BCL11A-targeted γ-globin gene induction by triterpenoid glycosides of Fagonia indica: A preclinical scientific validation of indigenous herb for the treatment of ß-hemoglobinopathies.

Pharmacological induction of fetal hemoglobin has proven to be a promising therapeutic intervention in ß-hemoglobinopathies by reducing the globin chain imbalance and inhibiting sickle cell polymerization. Fagonia indica has shown therapeutic relevance to ß-thalassemia. Therefore, we study the ethnopharmacological potential of Fagonia indica and its biomarker compounds for their HbF induction ability for the treatment of ß-thalassemia. Here, we identify, compound 8 (triterpenoid glycosides) of F. indica. as a prominent HbF inducer in-vitro and in-vivo. Compound 8 showed potent erythroid differentiation, enhanced cellular proliferation, ample accumulation of total hemoglobin, and a strong notion of γ-globin gene expression in K562 cultures. Compound 8 treatment also revealed strong induction of erythroid differentiation and fetal hemoglobin mRNA and protein in adult erythroid precursor cells. This induction was associated with simultaneous downregulation of BCL11A and SOX6, and overexpression of the GATA-1 gene, suggesting a compound 8-mediated partial mechanism involved in the reactivation of fetal-like globin genes. The in vivo study with compound 8 (10 mg/kg) in ß-YAC mice resulted in significant HbF synthesis demonstrated by the enhanced level of F-cells (84.14 %) and an 8.85-fold increase in the γ-globin gene. Overall, the study identifies compound 8 as a new HbF-inducing entity and provides an early "proof-of-concept" to enable the initiation of preclinical and clinical studies in the development of this HbF-inducing agent for ß-thalassemia.

Recent perspectives of pediatric ß-thalassemia.

Beta-thalassemia is a potentially lethal hereditary anemia, caused by reduced or absent expression of HBB polypeptide chains of adult hemoglobin (HbA: α2ß2). Current curative treatments options are limited to few patients, while alternative, chronic palliative therapy consisting of frequent transfusions coupled with iron chelation therapy, are costly. The above treatments also affect quality of life of patients. A search was conducted in the electronic databases like Medline, PubMed, etc. for screening studies reporting various aspects including gene therapy, prevention strategies, blood, transfusion and chelation therapy for the management of ß-thalassemia. Increased levels of fetal hemoglobin (HbF: α2γ2) were shown to lessen the severity of ß-thalassemia, highlighting the therapeutic potential of a gene-therapy-mediated increase in HBG1 and HBG2 (HBG) expression. The primary outcome of most of the above studies was the efficient management of ß-thalassemia, without any major complication. So, the present review is focused on the recent perspectives in the management of ß-thalassemia including combinatorial gene therapy for ß-thalassemia.

Association of plasma homocysteine level with vaso-occlusive crisis in sickle cell anemia patients of Odisha, India.

Vascular complications of sickle cell anemia (SCA) are influenced by many factors. Elevated plasma homocysteine (Hcy) is supposed to be an independent risk factor and is either genetic or nutritional origin. The present study evaluated the plasma Hcy level, MTHFR C677T gene polymorphism, effect of folic acid (FA) supplementation' and hemato-biochemical parameters in SCA and their effect on the vaso-occlusive crisis (VOC) in SCA patients of an Asian-Indian haplotype population. One hundred twenty cases of SCA (HbSS) and 50 controls with normal hemoglobin(HbAA) were studied. It was found that the plasma Hcy level is significantly higher (p < 0.0001) in patients with SCA (22.41 ± 7.8 µmol/L) compared to controls (13.2 ± 4.4 µmol/L). Moreover, patients without FA supplementation had a significantly (p < 0.001) higher Hcy level (27 ± 7 µmol/L) compared to those with supplementation (17.75 ± 5.7 µmol/L). Turkey-Kramer multiple comparison tests show that there is a significant difference (p < 0.05) in HbF percent, hemoglobin (Hb), platelet count, serum bilirubin (direct:Bil-D and total:Bil-T), aspartate transaminase (AST), lactate dehydrogenase (LDH), and plasma Hcy levels between mild and severe VOC. Between moderate VOC and severe VOC, there was a significant difference (p < 0.05) in HbF%, Bil-D, AST, Hcy. Pearson correlation revealed that plasma Hcy had a significantly (p < 0.05) positive correlation with AST, serum bilirubin (indirect and total), LDH, jaundice, stroke, VOC per year, and hospitalization per year whereas it was inversely correlated with HbF percentage, Hb level, and FA treatment. In the study population, increased plasma Hcy level, hemolysis, and platelet activation were found to influence VOC in SCA.

