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Haemoglobinopathies are the most common hereditary disorders in Greece. Although there is a successful national prevention program, established 35 years ago, there is lack of an official registry and collection of epidemiological data for haemoglobinopathies. This paper reports the results of the first National Registry for Haemoglobinopathies in Greece (NRHG), recently organized by the Greek Society of Haematology. NRHG records all patients affected by thalassaemia major (TM), thalassaemia intermedia (TI), "H" Haemoglobinopathy (HH) and sickle cell disease (SCD). Moreover, data about the annual rate of new affected births along with deaths, between 2000 and 2010, are reported. A total of 4,506 patients are registered all over the country while the number of affected newborns was significantly decreased during the last 3 years. Main causes for still having affected births are: (1) lack of medical care due to financial reasons or low educational level; (2) unawareness of time limitations for prenatal diagnosis (PD); due either to obstetricians’ malpractice or to delayed demand of medical care of couples at risk; and (3) religious, social or bioethical reasons. Cardiac and liver disorders consist main causes for deaths while life expectancy of patients lengthened after 2005 (p < 0.01). The NRHG of patients affected by haemoglobinopathies in Greece provides useful data about the haemoglobinopathies in the Greek population and confirms the efficacy of the National Thalassaemia Prevention Program on impressively decreasing the incidence of TM and sickle cell syndromes.

Objectives: beta-Thalassemias are inherited hemolytic anemias with a broad phenotype and an increased rate of vascular complications despite a near absence of coronary artery disease. Methods: We investigated the presence of endothelial dysfunction and subclinical atherosclerosis in non-transfusion-dependent patients with beta-thalassemia intermedia (beta-TI) by means of flow-mediated (FMD) and flow-independent (FID) brachial artery dilatation and carotid artery intima-media thickness. Results: In 20 nondiabetic young adults with beta-TI, FMD (6.6 +/- 3.7 vs. 10.3 +/- 3.1%; p = 0.002) and FID (14.0 +/- 4.7 vs. 18.0 +/- 5.6%; p = 0.02) were both lower relative to the values in 20 matched control subjects, whereas the intima-media thickness was increased (0.51 +/- 0.09 vs. 0.46 +/- 0.07 mm; p = 0.049). Fibrin generation, soluble endothelial activation markers, and proinflammatory proteins were higher in the patient group, while the plasma cholesterol level was lower. Conclusions: These findings indicate premature atherosclerosis among patients with beta-TI; this is in accord with the high incidence of noncoronary vascular episodes in beta-TI. Copyright (C) 2011 S. Karger AG, Basel Cardiology

Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) is an independent cardiovascular risk factor. We investigated the plasma levels of Lp-PLA(2) activity and mass as a function of plasma lipid levels, LDL subclass profile, and oxidative stress in patients with beta-thalassemia. Thirty-five patients with beta-thalassemia major (beta-TM) and 25 patients with beta-thalassemia intermedia (beta-TI) participated in the study. Lp-PLA(2) activity and mass were measured in total plasma, in apolipoprotein (apo) B-depleted plasma (HDL-Lp- PLA(2)), and in LDL subclasses. Lp-PLA(2) activity produced and secreted from peripheral blood monocytes in culture was also determined. Patients with beta-thalassemia are characterized by a predominance of small-dense LDL particles, increased oxidative stress, and very high plasma levels of Lp-PLA(2) mass and activity, despite low LDL-cholesterol levels. A significant positive correlation between plasma Lp-PLA(2) activity or mass and 8-isoprostane (8-epiPGF2a) and ferritin levels as well as intima-media thickness (IMT) values was observed. An increase in secreted and cell-associated Lp-PLA(2) activity from monocytes in culture was observed in both patient groups. The HDL-Lp-PLA(2) activity and mass as well as the ratio of HDL-Lp-PLA(2)/plasma Lp-PLA(2) were significantly higher in both patient groups compared with the control group. In conclusion, patients with beta-thalassemia exhibit high plasma Lp-PLA(2) levels, attributed to increased enzyme secretion from monocytes/macrophages and to the predominance of sdLDL particles in plasma. Plasma Lp-PLA(2) is correlated with carotid IMT, suggesting that this enzyme may be implicated in premature carotid atherosclerosis observed in beta-thalassemia.-Tselepis, A. D., G. Hahalis, C. C. Tellis, E. C. Papavasiliou, P. T. Mylona, A. Kourakli, and D. C. Alexopoulos. Plasma levels of lipoprotein-associated phospholipase A2 are increased in patients with beta-thalassemia. J. Lipid Res. 2010. 51: 3331-3341. Journal of Lipid Research

