Absolute Reticulocyte Count Acts as a Surrogate for Fetal Hemoglobin in Infants and Children with Sickle Cell Anemia

Hemoglobin switching is largely complete in humans by six months of age. Among infants with sickle cell anemia (HbSS, SCA), reticulocytosis begins early in life as fetal hemoglobin (HbF) is replaced by sickle hemoglobin (HbS). The objective of this study was to determine if absolute reticulocyte count (ARC) is related to HbF levels in a cohort of pediatric SCA patients. A convenience sample of 106 children with SCA between the ages of 1 month and 20 years who were not receiving hydroxyurea or monthly blood transfusions were enrolled in this observational study. Hematologic data, including ARC and HbF levels, were measured at steady state. F-cells were enumerated by flow cytometry. Initial studies compared infants with ARC greater than or equal to 200 K/μL (ARC ≥ 200) based upon the previously reported utility of this threshold as a predictive marker for SCA severity. Mean HbF and F-cell levels were significantly lower in the ARC ≥ 200 group when compared to the ARC < 200 group. Both HbF and F-cell percentages were negatively correlated to ARC in infants and in children between the ages of 1 and 9 years. However, the inverse relationship was lost after the age of 10 years. Overall, decreased expression and distribution of HbF during childhood SCA is well-correlated with increased reticulocyte production and release into the peripheral blood. As such, these data further support the clinical use of reticulocyte enumeration as a disease severity biomarker for childhood sickle cell anemia.     

Induction of foetal haemoglobin synthesis in erythroid progenitor stem cells: mediated by water‐soluble components of Terminalia catappa

Current novel therapeutic agents for the treatment of sickle cell anaemia (SCA) focus on increasing foetal haemoglobin (HbF) levels in SCA patients. Unfortunately, the only approved HbF‐inducing agent, hydroxyurea, has long‐term unpredictable side effects. Studies have shown the potential of plant compounds to modulate HbF synthesis in primary erythroid progenitor stem cells. We isolated a novel HbF‐inducing Terminalia catappa distilled water active fraction (TCDWF) from Terminalia catappa leaves that induced the commitment of erythroid progenitor stem cells to the erythroid lineage and relatively higher HbF synthesis of 9.2‐ and 6.8‐fold increases in both erythropoietin (EPO)‐independent and EPO‐dependent progenitor stem cells respectively. TCDWF was differentially cytotoxic to EPO‐dependent and EPO‐independent erythroid progenitor stem cell cultures as revealed by lactate dehydrogenase release from the cells. TCDWF demonstrated a protective effect on EPO‐dependent and not EPO‐independent progenitor cells. TCDWF induced a modest increase in caspase 3 activity in EPO‐independent erythroid progenitor stem cell cultures compared with a significantly higher (P?0.05) caspase 3 activity in EPO‐dependent ones. The results demonstrate that TCDWF may hold promising HbF‐inducing compounds, which work synergistically, and suggest a dual modulatory effect on erythropoiesis inherent in this active fraction. Copyright © 2014 John Wiley & Sons, Ltd.     

Mechanisms of human immunodeficiency virus type 1 inhibition by hydroxyurea

Virus life cycles depend on cellular factors. Therefore, targeting cellular in combination with viral enzymes could be an effective control in virus replication. In contrast to viral proteins, cellular proteins are not prone to mutations; therefore, viral escape is not expected from drugs inhibiting cellular factors. Hydroxyurea inhibits the cellular enzyme ribonucleotide reductase, thus reducing DNA synthesis. Furthermore, this drug potentiates the activity of nucleoside analogues, inhibits the escape of A-analogue resistant mutants, and increases the phosphorylation of T-analogues. Besides its antiviral activity, hydroxyurea effects the immune system by decreasing immune activation, inhibiting the expansion of CD8 cells and the depletion of CD4 cells. Hydroxyurea has been used in medicine for 40 years, is well tolerated, and it is the least expensive available anti-HIV-1 drug. These characteristics make hydroxyurea a primary candidate for use in combination therapies for the treatment of HIV-1 infection.     

