A comprehensive study of immune function and immunophenotyping of white blood cells from ß-thalassaemia/HbE patients on hydroxyurea supports the safety of the drug.

Hydroxyurea (HU) (hydroxycarbamide) is used as a therapeutic option in ß-thalassaemia to increase fetal haemoglobin, which results in a reduced requirement for blood transfusion. However, a potential serious adverse effect of HU is neutropenia. Abnormal neutrophil maturation and function in ß-thalassaemia/HbE patients are well documented. This raises questions about the effect of the drug with regards to the immune response these patients. This study investigated the effects of HU treatment on both innate and adaptive immunity in a cross-sectional study of 28 ß-thalassaemia/HbE patients who had received HU treatment (BE+HU) as compared with 22 ß-thalassaemia/HbE patients who had not received HU (BE-HU) and 26 normal subjects. The expression of PU.1 and C/EBPß, transcription factors, which are associated with neutrophil maturation, was significantly reduced in BE+HU patients as compared with BE-HU patients and normal subjects. Interestingly, C3bR expression on neutrophils and their oxidative burst activity in BE+HU were restored to close to normal levels when compared with BE-HU. There was no observed effect of HU on monocytes, myeloid derived suppressor cells (both granulocytic and monocytic subsets), CD4+ T cells, CD8+ T cells, complement levels and serum immunoglobulin levels in this study. The full immunophenotyping analysis in this study indicates that HU therapy in ß-thalassaemia/HbE patients does not significantly compromise the immune response.

How do we translate gaps and unmet needs of blood management for thalassemia into a collaborative implementation framework?

BACKGROUND AND OBJECTIVE: The mainstay of management for thalassemia is regular blood transfusions. However, gaps and unmet needs of blood services for thalassemia are still not clearly identified and addressed in Thailand, a country prevalent with thalassemia. What can be a collaborative implementation framework that helps advance practices and policies relating to blood management for thalassemia? METHODS: The first Blood & Beyond Roundtable Discussion was held in July 2022 to gather the current situation, gaps, and unmet needs of blood services for thalassemia from multidisciplinary experts and thalassemic patients. The Implementation Guide as suggested by the Centre for Effective Services was applied as a tool to consolidate information from the discussions and construct the collaborative implementation framework. RESULTS: The National Blood Center and hospitals in Thailand followed the missions specified in the National Blood Policy and the standard guidelines to ensure the best practice of blood management for thalassemia. However, there were six gaps and unmet needs identified from the discussions. After all discussion points were mapped onto the framework, an implementation plan comprised of five specific activities became clear and actionable. CONCLUSION: Without the complete information from both experts and patients, the implementation plan would not have been successfully constructed. The method that we employed to translate all information into the framework can be adapted by other countries to develop their own specific framework efficiently.

Erythropoietin and IGF-1 signaling synchronize cell proliferation and maturation during erythropoiesis.

Tight coordination of cell proliferation and differentiation is central to red blood cell formation. Erythropoietin controls the proliferation and survival of red blood cell precursors, while variations in GATA-1/FOG-1 complex composition and concentrations drive their maturation. However, clear evidence of cross-talk between molecular pathways is lacking. Here, we show that erythropoietin activates AKT, which phosphorylates GATA-1 at Ser310, thereby increasing GATA-1 affinity for FOG-1. In turn, FOG-1 displaces pRb/E2F-2 from GATA-1, ultimately releasing free, proproliferative E2F-2. Mice bearing a Gata-1(S310A) mutation suffer from fatal anemia when a compensatory pathway for E2F-2 production involving insulin-like growth factor-1 (IGF-1) signaling is simultaneously abolished. In the context of the GATA-1(V205G) mutation resulting in lethal anemia, we show that the Ser310 cannot be phosphorylated and that constitutive phosphorylation at this position restores partial erythroid differentiation. This study sheds light on the GATA-1 pathways that synchronize cell proliferation and differentiation for tissue homeostasis.

