An anthropometric and hematological comparison of sickle cell disease children from rural and urban areas

BACKGROUND:

Sickle cell disease (SCD) is a prevalent genetic disorder in India and the rural and urban areas experience distinctly different healthcare facilities. In view of this, a comparative study of SCD-SS pattern children of age 8–15 years from rural and urban areas of Wardha district of Central India was carried out using anthropometric and hematological parameters.

MATERIALS AND METHODS:

The data were collected using standard methods and the results showed a significant (P < 0.05) difference in the mean values for body weight, body mass index (BMI), hemoglobin, hematocrit, and white blood corpuscles (WBC). Statistical analysis of the data was done using SPSS 18.0 software. Individuals were screened by solubility test method. Sickle cell patterns (AS and SS) were determined by using electrophoresis technique.

RESULT:

The SCD-SS children from rural were significantly underweight than those from the urban area of Wardha district. BMI is a good indicator of nutritional status and BMI values of SCD children have less than desired.

CONCLUSION:

The study highlights an urgent need to conduct integrated investigations for SCD population of rural areas covering clinical, nutritional, and social aspects.     

P2X7 receptor activation induces cell death and microparticle release in murine erythroleukemia cells

Extracellular ATP induces cation fluxes in and impairs the growth of murine erythroleukemia (MEL) cells in a manner characteristic of the purinergic P2X7 receptor, however the presence of P2X7 in these cells is unknown. This study investigated whether MEL cells express functional P2X7. RT-PCR, immunoblotting and immunofluorescence staining demonstrated the presence of P2X7 in MEL cells. Cytofluorometric measurements demonstrated that ATP induced ethidium⁺ uptake into MEL cells in a concentration-dependent fashion and with an EC₅₀ of ∼ 154 μM. The most potent P2X7 agonist 2′- and 3′-0(4-benzoylbenzoyl) ATP, but not ADP or UTP, induced ethidium⁺ uptake. ATP-induced ethidium⁺ and YO-PRO-1²⁺ uptake were impaired by the P2X7 antagonist, A-438079. A colourmetric assay demonstrated that ATP impaired MEL cell growth. A cytofluorometric assay showed that ATP induced MEL cell death and that this process was impaired by A-438079. Finally, cytofluorometric measurements of Annexin-V binding and bio-maleimide staining demonstrated that ATP could induce rapid phosphatidylserine exposure and microparticle release in MEL cells respectively, both of which were impaired by A-438079. These results demonstrate that MEL cells express functional P2X7, and indicate that activation of this receptor may be important in the death and release of microparticles from red blood cells in vivo.     

The effect of hemolysis on plasma oxidation and nitration in patients with sickle cell disease

Abstract

This study aimed to determine the effect of haemolysis on plasma oxidation and nitration in sickle cell disease (SCD) patients. Blood was collected from haemoglobin (Hb)A volunteers and homozygous HbSS patients who had not received blood transfusions in the last 3 months. Haemolysis was characterised by low levels of haemoglobin and haptoglobin and high levels of reticulocyte, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), plasma cell-free haemoglobin, bilirubin, total lactate dehydrogenase (LDH) and dominance of LDH-1 isoenzyme. Plasma 8-isoprostane, protein carbonyl and nitrotyrosine levels were measured to evaluate oxidised lipids, oxidised and nitrated proteins, respectively. Plasma nitrite–nitrate levels were also determined to assess nitric oxide (NO) production in both SCD patients and controls. Markers of haemolysis were significantly evident in SCD patients compared to controls. Plasma 8-isoprostane, protein carbonyl and nitrotyrosine levels were markedly elevated in SCD patients compared to controls. Linear regression analysis revealed a significant inverse correlation between haemoglobin and reticulocyte counts and a significant positive correlation of plasma cell-free haemoglobin with protein carbonyl and nitrotyrosine levels. The obtained data shows that increased haemolysis in SCD increases plasma protein oxidation and nitration.     

Reduced plasma angiotensin II levels are reversed by hydroxyurea treatment in mice with sickle cell disease

Aims

Sickle cell disease (SCD) pathogenesis leads to recurrent vaso-occlusive and hemolytic processes, causing numerous clinical complications including renal damage. As vasoconstrictive mechanisms may be enhanced in SCD, due to endothelial dysfunction and vasoactive protein production, we aimed to determine whether the expression of proteins of the renin–angiotensin system (RAS) may be altered in an animal model of SCD.

Main methods

Plasma angiotensin II (Ang II) was measured in C57BL/6 (WT) mice and mice with SCD by ELISA, while quantitative PCR was used to compare the expressions of the genes encoding the angiotensin-II-receptors 1 and 2 (AT1R and AT2R) and the angiotensin-converting enzymes (ACE1 and ACE2) in the kidneys, hearts, livers and brains of mice. The effects of hydroxyurea (HU; 50–75 mg/kg/day, 4 weeks) treatment on these parameters were also determined.

Key findings

Plasma Ang II was significantly diminished in SCD mice, compared with WT mice, in association with decreased AT1R and ACE1 expressions in SCD mice kidneys. Treatment of SCD mice with HU reduced leukocyte and platelet counts and increased plasma Ang II to levels similar to those of WT mice. HU also increased AT1R and ACE2 gene expression in the kidney and heart.

Significance

Results indicate an imbalanced RAS in an SCD mouse model; HU therapy may be able to restore some RAS parameters in these mice. Further investigations regarding Ang II production and the RAS in human SCD may be warranted, as such changes may reflect or contribute to renal damage and alterations in blood pressure.     

