Volume 21 (2013): Issue 1 (March 2013)

The first description of microparticles dates back to 1967, when Wolf reported platelet membrane fragments in human plasma and called them “platelet dust”. These vesicles were later called microparticles and the knowledge about their characterization and function has advanced since then. The generation of microparticles represents a mechanism of intercellular communication, playing various roles in both physiological and pathological conditions. Besides other multiple roles in pathology such as inflammation, atherogenesis and cancer spreading, platelet-derived microparticles are involved in thrombogenesis. Tissue factor and phosphatidylserine are both exposed on the outer membrane of platelet-derived microparticles, providing catalytic procoagulant surfaces. The evaluation of microparticles may represent a possible investigation and diagnostic tool. Their enumeration and characterization is challenging and flow cytometry remains the most widely used method for the analysis of microparticles. The aim of the authors is to review the most relevant information on the main properties, mechanisms of generation, and clinical relevance of platelet-derived microparticles, since their evaluation is increasingly considered as a diagnostic biomarker.

Objective: The objectives of this study were: determination of serum hepcidin levels in patients with Rheumatoid arthritis (RA) with/without anemia and controls, and its correlation with disease activity and anemia parameters. Patients and Methods: 69 people were involved in our study: 54 patients and 15 healthy subjects (controls). Laboratory evaluation of anemia, iron parameters, serum hepcidin levels and disease activity was carried out. RA patients were divided in two groups: anemic group and non-anemic group (NA), according to hemoglobin levels (Hb). Soluble transferrin receptor-ferritin index (sTfR-F index) was used to classify anemia types: anemia of chronic disease (ACD) and anemia of chronic disease + iron deficiency anemia (ACD+IDA). Disease activity was evaluated using the following parameters: erythrocyte sedimentation rate (ESR), C-reactive protein (CPR), and disease activity score (DAS28). Results: ACD and ACD+IDA groups had significantly higher serum hepcidin concentrations than controls (p<0.001, p<0.001), and NA group (p=0.006, p=0.002). No difference in hepcidin levels was observed between ACD and ADC+IDA groups (p=0.85) and between NA and controls (p=0.66). ESR was significantly higher in ACD and ACD+IDA groups compared with NA group (p<0.001, p=0.002) and controls. (p<0.001, p<0.001).DAS 28 score was higher in anemic groups than NA group (ACD vs. NA, p=0.01), (ACD+IDA vs. NA, p=0.01) and no difference was observed between ACD and ACD+IDA In RA patients serum hepcidin concentration was significantly negatively correlated with hemoglobin (Hb) (r= -0.459, p<0.000) and serum iron( r=-0.357, p<0.01) and positively with disease activity variables: ESR (r=0.352, p<0.01) , CRP (r=0.369, p<0.01), DAS28 score (r=0.289, p<0.05). Conclusion: Hepcidin increases in RA patients with anemia and its levels correlate with Hb, serum iron, and disease activity variables.

Introduction. Since the introduction of the tyrosine kinase inhibitor (TKI) imatinib mesylate (IM) in the treatment of chronic myeloid leukemia (CML), a dramatic improvement in hematologic, cytogenetic and molecular responses was noted. Also, the overall survival increased significantly. Unfortunately, in certain patients, resistance to TKI develops relatively early, especially due to point mutations in the ABL kinase domain, among which the T315I mutation confers resistance to all three currently available TKIs (imatinib, dasatinib, nilotinib). Methods. We performed a prospective study on 74 patients diagnosed with chronic phase CML, for whom we analyzed the T315I mutation. Mutational analysis was performed using ARMS-PCR (with subsequent confirmation by direct sequencing) at regular intervals of 6 months or in case of suboptimal response, loss of response or progression. Correlations between the T315I mutation and disease characteristics, response to treatment and survival were analyzed. A comparative analysis between patients positive and negative for the mutation was performed. The patients were followed and evaluated according to European Leukemia Net (ELN) criteria. Results. T315I mutation was detected in 3 patients (4.05%) and its presence was correlated with younger age at diagnosis, second line TKI therapy, progressive disease and decreased survival from the moment of detection. Conclusions. ARMS-PCR is a sensitive, easy to use method for the detection of T315I mutation in chronic phase CML patients

Objectives: Reference values are fundamental for the interpretation of laboratory results, which are useful for medical decisions. Each laboratory has to have its own reference values classified according to age groups in order to interpret test results correctly. The expression “normal values” has been replaced by “reference values” because there are various variables that are considered to influence these values. The majority of “reference values” were established over two decades ago using obsolete medical devices and in many cases undefined populations; therefore, nowadays these intervals are not relevant anymore for modern testing technology in clinic laboratories. Methods and materials: The study was carried out at Sibiu Clinic Pediatric Hospital using the laboratory’s electronic archive. The samples were taken from hospitalized patients (children and teenagers) and outpatients registered between January and December, 2010. Blood sample testing was performed using the Sysmex XS 1000i analyzer. The reference values for hemoglobin was calculated based on results from a population sample of 9838 patients. The patients were classified into 3 age categories: 1 month - 2 years old; 2 - 10 years old; 10 - 18 years old. Reference values were determined after eliminating outliers,using the robust method to calculate 2.5 and 97.5 percentiles with the SPSS statistical software. Results and Conclusions: The results obtained differed from those specified in the Roche Diagnostics 2004 Guide but were found to be close to the results mentioned in Lothar Thomas’s publication, in Laboratory diagnostics.