Coordinated ß-globin expression and α2-globin reduction in a multiplex lentiviral gene therapy vector for ß-thalassemia.

A primary challenge in lentiviral gene therapy of ß-hemoglobinopathies is to maintain low vector copy numbers to avoid genotoxicity while being reliably therapeutic for all genotypes. We designed a high-titer lentiviral vector, LVß-shα2, that allows coordinated expression of the therapeutic ßA-T87Q-globin gene and of an intron-embedded miR-30-based short hairpin RNA (shRNA) selectively targeting the α2-globin mRNA. Our approach was guided by the knowledge that moderate reduction of α-globin chain synthesis ameliorates disease severity in ß-thalassemia. We demonstrate that LVß-shα2 reduces α2-globin mRNA expression in erythroid cells while keeping α1-globin mRNA levels unchanged and ßA-T87Q-globin gene expression identical to the parent vector. Compared with the first ßA-T87Q-globin lentiviral vector that has received conditional marketing authorization, BB305, LVß-shα2 shows 1.7-fold greater potency to improve α/ß ratios. It may thus result in greater therapeutic efficacy and reliability for the most severe types of ß-thalassemia and provide an improved benefit/risk ratio regardless of the ß-thalassemia genotype.

Effect of neonatal reticulocytosis on glucose 6-phosphate dehydrogenase (G6PD) activity and G6PD deficiency detection: a cross-sectional study.

BACKGROUND: Screening for G6PD deficiency in newborns can help prevent severe hemolysis, hyperbilirubinemia, and bilirubin encephalopathy, as recommended by the World Health Organization (WHO). It has been speculated that the presence of a high number of reticulocytes in newborns interferes with the diagnosis of G6PD deficiency since reticulocytes contain higher amounts of G6PD enzyme than mature erythrocytes. Therefore, the purposes of this study were to assess the effect of reticulocytosis in the determination of blood G6PD activity in Thai newborns by using a novel automated UV-based enzymatic assay and to validate the performance of this assay for the detection of G6PD deficiency in newborn samples. METHODS: The levels of reticulocytes and G6PD activity were measured in blood samples collected from 1,015 newborns. G6PD mutations were identified using TaqMan® SNP genotyping assay, PCR-restriction fragment length polymorphism (PCR-RFLP), and direct sequencing. The correlation between the levels of reticulocytes and G6PD activity was examined. The performance of the automated method was compared with that of the fluorescent spot test (FST) and the standard quantitative assay. RESULTS: The automated assay detected G6PD deficiency in 6.5% of the total newborn subjects compared to 5.3% and 6.1% by the FST and the standard method, respectively. The minor allele frequencies (MAFs) of G6PD ViangchanG871A, G6PD MahidolG487A, and G6PD UnionC1360T were 0.066, 0.005, and 0.005, respectively. The reticulocyte counts in newborns with G6PD deficiency were significantly higher than those in normal male newborns (p < 0.001). Compared with normal newborns after controlling for thalassemias and hemoglobinopathies, G6PD-deficient patients with the G6PD ViangchanG871A mutation exhibited elevated reticulocyte counts (5.82 ± 1.73%, p < 0.001). In a group of G6PD normal newborns, the percentage of reticulocytes was positively correlated with G6PD activity (r = 0.327, p < 0.001). However, there was no correlation between G6PD activity and the levels of reticulocytes in subjects with G6PD deficiency (r = -0.019, p = 0.881). The level of agreement in the detection of G6PD deficiency was 0.999, while the area under the receiver operating characteristic (AUC) curve demonstrated that the automated method had 98.4% sensitivity, 99.5% specificity, 92.4% positive predictive value (PPV), 99.9% negative predictive value (NPV), and 99.4% accuracy. CONCLUSIONS: We report that reticulocytosis does not have a statistically significant effect on the detection of G6PD deficiency in newborns by both qualitative and quantitative methods.

Thalassemia in Thailand.

