Inhibitory effect of beta zero-thalassaemia/haemoglobin E erythrocytes on Plasmodium falciparum growth in vitro

The growth of Plasmodium falciparum in erythrocytes from individuals with beta zero-thalassaemia or haemoglobin (Hb) E, or both, was assessed in vitro. A significant inhibitory effect on the growth of the parasite was found only with erythrocytes from individuals doubly heterozygous for beta zeros-thalassaemia and HbE. The inhibitory effect was particularly marked with erythrocytes from splenectomized beta zeros-thalassaemia/HbE patients. The protective effect was related to HbF, Hb levels and shape abnormalities of the variant erythrocytes.     

Comparison of erythrocyte antioxidative enzyme activities between two types of haemoglobin H disease.

The activities of erythrocyte antioxidative enzymes were measured in two groups of patients with different genotypes of haemoglobin (Hb) H disease: 21 with alpha-thalassaemia 1 or alpha-thalassaemia 2 (alpha-thalassaemia 1/2) and 21 with alpha-thalassaemia 1/Hb Constant Spring (HbCS). They were compared with 21 normal subjects. Both genotypes of Hb H disease had increased activities of erythrocyte superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase when compared with those of controls. Comparison of the two genotypes showed that subjects with alpha-thalassaemia 1/Hb CS, the more severe disease, had higher SOD and GSH-Px activities but lower catalase activity than those with alpha-thalassaemia 1/2. This indicates that there are compensatory mechanisms in Hb H erythrocytes to cope with increased generation of oxygen free radicals as a result of increased excess beta chain.     

The instability of the membrane skeleton in thalassemic red blood cells

The thalassemias are a heterogeneous group of disorders characterized by accumulation either of unmatched alpha or beta globin chains. These in turn cause the intramedullary and peripheral hemolysis that leads to varying anemia. A partial explanation for the hemolysis came our of our studies on material properties that showed that beta-thalassemia (beta- thal) intermedia ghosts were very rigid but unstable. A clue to this instability came from the observation that the spectrin/band 3 ratio was low in red blood cells (RBCs) of splenectomized beta-thal intermedia patients. The possible explanations for the apparent decrease in spectrin content included deficient or defective spectrin synthesis in thalassemic erythroid precursors or globin chain-induced membrane changes that lead to spectrin dissociation from the membrane during ghost preparation. To explore the latter alternative, samples from different thalassemic variants were obtained, ie, beta-thal intermedia, HbE/beta-thal, HbH (alpha-thal-1/alpha-thal-2), HbH/Constant Spring (CS), and homozygous HbCS/CS. We searched for the presence of spectrin in the first lysate of the standard ghost preparation. Normal individuals and patients with autoimmune hemolytic anemia, sickle cell anemia, and anemia due to chemotherapy served as controls. Using gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, no spectrin was detected in identical aliquots of the supernatants of normals and these control samples. Varying amounts of spectrin were detected in the first lysate supernatants of almost all thalassemic patients. The identification of spectrin was confirmed by Western blotting using an affinity-purified, monospecific, rabbit polyclonal antispectrin antibody. Relative amounts of spectrin detected were as follows in decreasing order: splenectomized beta-thal intermedia including HbE/beta-thal; HbCS/CS; nonsplenectomized beta-thal intermedia, HbH/CS; and, lastly, HbH. These findings were generally confirmed when we used an enzyme-linked immunosorbent assay technique to measure spectrin in the first lysate. Subsequent analyses showed that small amounts of actin and band 4.1 also appeared in lysates of thalassemic RBCs. Therefore, the three major membrane skeletal proteins are, to a varying degree, unstably attached in severe thalassemia. From these studies we could postulate that membrane association of abnormal or partially oxidized alpha- globin chains has a more deleterious effect on the membrane skeleton than do beta-globin chains.     

Reduced deformability of thalassemic erythrocytes and erythrocytes with abnormal hemoglobins and relation with susceptibility to Plasmodium falciparum invasion.

A number of genetically variant erythrocytes showed decreased deformability of both intact cells and membranes prepared therefrom as measured by laser diffractometry. Erythrocytes associated with minor or no clinical symptoms (eg, alpha-thalassemia traits, hemoglobin [Hb] E trait, Hb Constant Spring trait), which showed only a minimal decrease in deformability, were, in general, invaded efficiently by the malarial parasite Plasmodium falciparum. Other variant erythrocytes (beta-thalassemia/Hb E, homozygous Hb E, homozygous Hb Constant Spring, Hb H, Hb H/Hb Constant Spring) with low deformability showed different degrees of reduction in invasion susceptibility, most of which were less than proportional with deformability decrease. It is concluded that parasite invasion is only weakly related to gross cell deformability, which in turn depends on various factors other than membrane deformability.     

