factors for achieving optimal response on TKIs therapy.

Evaluation of platelet activation in leukocyte-depleted platelet concentrates during storage

Structural and functional changes in platelets during storage can lead to the loss of platelet reactivity and response. Our aim was to evaluate leukocyte-depleted platelet concentrates on storage days 0, 3 and 5, obtained by in-line filtration. In non-filtered platelet concentrates (NF-PC) group, 180 whole blood units were collected with quadruple blood bags and then compared to another group of 180 whole blood units (leukocyte-depleted platelet concentrates [LD-PC]), collected in Imuflex Whole Blood Filter Saving Platelets (WB-SP) bags with an integrated leukoreduction filter, with regard to the platelet quality and characteristics. The efficacy of the two techniques for platelet concentrate preparation was evaluated by white blood cell (WBC) and platelet count on day 0. The partial pressure of oxygen (pO2), pH, platelets positive for P-selectin (CD62P), CD63, cluster of differentiation 42b (CD42b), phosphatidylserine (PS), and mitochondrial membrane potential (MMP) were analyzed during the storage in both groups. A significantly lower WBC count and higher platelet count was observed in LD-PC compared to NF-PC group, indicating the overall efficacy of the first technique. During the 5-day storage, pH and pO2 decreased in both groups. In LD-PC group, higher pH, increased pO2 and decreased platelet surface expression of CD62P, CD63 and PS were observed compared to NF-PC group. In both groups, the percentage of CD42b positive platelets and MMP did not change significantly during the 5-day period. The assessment of different markers of platelet activation may be an effective tool in evaluating the quality of platelets during storage. A better understanding of platelet activation may provide new insights for developing a novel therapeutic approach in the manipulation of platelet aggregation.

The significance of soluble transferrin receptors in diagnosing iron deficiency anemia

Abstract Introduction. In recent years, determination of soluble transferrin receptor levels has been emerging as a test that can reliably indicate iron deficiency in various states, and that is non-invasive and easy to use. The aim of this study was: to determine reference values of soluble transferrin receptor concentrations in serums in our population, to examine the reliability of this method in the diagnosis of anemia due to iron deficiency and associated iron deficiency in anemia accompanying malignant hemopathies, and to identify possible limitations of the test in certain conditions. Material and Methods. The prospective research included 86 patients with anemia: 46 patients with iron deficiency anemia, and 40 patients with malignant hemopathies. The control group consisted of 40 healthy persons aging over 18. Results. Ferritin values were reduced in 76.1% of patients, while higher levels of soluble transferrin receptors appeared in 100% of patients with iron deficiency anemia. In patients with reduced serum ferritin levels, the soluble transferrin receptor/log ferritin index was statistically significantly higher than in patients in whom ferritin concentration was in the normal range (p <0.001). ROC analysis of patients with iron deficiency anemia showed that the soluble transferrin receptor/log ferritin index (AUC 0.977) and levels of soluble transferrin receptors (AUC 0.931) occupied the largest area under the curve. The best diagnostic parameter for detecting iron deficiency in patients with malignant hemopathies by ROC analysis is the soluble transferrin receptor/log ferritin index (AUC 0.770). Conclusion. Soluble transferrin receptors are useful in the diagnosis of iron deficiency anemia, especially when ferritin values are not reduced. The calculation of soluble transferrin receptor/log ferritin index is even more reliable. In patients with malignant hemopathies, the associated iron deficiency could be best indicated by soluble transferrin receptor/log ferritin index.

Preparation of patients with hemophilia A for oral surgery

Hemophilia A is an inherited disease characterized by deficiency of coagulation factor VIII and bleeding tendency. It is transmitted through the X chromosome. Hemophilia A is characterized by excessive bleeding in various tissues of the body, including soft tissue hematomas and hemarthrosis. In formulating the diagnosis of hemophilia A, in addition to a well-taken medical history and physical examination, laboratory tests should also be carried out and analyzed. Tooth extraction is the most common surgical procedures in patients with hemophilia. Hematological preparation implies the application of a concentrated factor VIH for one to two days prior to the intervention to achieve a desired level of factor VIII needed for the operation. In tooth extraction, this level has to be 50% before and after the tooth extraction for 5 days, with the application of antifibrinolytic agents. In oral surgical interventions the desired level of factor VIII is 50-80% preoperatively, 30-80% for 5 days after surgery, and 30% up to 14 days, also with the use of antifibrinolytic therapy. Patients with hemophilia and inhibitors are prepared for intervention through the application of recombinant FVIIa at the dose of 120mcg/kg, repeated every 2 hours for the period of 7-10 days after the intervention. It is necessary to apply antifibrinolytic agents and local hemostatic measures. Measures of local hemostasis are unavoidable in the case of oral surgical interventions in patients with hemophilia A. Implementation of these procedures in oral surgery has the role of minimizing the possibility of intra- and postoperative bleeding in patients with hemophilia A. For this purpose, the following are mostly used: absorbable suture thread, preparations of collagen, oxycellulose, gelatin, fibrin glue, with topical application of tranexamic or epsilon aminocaproic acid. Conclusion: Close cooperation between hematologists and oral surgeons is essential in order to minimize unwanted complications in patients with hemophilia A.