Nitric oxide production from hydroxyurea

Hydroxyurea is a relatively new treatment for sickle cell disease. A portion of hydroxyurea's beneficial effects may be mediated by nitric oxide, which has also drawn considerable interest as a sickle cell disease treatment. Patients taking hydroxyurea show a significant increase in iron nitrosyl hemoglobin and plasma nitrite and nitrate within 2 h of ingestion, providing evidence for the in vivo conversion of hydroxyurea to nitric oxide. Hydroxyurea reacts with hemoglobin to produce iron nitrosyl hemoglobin, nitrite, and nitrate, but these reactions do not occur fast enough to account for the observed increases in these species in patients taking hydroxyurea. This report reviews recent in vitro studies directed at better understanding the in vivo nitric oxide release from hydroxyurea in patients. Specifically, this report covers: (1) peroxidase-mediated formation of nitric oxide from hydroxyurea; (2) nitric oxide production after hydrolysis of hydroxyurea to hydroxylamine; and (3) the nitric oxide-producing structure-activity relationships of hydroxyurea. Results from these studies should provide a better understanding of the nitric oxide donor properties of hydroxyurea and guide the development of new hydroxyurea-derived nitric oxide donors as potential sickle cell disease therapies.     

Genome Wide Association Study of Fetal Hemoglobin in Sickle Cell Anemia in Tanzania

Background

Fetal hemoglobin (HbF) is an important modulator of sickle cell disease (SCD). HbF has previously been shown to be affected by variants at three loci on chromosomes 2, 6 and 11, but it is likely that additional loci remain to be discovered.

Methods and Findings

We conducted a genome-wide association study (GWAS) in 1,213 SCA (HbSS/HbSβ⁰) patients in Tanzania. Genotyping was done with Illumina Omni2.5 array and imputation using 1000 Genomes Phase I release data. Association with HbF was analysed using a linear mixed model to control for complex population structure within our study. We successfully replicated known associations for HbF near BCL11A and the HBS1L-MYB intergenic polymorphisms (HMIP), including multiple independent effects near BCL11A, consistent with previous reports. We observed eight additional associations with P<10⁻⁶. These associations could not be replicated in a SCA population in the UK.

Conclusions

This is the largest GWAS study in SCA in Africa. We have confirmed known associations and identified new genetic associations with HbF that require further replication in SCA populations in Africa.     

Hydroxyurea in the treatment of HIV-1 infection: toxicity and side effects

Hydroxyurea has been used in numerous clinical trials for the treatment of HIV-1 infection, almost always in combination with didanosine with or without other antiretrovirals. Due to its inhibition of DNA synthesis, the main side effect of hydroxyurea is myelosuppression. When administered at the dosage of 1000 mg/day in asymptomatic, moderately-immunosuppressed HIV-1 infected patients, its tolerability profile appears to be quite favourable, with rare, reversible episodes of peripheral blood cytopenia that seldom require therapy discontinuation. Higher dosages of hydroxyurea and its use in advanced, heavily-pretreated patients may increase the likelihood of more severe side effects or newer toxicities developing. So far hydroxyurea-containing long therapy courses, up to 3 years, have not elicited any significant toxicity and appear to be safe as in onco-hematologic patients.     

Comparison of in-vitro and in-vivo response to fetal hemoglobin production and γ-mRNA expression by hydroxyurea in Hemoglobinopathies

BACKGROUND:

Hydroxyurea, which induces Fetal hemoglobin (HbF) synthesis, is the only drug widely used in different hemoglobinopathies; however, the response is very variable. We compared the efficacy of hydroxyurea in-vitro in erythroid cultures and in-vivo in the same patients with different hemoglobinopathies to induce HbF production and enhance γ-messenger RNA expression.