Nitrate and nitrite in saliva and urine of inhabitants of areas of low and high incidence of cholangiocarcinoma in Thailand

Cholangiocarcinoma is one of the main liver diseases in northeast Thailand. Associations with exposure to liver fluke and N-nitrosodimethylamine in formation of the tumour have been demonstrated in animals. This study was carried out to compare possible endogenous formation of N-nitroso compounds in inhabitants of areas with low and high incidences of cholangiocarcinoma by examining the levels of nitrate and nitrite in their saliva and urine. Thirty-two subjects (16 males and 16 females) living in the north-east (high incidence) and 12 volunteers (6 males and 6 females) in Bangkok (low incidence) were allowed to take regular meals, and their saliva and urine were collected before, and 30, 60 and 120 min after each meal. Nitrate and nitrite concentrations in saliva of the group in the high-incidence area were significantly higher than those of the group in Bangkok: salivary nitrate was 2-2.8 times higher and nitrite 2-5.6 times higher in the north-eastern group when compared with levels at each corresponding time interval in the low-incidence group. Nitrate levels in urine were also significantly higher in the north-eastern group at some time intervals, but urinary nitrite levels were similar and very low in both groups throughout the day. This finding may indicate a greater possibility of in-vivo formation of N-nitroso compounds in the north-east area than in Bangkok and might be associated with the occurrence of cholangiocarcinoma in north-east Thailand.     

Immunization with a live attenuated dengue-2-virus candidate vaccine (16681-PDK 53): clinical, immunological and biological responses in adult volunteers

A live dengue-2 (DEN-2) candidate vaccine (strain 16681-PDK 53), attenuated by passage in primary dog kidney cells, was tested in ten adult volunteers for evaluation of the safety, infectivity and immunogenicity of a dose of 1.9-2.7 × 10⁴ plaque-forming units. Five of the volunteers were nonimmune to either dengue or Japanese encephalitis (JE) viruses; the other five were nonimmune to dengue but immune to JE. After receiving 1.0 ml of the vaccine subcutaneously, all ten volunteers developed neutralizing antibodies to DEN-2 which were maintained for at least one and a half years. None of the subjects developed abnormal signs or symptoms and the results of clinical chemistry investigations were within normal range throughout the 21 days of observation after the immunization. Virus isolated from one viraemic volunteer retained the small-plaque and temperature-sensitive growth characteristics of the vaccine virus in vitro. Further testing of this candidate vaccine in humans is indicated.     

Opisthorchis viverrini infection and cholangiocarcinoma. A prospective, case-controlled study

During a prospective, 24-mo case-controlled study, 551 patients from northeastern Thailand were independently evaluated for Opisthorchis viverrini infection, hepatobiliary tract disease, and hepatic carcinoma to determine whether there was any association between hepatic carcinoma and O. viverrini infection. Stool examination by the formalin-ether concentration method revealed O. viverrini ova in 389 (70.6%) patients. Of the 551 patients, 72 (13.1%) had both clinical and laboratory evidence of hepatobiliary tract disease, chronic liver disease, or hepatic carcinoma, alone or in combination. Of these 72 patients, 28 (38.9%) had a liver biopsy that revealed cholangiocarcinoma in 7 patients with O. viverrini ova in their stools, and in 4 patients without. In another patient with ova in the stool combined hepatocellular carcinoma and cholangiocarcinoma was found. In the 4 patients with cholangiocarcinoma who had no O. viverrini ova in their stools, ova were detected in the bile fluid aspirated from the intrahepatic biliary tree during exploratory laparotomy. An additional patient with clinically suspected cholangiocarcinoma and O. viverrini ova in stool had a left supraclavicular lymph node biopsy specimen taken that revealed metastatic adenocarcinoma; this adenocarcinoma was interpreted as compatible with cholangiocarcinoma. Cholangiocarcinoma, therefore, was found only in patients with O. viverrini ova in stool or in the intrahepatic biliary tree. Statistical analysis revealed that patients with known O. viverrini infection had a higher incidence of cholangiocarcinoma than did patients without such infection (X2 test, p less than 0.05).     

Enhanced maturation and proliferation of beta-thalassemia/Hb E erythroid precursor cells in culture

Upon erythroid cell maturation in vivo, beta-thalassemic erythroid cells accumulate unmatched unstable alpha-globin chains that are believed to be a causal factor in such cell destruction. This study showed that beta-thalassemia/Hb E erythroid precursor cells from peripheral blood had accelerated maturation, and could mature to the terminal erythroid stage. During the early period of cell culture, erythroid precursor cells derived from subjects with the more severe form of beta-thalassemia/Hb E had higher rate of erythroid maturation. In addition, peripheral blood mononuclear cells from beta-thalassemia/Hb E subjects had higher erythroid proliferative potential than cells derived from normal controls. Erythroid proportion in the more severe beta-thalassemia/Hb E cases was less than that of the milder cases. Premature apoptosis was not observed during the 15 days of erythroid cell culture from both beta-thalassemia/Hb E and normal subjects.     

Icsh activities and Asia

The International Council for Standardization in Haematology (ICSH), an international organization promoting international agreement on hematological testing, is now restructuring to strengthen its activities. In Asia, a diversity of testing methods exists and the resulting testing levels make it difficult to compare test results internationally among Asian countries. Fortunately, the ICSH is considering regionalizing its organization to 5 sub-societies to increase its activity, and we have been able to establish a new society, ICSH-Asia, under the ICSH umbrella.