Background: Patients with β-thalassemia major (β-TM) demonstrate an increased incidence of vascular complications, which are thought to result from a procoagulant/proinflammatory environment. We investigated the arterial vasorelaxing capacity and sought for early carotid atherosclerosis and underlying pathophysiological correlates in these transfusion-dependent patients. Methods and results: The vasodilatory properties of the brachial artery and the carotid intima-media thickness (IMT) were examined with ultrasonography in 35 non-diabetic young adults with β-TM (patient group) and 35 control subjects (control group). Among thalassemic patients, both endothelium-dependent (FMD) and -independent dilatation (FID) as well as their ratio was impaired, whereas IMT was increased (p < 0.01). Patients on optimal, as compared with those on non-optimal chelation treatment had a non-significantly lower IMT. Vasodilatory capacity in the patient group was inversely correlated with IMT and independently associated either with the quality of chelation therapy (FMD) or serum ferritin levels (FID). Plasma concentrations of D-dimers, circulating markers of endothelial activation, inflammation and apoptosis were higher, while plasma cholesterol and fibrinogen levels were lower-than-normal in the patient group. Independent predictors of IMT among thalassemic patients were tumor necrosis factor-α levels and age. Conclusions: Young adults with β-TM exhibit both a global impairment of arterial vasorelaxation and early carotid atherosclerosis. A procoagulant/proinflammatory state in these transfusion-dependent patients may overwhelm atheroprotective mechanisms, including an optimal chelation regimen, and promote vascular injury and atherogenesis. © 2007 Elsevier Ireland Ltd. All rights reserved.

Background: Patients with beta-thalassemia major (beta-TM) demonstrate an increased incidence of vascular complications, which are thought to result from a procoagulant/proinflammatory environment. We investigated the arterial vasorelaxing capacity and sought for early carotid atherosclerosis and underlying pathophysiological correlates in these transfusion-dependent patients. Methods and results: The vasodilatory properties of the brachial artery and the carotid intima-media thickness (IMT) were examined with ultrasonography in 35 non-diabetic young adults with beta-TM (patient group) and 35 control subjects (control group). Among thalassemic patients, both endothelium-dependent (FMD) and -independent dilatation (FID) as well as their ratio was impaired, whereas IMT was increased (p

Patients with beta-thalassemia major (beta-TM) demonstrate an increased incidence of vascular complications, which are thought to result from a procoagulant/proinflammatory environment. We investigated the arterial vasorelaxing capacity and sought for early carotid atherosclerosis and underlying pathophysiological correlates in these transfusion-dependent patients.The vasodilatory properties of the brachial artery and the carotid intima-media thickness (IMT) were examined with ultrasonography in 35 non-diabetic young adults with beta-TM (patient group) and 35 control subjects (control group). Among thalassemic patients, both endothelium-dependent (FMD) and -independent dilatation (FID) as well as their ratio was impaired, whereas IMT was increased (p0.01). Patients on optimal, as compared with those on non-optimal chelation treatment had a non-significantly lower IMT. Vasodilatory capacity in the patient group was inversely correlated with IMT and independently associated either with the quality of chelation therapy (FMD) or serum ferritin levels (FID). Plasma concentrations of D-dimers, circulating markers of endothelial activation, inflammation and apoptosis were higher, while plasma cholesterol and fibrinogen levels were lower-than-normal in the patient group. Independent predictors of IMT among thalassemic patients were tumor necrosis factor-alpha levels and age.Young adults with beta-TM exhibit both a global impairment of arterial vasorelaxation and early carotid atherosclerosis. A procoagulant/proinflammatory state in these transfusion-dependent patients may overwhelm atheroprotective mechanisms, including an optimal chelation regimen, and promote vascular injury and atherogenesis.