Thailand has a population of 66.2 million with 30.0-40.0% of them carrying thalassemia genes. Interaction of these thalassemia genes lead to more than 60 genotypes with a wide spectrum of clinical severity from asymptomatic to lethal. Estimation based on gene frequencies and number of babies born each year, there will be about 1.2% babies born with severe cases of thalassemia each year. Further estimation revealed that 1.0% of the Thai population have thalassemia disease, which is a big health problem for the country. Thalassemia prevention and control programs were introduced using post conception screening in couples and prenatal diagnosis (PND) for the prevention of new thalassemic births. Moreover, the majority of existing cases are undergoing supportive treatment with regular blood transfusions and iron chelation. Curative treatment by hematopoietic stem cell transplantation (HSCT) is available but is limited to a minority of the patients.

Clinical Severity of ß-Thalassemia Pediatric Patients in Myanmar.

ß-Thalassemia (ß-thal) is highly prevalent in Myanmar, but limited data are available on the molecular basis and the clinical manifestations in Myanmar patients. In this study, we investigated the clinical features and ß-globin gene abnormalities in 15 homozygous ß-thal and 60 Hb E (HBB: c.79G>A)/ß-thal pediatric patients who attended Yangon Children Hospital, the biggest thalassemia day care unit center in Myanmar. Eight different ß0-thal mutations were identified, with four accounting for 88.9% of alleles studied (excluding the Hb E variant). A genotype-phenotype correlation was found; all homozygous ß0-thalassemias had severe clinical courses, whereas the highly variable disease severity was demonstrated among Hb E/ß0-thal patients. Interactions of IVS-I-1 (G>T) (HBB: c0.92+1G>T) ß0-thal with Hb E are associated with milder clinical symptoms. The number of mildly affected Hb E/ß-thal patients was lower than expected, suggesting that there may be a considerable number of patients in the population who have either not been admitted to hospital or diagnosed with carrying the disease. Although the clinical severity in the Myanmar ß-thal patients seems to be similar to that in other populations, the levels of hemoglobin (Hb) appears to be very low. These findings indicate the need for the improvement of patient management and the development of prevention and control programs for ß-thal in Myanmar.

Increased susceptibility to dextran sulfate-induced mucositis of iron-overload ß-thalassemia mice, another endogenous cause of septicemia in thalassemia.

Enterocyte damage and gut dysbiosis are caused by iron-overload in thalassemia (Thl), possibly making the gut vulnerable to additional injury. Hence, iron-overload in the heterozygous ß-globin deficient (Hbbth3/+) mice were tested with 3% dextran sulfate solution (DSS). With 4 months of iron-gavage, iron accumulation, gut-leakage (fluorescein isothiocyanate dextran (FITC-dextran), endotoxemia, and tight junction injury) in Thl mice were more prominent than WT mice. Additionally, DSS-induced mucositis in iron-overloaded mice from Thl group was also more severe than the WT group as indicated by mortality, liver enzyme, colon injury (histology and tissue cytokines), serum cytokines, and gut-leakage (FITC-dextran, endotoxemia, bacteremia, and the detection of Green-Fluorescent Producing Escherichia coli in the internal organs after an oral administration). However, Lactobacillus rhamnosus GG attenuated the disease severity of DSS in iron-overloaded Thl mice as indicated by mortality, cytokines (colon tissue and serum), gut-leakage (FITC-dextran, endotoxemia, and bacteremia) and fecal dysbiosis (microbiome analysis). Likewise, Lactobacillus conditioned media (LCM) decreased inflammation (supernatant IL-8 and cell expression of TLR-4, nuclear factor κB (NFκB), and cyclooxygenase-2 (COX-2)) and increased transepithelial electrical resistance (TEER) in enterocytes (Caco-2 cells) stimulated by lipopolysaccharide (LPS) and LPS plus ferric ion. In conclusion, in the case of iron-overloaded Thl, there was a pre-existing intestinal injury that wask more vulnerable to DSS-induced bacteremia (gut translocation). Hence, the prevention of gut-derived bacteremia and the monitoring on gut-leakage might be beneficial in patients with thalassemia.

