Eltrombopag for the Treatment of Immune Thrombocytopenia: The Aegean Region of Turkey Experience.

OBJECTIVE: Immune thrombocytopenia (ITP) is an immune-mediated disease characterized by transient or persistent decrease of the platelet count to less than 100x109/L. Although it is included in a benign disease group, bleeding complications may be mortal. With a better understanding of the pathophysiology of the disease, thrombopoietin receptor agonists, which came into use in recent years, seem to be an effective option in the treatment of resistant cases. This study aimed to retrospectively assess the efficacy, long-term safety, and tolerability of eltrombopag in Turkish patients with chronic ITP in the Aegean region of Turkey. MATERIALS AND METHODS: Retrospective data of 40 patients with refractory ITP who were treated with eltrombopag in the Aegean region were examined and evaluated. RESULTS: The total rate of response was 87%, and the median duration of response defined as the number of the platelets being over 50x109/L was 19.5 (interquartile range: 5-60) days. In one patient, venous sinus thrombosis was observed with no other additional risk factors due to or related to thrombosis. Another patient with complete response and irregular follow-up for 12 months was lost due to sudden death as the result of probable acute myocardial infarction. CONCLUSION: Although the responses to eltrombopag were satisfactory, patients need to be monitored closely for overshooting platelet counts as well as thromboembolic events.

Molecular Evaluation of t(14;18)(bcl-2/IgH) Translocation in Follicular Lymphoma at Diagnosis Using Paraffin-Embedded Tissue Sections.

OBJECTIVE: Follicular lymphoma (FL) is one of the most common lymphomas, and is characterized by t(14;18)(q32;q21) in more than 80% of patients. The aim of this study was to determine the rate of t(14;18) positivity based onthe detection of mbr or mcr in paraffin-embedded tissue samples. MATERIAL AND METHODS: The study included 32 paraffin-embedded tissue samples collected from 32 consecutive FL patients that were diagnosed and followed-up at our hospital between 1999 and 2006. The MBR breakpoint wasidentified based on real-time PCR using a LightCycler v.2.0 t(14;18) Quantification Kit (MBR), multiplex PCR, and seminestedPCR. To identify the mcr breakpoint, real-time PCR was performed using specific primers and the FastStart DNAMaster SYBR Green I Kit. To detect t(14;18) via fluorescence in situ hybridization (FISH) nuclei from paraffin-embeddedtissue sections were extracted and used together with LSI IgH (immunoglobulin heavy chain) (spectrum green)/bcl-2(B-cell leukemia-lymphoma 2) (spectrum orange) probes. RESULTS: The DNA and nuclei isolation success rate for B5 formalin-fixed, paraffin-embedded tissue sections (n = 12)was 42% and 33%, respectively, versus 95% and 60%, respectively, for 20 tissue sections fixed in formalin only. In all,24 paraffin-embedded tissue sections were analyzed and mbr positivity was observed in the DNA of 82.14% via seminested PCR, in 53.57% via multiplex PCR, and in 28.57% via real-time PCR. We did not detect mcr rearrangementin any of the samples. In all, 15 of 16 patients (93.75%) whose nuclei were successfully isolated were observed to bet(14;18) positive via the FISH method. CONCLUSION: Semi-nested PCR and FISH facilitated the genetic characterization of FL tumors. As such, FISH and PCR complement each other and are both essential for detecting t(14;18) translocation.

The effect of hormone replacement treatment on thrombin-activatable fibrinolysis inhibitor activity levels in patients with Hashimoto thyroiditis.