Background. A high occurrence of translocation t(4;11)(q21;q23) was reported in infant acute lymphoblastic leukemia (ALL) leading to the fusion of the mixed lineage leukemia (MLL) gene on chromosome 11 and the AF4 gene on chromosome 4. More than 50 distinct MLL-AF4 types of fusion have been previously identified, none of those reported matching the peculiarities found in an infant ALL case to be reported below. Materials and methods. Molecular tests were performed for the detection of TEL-AML1, BCR-ABL(p190), E2A-PBX1, and MLL-AF4 in the peripheral blood sample of a 21 days new-born boy suspected of ALL. An unexpected MLL-AF4 fragment was identified, further purified, and later analyzed by sequencing. Flow cytometry analyses were carried out at diagnosis and relapse on a FACSCanto-II cytometer (Becton-Dickinson). Results. The patient was found to be positive for the MLL-AF4 transcript, with an uncommonly long-sized product and a previously undescribed sequence (in-frame fusion between exon 12 of MLL and exon 4 of the AF4 gene). The immunophenotypic analyses also showed a particular development: while at diagnosis a dominant malignant clone displaying a B lymphoid precursor phenotype was described, at relapse a malignant monocytoid population predominantly expanded. The presence of MLL-AF4 e12-e4 transcript was still manifest at relapse, without other transcript characteristic for myeloid lineage. Conclusions. To our knowledge, this is the first report of a MLL-AF4 rearrangement revealing this complex transcript with new breakpoints in MLL. Its early detection may predict an immunophenotypic switch and may assist the clinicians in designing optimized therapies.

De novo acute myeloid leukemias (AML) represent a heterogeneous group of clonal hematopoietic disorders in which chromosomal abnormalities are detected in a majority of patients. At present, cytogenetic changes are recognized as important diagnostic markers and prognosis determinants. Complex karyotype changes are associated with resistance to treatment and unfavorable evolution. We report on an AML case with complex karyotype changes characterized by molecular genetic techniques (fluorescence in situ hybridization - FISH and array-based comparative genomic hybridization - array-CGH) and an extremely poor outcome. A 72 year-old female patient was admitted for genetic investigations with a clinical diagnosis of AML. Classical and molecular cytogenetic tests as well as array-CGH were performed. Complex chromosomal abnormalities were identified at diagnosis, consisting of genomic imbalances involving chromosomes 6, 7, 9, and 17. AML with complex karyotype changes is a heterogeneous disease, as a variety of genomic abnormalities are detected, involving virtually all chromosomes. The pathogenesis of AML with complex karyotype is poorly understood. The complexity of karyotypic changes in our case highlights the importance of using complementary genetic investigation in order to obtain a comprehensive view of AML genome.

We present the case of a 77 year old patient with a primary breast carcinosarcoma composed mostly of an osteogenic sarcoma of fibroblastic type in which only immunohistochemical analysis disclosed the presence of a minor malignant epithelial component. The malignant mesenchymal component derives from dedifferentiation of myoepithelial cells since myoepithelial markers are positive. Also, like the majority of the other metaplastic carcinomas in the breast, carcinosarcoma is a basal type of tumor that will not respond to endocrine drugs or Her2/neu therapy.

The aim of the present study is to explore the use of salivary 8-hydroxideoxyguanosine (8-OHdG) and Interleukin-1 (IL-1) gene polymorphism in the diagnosis of the patients with aggressive periodontitis. The correlation between salivary 8-OHdG level and clinical parameters was analyzed, at the same time as the use of 8- OHdG level and IL-1gene polymorphism in patients with aggressive periodontitis. Eighteen patients suffering from aggressive periodontitis and 18 healthy subjects without any sign of periodontitis were enrolled into the study after clinical examination. The analysis of genetic polymorphism of IL-1 gene was carried out from oral swabs by using the GenoType IL-1 test; the 8-OHdG biomarker was quantified from saliva samples by using an ELISA competition test. The salivary level of 8-OHdG in the control group was 0.70±0.54 ng/mL and in aggressive periodontitis, 6.93±2.90 ng/mL (p<0.001). A positive genotype consisting of allele 2 (Thymine/Thymine) was found with lower prevalence in healthy subjects - 5.56% - when compared to aggressive periodontitis, respectively 72.22 % (p<0.001). Our study demonstrated that the salivary level of the 8-OHdG biomarker and IL-1 gene polymorphism can be used in the evaluation of the oro-dental status at patients with aggressive periodontitis