Association of the Degree of Erythroid Expansion and Maturation Arrest with the Clinical Severity of ß0-Thalassemia/Hemoglobin E Patients.

INTRODUCTION: ß-Thalassemia/hemoglobin E represents one-half of all the clinically severe ß-thalassemias worldwide. Despite similar genetic backgrounds, patients show clinical heterogeneity ranging from nearly asymptomatic to transfusion-dependent thalassemia. The underlying disease modifying factors remain largely obscure. METHODS: To elucidate the correlation between ineffective erythropoiesis and ß0-thalassemia/hemoglobin E (HbE) disease severity, in vitro culture of erythroid cells derived from patients with different clinical symptoms was established. Cell proliferation, viability, and differentiation were investigated. To identify potential molecular mechanisms leading to the arrested erythroid maturation, the expression levels of erythropoiesis modifying factors were measured. RESULTS: The ß0-thalassemia/HbE cells exhibited enhanced proliferation, limited differentiation, and impaired erythroid terminal maturation but did not show accelerated erythroblast differentiation and increased cell death. Erythroblasts derived from mild patients showed the highest proliferation rate with a faster cell division time, while erythroblasts derived from severe patients displayed extremely delayed erythroid maturation. Downregulation of growth differentiation factor 11 and FOXO3a was observed in mild ß0-thalassemia/HbE erythroblasts, while upregulation of heat shock protein 70 and activin receptor 2A was revealed in severe erythroblasts. DISCUSSION/CONCLUSION: The degree of erythroid expansion and maturation arrest contributes to the severity of ß0-thalassemia/HbE patients, accounting for the disease heterogeneity. The findings suggest a restoration of erythroid maturation as a promising targeted therapy for severe patients.

Consumption of Sinlek Rice Drink Improved Red Cell Indices in Anemic Elderly Subjects.

Iron fortifications are used for the treatment of iron-deficiency anemia; however, iron dosing may cause oxidative damage to the gut lumen. Thai Sinlek rice is abundant in iron and contains phytochemicals. We aimed at evaluating the effect of an iron-rice (IR) hydrolysate drink (100 mL/serving) on neurological function, red cell indices and iron status in elders. Healthy elderly subjects were divided into three non-anemic groups and one anemic group. The non-anemic groups consumed one WR (2 mg iron/serving) and two IR drinks (15 and 27 mg iron/serving) (groups A, B and D, respectively), while the anemic group consumed one IR drink (15 mg iron serving) (group C) every day for 30 days. There were no significant differences in the MMSE Thai 2002 and PHQ9 test scores for members of all groups, while the nutrition scores and body weight values of group D subjects were significantly increased. Hemoglobin (Hb) and mean corpuscular hemoglobin concentrations increased significantly only in group C. Serum iron and transferrin saturation levels tended to increase in group A, while these levels were decreased in members of group C. Serum antioxidant activity levels were increased in all groups, and were highest in group C. Thus, consumption of an IR drink for 15 days functioned to increase Hb and antioxidant capacity levels in anemic elders.

Chemical Analysis, Toxicity Study, and Free-Radical Scavenging and Iron-Binding Assays Involving Coffee (Coffea arabica) Extracts.