Sickle cell maculopathy: Identification of systemic risk factors, and microstructural analysis of individual retinal layers of the macula.

PURPOSE: To identify systemic risk factors for sickle cell maculopathy, and to analyze the microstructure of the macula of Sickle Cell Disease (SCD) patients by using automated segmentation of individual retinal layers. METHODS: Thirty consecutive patients with SCD and 30 matched controls underwent spectral-domain optical coherence tomography (SD-OCT) and automated thickness measurement for each retinal layer; thicknesses for SCD patients were then compared to normal controls. Demographic data, systemic data, and lab results were collected for each SCD patient; multivariate logistic regression analysis was used to identify potential risk factors for sickle cell maculopathy. RESULTS: Ongoing chelation treatment (p = 0.0187) was the most predictive factor for the presence of sickle cell maculopathy; the odds were 94.2% lower when chelation was present. HbF level tended to influence sickle cell maculopathy (p = 0.0775); the odds decreased by 12.9% when HbF increased by 1%. Sickle cell maculopathy was detected in 43% of SCD patients as patchy areas of retinal thinning on SD-OCT thickness map, mostly located temporally to the macula, especially in eyes with more advanced forms of sickle cell retinopathy (p = 0.003). In comparison to controls, SCD patients had a subtle thinning of the overall macula and temporal retina compared to controls (most p<0.0001), involving inner and outer retinal layers. Thickening of the retinal pigment epithelium was also detected in SCD eyes (p<0.0001). CONCLUSIONS: Chronic chelation therapy and, potentially, high levels of HbF are possible protective factors for the presence of sickle cell maculopathy, especially for patients with more advanced forms of sickle cell retinopathy. A subtle thinning of the overall macula occurs in SCD patients and involves multiple retinal layers, suggesting that ischemic vasculopathy may happen in both superficial and deep capillary plexi. Thinning of the outer retinal layers suggests that an ischemic insult of the choriocapillaris may also occur in SCD patients.

A1M Ameliorates Preeclampsia-Like Symptoms in Placenta and Kidney Induced by Cell-Free Fetal Hemoglobin in Rabbit.

Preeclampsia is one of the most serious pregnancy-related diseases and clinically manifests as hypertension and proteinuria after 20 gestational weeks. The worldwide prevalence is 3-8% of pregnancies, making it the most common cause of maternal and fetal morbidity and mortality. Preeclampsia lacks an effective therapy, and the only "cure" is delivery. We have previously shown that increased synthesis and accumulation of cell-free fetal hemoglobin (HbF) in the placenta is important in the pathophysiology of preeclampsia. Extracellular hemoglobin (Hb) and its metabolites induce oxidative stress, which may lead to acute renal failure and vascular dysfunction seen in preeclampsia. The human endogenous protein, α1-microglobulin (A1M), removes cell-free heme-groups and induces natural tissue repair mechanisms. Exogenously administered A1M has been shown to alleviate the effects of Hb-induced oxidative stress in rat kidneys. Here we attempted to establish an animal model mimicking the human symptoms at stage two of preeclampsia by administering species-specific cell-free HbF starting mid-gestation until term, and evaluated the therapeutic effect of A1M on the induced symptoms. Female pregnant rabbits received HbF infusions i.v. with or without A1M every second day from gestational day 20. The HbF-infused animals developed proteinuria and a significantly increased glomerular sieving coefficient in kidney that was ameliorated by co-administration of A1M. Transmission electron microscopy analysis of kidney and placenta showed both intracellular and extracellular tissue damages after HbF-treatment, while A1M co-administration resulted in a significant reduction of the structural and cellular changes. Neither of the HbF-treated animals displayed any changes in blood pressure during pregnancy. In conclusion, infusion of cell-free HbF in the pregnant rabbits induced tissue damage and organ failure similar to those seen in preeclampsia, and was restored by co-administration of A1M. This study provides preclinical evidence supporting further examination of A1M as a potential new therapy for preeclampsia.