Sickle cell disease is characterized by vaso-occlusive episodes, mainly in the small vessels, resulting in tissue ischemia, multi-organ failure, and, occasionally, death. Hydroxyurea (HU) is an agent with important and effective role in the treatment of patients suffering from this disease. The purpose of this study was to estimate the effect of HU on the deformability of the red blood cell's membrane (RBCM) in an effort to possibly improve the rheological properties of the RBCs of patients with sickle cell anemia (SCA), as well as to investigate the mechanical and rheological properties of these cells using micropipette and filtration techniques. The rigidity index, IR, which is a measure of cell rigidity and the elastic shear modulus, mu, which is a measure of cell's membrane deformability (CMd), of the RBCs from normal subjects, used as normal controls, were found significantly lower as compared to those of patients with SCA, regardless the treatment with HU. Patients under treatment with HU exhibited values better than those of untreated patients, in both, IR as well as mu, although still worse than the values of normal controls.

Objectives A molecular study was carried out of beta-thalassemia intermedia patients, compound heterozygotes for mutations usually found in beta-thalassemia major, with high levels of HbF in the absence of hereditary persistence of fetal hemoglobin (HPFH) syndrome. Our objective was to locate cis-DNA structures, DNA haplotypes, motifs, or polymorphisms that may correlate with the presence of high HbF. Methods Allele-specific oligonucleotide (ASO) hybridization was used for the detection of mutations and restriction fragment length polymorphism (RFLP) analysis and automated sequencing for motifs, haplotypes, and polymorphisms. Southern blot was used for investigating alpha-thalassemia and/or alpha- or gamma-globin genes triplications. RNA extracted from burst forming unit-erythroid (BFU-e) colonies of peripheral blood mononuclear cell cultures was used in reverse transcriptase-polymerase chain reaction (RT-PCR) to investigate intergenic transcription. Results We established that (i) the combination: T haplotype of the Agamma-delta-globin intergenic region, the motif (TA)9N10(TA)10 in the HS2 site of locus control region (LCR), and TAG pre-Ggamma haplotype is sufficient but not necessary for high HbF, (ii) the genetic determinant(s) for high HbF involves an element associated with this combination and must be present in the specific R haplotype occurring in beta-thalassemia intermedia and (iii) the genetic determinant(s) for high HbF does not involve the abolition of intergenic transcription in the Agamma-delta-globin intergenic region. Conclusions The genetic determinant(s) of high HbF in the absence of HPFH is linked to intergenic haplotype T and does not disrupt intergenic transcription.

Deferasirox (ICL670) is a once-daily oral iron chelator developed for the treatment of chronic iron overload from blood transfusions. A comparative phase 3 trial was conducted to demonstrate the efficacy of deferasirox in regularly transfused patients with beta-thalassemia aged 2 years or older. Patients were randomized and received treatment with deferasirox (n = 296) or deferoxamine (n = 290), with dosing of each according to baseline liver iron concentration (LIC). The primary endpoint was maintenance or reduction of LIC; secondary endpoints included safety and tolerability, change in serum ferritin level, and net body iron balance. In both arms, patients with LIC values of 7 mg Fe/g dry weight (dw) or higher had significant and similar dose-dependent reductions in LIC and serum ferritin, and effects on net body iron balance. However, the primary endpoint was not met in the overall population, possibly due to the fact that proportionally lower doses of deferasirox relative to deferoxamine were administered to patients with LIC values less than 7 mg Fe/g dw. The most common adverse events included rash, gastrointestinal disturbances, and mild nonprogressive increases in serum creatinine. No agranulocytosis, arthropathy, or growth failure was associated with deferasirox administration. Deferasirox is a promising once-daily oral therapy for the treatment of transfusional iron overload.