We aimed to analyze the chemical compositions in Arabica coffee bean extracts, assess the relevant antioxidant and iron-chelating activities in coffee extracts and instant coffee, and evaluate the toxicity in roasted coffee. Coffee beans were extracted using boiling, drip-filtered and espresso brewing methods. Certain phenolics were investigated including trigonelline, caffeic acid and their derivatives, gallic acid, epicatechin, chlorogenic acid (CGA) and their derivatives, p-coumaroylquinic acid, p-coumaroyl glucoside, the rutin and syringic acid that exist in green and roasted coffee extracts, along with dimethoxycinnamic acid, caffeoylarbutin and cymaroside that may be present in green coffee bean extracts. Different phytochemicals were also detected in all of the coffee extracts. Roasted coffee extracts and instant coffees exhibited free-radical scavenging properties in a dose-dependent manner, for which drip coffee was observed to be the most effective (p < 0.05). All coffee extracts, instant coffee varieties and CGA could effectively bind ferric ion in a concentration-dependent manner resulting in an iron-bound complex. Roasted coffee extracts were neither toxic to normal mononuclear cells nor breast cancer cells. The findings indicate that phenolics, particularly CGA, could effectively contribute to the iron-chelating and free-radical scavenging properties observed in coffee brews. Thus, coffee may possess high pharmacological value and could be utilized as a health beverage.

Gut leakage enhances sepsis susceptibility in iron-overloaded ß-thalassemia mice through macrophage hyperinflammatory responses.

Iron overload induces intestinal-permeability defect (gut leakage), and gut translocation of organismal molecules might enhance systemic inflammation and sepsis severity in patients with thalassemia (Thal). Hence, iron administration in Hbbth3/+ mice, heterozygous ß-globin-deficient Thal mice, was explored. Oral iron administration induced more severe secondary hemochromatosis and gut leakage in Thal mice compared with wild-type (WT) mice. Gut leakage was determined by 1) FITC-dextran assay, 2) spontaneous serum elevation of endotoxin (LPS) and (1→3)-ß-d-glucan (BG), molecular structures of gut-organisms, and 3) reduction of tight-junction molecules with increased enterocyte apoptosis (activated caspase-3) by immunofluorescent staining. Iron overload also enhanced serum cytokines and increased Bacteroides spp. (gram-negative bacteria) in feces as analyzed by microbiome analysis. LPS injection in iron-overloaded Thal mice produced higher mortality and prominent cytokine responses. Additionally, stimulation with LPS plus iron in macrophage from Thal mice induced higher cytokines production with lower ß-globin gene expression compared with WT. Furthermore, possible gut leakage as determined by elevated LPS or BG (>60 pg/mL) in serum without systemic infection was demonstrated in 18 out of 41 patients with ß-thalassemia major. Finally, enhanced LPS-induced cytokine responses of mononuclear cells from these patients compared with cells from healthy volunteers were demonstrated. In conclusion, oral iron administration in Thal mice induced more severe gut leakage and increased fecal gram-negative bacteria, resulting in higher levels of endotoxemia and serum inflammatory cytokines compared with WT. Preexisting hyperinflammatory cytokines in iron-overloaded Thal enhanced susceptibility toward infection.NEW & NOTEWORTHY Although the impact of iron accumulation in several organs of patients with thalassemia is well known, the adverse effect of iron accumulation in gut is not frequently mentioned. Here, we demonstrated iron-induced gut-permeability defect, impact of organismal molecules from gut translocation of, and macrophage functional defect upon the increased sepsis susceptibility in thalassemia mice.

Increased ferritin levels in non-transfusion-dependent ß°-thalassaemia/HbE are associated with reduced CXCR2 expression and neutrophil migration.

Severe bacterial infection is a major complication causing morbidity and mortality in ß-thalassaemia/HbE patients. Innate immunity constitutes the first line of defence against bacterial infection. This study aimed to comprehensively investigate the innate immune phenotype and function related to factors predisposing to infection in non-transfusion-dependent (NTD) ß°-thalassaemia/HbE patients. Twenty-six patients and 17 healthy subjects were recruited to determine complement activity (C3, C4, mannose-binding lectin and CH50) and surface receptor expression including markers of phagocytosis (CD11b, CD16 and C3bR), inflammation (C5aR) and migration (CD11b, CXCR1 and CXCR2) on neutrophils and monocytes. In addition, phagocytosis and oxidative burst activity of neutrophils and monocytes against Escherichia coli and neutrophil migration were examined. Decreased C3 and surface expression of CD11b and C3bR on neutrophils were found in patients. However, phagocytosis of neutrophils in patients was still in the normal range. Interestingly, patients displayed a significant reduction of surface expression of CXCR2 [1705 ± 217 mean fluorescent intensity (MFI)] on neutrophils, leading to impaired neutrophil migration (9·2 ± 7·7%) when compared to neutrophils from healthy subjects (2261 ± 627 MFI and 27·8 ± 9% respectively). Moreover, surface expression of CXCR2 on neutrophils was associated with splenectomy status, serum ferritin and haemoglobin levels. Therefore, impaired neutrophil migration could contribute to the increased susceptibility to infection seen in NTD ß°-thalassaemia/HbE patients.