Radically reduced ex vivo cell activation by using "in-line" filtered whole blood as a source of platelet concentrate.

Dear Sir, Factors affecting the quality of platelet concentrates, obtained from whole blood (WB) are: (i) the processing method used (originating from platelet-rich plasma or buffy coat-derived platelet concentrates [BC-PC]); (ii) the ex vivo manipulation applied (such as cryopreservation, pathogen reduction); (iii) cell storage conditions/duration; and (iv) quantity of residual white blood cells, particularly mononuclear cells1,2. As described previously3, higher mononuclear counts in platelet concentrates are associated with ex vivo cytokine release and cell/cytokine-mediated adverse events. Furthermore, mononuclear cells result in increased platelet surface antigen/marker expression and platelet activation ex vivo, followed by morphological and ultrastructural changes. Commonly, GP140/CD62p expression ≤40% correlates with acceptable clinical efficacy of platelet concentrates. However, discharge of alpha-granules and elevated GP140/CD62p expression (≥50–60%) can lead to decreased platelet recovery and impaired function. A high level of GP53/CD63 expression (due to the release of the contents of lysosomes) is typically detectable only following major platelet activation1,2,4. The aim of this research was to compare the quality of non-filtered platelet concentrates (NF-PC or control group) with “in-line-leucodepleted” platelet concentrates (LD-PC or study group) determined on the basis of residual mononuclear cell count and expression of activation markers (GP/140/CD62p and GP53/CD63), directly after collection and processing of whole blood (day 0), as well as on the 3rd and 5th days of storage of the platelet concentrates (day 3 and day 5). We assumed that the use of “in-line” filtered whole blood (as an optimised source of platelet concentrate) could radically prevent ex vivo platelet activation in LD-PC. For the preparation of NF-PC, whole blood (450±45 mL) from donors, non-reactive for infectious disease markers (hepatitis B and C, AIDS and syphilis) in serological screening tests (Biokit S.A, Llissa de Munt, Spain and Ortho Clinical Diagnostics, Raritan, JS, USA) and with normal clinical data, was collected in quadruple bags (Macopharma, Tourcoing, France). This bag system contains 63 mL citrate phosphate dextrose (CPD) and 100 mL saline-adenine-glucose-mannitol (SAGM) solutions. For the LD-PC, the same volume of whole blood from donors with normal clinical and laboratory findings was collected into Imuflex WB-SP bags (Terumo, Tokio, Japan) with CPD, SAGM, and an “in-line” filter that “saves” (not retains platelets)5. NF-PC were obtained from whole blood using a “standard buffy-coat” method. Non-filtered whole blood (total=180 units) was initially centrifuged with a “hard-spin” technique (3,890×g for 10 minutes, at 20±2 °C) in a Cryofuge 8500 (Heraeus, Langenselbold, Germany) and then processed using a T-ACE device (Terumo, Japan). NF-BC units were obtained from the separated buffy coat units were obtained following “light-spin” centrifugation (310×g for 7 minutes, at 20±2 °C) and second (manual) processing, as described previously2,3. Six NF-PC were pooled using a sterile connection system (TSCD, Terumo, Tokio, Japan) and stored on a shaker (Teknolabo Instruments Srl, Milan, Italy) at 22±2 °C for 5 days. In the same manner “in-line” filtered whole blood (total=180 units) underwent “hard-spin” centrifugation. The leucocyte-poor buffy-coat (platelet rich, but leucoreduced cell layer) was separated on a T-ACE device. The second (“light-spin”) centrifugation of these “leucocyte-poor buffy coats” was performed as described above. LD-PC were obtained by processing; the pooling and storage procedures for LD-PC were completed in the same manner as for the NF-PC. Standard haematological parameters were analysed on a Beckman-ACT device (Beckman-Coulter, Fullerton, CA, USA). The residual white blood cell count in LD-PC units was quantified manually in a Negeotte haemocytometer chamber, as described previously3. The platelet activation markers (GP140/CD62p and GP53/ CD63) were investigated using monoclonal antibodies (Becton Dickinson, San Jose, CA, USA) and flow cytometry. Briefly, samples of platelet concentrates (1×106 cells per tube) were incubated with fluorescein isothiocyanate-labelled monoclonal antibodies, in accordance with the manufacturer’s recommendations and instructions. Non-specific binding was detected using control cells, which were incubated with phosphate-buffered saline alone. After incubation, cells were washed and analysed on an Epics XL (Beckman-Coulter). The results were calculated and expressed as percentage of total cell number4. Differences between groups were analysed by Student’s t-test and were considered as statistically significant at p≤0.05. Our preclinical study confirms that the mononuclear cell count was lower in LD-PC (0.05±0.02×106/unit) than in NF-PC (5.32±1.7×106/unit) (t=6.693; p<0.001) (Table I). Table I The expression of platelet activation markers in NF-PC and LD-PC during 5 days of storage. The levels of GP140/CD62p and GP53/CD63 expression in both NF-PC and LD-PC over the storage period are shown in Table I. GP140/CD62p