Haemoglobinopathies in Southeast Asia.

In Southeast Asia α-thalassaemia, β-thalassaemia, haemoglobin (Hb) E and Hb Constant Spring (CS) are prevalent. The abnormal genes in different combinations lead to over 60 different thalassaemia syndromes, making Southeast Asia the locality with the most complex thalassaemia genotypes. The four major thalassaemic diseases are Hb Bart's hydrops fetalis (homozygous α-thalassaemia 1), homozygous β-thalassaemia, β-thalassaemia/Hb E and Hb H diseases. α-Thalassaemia, most often, occurs from gene deletions whereas point mutations and small deletions or insertions in the β-globin gene sequence are the major molecular defects responsible for most β-thalassaemias. Clinical manifestations of α-thalassaemia range from asymptomatic cases with normal findings to the totally lethal Hb Bart's hydrops fetalis syndrome. Homozygosity of β-thalassaemia results in a severe thalassaemic disease while the patients with compound heterozygosity, β-thalassaemia/Hb E, present variable severity of anaemia, and some can be as severe as homozygous β-thalassaemia. Concomitant inheritance of α-thalassaemia and increased production of Hb F are responsible for mild clinical phenotypes in some patients. However, there are still some unknown factors that can modulate disease severity in both α- and β-thalassaemias. Therefore, it is possible to set a strategy for prevention and control of thalassaemia, which includes population screening for heterozygotes, genetic counselling and foetal diagnosis with selective abortion of affected pregnancies.

Nitric oxide and caspase 3 mediated cytokine induced apoptosis in acute leukemia.

Background: Leukemia is characterized by the uncontrolled accumulation of white blood cells. Recently, cytokines have been used in immunotherapy, which is a new strategy for leukemia treatment. Objective: To investigate the effect of cytokines on induction of apoptosis in acute leukemic cell lines; HL-60, MV4-11, K-562 and Molt-4 and in addition, to study the involvement of nitric oxide (NO) in apoptotic pathways. Methods: Leukemic cell lines were incubated with cytokines; interleukin-1β, tumor necrosis factor-a, and interferon-γ in various concentrations and for variable periods of time. The percent apoptosis and caspase 3 activation were examined by flow cytometry. Moreover, NO production and inducible nitric oxide synthase (iNOS) mRNA were measured by using Griess method and Real-time PCR, respectively. Results: Cytokines caused a time and dose-dependent induction of apoptosis in leukemic cell lines. The highest cell apoptosis was found in K-562 treated with 40 U/ml interferon-γ for 48 hours; this correlated with the result of cell growth inhibition and caspase 3 activation. NO and iNOS mRNA were increased in cytokines treated cells. Moreover, apoptosis was reduced by SMT, an iNOS inhibitor, which confirms the possible involvement of NO in the apoptotic pathway. Conclusion: Cytokines especially interferon-g induced apoptosis in acute leukemia via NO and caspase 3 pathway.

Modification of platelet shape change parameter by oxidized lipoprotein from beta-thalassemia/Hemoglobin E.

BACKGROUND: Beta-thalassemia/Hemoglobin E (beta-thal/Hb E) is a congenital hemolytic anemia that is prevalent in Thailand Pulmonary arterial occlusion is the cause of morbidity and mortality in these patients. Abnormality of platelets has been implicated as pathogenesis of this condition. However the blood-borne factors that induce platelet activation are not identified Recently, oxidized low-density lipoproteins (ox-LDLs) had been identified in thalassemic blood. OBJECTIVE: Identify whether oxidized LDL is the blood bone factor that induce platelet activation in beta-thal/Hb E patients. MATERIAL AND METHOD: Platelet activation was measured by monitoring platelet shape change parameter using plasma-free human platelets. The shape change parameter was monitored following exposure to normal LDL, oxidized LDL, and thalassemic LDL. RESULTS: Oxidized LDL, but not the native LDL and thalassemic LDL, showed platelet activation activity. Oxidation of thalassemic LDL with copper give rise to oxidized LDL with platelet activating activity. However less copper was needed by LDL from splenectomized beta-thal/Hb E patients than those from nonsplencectomized beta-thal/Hb E patients. CONCLUSION: LDL from splenectomized beta-thal/Hb E patients is more susceptible for oxidation and gives rise to oxidized-LDL that plays an important role in thrombosis event in these patients.

Role of interleukin-3 and signaling pathways on beta-thalassemia/HbE erythroid progenitor cell in culture.

In order to study the role of the cytokine interleukin-3 (IL-3) and its signaling pathways in erythropoiesis of beta-thalassemia/HbE erythroid progenitor cells, CD34 positive cells were isolated from peripheral blood of patients and healthy subjects. After culturing the cells in the presence or absence of IL-3, cell viability was measured by trypan blue staining and apoptotic cells were analyzed by flow cytometry. After 7 days of culture the highest percent erythroid progenitor cell viability was obtained with cells from healthy subjects, while the lowest percentage was found in those from splenectomized beta-thalassemia/HbE. Viability of beta-thalassemia/HbE erythroid progenitor cells in the presence of IL-3 was higher than that of nonsupplemented cells. In addition, specific inhibitors of protein kinase C (Ro-318220), phospholipase C (U-73122) and Janus kinase 2 (AG-490) were used to investigate the involvement of signaling pathways in erythropoiesis. Percent apoptosis of erythroid progenitor cells from splenectomized beta-thalassemia/HbE subjects treated with RO-318220 was higher than those of nonsplenectomized beta-thalassemia/HbE and healthy subjects. Treatment with U-73122 resulted in enhanced percent apoptotic cells from normal and beta-thalassemia/HbE subjects. All these effects were independent of IL-3 treatment.