UNC0638 induces high levels of fetal hemoglobin expression in ß-thalassemia/HbE erythroid progenitor cells.

Increased expression of fetal hemoglobin (HbF) improves the clinical severity of ß-thalassemia patients. EHMT1/2 histone methyltransferases are epigenetic modifying enzymes that are responsible for catalyzing addition of the repressive histone mark H3K9me2 at silenced genes, including the γ-globin genes. UNC0638, a chemical inhibitor of EHMT1/2, has been shown to induce HbF expression in human erythroid progenitor cell cultures. Here, we report the HbF-inducing activity of UNC0638 in erythroid progenitor cells from ß-thalassemia/HbE patients. UNC0638 treatment led to significant increases in γ-globin mRNA, HbF expression, and HbF-containing cells in the absence of significant cytotoxicity. Moreover, UNC0638 showed additive effects on HbF induction in combination with the immunomodulatory drug pomalidomide and the DNMT1 inhibitor decitabine. These studies provide a scientific proof of concept that a small molecule targeting EHMT1/2 epigenetic enzymes, used alone or in combination with pomalidomide or decitabine, is a potential therapeutic approach for HbF induction. Further development of structural analogs of UNC0638 with similar biological effects but improved pharmacokinetic properties may lead to promising therapies and possible clinical application for the treatment of ß-thalassemia.

Visual genotyping of thalassemia by using pyrrolidinyl peptide nucleic acid probes immobilized on carboxymethylcellulose-modified paper and enzyme-induced pigmentation.

A simple probe pair was designed for the detection of hemoglobin E (HbE) genotype, a single-point mutation that leads to abnormal red blood cells commonly found in South East Asia. The key to differentiation is the use of a conformationally constrained peptide nucleic acid (PNA) that was immobilized on carboxymethylcellulose-modified paper. This was then used for target DNA binding and visualization by an enzyme-catalyzed pigmentation. The biotinylated target DNA bound to the immobilized probe was visually detected via alkaline phosphatase-linked streptavidin. This enzyme conjugate catalyzed the dephosphorylation of the substrate 5-bromo-4-chloro-3-indolyl phosphate, leading to a series of reactions that generate an intense, dark blue pigment. The test was validated with 100 DNA samples, which shows good discrimination among different genotypes (normal, HbE, and heterozygous) with 100% accuracy when optimal conditions of analysis were applied. The method does not require temperature control and can be performed at ambient temperature. This is an attractive feature for diagnosis in primary care, which accounts for a large part of affected population. Graphical abstract Schematic representation of a paper-based sensor for the detection of the gene Hemoglobin E. The interaction between an immobilized peptide nucleic acid and a DNA target leads to enzymatic pigmentation, allowing simple visual readout with up to 100% accuracy.

Micromapping of Thalassemia and Hemoglobinopathies Among Laos, Khmer, Suay and Yer Ethnic Groups Residing in Lower Northeastern Thailand.