The difference in light-emission kinetics between the Ca(2+)-triggered bioluminescent reaction of the photoprotein aequorin (AEQ) and the alkaline phosphatase (ALP)-catalyzed chemiluminescent hydrolysis of dioxetane aryl phosphate substrates was exploited for the analysis of both alleles of biallelic polymorphisms in a single microtiter well. The genotyping of the IVS-1-110 locus of the human beta-globin gene was chosen as a model. Genomic DNA, isolated from whole blood, was first subjected to polymerase chain reaction using primers flanking the polymorphic site. A single oligonucleotide-ligation reaction employing two allele-specific probes, labeled with biotin and digoxigenin, and a common probe carrying a characteristic tail was then performed. The ligation products were captured in a microtiter well through hybridization of the tail with an immobilized complementary oligonucleotide. The products were detected by adding a mixture of streptavidin-aequorin complex and antidigoxigenin-alkaline phosphatase conjugate. AEQ was measured first by adding Ca(2+) and integrating the signal for 3s followed by the addition of the substrate for ALP. The ratio of the luminescence signals obtained from ALP and AEQ gives the genotype of each sample. The coefficient of variation of the dual assay ranged from 7 to 11% for each allele. The reproducibility of the ALP/AEQ signal ratio was about 14%. The proposed assay allows for many samples to be screened in parallel in a single microtiter plate, for single-nucleotide polymorphisms.

Purpose: Patients with beta-thalassemia (beta-tha) represent a group with lifelong transfusion-dependent anemias. This study aimed to describe the conjunctival changes and tear film parameters in these patients. Methods: A total of 52 patients (104 eyes) with beta-tha major and 22 normal control subjects (44 eyes) were studied during 1999 through 2000. Tear film break-up time (BUT), Schirmer test, rose Bengal staining, and cytologic evaluation of the conjunctival epithelium were performed in all subjects. The Papanicolaou and May-Grumwald-Giemsa staining procedures were performed on all smears. Patients and control subjects were compared for tear function parameters and conjunctival changes. Results: The BUT, Schirmer test, and rose Bengal staining values were significantly lower (P < 0.001) in beta-tha patients than in control subjects. Keratinized cells were observed in conjunctival samples in 41% of patients, with a decrease in the number of goblet cells per slide in 64% of patients. In 9% of beta-tha patients, there were a slightly greater number of inflammatory cells than in control eyes. Conclusion: Ocular surface disorder of these patients was characterized by goblet cell loss and conjunctival squamous metaplasia. Our findings were correlated positively with the variable age. Epithelial damage by toxic reaction and disorder of tear quality and quantity are implicated as important factors in the pathogenesis of the ocular surface disease in beta-tha patients.

Anemia of chronic disease is a common feature of giant cell arteritis (GCA). It has been shown that constitutive hematopoiesis is regulated in the bone marrow microenvironment by direct cell-to-cell contact between hematopoietic progenitor cells and marrow stromal cells. Since cytokines are of crucial significance in this process, we decided to examine the role of stem cell factor (SCF), granulocyte macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL-3), granulocyte colony stimulating factor (G-CSF), and interleukin 1 alpha (IL-1 alpha), known for their stimulatory effects on in vitro hematopoiesis; and transforming growth factor beta (TGF beta), interferon gamma (IFN gamma) and tumor necrosis factor alpha (TNF-alpha), known for their inhibitory effect, in the anemia of GCA.The expression of these cytokines was examined in the marrow stromal cells from 6 GCA patients with anemia of chronic disease and in 6 healthy individuals by the reverse transcription mediated polymerase chain reaction (RT-PCR).Consistent constitutive gene expression was detected in the marrow stromal cells of all control samples for SCF, GM-CSF, IL-1 alpha, TGF beta and TNF-alpha, while only 2/6 expressed IFN gamma. None of the controls expressed either IL-3 or G-CSF. In all GCA patients SCF, TGF beta and TNF-alpha were expressed, but the level of expression was reduced compared to the control group. IL-1 alpha was expressed in 3/6 patients, and IFN gamma in 2/6. More importantly, GM-CSF expression was not detected in any patient, in marked contrast with the controls.The combination of the defective expression of SCF and GM-CSF, cytokines with a known stimulatory effect on in vitro hematopoiesis, may contribute to the pathogenesis of anemia in GCA.