BACKGROUND: Hypothyroid patients have increased risk of cardiovascular diseases, and several mechanisms have been considered responsible in these patients. Although, a few studies demonstrated fibrinolytic system changes in hypothyroid patients, there is no study demonstrating TAFI activity in hypothyroid Hashimoto's thyroiditis patients. The aim of this study was to evaluate TAFI activity status and the effect of L thyroxin hormone replacement treatment on fibrinolytic system in this patient group. METHODS: Thirty patients with hypothyroid Hashimoto thyroiditis (all were female and the mean age was 44.3+/-14.6 years, ranging between 17-68 years) were enrolled to study. Their TSH levels were high (27.2+/-5.2 mU/L) and Free T3 and Free T4 hormone levels were below than normal. In this study, euthyroid 20 healthy volunteers (mean age 32.5+/-4.9 years, range 26-42 years) were adopted. L-thyroxin treatment before and after TAFI activity levels were measured in patients. RESULTS: In the control group, TAFI activity levels were 9.6+/-0.4 microg/mL. In patients with L-thyroxin before and after treatment there were high levels of TAFI activity value of 14.2+/-0.9 and 12.9+/-0.8 microg/mL, respectively. In the patient group, after L-thyroxin treatment TAFI activity levels were decreased but they were not statistically significant (p=0.187). When compared to the control group, high levels of TAFI activity were observed in the patient group (p<0.0001). CONCLUSION: Our data demonstrated that in Hashimoto thyroiditis, patients have high levels of TAFI activity compared to controls. A high level of TAFI activity suggests fibrinolytic deficit or thrombotic tendency in hypothyroid patients and this deficit is persistent after L-thyroxine replacement.

Presence of factors that activate platelet aggregation in mitral stenotic patients' plasma.

BACKGROUND: Although the association between mitral stenosis (MS) and increased coagulation activity is well recognized, it is unclear whether enhanced coagulation remains localized in the left atrium or whether this represents a systemic problem. To assess systemic coagulation parameters and changes in platelet aggregation, we measured fibrinogen levels and performed in vitro platelet function tests in plasma obtained from mitral stenotic patients' and from healthy control subjects' peripheral venous blood. METHODS: Sixteen newly diagnosed patients with rheumatic MS (Group P) and 16 healthy subjects (Group N) were enrolled in the study. Platelet-equalized plasma samples were evaluated to determine in vitro platelet function, using adenosine diphosphate (ADP), collagen and epinephrine in an automated aggregometer. In vitro platelet function tests in group N were performed twice, with and without plasma obtained from group P. RESULTS: There were no significant differences between the groups with respect to demographic variables. Peripheral venous fibrinogen levels in Group P were not significantly different from those in Group N. Adenosine diphosphate, epinephrine and collagen-induced platelet aggregation ratios were significantly higher in Group P than in Group N. When plasma obtained from Group P was added to Group N subjects' platelets, ADP and collagen-induced, but not epinephrine-induced, aggregation ratios were significantly increased compared to baseline levels in Group N. CONCLUSION: Platelet aggregation is increased in patients with MS, while fibrinogen levels remain similar to controls. We conclude that mitral stenotic patients exhibit increased systemic coagulation activity and that plasma extracted from these patients may contain some transferable factors that activate platelet aggregation.

Involvement of protein phosphatase 2A in interferon-alpha-2b-induced apoptosis in K562 human chronic myelogenous leukaemia cells.

Interferon-alpha (IFN-alpha)-2b is known to have antiproliferative effects on hematological malignant cells, especially chronic myelogenous leukaemia (CML). However, it can induce cytogenetical remissions in a very small percentage of the patients. Also during interferon therapy, resistance can emerge in the CML clones. K562 is an in vitro model cell line transformed from a Ph positive CML patient. It can be induced to differentiate to granulocytic and/or monocytic lineages with certain molecules. IFN-alpha-2b generally exerts its effects on CML cells by Janus family kinases (Jak/Stat) pathway, mostly through tyrosine kinase system. However, there is almost no data on the relevance of serine/threonine (Ser/Thr) protein phosphatase (PP) system in the interferon induced signal transduction pathways. In this study, we investigated serine/threonine protein phosphatases in the IFN-alpha-2b induced K562 cytotoxicity. Trypan blue dye exclusion test and MTT assay were utilised for determining cytotoxicity. IC(50) of IFN-alpha-2b on K562 cells was found to be 600IU/ml. However, no differentiation was determined by analysis of cell surface antigen expressions. Serine/threonine protein phosphatase inhibitors calyculin A (Cal A) and okadaic acid (OKA) augmented the IFN-alpha-2b induced cytotoxicity. Apoptosis assay by the mono-oligonucleosome detection and acridine orange/propidium iodide dye revealed marked apoptosis underlying cytotoxicity. Phosphatase enzyme assay revealed a gradual increase in protein phosphatase 2A (PP2A) activity during interferon induced cytotoxicity. Conversely, immunoblots showed no change in the expression of PP2A catalytic and regulatory subunits. In conclusion, PP2A plays a role in IFN-alpha-2b induced apoptosis of K562 cells and should be investigated as a new window furthermore.