Northeastern (NE) Thailand is one of the areas with a prevalence of thalassemias and hemoglobinopathies. Data on the prevalence of the diseases in minorities in the region has been limited. This study aimed to survey the thalassemias and hemoglobinopathies that take into account ethnicity. Four ethnic groups, including Laos (n = 162), Khmer (n = 145), Suay (n = 134), and Yer (n = 101) inhabiting the lower region of NE Thailand, were selected to represent the study populations. The results demonstrated that an extremely high prevalence of Hb E (HBB: c.79G>A) (>50.0%) was observed in the Khmer, Suay and Yer ethnic groups. The highest prevalence of α+-thalassemia (α+-thal) [-α3.7 (rightward)] deletion was found in the Khmer ethnic group (48.28%). The -α4.2 (leftward) deletion (α+-thal) was restricted to the Yer ethnic group. Yer and Suay had a high incidence of Hb Constant Spring (Hb CS; HBA2: c.427T>C) as well as Hb Paksé (HBA2: c.429A>T). As the prevalence of α0-thalassemia (α0-thal) is relatively high in Suay (7.46%), couples who are members of Suay ethnic population should be urged to undergo hematological screening before planning a pregnancy to control the Hb Bart's hydrops fetalis. Micromapping of thalassemias and hemoglobinopathies herein described will be helpful in genetic counseling and public education campaigns, which should be carried out in appropriate languages, with exhibitions at the village levels. This information will be of benefit for the long-term effort to reduce the burden of severe thalassemia disease in the region.

Genotype-Phenotype Correlation of ß-Thalassemia in Malaysian Population: Toward Effective Genetic Counseling.

Effective prevention of ß-thalassemia (ß-thal) requires strategies to detect at-risk couples. This is the first study attempting to assess the prevalence of silent ß-thal carriers in the Malaysian population. Hematological and clinical parameters were evaluated in healthy blood donors and patients with ß-thal trait, Hb E (HBB: c.79G>A)/ß-thal and ß-thal major (ß-TM). ß-Globin gene sequencing was carried out for 52 healthy blood donors, 48 patients with Hb E/ß-thal, 34 patients with ß-TM and 38 patients with ß-thal trait. The prevalence of silent ß-thal carrier phenotypes found in 25.0% of healthy Malaysian blood donors indicates the need for clinician's awareness of this type in evaluating ß-thal in Malaysia. Patients with ß-TM present at a significantly younger age at initial diagnosis and require more blood transfusions compared to those with Hb E/ß-thal. The time at which genomic DNA was extracted after blood collection, particularly from patients with ß-TM and Hb E/ß-thal, was found to be an important determinant of the quality of the results of the ß-globin sequencing. Public education and communication campaigns are recommended as apparently healthy individuals have few or no symptoms and normal or borderline hematological parameters. ß-Globin gene mutation characterization and screening for silent ß-thal carriers in regions prevalent with ß-thal are recommended to develop more effective genetic counseling and management of ß-thal.

Anti-Platelet Aggregation and Anti-Cyclooxygenase Activities for a Range of Coffee Extracts (Coffea arabica).

Coffee is rich in caffeine (CF), chlorogenic acid (CGA) and phenolics. Differing types of coffee beverages and brewing procedures may result in differences in total phenolic contents (TPC) and biological activities. Inflammation and increases of platelet activation and aggregation can lead to thrombosis. We focused on determining the chemical composition, antioxidant activity and inhibitory effects on agonist-induced platelet aggregation and cyclooxygenase (COX) of coffee beverages in relation to their preparation method. We prepared instant coffee and brewed coffee beverages using drip, espresso, and boiling techniques. Coffee extracts were assayed for their CF and CGA contents using HPLC, TPC using colorimetry, platelet aggregation with an aggregometer, and COX activity using ELISA. The findings have shown all coffee extracts, except the decaffeinated types, contained nearly equal amounts of CF, CGA, and TPC. Inhibitory effects of coffee extracts on platelet aggregation differed depending on the activation pathways induced by different agonists. All espresso, drip and boiled coffee extracts caused dose dependent inhibition of platelet aggregation induced by ADP, collagen, epinephrine, and arachidonic acid (ARA). The most marked inhibition was seen at low doses of collagen or ARA. Espresso and drip extracts inhibited collagen-induced platelet aggregation more than purified caffeine or CGA. Espresso, boiled and drip coffee extracts were also a more potent inhibitors of COX-1 and COX-2 than purified caffeine or CGA. We conclude that inhibition of platelet aggregation and COX-1 and COX-2 may contribute to anti-platelet and anti-inflammatory effects of espresso and drip coffee extracts.