A mass spectrometry assay for detection of endogenous and lentiviral engineered hemoglobin in cultured cells and sickle cell disease mice

Sickle cell disease (SCD) results from a sequence defect in the β-globin chain of adult hemoglobin (HbA) leading to expression of sickle hemoglobin (HbS). It is traditionally diagnosed by cellulose-acetate hemoglobin electrophoresis or high-performance liquid chromatography. While clinically useful, these methods have both sensitivity and specificity limitations. We developed a novel mass spectrometry (MS) method for the rapid, sensitive and highly quantitative detection of endogenous human β-globin and sickle hβ-globin, as well as lentiviral-encoded therapeutic hβAS3-globin in cultured cells and small quantities of mouse peripheral blood. The MS methods were used to phenotype homozygous HbA (AA), heterozygous HbA–HbS (AS) and homozygous HbS (SS) Townes SCD mice and detect lentiviral vector-encoded hβAS3-globin in transduced mouse erythroid cell cultures and transduced human CD34⁺ cells after erythroid differentiation. hβAS3-globin was also detected in peripheral blood 6 weeks post-transplant of transduced Townes SS bone marrow cells into syngeneic Townes SS mice and persisted for over 20 weeks post-transplant. As several genome-editing and gene therapy approaches for severe hemoglobin disorders are currently in clinical trials, this MS method will be useful for patient assessment before treatment and during follow-up.     

A justice‐based argument for including sickle cell disease in CRISPR/Cas9 clinical research

CRISPR/Cas9 is quickly becoming one of the most influential biotechnologies of the last five years. Clinical trials will soon be underway to test whether CRISPR/Cas9 can edit away the genetic mutations that cause sickle cell disease (SCD). This article will present the background of CRISPR/Cas9 gene editing and SCD, highlighting research that supports the application of CRISPR/Cas9 to SCD. While much has been written on why SCD is a good biological candidate for CRISPR/Cas9, less has been written on the ethical implications of including SCD in CRISPR/Cas9 research. This article will argue that there is a strong case in favor of including SCD. Three benefits are achieving distributive justice in research, continuing to repair the negative relationship between patients with SCD and the health‐care system, and benefit‐sharing for those who do not directly participate in CRISPR/Cas9 research. Opponents will argue that SCD is a risky candidate, that researchers will not find willing participants, and that the burden of SCD is low. Of this set of arguments, the first gives pause. However, on balance, the case in favor of including SCD in CRISPR/Cas9 research is stronger than the case against. Ultimately, this article will show that the historic and sociopolitical injustices that impede progress in treating and curing SCD can be alleviated through biotechnology.     

A path analysis of the causal relationships between red cell glycolytic intermediates, ADP and ATP in sickle cell anemia

The statistical relationships among the glycolytic intermediates (GI)) of the Embden-Meyerhof pathway, adenine nucleotides (ANs) and various hematological measures were estimated for 34 sickle cell anemia patients. Heterogeneity in linear and quadratic regressions of hemoglobin and hematocrit, both singly and jointly, on the GI and AN variables implied 1) that any single formula to standardize optical density measures of the GIs and ANs on a per gram hemoglobin or per liter cell water basis would not uniformly remove hemoglobin and hematocrit effects: 2) that ignoring significant hematological effects could bias the estimates of correlation among GIs and ANs; and 3) that hemoglobin and hematocrit measures do not reflect the same source of variability. The correlations among the GIs and ANs, after adjustment for hematological variability, were analyzed by path analysis to determine which of five proposed path models for cause and effect relationships were compatible with the data. AMP had a greater influence on ADP (coefficient of determination (CD) = 23%) than all the GIs together, while G6P and ADP influenced ATP variability the most (CD = 33% and 12%). The contributions of unknown factors to ADP and ATP variability were large for all models (CD = 56--77%) possibly due to stress of sickle cell disease. The path model with AMP and the four GIs (G6P, F6P, FDP, DHAP) influencing ADP variation, and the same GIs and ADP influencing ATP was the model most compatible with the data.     

TNF-alpha and IL-8: serum levels and gene polymorphisms (-308G>A and -251A>T) are associated with classical biomarkers and medical history in children with sickle cell anemia

Sickle cell anemia (SCA) is a disorder characterized by a heterogeneous clinical outcome. In the present study, we investigated the associations between Tumor Necrosis Factor-alpha (TNF-alpha) −308G>A and Interleukin 8 (IL-8) −251A>T gene polymorphisms, medical history and classical biomarkers in children with steady-state SCA. In total, 210 SCA patients aged 2–21 years and 200 healthy controls were studied. Gene polymorphisms, beta^S-globin haplotypes and a 3.7-kb deletion in alpha2-thalassemia (α₂-thal^{3.7 kb}) were investigated by PCR/RFLP analysis, and cytokine levels were determined by ELISA. Splenomegaly (p = .032) was more prevalent among children younger than 5 years of age. The A allele of the TNF-alpha −308G>A gene polymorphism and the presence of α₂-thal^{3.7 kb} were associated with an increase risk of splenic sequestration events (p = .001; p = .046), while the T allele of the IL-8 −251A>T gene polymorphism was considered to be a protective factor for splenomegaly events (p = .032). Moreover, the A allele of the TNF-alpha −308G>A gene polymorphism was associated with high TNF-alpha levels (p = .021), and the hemoglobin F and hemoglobin S haplotypes were correlated with serum levels of IL-8. The logistic regression analysis showed significant effects of the TNF-alpha and IL-8 gene polymorphisms, beta^S-globin gene haplotypes and α₂-thal^{3.7 kb} on the occurrence of splenic sequestration events. Our study emphasizes that the identification of new genetic and immunological biomarkers and their associations with classical markers is an important strategy to elucidate the underlying causes of different SCA phenotypes and their effects on patient outcome.