Platelet dysfunction in lean women with polycystic ovary syndrome and association with insulin sensitivity.

Platelet dysfunction and its association with insulin resistance and/or hyperandrogenemia were evaluated in 50 women with polycystic ovary syndrome (PCOS), 50 women with non-classic congenital adrenal hyperplasia (NC-CAH), and 30 women in the control group. Agonist-induced platelet aggregation was measured. Women with PCOS had significantly higher levels of platelet aggregations induced by ADP (77.4 +/- 3.3 vs. 67.3 +/- 2.8), collagen (79.7 +/- 1.8 vs. 69.1 +/- 3.9), and epinephrine (84.7 +/- 2.6 vs. 67.8 +/- 3.8), compared with controls. However platelet aggregations of women with NC-CAH because of ADP (68.2 +/- 4.22), collagen (69.5 +/- 5.4), or epinephrine (68.6 +/- 4.3) were similar to those in the control group. There were negative correlations between aggregations induced by agonists and the insulin sensitivity in women with PCOS. These correlations also appeared significant after androgen levels with covariance analysis were excluded. These covariance analyses were performed because serum androgen levels might affect platelet function. Any significant correlations were not found between androgen levels and agonist-induced platelet aggregation in women with NC-CAH. We conclude that platelet dysfunction may be an important reason for the possible cardiovascular heart diseases in women with PCOS.

Cytotoxic and inhibitory effects of 4,4'-dihydroxy chalcone (RVC-588) on proliferation of human leukemic HL-60 cells.

Chalcones have been identified as interesting compounds with cytotoxic and tumor reducing properties. In the present study, the biological activity of synthetic chalcones on myeloid leukemic cells was investigated. Human myeloid HL-60 leukemia cells were exposed to 1-20 micro M chemicals for 0-96h. The viability of the cells was measured using trypan blue dye exclusion method. 4,4'-Dihydroxy chalcone (RVC-588) was selected for further experiments to determine characteristics of cytotoxicity among other compounds. The data show that cell viability decreased after treatment and IC(50) value was approximately 2 micro M for RVC-588. Cell differentiation was analyzed with cytofluorometry by changes in expression of glicoprotein surface markers CD11b/Mac-1, CD11c and CD14 together with morphological analysis. A maximum level of expression changes was determined at 72h but these changes were not statistically significant to show the differentiation of HL-60 cells to mature myeloid and/or monocytoid cells. Apoptotic DNA degradation was evaluated and quantitated using sensitive enzyme-linked immunoabsorbant (ELISA) method. Using this technique, a maximum level of apoptosis 1.2-fold higher than control was observed in cultures exposed for 48h to 2 micro M RVC-588. The DNA ladder assay was subsequently used to determine DNA breaks qualitatively. After 24h, the cells exposed to 2 micro M RVC-588 was shown to have cytotoxic-late apoptotic ladder pattern compared to control cells. These data demonstrate that RVC-588 has a high cytotoxic and antitumor activity in HL-60 cells among other chemicals we synthesized. Although the mechanism by which RVC-588 initiated cell death in these cells is presently not known and apoptotic mechanisms are likely to play less role compared to other chalcone analogues reported previously.

Vena cava superior syndrome due to sternal plasmacytoma in the course of systemic myeloma.

Here we present a patient with multiple myeloma course. The first clinical manifestation was superior vena cava syndrome due to a huge sternal plasmocytoma. The patient was treated with a combination of high dose radiotherapy and autologous peripheral blood stem cell transplantation. We suggest careful evaluation of these patients with respect to aggressive therapy.