Effects of green tea on iron accumulation and oxidative stress in livers of iron-challenged thalassemic mice.

Liver is affected by secondary iron overload in transfusions dependent b-thalassemia patients. The redox iron can generate reactive oxidants that damage biomolecules, leading to liver fibrosis and cirrhosis. Iron chelators are used to treat thalassemias to achieve negative iron balance and relieve oxidant-induced organ dysfunctions. Green tea (GT) (Camellia sinensis) catechins exhibit anti-oxidation, the inhibition of carcinogenesis, the detoxification of CYP2E1-catalyzed HepG2 cells and iron chelation. The purpose of this study was to investigate the effectiveness of GT in iron-challenged thalassemic mice. Heterozygous BKO type-thalassemia (BKO) mice (C57BL/6) experienced induced iron overload by being fed a ferrocene-supplemented diet (Fe diet) for 8 weeks, and by orally being given GT extract (300 mg/kg) and deferiprone (DFP) (50 mg/kg) for a further 8 weeks. Liver iron content (LIC) was analyzed by TPTZ colorimetric and Perl's staining techniques. Concentrations of liver reduced glutathione (GSH), collagen and malondialdehyde (MDA) were also measured. Dosages of the GT extract and DFP lowered LIC in the Fe diet-fed BKO mice effectively. The extract did not change any concentrations of liver glutathione, collagen and MDA in the BKO mice. Histochemical examination showed leukocyte infiltration in the near by hepatic portal vein and high iron accumulation in the livers of the iron-loaded BKO mice, however GT treatment lowered the elevated iron deposition. In conclusion, green tea inhibits or delays the deposition of hepatic iron in regularly iron-loaded thalassemic mice effectively. This will prevent the iron-induced generation of free radicals via Haber-Weiss and Fenton reactions, and consequently liver damage and fibrosis. Combined chelation with green tea would be investigated in beta-thalassemia patients with iron overload.

Flow cytometric method for simultaneous assay of foetal haemoglobin containing red cells, reticulocytes and foetal haemoglobin containing reticulocytes.

Level of foetal haemoglobin (HbF) containing red cells (F cells) is a parameter for monitoring sickle cell anaemia (SS) patients undergoing treatment with HbF modulating drugs (including hydroxyurea (HU)). One convenient technique for F cell assay is flow cytometry. A flow cytometric method for the simultaneous assay of F cells, reticulocytes and HbF-containing reticulocytes (F reticulocytes) is described in this paper. These three parameters can be obtained within 2 h using double colour staining flow cytometry. Glutaraldehyde fixation, Triton X-100 permeabilization, monoclonal antibody to HbF Tri-colour conjugate (MoAb-HbF-TC; deep-red fluorescence) immuno-staining and thiazole orange (TO; green fluorescence) are employed. The red cell gate was set on forward scatter (FSC) and logarithmic side scatter (logSSC) for 50 000 cells on the flow cytometer. Fluorescent signals were acquired from fluorescent channel 1 (FL1; green) and (FL4; deep-red). Coefficient of variation percent (%CVs) of intra- and inter-assay were less than 9% and 15%, respectively. EDTA, citrate, heparin and CTAD anticoagulants are all suitable; the samples can be stored at 4 degrees C for up to 6 days. The method is a simple, rapid, convenient, reproducible and useful way of determining F cell, reticulocyte and F reticulocyte levels in sickle cell and thalassaemic patients.

Technical Report: Triple-Colour Staining Flow Cytometry for Co-Distribution of Thrombospondin Receptor (CD36), Ribonucleic Acid (RNA) and Fetal Haemoglobin (HbF) in Sickle Red Blood Cells.