MATERIALS AND METHODS:

A total of 24-patients with different Hemoglobinopathies were given hydroxyurea and their response was studied in-vivo and in-vitro on mononuclear cells collected from them simultaneously.

RESULTS:

A total of 57.7% of patients (responders) showed no further crisis or transfusion requirements after hydroxyurea therapy with a mean increase in fetal cells (F-cells) of 63.8 ± 59.1% and γ-mRNA expression of 205.5 ± 120.8%. In-vitro results also showed a mean increase in F-cells of 27.2 ± 24.7% and γ-mRNA expression of 119.6% ± 65.4% among the treated cells. Nearly 19.0% of the partial-responders reduced their transfusion requirements by 50% with a mean increase in F-cells of 61.2 ± 25.0% and 28.4 ± 25.3% and γ-mRNA-expression of 21.0% ± 1.4% and 80.0% ± 14.1% in-vivo and in-vitro respectively. The non-responders (15.3%) showed no change in their clinical status and there was no significant increase in F-cells levels and γ-mRNA expression in-vivo or in-vitro.

CONCLUSION:

Thus, this method may help to predict the in-vivo response to hydroxyurea therapy; however, a much larger study is required.     

Widespread Natural Occurrence of Hydroxyurea in Animals

Here we report the widespread natural occurrence of a known antibiotic and antineoplastic compound, hydroxyurea in animals from many taxonomic groups.Hydroxyurea occurs in all the organisms we have examined including invertebrates (molluscs and crustaceans), fishes from several major groups, amphibians and mammals. The species with highest concentrations was an elasmobranch (sharks, skates and rays), the little skate Leucoraja erinacea with levels up to 250 μM, high enough to have antiviral, antimicrobial and antineoplastic effects based on in vitro studies. Embryos of L. erinacea showed increasing levels of hydroxyurea with development, indicating the capacity for hydroxyurea synthesis. Certain tissues of other organisms (e.g. skin of the frog (64 μM), intestine of lobster (138 μM) gills of the surf clam (100 μM)) had levels high enough to have antiviral effects based on in vitro studies. Hydroxyurea is widely used clinically in the treatment of certain human cancers, sickle cell anemia, psoriasis, myeloproliferative diseases, and has been investigated as a potential treatment of HIV infection and its presence at high levels in tissues of elasmobranchs and other organisms suggests a novel mechanism for fighting disease that may explain the disease resistance of some groups. In light of the known production of nitric oxide from exogenously applied hydroxyurea, endogenous hydoxyurea may play a hitherto unknown role in nitric oxide dynamics.     

Percutaneous cryotherapy for metastatic bladder cancer: Experience with 23 patients

Bladder cancer is the most common malignancy of the urinary tract and in many patients is metastatic at diagnosis. Chemotherapy is the standard treatment for these patients but has serious side effects and in many patients is not tolerated. To avoid the side effects of systemic chemotherapy, patients with late stage bladder cancer have sought cryotherapy in our hospital. We reviewed data for the past 4 years to evaluate the safety and efficiency of percutaneous cryotherapy in 23 patients. Within 3 days after cryosurgery, all complications of bladder cancer (e.g. hematuria, urinary irritation, hypogastralgia, lumbago) had decreased to some degree. No new complications (e.g. bladder perforation) occurred and all complications had disappeared completely after 2 weeks. The progression-free survival (PFS) of these patients was 14 ± 8 months. There was no effect on PFS of tumor location or histopathology; however, differentiation status and tumor size influenced the therapeutic effect of percutaneous cryoablation. In conclusion, percutaneous cryotherapy may be a safe and efficacious therapeutic option in the treatment of metastatic bladder cancer.     