One of the major determinants of erythrocyte survival is membrane deformability, and an important intrinsic parameter of membrane deformability is the shear elastic modulus (mu) with higher mu values corresponding to increased membrane rigidity. Using a micropipette technique, we determined the shear elastic modulus of erythrocytes from 21 patients with myelodysplastic syndromes (MDS). Ten thalassemic patients and 15 healthy subjects served as controls. The shear elastic modulus of MDS erythrocytes was very significantly increased in all the patients studied, reflecting the rigidity of the membrane; the value of mu was also significantly higher in comparison with thalassemic cells. These data point to a fundamental change in the mechanical properties of the erythrocyte membrane in MDS. Biochemical studies of the membrane composition are clearly needed.

We determined nine immune function parameters at diagnosis in patients with myelodysplastic syndromes (MDS) and correlated the results with the FAB classification and prognosis by univariate and multivariate analyses. Patients with refractory anaemia (RA) and refractory anaemia with ring sideroblasts (RAS) tended to have a higher CD4/CD8 ratio and a lower amount of γ-globulins and soluble interleukin-2 receptors in serum in comparison to those suffering from the other three subgroups of MDS. FAB classification, neutrophil and CD8 + T-cell number had the best discriminatory capacity for predicting survival less than 1 year, and FAB classification, neutrophil number and serum TNF levels were predictors for conversion to acute leukaemia. The frequent occurrence of infections, on the other hand, could be better predicted by the absolute numbers of neutrophils and CD4 + cells and by the skin test score.

IL17-producing T cells have been recently described as a distinct T cell helper population (Th17 cells) characterized by expression of membrane CD4 and IL23R and intracellular expression of the orphan nuclear receptor RORgt. In Th17 cells the transcription factor RORgt induces the transcription of IL17 gene, whereas in Th1 cells the transcription factor Tbet is responsible for the transcription of IFNg gene. Th1 along with Th17 cells are thought to contribute to the pathogenesis of autoimmune diseases. In murine models Th17 cells are fully polarized. In humans a proportion of Th17 cells are also positive for interferon gamma (IFN-g); they are named Th1/17 cells and their function is yet unclear. In patients with colitis and seronegative arthritis Th17 cells are increased. The induction of Th17 and Th1/17 in patients with MDS has not been previously evaluated. To examine the expression of Th17 and Th1/17 cells in this disease, peripheral blood mononuclear cells (PBMC) from patients with MDS were cultured in vitro for 6 days in RPMI-1640, 15% FBS supplemented with PHA (0.1 μg/mL) and IL-2 (10 ng/mL). Percentages of CD4+IL23R+IL-17+ T cells (Th17) and CD4+IL23R+IL17+IFN-g+ T cells (Th1/17) in patients with MDS were determined by flow cytometry: Th17 cells were markedly increased in patients (n=30) compared to healthy controls (n=15), (17.5% ± 3.4 vs 2.5% ± 0.4, p=0.008). Th1/Th17 cells were also significantly increased in MDS patients compared to controls (15.17% ± 2.80 vs 2.56% ± 0.80, p=0.008). None of the patients had been on immunosuppressive treatment or transfused before sampling. In multi-transfused patients with no underlying hematologic disease examined (n=3) the Th17 and Th1/17 populations were comparable to those of healthy donors. In patients with MDS the majority of the Th17 cells expressed also IFNg (90.07% ± 2.87) whereas in healthy controls only 59.7% ± 5.5 of the Th17 cells were also positive for IFNg (p CD4+IL23R+IL-17+ T cells and CD4+IL23R+IL17+IFN-g+ T cells are increased in most patients with MDS. T cells have recently been implicated in MDS pathogenesis. Although more studies are needed in order to define the role of Th17 and Th1/17 cells in the pathogenesis of MDS, our in vitro data with the kinase inhibitors may suggest a probable therapeutic target for patients with MDS.