Red blood cells (RBCs) from sickle cell patients (SS) express thrombospondin receptor (CD36), contain ribonucleic acid (RNA, recognised as reticulocytes) and fetal haemoglobin (HbF, defined as F cells) in a higher proportion than RBCs from healthy individuals. The co-distribution of CD36, RNA and HbF on the same RBCs has not been demonstrated due to a lack of detection methods. A triple-colour staining flow cytometry for the co-distribution of CD36, RNA and HbF was developed. The method can simultaneously determine CD36-expressing RBCs (CD36 cells), RNA-bearing RBCs (reticulocytes), HbF-bearing RBCs (F cells), CD36-expressing reticulocytes (CD36 reticulocytes), CD36-expressing-F cells (CD36-F cells), HbF-bearing reticulocytes (F reticulocytes) and CD36-expressing-F reticulocjrtes (CD36-F reticulocytes). Mouse monoclonal antibody against CD36 (MoAb-CD36), antibodagainst mouse-immunoglobulin conjugated to biotin (Ab-Molg-Bi), streptavidin conjugated to rhodamine phycoerythrin (StA-RFE), MoAb against HbF conjugated to Tri-Colour® (MoAb-HbF-TC), Thiazole orange (TO), Glutaraldehyde and Triton X-100 were used. The procedure takes approximately 7 hours. The numbers of CD36 cells, reticulocytes and F cells obtaining from single and triple staining were well correlated and not significantly different. Intra- and inter-assay coefficient of variation percents (%CVs) of the triple-colour staining were less than 10 and 15% respectively. EDTA blood samples stored at 4°C for less than 3 days are suitable. The method trial was then employed on blood samples from SS and healthy individuals. The method is reproducible, objective and applicable for determination of co-distribution of other membrane and intracellular markers in RBCs.

Simplified flow cytometric method for fetal hemoglobin containing red blood cells.

Assay of fetal hemoglobin (HbF) and/or HbF containing red blood cells (F+ cells) is essential for monitoring sickle cell and thalassemic patients, especially during treatment with HbF stimulators. Some previous flow cytometric methods contain several washing steps. This simplified method contains no washing step and takes less than an hour to perform. The %F+ cells in five mixtures of fetal red blood cells with adult red blood cells were nonsignificantly different in the original and simplified procedure. The %F+ cells of 12 patients compared in these two procedures were also not significantly different. The intra- and interassay %CVs do not exceed 3% and 7% respectively. EDTA, citrate, or heparin is suitable as anticoagulant and the samples can be stored at 4 degrees C for up to 2 weeks. The %F+ cells and %HbF [by high-performance liquid chromatography (HPLC)] of 83 samples were highly significantly correlated regardless of diagnosis. In conclusion, this new simplified flow cytometric method for F+ cells is simple, convenient, rapid, reproducible, and could be applied for monitoring sickle cell and thalassemic patients as an alternative to HPLC, where this is unavailable. It can also be applied as a fetal cell assay in fetomaternal hemorrhage.

Risk factors for HIV-1 transmission from HIV-seropositive male blood donors to their regular female partners in northern Thailand.

OBJECTIVE: To describe risks for HIV transmission from male blood donors to their regular female sex partners in Chiang Mai, Thailand. DESIGN: Cross-sectional study. METHODS: From March 1992 through September 1995, 405 HIV-seropositive male blood donors (index cases) and their regular female partners were enrolled in the study. Women with risk factors for HIV infection other than sexual contact with the index male were excluded. Couples were interviewed and examined; specimens were collected for laboratory analysis. RESULTS: Overall, 46% of the 405 women enrolled were HIV-positive. Ninety-eight per cent of male index cases had a history of sex with a female prostitute; 1.5% reported always using condoms with their regular partner. History of sexually transmitted disease (STD) and swollen inguinal lymph nodes in the female partner were associated with an increased risk of HIV infection in the female. History in the female of genital herpes [odds ratio (OR), 3.46; 95% confidence interval (CI), 1.50-8.78], gonorrhea or chlamydia infection (OR, 2.71; 95% CI, 1.39-5.53), and stable relationship of longer than 24 months (OR, 2.28; 95% CI, 1.02-5.09) were associated with an increased risk of HIV infection in the female. Consistent condom use in the past 2 years (OR, 0.10; 95% CI, 0.01-0.79) was associated with a decreased risk of HIV infection in the female. CONCLUSIONS: Married women in northern Thailand who appear otherwise to be at low risk for HIV infection may be exposed to this virus by their husbands. High rates of sex with commercial sex workers among men and low use of condoms within stable relationships may be important factors promoting the transmission of HIV in married couples. Programs to increase the regular use of condoms among married couples could be an important public health intervention to prevent transmission of HIV and other types of STD in northern Thailand.