Evaluation of the mutagenic activity of hydroxyurea on the G1-S-G2 phases of the cell cycle: an in vitro study

Hydroxyurea is considered an antineoplastic drug, which also plays an important role in the treatment of sickle cell anemia patients. We evaluated and compared the clastogenic and cytotoxic effects of hydroxyurea, using chromosomal aberrations and mitotic index, respectively, as endpoints. In vitro short-term cultures of lymphocytes were exposed to several concentrations of this drug, at various cell cycle phases. There was a significant increase in the cytotoxicity of hydroxyurea at G1 and G1/S as well in the G2 phase of the cell cycle. Hydroxyurea did not significantly increase chromosome aberrations. There was an S-dependent cytotoxic effect of hydroxyurea, which is expected based on the known activity of hydroxyurea as an inhibitor of ribonucleotide reductase.     

Hydroxyurea-Increased Fetal Hemoglobin Is Associated with Less Organ Damage and Longer Survival in Adults with Sickle Cell Anemia

Background

Adults with sickle cell anemia (HbSS) are inconsistently treated with hydroxyurea.

Objectives

We retrospectively evaluated the effects of elevating fetal hemoglobin with hydroxyurea on organ damage and survival in patients enrolled in our screening study between 2001 and 2010.

Methods

An electronic medical record facilitated development of a database for comparison of study parameters based on hydroxyurea exposure and dose. This study is registered with ClinicalTrials.gov, number NCT00011648.

Results

Three hundred eighty-three adults with homozygous sickle cell disease were analyzed with 59 deaths during study follow-up. Cox regression analysis revealed deceased subjects had more hepatic dysfunction (elevated alkaline phosphatase, Hazard Ratio = 1.005, 95% CI 1.003–1.006, p<0.0.0001), kidney dysfunction (elevated creatinine, Hazard Ratio = 1.13, 95% CI 1.00–1.27, p = 0.043), and cardiopulmonary dysfunction (elevated tricuspid jet velocity on echocardiogram, Hazard Ratio = 2.22, 1.23–4.02, p = 0.0082). Sixty-six percent of subjects were treated with hydroxyurea, although only 66% of those received a dose within the recommended therapeutic range. Hydroxyurea use was associated with improved survival (Hazard Ratio = 0.58, 95% CI 0.34–0.97, p = 0.040). This effect was most pronounced in those taking the recommended dose of 15–35 mg/kg/day (Hazard Ratio 0.36, 95% CI 0.17–0.73, p = 0.0050). Hydroxyurea use was not associated with changes in organ function over time. Further, subjects with higher fetal hemoglobin responses to hydroxyurea were more likely to survive (p = 0.0004). While alkaline phosphatase was lowest in patients with the best fetal hemoglobin response (95.4 versus 123.6, p = 0.0065 and 96.1 versus 113.6U/L, p = 0.041 at first and last visits, respectively), other markers of organ damage were not consistently improved over time in patients with the highest fetal hemoglobin levels.

Conclusions

Our data suggest that adults should be treated with the maximum tolerated hydroxyurea dose, ideally before organ damage occurs. Prospective studies are indicated to validate these findings.     

Evidence for four nonallelic structural genes for the γ chain of human fetal hemoglobin

An abnormal human fetal hemoglobin not only may be either a^Gγ- or an^Aγ-chain variant but also may be present in a different proportion of the total fetal hemoglobin.^Gγ-Chain variants contribute either about one-fourth or one-eighth to the total production of HbF in the heterozygote, whereas the^Aγ-chain variants approximate either one-eighth or one-sixteenth of the total HbF. These observations may indicate the presence of four nonallelic Hb_γ structural genes (termed ) which produce γ chains in an approximate ratio of 4 : 2 : 2 : 1. HbF Malta I is considered to be the product of a mutant of the locus, an undefined HbF_x that of the locus, HbF Hull and HbF Jamaica products of mutated loci, and the newly discovered HbF Malta II a mutant of the gene. This work was supported in part by grants HL-05168 and HL-02558 from the National Institutes of Health, U.S. Public Health Service.     