Prenatal diagnosis of thalassemia and hemoglobinopathies: 20 years experience at Siriraj Hospital.

Objective: To show the experience of prenatal diagnosis of Thalassemia and hemoglobinopathies in Siriraj Hospital. Methods: Hb Bart’s hydrops fetalis can be detected by DNA study from polymerase chain reaction (PCR) product in the first trimester of pregnancy either by chorionic villus sampling (CVS) or aminocentesis but in late pregnancy it can be detected unambiguous by ultrasonography at 18-20 weeks gestation, the suspected cases are confirmed by fetal blood sampling and Hb electrophoresis. Prenatal diagnosis (PND) for β-thalassemia diseases can be done at early pregnancy by direct visualization of the PCR products on electrophoresis or by dot blot analysis of amplified DNA with a set of HRP-labeled oligonucleotide probes complementary to the mutations. If the mutation is unknown. The couples have to wait for Hb analysis by HPLC or in vitro globins chain analysis from fetal blood in the second trimester. Results: The results of PND at Siriraj Hospital are summarized as Hb Bart’s Hydrops fetalis 228 cases, Homozygous Beta-Thalassemia 126 cases, and Beta Thalassemia/Hb E disease 550 cases. There are various methods of sampling namely chorionic villous sampling, amniocentesis, fetal blood sampling, ultrasound, or even combined method. There are minimal incidences of fetal loss 9 out 904 cases which comparatively give us one of the best center for prenatal diagnosis in Asia. Conclusion: Of the 904 pregnancies, the diagnosis were obtained in 891 pregnancies in which had 5 fetal loss from dead fetus in utero after fetal blood sampling in the second trimester. The other complication occurred after sampling failure.

Prevention and control of thalassemia in Asia.

Thalassemia and abnormal hemoglobin are the most common inherited diseases. The only treatment readily available in most countries is regular blood transfusion and iron chelation that is recommended in severely anemic patients with iron overload. In the last 20 years there has been much progress in terms of diagnosing, preventing and managing thalassemia. This has lead to the success of prevention and control of thalassemia in many Mediterranean countries such as Cyprus, Italy and Greece. Objective: To introduce approaches that may be applied for the control of thalassemia in developing countries including Asia where thalassemias are very prevalent.

Genetic polymorphisms and implications for human diseases.

After the sequencing of the human genome is done, enormous genomic information and high-throughput profiling technologies are used. Increased attention has been paid to applying this knowledge to get better understanding of inherited diseases and complex disorders. Single nucleotide polymorphisms (SNPs) are DNA sequence variations that occur when a single nucleotide in the genome sequence is altered SNPs are an important tool for the study of the human genome. Application of SNPs analysis to human disease permits exploration of the influence of genetic polymorphisms on disease susceptibility, drug sensitivity/resistance, and ultimately health care. Databases of SNPs provide a powerful resource for association studies that try to establish a relationship between a phenotype and regions of the genome. Genomic approaches have garnered so much attention and investment because they offer the potential to provide better understanding of genetic factors in human health and disease, as well as more-precise definitions of the non-genetic factors involved.

Ultrastructural features of sidero - apoptotic hepatocytes in thalassemia.

Liver tissues from 20 patients with thalassemia were investigated by light and electron microscopy, with focus on ultrastructure of sidero-apoptosis. The results of light microscopy were compared. Sidero-apoptosis was found in 82% in b thal/Hb E disease, 67% in thalassemia Hb H, 100% in and b-thal major, respectively. Regardless of the type of hemoglobin, sidero-apoptosis was present in various degrees. The main criteria used for identification of sidero-apoptosis were marked condensation of chromatin against the nuclear membrane, and fragmentation into nuclear fragments of varying sizes and structures into membrane-bounded sidero-apop-totic bodies. These bodies may be phagocytosed by macrophages or neighboring cells or remain free. However, the cell may also shrink into a dense, rounded mass, as a single sidero-apoptotic body. Sidero-apoptoic bodies were similar to acidophilic bodies, or Councilman bodies, commonly found in viral hepatitis. This preliminary report suggestsd that sidero-apoptosis is a features of human liver pathology in thalassemia and may be due to hepatotoxicity of iron overload. In addition, many factors or other agents-hormone, killer cells, cytokines, various physical, chemical and viral agents should be considered. We propose the term "sidero-apoptosis" to describe the features resenbling acidophilic bodies commonly seen in viral hepatitis but with a heavy depositi of iron.

Electron microscopical and immunohistochemical studies of liver tissue in thalassaemic patients: Emphasis on iron and hepatitis B virus.

Fine structure and immunohistochemical studies were investigated in 30 thalassaemic patients. They were diagnosed as having beta thalassemis/haemoglobin E in 19 cases, haemoglobin H disease in 7, and beta thalassemia major in 4. Regardless of the type of haemoglobin, lysosomal haemosiderin was present in various degrees in all cases and some were associated with ferritin molecules. The ferritin molecules were found in both cell sap and lysosome, in four forms: paracrystalline arrangement, hexagonal pattern, finger print fashion and unorganized form. By peroxidase-anti peroxidase (PAP) technique for the detection of hepatitis B surface (HBs) and hepatitis B core (HBc) antigens, HBsAg was found in 1 case of beta thalassemia/Hb E. Ferro-acidophilic bodies (FAB) and ferro-acidophilic degeneration (FAD) were also found. FAB and FAD were probably due to iron hepatotoxicity, because no viral particles were found in this study.