PIP: The risk factors for HIV transmission from infected male blood donors to their regular female sex partners were investigated in a cross-sectional study conducted in Chiang Mai, Thailand, in 1992-95. During the 3.5-year study period, 405 couples were recruited. 98% of male blood donors reported a history of sex with female prostitutes. Only 28 men (7%) were aware of their seropositivity prior to notification by the blood bank, and just 1.5% always used condoms with their regular sex partner. 187 (46%) of the 405 female sex partners--none of whom had HIV risk factors other than sexual contact with their husbands--were HIV-positive at baseline. In the multivariate analysis, three variables were associated with a significantly increased risk of HIV in female partners: history of genital herpes (odds ratio (OR), 3.46; 95% confidence interval (CI), 1.50-8.78), history of gonorrhea or chlamydia (OR, 2.71; 95% CI, 1.39-5.53), and a stable relationship of at least 2 years' duration (OR, 2.28; 95% CI, 1.02-5.09); consistent condom use in the past 2 years was significantly associated with a decreased risk of HIV (OR, 0.10; 95% CI, 0.01-0.79). Medroxyprogesterone acetate injection and oral contraceptive use were not associated with HIV risk. These findings confirm a high risk of HIV transmission through monogamous sexual relationships in Thailand. Recommended are campaigns to increase regular condom use among married couples.

Infectious disease markers in blood donors in northern Thailand.

BACKGROUND: A major epidemic of human immunodeficiency virus type 1 (HIV-1) infections that are primarily due to heterosexual transmission has developed in Thailand since 1988. The epidemic has been most severe in northern Thailand. The blood banks in Chiang Mai began screening donors for HIV-1 antibodies in February 1988 and for p24 antigen in April 1992. STUDY DESIGN AND METHODS: The trends of HIV-1 antibody prevalence were analyzed by type of donor (i.e., paid, replacement, and voluntary) for the period of 1988 through 1993. In addition, the prevalence of HIV-1 p24 antigen and of antibodies to syphilis, hepatitis B surface antigen, and hepatitis C virus was evaluated among blood donors at Chiang Mai University Hospital and the Thai Red Cross blood banks in Chiang Mai. RESULTS: The prevalence of HIV-1 antibodies increased from 0.84 percent in 1988 to 4.04 percent in 1991. Seropositivity was highest in paid professional donors. After discontinuation of the use of paid donors in 1993, HIV-1 antibody prevalence decreased to 3.34 percent. Antibody prevalence in replacement donors increased from 0.56 percent in 1988 to 5.82 percent in 1991. Among 44,446 donors tested, 7 (0.016%) were HIV-1 p24 antigen positive but antibody negative. CONCLUSION: The exclusion of paid donors and the use of p24 antigen testing are justified in northern Thailand. Additional strategies to exclude donors at very high risk and to attract those at low risk for infection should be developed and evaluated to increase blood transfusion safety in this and other, similar populations.

PIP: A major epidemic of human immunodeficiency virus type 1 (HIV-1) infections has developed in Thailand since 1988. The blood banks in Chiang Mai began screening donors for HIV-1 antibodies in February 1988 and for p24 antigen in April 1992. The trends of HIV-1 antibody prevalence were analyzed by type of donor (i.e., paid, replacement, and voluntary) for the period of 1988 through 1993. In addition, the prevalence of HIV-1 p24 antigen and of antibodies to syphilis, hepatitis B surface antigen, and hepatitis C virus was evaluated among blood donors at Chiang Mai University Hospital and the Thai Red Cross blood banks in Chiang Mai. The prevalence of HIV-1 antibodies in donor sera increased from 0.84% in 1988 to 3.23% in 1989. It continued to increase in subsequent years, reaching a maximum of 4.04% in 1991; the prevalence declined slightly in 1992 and 1993. In the first year of screening, the prevalence of HIV antibodies was highest in paid professional donors. The use of paid donors was discontinued on July 1, 1992, largely because of their high rates of HIV seropositivity. This action lowered the prevalence of HIV infection in the overall donor population in 1992 to 3.61%, from the peak of 4.04% in 1991. Antibody prevalence in replacement donors increased from 0.56% in 1988 to 5.82% in 1991. Screening for p24 antigen, introduced on April 29, 1992, identified 48 infected donors (among the 44,446 donors tested in 1992 and 1993) who were HIV p24 antigen positive. Altogether, 7 (0.016%) donors tested repeatedly reactive for p24 antigen, had positive p24 antigen neutralization tests, and were negative for antibodies to HIV. The exclusion of paid donors and the use of p24 antigen testing are justified in northern Thailand. Additional strategies to exclude donors at very high risk and to attract those at low risk for infection should be developed.