Association of Xmn1 −158 γG variant with severity and HbF levels in β-thalassemia major and sickle cell anaemia

Haemoglobinopathies including β-thalassemia and sickle cell anaemia (SCA) are considered to be classical monogenic diseases. There is considerable clinical variability between patients inheriting identical β-globin mutations. The reasons for this variability are not well understood. Previous studies have suggested that a variety of genetic determents influence different clinical phenotypes. The genetic variants that modulate HbF levels have a very strong impact on ameliorating the clinical phenotype. In the present study 6,500 blood samples from suspected cases were analysed using HPLC, ARMS-PCR, RDB techniques. Patients with β-thalassemia and SCA were classified into mild, moderate, severe according to the severity score based on Hb levels, age of onset, age at which patients received their first blood transfusion, the degree of growth retardation and splenectomy. Patients with β-thalassemia and SCA were analysed for Xmn1 polymorphism and association between this polymorphism and severity of β-thalassemia and SCA was evaluated. We found a significant difference in genotypic and allelic frequencies of Xmn1 polymorphism between mild and moderate and mild and severe cases. There was a significant difference in high and low percentage of HbF in CC, CT and TT bearing individuals. The TT bearing individuals were found to have a high percentage of HbF in β-thalassemia as well as SCA. This study confirms that increased γ^G-globin expression associated with Xmn1 polymorphism ameliorates the clinical severity in β-thalassemia as well as SCA in the study population.     

Differential effects of hydroxyurea and zileuton on interleukin-13 secretion by activated murine spleen cells: implication on the expression of vascular cell adhesion molecule-1 and vasoocclusion in sickle cell anemia

BACKGROUND: Interleukin-13 (IL-13), a TH2 cytokine, upregulates the expression of vascular cell adhesion molecule-1 on endothelial cells, a factor involved in vasoocclusion in sickle cell disease (SCD). Hydroxyurea improves clinical status of SCD patients in part by induction of fetal hemoglobin. Its effect on IL-13 secretion has not been investigated. OBJECTIVE: To determine whether hydroxyurea and zileuton, a hydroxyurea derivative with antiinflammatory properties, affect IL-13 secretion. METHODS: We measured IL-13 in the supernatant of murine spleen cells incubated without and with hydroxyurea, zileuton (10 microg/ml), concanavalin A (2.5 microg/ml), and anti-CD3 (50 ng/ml) (n=8). RESULTS: Hydroxyurea and zileuton do not affect baseline IL-13 secretion. Unexpectedly, hydroxyurea increases IL-13 levels above baseline (120%, 216.5%, [p<0.05] after 24 h and 48 h, respectively) in lymphocytes activated by anti-CD3, while zileuton reduces them by 59%-78% (p<0.005). In lymphocytes activated by concanavalin A, hydroxyurea and zileuton reduce IL-13 secretion by 24-36% and 50-87%, respectively (p<0.05). Hydroxyurea, but not zileuton, significantly inhibits spleen cell proliferative responses to mitogens (p<0.005). CONCLUSION: Data suggest that hydroxyurea up-regulates IL-13 secretion in anti-CD3-activated lymphocytes through gene activation but not by altered cell proliferation. Increased IL-13 secretion may contribute to unresponsiveness of certain SCD patients to hydroxyurea. The potential benefit of zileuton in the management of vasoocclusion is discussed.     