Immunostick Test for Detecting ζ-Globin Chains and Screening of the Southeast Asian α-Thalassemia 1 Deletion.

BACKGROUND: Couples who carry α-thalassemia-1 deletion are at 25% risk of having a fetus with hemoglobin Bart's hydrops fetalis. Southeast Asian deletion (--(SEA)) is the most common type of α-thalassemia 1 among Southeast Asian populations. Thus, identification of the (--(SEA)) α-thalassemia 1 carrier is necessary for controlling severe α-thalassemia in Southeast Asian countries. RESULTS: Using our generated anti ζ-globin chain monoclonal antibodies (mAbs) clones PL2 and PL3, a simple immunostick test for detecting ζ-globin chain presence in whole blood lysates was developed. The procedure of the developed immunostick test was as follows. The immunostick paddles were coated with 50 µg/mL of mAb PL2 as capture mAb, or other control antibodies. The coated immunostick was dipped into cocktail containing tested hemolysate at dilution of 1:500, 0.25 µg/mL biotin-labeled mAb PL3 and horseradish peroxidase-conjugated streptavidin at dilution of 1:1000. The immunostick was then dipped in precipitating substrate and the presence of ζ-globin chain in the tested sample was observed by the naked eye. Upon validation of the developed immunostick test with various types of thalassemia and normal subjects, 100% sensitivity and 82% specificity for detection of the (--(SEA)) α-thalassemia-1 carriers were achieved. The mAb pre-coated immunostick can be stored at room temperature for at least 20 weeks. CONCLUSION: In this study, a novel simple immunostick test for the screening of (--(SEA)) α-thalassemia 1 carriers was presented. The developed immunostick test, within a single test, contains both positive and negative internal procedural controls.

Reduced PU.1 expression underlies aberrant neutrophil maturation and function in ß-thalassemia mice and patients.

ß-Thalassemia is associated with several abnormalities of the innate immune system. Neutrophils in particular are defective, predisposing patients to life-threatening bacterial infections. The molecular and cellular mechanisms involved in impaired neutrophil function remain incompletely defined. We used the Hbbth3/+ ß-thalassemia mouse and hemoglobin E (HbE)/ß-thalassemia patients to investigate dysregulated neutrophil activity. Mature neutrophils from Hbbth3/+ mice displayed a significant reduction in chemotaxis, opsonophagocytosis, and production of reactive oxygen species, closely mimicking the defective immune functions observed in ß-thalassemia patients. In Hbbth3/+ mice, the expression of neutrophil CXCR2, CD11b, and reduced NAD phosphate oxidase components (p22phox, p67phox, and gp91phox) were significantly reduced. Morphological analysis of Hbbth3/+ neutrophils showed that a large percentage of mature phenotype neutrophils (Ly6GhiLy6Clow) appeared as band form cells, and a striking expansion of immature (Ly6GlowLy6Clow) hyposegmented neutrophils, consisting mainly of myelocytes and metamyelocytes, was noted. Intriguingly, expression of an essential mediator of neutrophil terminal differentiation, the ets transcription factor PU.1, was significantly decreased in Hbbth3/+ neutrophils. In addition, in vivo infection with Streptococcus pneumoniae failed to induce PU.1 expression or upregulate neutrophil effector functions in Hbbth3/+ mice. Similar changes to neutrophil morphology and PU.1 expression were observed in splenectomized and nonsplenectomized HbE/ß-thalassemia patients. This study provides a mechanistic insight into defective neutrophil maturation in ß-thalassemia patients, which contributes to deficiencies in neutrophil effector functions.