The present study aims to identify and analyse the cardiometabolic risk factors associated with the metabolic syndrome in overweight children and adolescents. The study group included 163 overweight children and adolescents, average age: 13.02 ± 3.42 years. The following evaluations were performed: anthropometrical measurements, blood pressure measurements, biochemical tests investigating the lipid and carbohydrate metabolism. Metabolic syndrome was identified in 48 subjects (29.4%). The risk to develop MS was found to be higher in males and within the 13-18 age group. The most common cardiometabolic risk factors were abdominal obesity (75.5%) and high blood pressure (41.1%), followed by low HDL-cholesterol (35%), increased fasting blood glucose (23.3%) and hypertriglyceridemia (17.8%.). The variables under analysis exhibited significant correlations with the number of metabolic syndrome diagnosis criteria. The metabolic syndrome prevalence in the paediatric population affected by excess body weight has reached high values in our geographical area. It is thus justified to initiate screening activities for the early detection and adequate treatment of the modifiable cardiometabolic risk factors, contributing to the prevention of long-term complications.

Adenylat kinase is an ubiquitous enzyme found in prokaryotes and eukaryotes. In the present study we examined the inhibition of Streptococcus pneumoniae adenylate kinase (AKSP) by six 5-arylidene-thiazolidin-4- on-2-thione derivates using 2, 4-dinitrophenylhydrazine colorimetric assay. Inhibition of AKSP activity with synthetic compounds was performed against recombinant enzyme over-expressed in E. coli. The compound C₁₀H₆BrNOS₂ with the bromine atom in -ortho position has shown the most efficient inhibitory activity; I50 value (the inhibitor concentration that leads to 50% activity inhibition) was 0.067mM.

Cathelicidin LL37 is an innate immunity antimicrobial peptide involved in the immune modulation of IFN-Abstract Cathelicidin LL37 is an innate immunity antimicrobial peptide involved in the immune modulation of IFN-γ, the key cytokine of T helper cell type 1 (Th1) response. The role of LL37 in viral hepatitis inflammation is unknown. We assessed the serum variations of LL37 and the Th1 response in hepatitis C virus (HCV), hepatitis B virus (HBV) and hepatitis D virus (HDV) infections. The LL37 level (Elisa detection) and Th1 response (defined by IFN-γ level, CD4+ and CD8+ T cell count) were analyzed in 87 patients: 65 hepatitis patients (34 HCV, 18 HBV, 13 HDV) and 22 healthy controls. The subjects, 33 males/ 54 women aged 20-64 years, were selected at "Matei Bals" Institut, Bucharest, Romania. Hepatitis patients were classified according to viral etiology and viral replication as active cases (detectable viremia) versus negative cases (undetectable viremia). Student T test and Mann Whitney analysis were applied. High levels of LL37 (138.09±88.45ng/ml, p=0.045) and IFN-γ (69.82 pg/ml, p=0.005) were detected in the whole group of hepatitis. Active HCV hepatitis presented a significant increase in LL37 level (155.15±78.84ng/ml, p=0.014) and Th1 response by comparison with inactive HCV hepatitis. Conversely active HBV patients displayed low LL37 levels (76.75ng/ml, p=0.009) and no Th1 dominant response by comparison with inactive B hepatitis. High levels of LL37 up to 171.01±72.08 ng/ml and a moderate Th1 response defined HDV patients. Our results highlights increased levels of the cathelicidin LL37 in all viral hepatitis correlated with a strong and concordant immune response in active HCV hepatitis. , the key cytokine of T helper cell type 1 (Th1) response. The role of LL37 in viral hepatitis inflammation is unknown. We assessed the serum variations of LL37 and the Th1 response in hepatitis C virus (HCV), hepatitis B virus (HBV) and hepatitis D virus (HDV) infections. The LL37 level (Elisa detection) and Th1 response (defined by IFN-γ level, CD4+ and CD8+ T cell count) were analyzed in 87 patients: 65 hepatitis patients (34 HCV, 18 HBV, 13 HDV) and 22 healthy controls. The subjects, 33 males/ 54 women aged 20-64 years, were selected at "Matei Bals" Institut, Bucharest, Romania. Hepatitis patients were classified according to viral etiology and viral replication as active cases (detectable viremia) versus negative cases (undetectable viremia). Student T test and Mann Whitney analysis were applied. High levels of LL37 (138.09±88.45ng/ml, p=0.045) and IFN-γ (69.82 pg/ml, p=0.005) were detected in the whole group of hepatitis. Active HCV hepatitis presented a significant increase in LL37 level (155.15±78.84ng/ml, p=0.014) and Th1 response by comparison with inactive HCV hepatitis. Conversely active HBV patients displayed low LL37 levels (76.75ng/ml, p=0.009) and no Th1 dominant response by comparison with inactive B hepatitis. High levels of LL37 up to 171.01±72.08 ng/ml and a moderate Th1 response defined HDV patients. Our results highlights increased levels of the cathelicidin LL37 in all viral hepatitis correlated with a strong and concordant immune response in active HCV hepatitis.