Disinfection and toxicological assessments of pulsed UV and pulsed-plasma gas-discharge treated-water containing the waterborne protozoan enteroparasite Cryptosporidium parvum

We report for the first time on the comparative use of pulsed-plasma gas-discharge (PPGD) and pulsed UV light (PUV) for the novel destruction of the waterborne enteroparasite Cryptosporidium parvum. It also describes the first cyto-, geno- and ecotoxicological assays undertaken to assess the safety of water decontaminated using PPGD and PUV. During PPGD treatments, the application of high voltage pulses (16 kV, 10 pps) to gas-injected water (N₂ or O₂, flow rate 2.5 L/min) resulted in the formation of a plasma that generated free radicals, ultraviolet light, acoustic shock waves and electric fields that killed ca. 4 log C. parvum oocysts in 32 min exposure. Findings showed that PPGD-treated water produced significant cytotoxic properties (as determined by MTT and neutral red assays), genotoxic properties (as determined by comet and Ames assays), and ecotoxic properties (as determined by Microtox™, Thamnotox™ and Daphnotox™ assays) that are representative of different trophic levels in aquatic environment (p < 0.05). Depending in part on the type of injected gas used, PPGD-treated water became either alkaline (pH ≤ 8.58, using O₂) or acidic (pH ≥ 3.21, using N₂) and contained varying levels of reactive free radicals such as ozone (0.8 mg/L) and/or dissociated nitric and nitrous acid that contributed to the observed disinfection and toxicity. Chemical analysis of PPGD-treated water revealed increasing levels of electrode metals that were present at ≤ 30 times the tolerated respective values for EU drinking water. PUV-treated water did not exhibit any toxicity and was shown to be far superior to that of PPGD for killing C. parvum oocysts taking only 90 s of pulsing [UV dose of 6.29 μJ/cm²] to produce a 4-log reduction compared to a similar reduction level achieved after 32 min PPGD treatment as determined by combined in vitro CaCo-2 cell culture-qPCR.     

Long-term Effects of Hydroxyurea in Psoriasis

Hydroxyurea is an effective treatment for psoriasis but consistently produces macrocytosis in peripheral blood with a fall in haemoglobin levels and white cell counts. In this long-term study of 16 patients anaemia and leukopenia have proved frequent and troublesome side effects requiring discontinuance of treatment in many cases. In view of these findings hydroxyurea cannot be recommended as the drug of first choice even for treating severe and intractable psoriasis, and it is our opinion that methotrexate is safer and at least as effective in these circumstances. The clearest indications for the use of hydroxyurea appear to be in life-ruining psoriasis when methotrexate treatment has either failed or is contraindicated because of liver damage. A great deal more investigative work is required before these indications can be generally extended.     

Comparison of the effects of eFSH and deslorelin treatment regimes on ovarian stimulation and embryo production of donor mares in early vernal transition

The objective was to compare the effects of eFSH and deslorelin treatment regimes on ovarian stimulation and embryo production of donor mares in early spring transition. Starting January 30th, mares kept under ambient light were examined by transrectal ultrasonography. When a follicle ≥25 mm was detected, mares were assigned to one of two treatment groups, using a sequential alternating treatment design. In the eFSH group, mares (n = 18) were treated twice daily with eFSH (12.5 mg im) until they achieved a follicle ≥35 mm; hCG was given 36 h later. In the deslorelin group, mares (n = 18) were treated twice daily with deslorelin (63 μg im) until a follicle ≥35 mm was detected, and then they were given hCG. Estrous mares were inseminated with fresh semen. Eight days after ovulation, embryo recovery attempts were performed. In each group, 14/18 (78%) mares ovulated following the eFSH or deslorelin treatment regimes. The mean (95% CI) interval from treatment initiation to ovulation was 8.2 d (7.3, 8.9) and 7.2 d (6.2, 8.1) in the eFSH and deslorelin groups, respectively. In the eFSH group, the number of ovulations was significantly higher (mean ± S.E.M.; 3.4 ± 0.4 vs. 1.1 ± 0.1 ovulations), and more embryos were recovered (2.6 ± 0.5 vs. 0.4 ± 0.2 embryos/recovery attempt). We concluded that eFSH and deslorelin treatment regimes were equally effective in inducing ovulation in early transitional mares, within a predictable time of treatment; however, the eFSH regime increased the number of ovulations and embryos recovered per mare.