Effects of iron supplements and perinatal factors on fetal hemoglobin disappearance in LBW infants.

BACKGROUND: The homeostatic mechanisms of iron metabolism and erythropoiesis in infants are unclear. Infants synthesize both fetal hemoglobin (HbF) and adult hemoglobin (HbA), and it is not known how the hemoglobin switch is regulated. We hypothesized that iron supplements to infants affect the disappearance of HbF. METHODS: We randomized 285 low-birth-weight infants (2,000-2,500 g) into three intervention groups receiving 0, 1, or 2 mg/kg/d of iron supplements from 6 wk to 6 mo of age. In the present secondary analysis, we analyzed iron status, total hemoglobin (Hb), and HbF fraction at 6 wk, 12 wk, and at 6 mo and calculated absolute levels of HbF. RESULTS: We observed dose-dependent increased levels of Hb in iron-supplemented groups at 6 mo of age. However, for absolute HbF concentration, there was no similar effect of intervention. Mean (SD) HbF was 81.2 (16.8), 37.0 (13.8), and 8.1 (5.6) g/l at 6 wk, 12 wk, and 6 mo, respectively, similar in all groups. In linear regression analyses, postconceptional age turned out as the major predictor of HbF, independent of gestational age at birth. CONCLUSION: Our hypothesis was rejected. Instead, we confirmed a close correlation to postconceptional age, supporting a genetically programmed switch, insensitive to most environmental factors including birth.

Alterations of the arginine metabolome in sickle cell disease: a growing rationale for arginine therapy.

Low global arginine bioavailability (GAB) is associated with numerous complications of SCD including early mortality. Mechanisms of arginine dysregulation involve a complex paradigm of excess activity of the arginine-consuming enzyme arginase, elevated levels of asymmetric dimethylarginine, altered intracellular arginine transport, and nitric oxide synthase dysfunction. Restoration of GAB through exogenous supplementation is therefore, a promising therapeutic target. Studies of arginine therapy demonstrate efficacy in treating patients with leg ulcers, pulmonary hypertension risk, and pain. Co-administration with hydroxyurea increases levels of nitrite and fetal hemoglobin. Addressing the alterations in the arginine metabolome may result in new strategies for treatment of SCD.

A new unstable variant of the fetal hemoglobin HBG2 gene: Hb F-Turritana [(G) γ64(E8)Gly→Asp, HBG2:c.194G>A] found in cis to the Hb F-Sardinia gene [(A) γ(E19)Ile→Thr, HBG1:c.227T>C].

A new variant of the fetal hemoglobin (Hb) was observed in a newborn baby subjected to phototherapy due to jaundice, by means of electrophoretic and chromatographic techniques. The variant Hb resulted unstable by the isopropanol stability test. After HBG2 gene sequencing, the G to A transversion at codon 64, position eight of the E helix, was found, which corresponds to the Asp for Gly amino acid substitution. The new variant was called Hb F-Turritana [(G) γ64(E8)Gly→Asp, HBG2:c.194G>A]. Incoming aspartic acid residue, bulky and negatively charged, may be responsible for alteration of the heme pocket steric configuration and for instability. The new abnormal HBG2 gene was found to be associated in cis with the mutated HBG1 gene, which characterizes the Hb F-Sardinia [(A) γ (E19)Ile→Thr, HBG1:c.227T>C] variant.

Hematologic, biochemical, and cardiopulmonary effects of L-arginine supplementation or phosphodiesterase 5 inhibition in patients with sickle cell disease who are on hydroxyurea therapy.