expression (calculated as a percentage) on day 0 was significantly higher in NF-PC than in LD-PC (t=10.642; p<0.001). Significant differences in GP140/CD62p expression between NF-PC and LD-PC (“intergroup” disparity) were also observed on day 3 (t=12.754; p<0.001) and day 5 (t=10.724; p<0.001). A marked linear increase of GP140/CD62p expression was confirmed in both investigated groups during the 5 days of storage (p<0.001). However, GP53/CD63 expression was significantly higher at each measurement (day 0, day 3, and day 5) in NF-PC than in LD-PC (p<0.001). Finally, GP53/CD63 expression increased significantly during the 5 days of storage in both NF-PC and LD-PC (p<0.001). As stated, the aim of this study was to determine the effect of leucodepletion on the expression of platelet activation markers in platelet concentrates. The results obtained showed that mononuclear cell count was significantly lower in LD-PC than in NF-PC (prepared by the “standard buffy-coat” method). This is consistent with research conducted by Paunovic et al., who showed that the “in-line” WB-SP filter (which “saves” platelets) achieves optimised, practical leucodepletion of blood products-packed (resuspended) red blood cells and platelet concentrates5. Our earlier research showed better quantitative and qualitative platelet recovery -that is, superior hypotonic shock response, aggregation ability, morphological score of platelets, ultrastructural properties (intact microtubules, minimal membrane changes, etc.)- in platelet concentrates treated by multiple, but optimised ex vivo manipulations2–4. The quality of platelet concentrates for transfusion is optimal when no activation markers are expressed, since activated platelets have inferior quantitative and qualitative recovery, and could promote prothrombotic and procoagulant events. Generally, platelet activation is accompanied by increased expression of GP140/CD62p and GP53/CD63 antigens on the platelet membrane. The expression of these antigens rises progressively during storage, and correlates with the degree of cell activation in platelet concentrates. The structural and adhesive glycoprotein GP140/CD62p (or P-selectin) is an integral constituent of the membrane of alpha-granules of “resting” platelets and Weibel-Palade bodies of endothelial cells. Its expression on the platelet surface upon alpha-granule exocytosis has been used as an ex vivo marker of platelet secretion and activation. In fact, GP140/CD62p expression is a marker of the platelet storage-lesion. Thus, GP140/CD62p expression is extensively used to evaluate platelet concentrate quality following whole blood processing, filtering, liquid-state storage or cryopreservation. Nevertheless, despite numerous investigations, the clinical relevance of GP140/CD62p expression levels or their value as an ex vivo measure of in vivo platelet activity or viability remains uncertain1,2,4. GP53/CD63 was the first identified marker of platelet activation that is elevated on the cell surface following releasing from granules2,4. Our recent study established that GP53/CD63 expression was really lower in LD-PC over the entire storage period. The expression of GP53/CD63 was also four times higher in NF-PC on day 5 than on day 0, which is in expected correlation. In summary, the use of optimised (with regards to timing) and effective (with regards to degree) “in-line” leucodepletion (Imuflex WB-SP system) undoubtedly reduces white blood cell and mononuclear cell counts. In the LD-PC investigated, GP140/CD62p and GP53/CD63 expression was less during the 5 days of storage, a finding strongly correlated with lower platelet activation. In accordance with our earlier research, we speculate that the use of LD-PC, with improved ex vivo cell quality, could have a better clinical effect.

The effect of a leukodepletion model on the activation stage of platelets

The preparation of thrombocyte concentrates with filtration before storage (in-line) makes it possible to avoid the presence of mononuclear cells in the concentrate and proinflammatory cytokines. Therefore, this filtration may result with decreased activation of trombocyte receptors in vitro, which may improve therapeutic efficiancy. Methods. We compared two groups, each with 30 therapeutic doses of concentrated thrombocytes. We prepared the first group using the classic model from the buffy coat and the other with concentrated thrombocyte samples filtrated during sampling, so-called in-line, with the WBC filter Imuflex (Terumo). Mononuclear cells (MNC), thrombocyte, and erythrocyte counts in the units of concentrated thrombocytes were obtained on an automatic cell counter, and we used flow cytometry to measure the expression of surface thrombocyte receptors. The results demonstrated that the trombocytes prepared with pre-storage filtration contained a very low level of mononuclear cells and markedly reduced trombocyte receptors. Conclusion. The number of MNC and expression of surface thrombocyte receptors were markedly lower in the concentrated thrombocyte units prepared with in-line filtration. The thrombocytes prepared in this way contain fewer mononuclear cells, are of higher quality, are more functional, and may produce a better therapeutic effect in vivo.