OBJECTIVES: Fetal hemoglobin (HbF) induction involves NO-cGMP signaling pathways. L-arginine, an NO precursor, and the phosphodiesterase (PDE) 5 inhibitor sildenafil, which potentiates cGMP, were studied in adults with sickle cell disease (SCD) who were stably on HU. METHODS: Twenty four courses of L-arginine (0.1-0.2 g/kg divided TID) or sildenafil (25-100 mg TID), assigned based on gender due to concerns about sildenafil-related priapism, were successfully completed. Biochemical assays, pulmonary pressures, and cardiopulmonary exercise capacity are reported from patients in whom serial values are available. Hematologic responses are reported in 14 subjects with HbSS who had stable baseline HbF levels. RESULTS: L-arginine increased plasma arginine and ornithine, but not citrulline, suggesting diversion by plasma arginase from NO, and citrulline, generation. Glutathione increased only in patients on L-arginine. Sildenafil increased plasma cGMP and citrulline, but not other amino acids. Pulmonary pressures and 6-min walk distances improved only in patients on sildenafil. In subjects with stable baseline HbF levels, HbF levels changed little from a normalized baseline on l-arginine, decreasing by 2.9 +/- 16.1%, n = 6; P = n.s., but increased on sildenafil, by 7.5 +/- 11.7%, n = 8, P < 0.05. Absolute reticulocyte counts initially decreased in patients on sildenafil. CONCLUSIONS: L-arginine, at doses that increase plasma arginine levels, altered redox potential in red cells. The lack of clinically detectable efficacy of L-arginine may be due to increased arginine metabolism in SCD patients. In vivo augmentation of the cyclic nucleotide pathway by PDE inhibition may induce HbF slightly, but strikingly improves hemodynamic and functional status in SCD.

The pharmacotherapy of sickle cell disease.

Sickle cell disease (SCD) is characterized by acute and chronic complications, which can affect almost any organ system in the body. Recently, there has been remarkable progress in the pharmacotherapy of SCD, and therapeutic options have been greatly expanded beyond red blood cell transfusion and hematopoietic stem cell transplantation. The most successful approach has been the stimulation of Hb F production through drugs such as hydroxycarbamide, which reduces the frequency of vaso-occlusive pain events and is the only therapeutic agent at present approved by the US Food and Drug Administration for SCD. Hydroxycarbamide is still greatly underused, but its indications are being broadened through a number of Phase II and III clinical trials. Other Hb F-promoting agents such as decitabine and butyrate have been studied less extensively. Another approach is the use of agents that reduce red cell dehydration through blockage of ion channels in the red cell membrane. Also under investigation is manipulation of nitric oxide (NO) metabolism to counteract the effects of chronic hemolysis. As yet, unproven drugs include agents that target coagulation, inflammation and adhesion to vascular endothelium. In the near future we expect to see exploration of the efficacy of drug combinations and an expansion in the use of the proven therapeutic, hydroxycarbamide.

Modern treatment of thalassaemia intermedia.

The term thalassaemia intermedia includes a large spectrum of conditions of varying severity. Blood transfusion and chelation are necessary in some patients, especially during childhood, in order to promote growth and prevent bone deformities. Alloimunisation, however, is frequent and can be difficult to control. Splenectomy is usually needed at some time because of hypersplenism and mechanical encumbrance. Reactivation of HbF is possible only in a small proportion of patients: hydroxycarbamide (also known as hydroxyurea) appears to be the most effective drug for this purpose. Antioxidant agents, although theoretically useful, do not improve haemoglobin levels. Stem cell transplantation is an option limited to the severe forms. Gene therapy and other molecular approaches are subjects of intense study. Numerous complications, including pulmonary hypertension, thrombotic events, pseudoxanthoma elasticum and osteoporosis, have been described and all contribute to complicate the treatment of a disease that represents a significant burden for the patients and their families.

[Clinical observation on effects of Yisui Shengxue Granule in treating 156 patients with beta-thalassemia].

OBJECTIVE: To study the clinical effect and security of Yisui Shengxue Granule (YSG) in treating beta-thalassemia. METHODS: One hundred and fifty-five patients with beta-thalassemia from high-incidence area in Guangxi Zhuang Autonomous Region were treated with YSG for 3 months. Clinical symptoms and levels of hemoglobin (Hb), red blood cell (RBC), reticulocyte (Ret) and hemoglobin F (HbF) were observed before and after treatment, and the adverse reaction occurred in the therapeutic course was observed as well. A 3-6 months follow-up study was performed after withdrawal of YSG. RESULTS: Levels of Hb, RBC, Ret and HbF obviously elevated, and clinical symptoms markedly improved in patients after treatment since the 1st month to the 3rd month (all P < 0.01). Dynamical observation showed that the improvement of symptoms was in accordance with the elevation of hemorrheological indexes. The treatment was effective in 145 patients and ineffective in 11, and the total effective rate being 92.9%, without any adverse reaction occurred. Follow-up study showed the therapeutic effect could be sustained for 3 to 4 months. CONCLUSION: YSG has evident effect on alpha-thalassemia without obvious adverse reaction, which provides a definite basis for treatment of beta-thalassemia with TCM.

Neonatal hyperbilirubinemia in Japanese neonates: analysis of the heme oxygenase-1 gene and fetal hemoglobin composition in cord blood.

Neonatal hyperbilirubinemia is frequent and severe in Japanese infants. Although the G71R mutation of the bilirubin uridine diphosphate-glucuronosyltransferase gene is associated with severe neonatal hyperbilirubinemia in this population, it accounts for only half of the neonates with severe hyperbilirubinemia. It was suggested that increased bilirubin production would also be associated with severe neonatal hyperbilirubinemia in Japanese infants. To elucidate the genetic factors causing severe hyperbilirubinemia in these patients, we studied two notable factors associated with bilirubin production: heme oxygenase-1, a key enzyme of heme metabolism, and fetal Hb composition, a factor possibly associated with heme load in neonates. We first determined the sequences of promoter and all coding regions of the heme oxygenase-1 gene in Japanese neonates who had undergone phototherapy, but found no mutation except for the polymorphic (GT)n repeats in the promoter region. These repeats modulate the transcription of the heme oxygenase-1 gene, and the longer repeat sequences are known to reduce the transcription. We detected a significant difference in the allele frequencies of each number of (GT)n repeats between Japanese and German populations. However, we could not find a relation between those polymorphisms and neonatal hyperbilirubinemia. We next analyzed the state of Hb switching of the gamma- to beta-globin chain and the phenotype of gamma-globin chain isoforms in cord blood. We found no relation between fetal Hb composition and neonatal hyperbilirubinemia. Further studies are required to elucidate genetic or environmental factors in neonatal hyperbilirubinemia in Japanese infants.

Sudden infant death syndrome: hypothalamic failure to sense elevated blood pyrogens.

Sudden infant death syndrome (SIDS) is frequently associated with a mild infection, the incidence peaking during the third month of life. We hypothesize that the neonatal immaturity of both the acute febrile response and hypothalamus promote neonatal protection from SIDS. Vagal afferents modify the febrile response. Vagotomized rodents displayed a loss of febrile responsiveness in a 'non-sensing' brain. The failure of a 'non- sensing' brain to react to elevated blood pyrogens leads to failure of the febrile response and to a shock-like state. SIDS infants may appear well yet, within hours of this observation, may be found dead. There is a mismatch between the acute febrile response and hypothalamic hypoactivation. The discrepancy increase wtih development. There is an elevated cytokine response in endothelial cells which induces nitric oxide (NO) production and retarded development of the hypothalamus. Cigarette smoke also induces NO production and retards hypothalamic development by augmented apoptosis. Zinc inhibits this effect in mouse thymocytes. Fetal haemoglobin (HbF) induces hypoxia which is a stimulator of the immune response, while vasodilator gases (carbon monoxide (CO), NO) reduce hypothalamic function. The hypothalamic failure to sense elevated blood pyrogens induces toxic shock--a feature of SIDS.