Investigation of sickle-cell haemoglobin polymerisation under electrochemical control.

We describe an electrochemistry-based technique to control and monitor the polymerisation of sickle-cell haemoglobin (HbS). The polymerisation was monitored as a change in turbidity during the depletion of oxygen in a small volume custom-built thin-layer electrochemical cell. The cell allowed the investigation of HbS polymerisation as a function of HbS concentration, temperature and solution pH. We confirm that the oxygen was efficiently depleted using finite-element modelling to accurately recreate the electrochemical thin-layer cell. Understanding the nucleation and growth of HbS polymerisation will provide a better understanding of the pathophysiology of sickle-cell disease in vivo, and thus help improve therapeutic strategies for this common and frequently disabling disorder.

Hemoglobin ontogenesis: test of the gene excision hypothesis.

The gene excision hypothesis of hemoglobin ontogenesis was tested in persons with HbSC disease, with the use of monospecific fluorescent antibodies for the identification of hemoglobins S, C, and F in individual erythrocytes. The results are incompatible with the prediction that only one gamma- or beta-globin gene may be active in any single chromosome and provide further evidence for incomplete repression of gamma-globin genes lying cis to active beta-globin genes.

Erythroid progenitors circulating in the blood of adult individuals produce fetal hemoglobin in culture.

Erythroid colonies, raised from erythroid stem cells circulating in the peripheral blood of normal adult individuals, synthesize considerable amounts of fetal hemoglobin. In cultures from persons with sickling disorders, amounts of hemoglobin F that are known to inhibit sickling in vivo are produced. The results provide evidence that primitive erythroid progenitors are able to express the hemoglobin F production program and that cultures of mononuclear cells of the adult blood can be used to investigate the mechanisms involved in regulation of gamma-globin gene switching.

Theoretical Simulation of Red Cell Sickling Upon Deoxygenation Based on the Physical Chemistry of Sickle Hemoglobin Fiber Formation.

The polymerization of the mutant hemoglobin S upon deoxygenation to form fibers in red blood cells of patients suffering from sickle-cell anemia results in changes in cell shape and rigidity, also known as sickling, which underlie the pathology of the disease. While much has been learned about the fundamental physical chemistry of the polymerization process, transferring these insights to sickling of red cells under in vivo conditions requires being able to monitor, and ultimately predict, the time course of cellular sickling under physiological conditions of deoxygenation. To this end, we have developed an experimental technique for tracking the temporal evolution of the sickling of red blood cells under laboratory deoxygenation conditions, based on the automated analysis of sequences of microscope images and machine-learning analysis to characterize cell morphology. As an aid in the quantitative understanding of these experiments, we have developed a computational framework for simulating the time dependence of sickling in populations of red blood cells which incorporates the current theoretical and empirical understanding of the physical chemistry of the sickling process. In order to apply these techniques to our experiments, we have theoretically determined the time course of deoxygenation by solving the diffusion equation for oxygen in our experimental geometry. With this combined description, we are able to reproduce our experimentally observed kinetics of sickling, suggesting that our theoretical approach should be applicable to physiological deoxygenation scenarios.

Hemoglobin biosynthesis in individual erythropoietic bursts in culture. Studies of adult peripheral blood.

By using a methylcellulose clonal assay, we cultured peripheral blood erythropoietic precursors from a patient with sickle cell anemia, a patient with sickle cell hemoglobin C disease, and a normal volunteer. We then analyzed the synthetic rates of adult and fetal hemoglobins (Hb) in individual erythropoietic bursts. Bb were labeled with 14C-amino acids in culture, separated by slab gel isoelectric focusing techniques, and quantitated by fluorographic methods. All bursts exhibited both fetal and adult Hb in varying ratios. Frequency distributions of the individual burst differing in percentage of BbF biosynthesis approached normal distributions. Further stimulation of HbF synthesis by higher erythropoietin in culture was associated with increased HbF biosynthesis in individual bursts. Augmentation of human HbF synthesis in culture appears to be controlled by qualitative intracellular changes rather than by changes in cellular population.

In vivo demonstration of red cell-endothelial interaction, sickling and altered microvascular response to oxygen in the sickle transgenic mouse.

Intravascular sickling, red cell-endothelium interaction, and altered microvascular responses have been suggested to contribute to the pathophysiology of human sickle cell disease, but have never been demonstrated under in vivo flow. To address this issue, we have examined a transgenic mouse line, alphaHbetaSbetaS-Antilles [betaMDD] which has a combined high (78%) expression of beta S and beta S-Antilles globins. In vivo microcirculatory studies using the cremaster muscle preparation showed adhesion of red cells, restricted to postcapillary venules, in transgenic mice but not in control mice. Electron microscopy revealed distinct contacts between the red cell membrane and the endothelium surface. Some red cells exhibiting sickling were regularly observed in the venular flow. Infusion of transgenic mouse red cells into the ex vivo mesocecum vasculature also showed adhesion of mouse red cells exclusively in venules. Under resting conditions (pO2, 15-20 mmHg), there were no differences in the cremaster microvascular diameters of control and transgenic mice; however, transgenic mice showed a drastic reduction in microvascular red cell velocities (Vrbc) with maximal Vrbc decrease (> 60%) occurring in venules, the sites of red cell adhesion and sickling. Local, transient hyperoxia (pO2, 150 mmHg) resulted in striking differences between control and transgenic mice. In controls, oxygen caused a 69% arteriolar constriction, accompanied by 75% reduction in Vrbc. In contrast, in transgenic mice, hyperoxia resulted in only 8% decrease in the arteriolar diameter and in 68% increase in VrBC; the latter is probably due to an improved flow behavior of red cells as a consequence of unsickling. In summary, the high expression of human sickle hemoglobin in the mouse results not only in intravascular sickling but also red cell-endothelium interaction. The altered microvascular response to oxygen could be secondary to blood rheological changes, although possible intrinsic differences in the endothelial cell/vascular smooth muscle function in the transgenic mouse may also contribute. These sickle transgenic mice could serve as a useful model to investigate vasoocclusive mechanisms, as well as to test potential therapies.

Sickle cell anemia in the newborn.

The clinical presentation of homozygous sickle cell disease is unusual in the neonatal period. Recently, we have encountered a newborn infant whose disease was apparent at birth and who died at 5 days of age. The findings at autopsy suggested a sickle cell crisis with multisystem involvement that was present prior to birth. Laboratory findings confirmed homozygous sickle cell disease without the presence of elevated levels of hemoglobin S. The cause of the unusually severe clinical course of the disease in our patient is the object of the discussion.

Acute Chest Syndrome: the role of erythro-exchange in patients with sickle cell disease in Sicily.

Acute Chest Syndrome (ACS) describes a syndrome characterized by the presence of a new pulmonary infiltrate on a chest X-ray, fever, and respiratory symptoms and is the leading cause of death and hospitalization in sickle cell disease (SCD). We studied 21 patients affected by SCD (13 HbSbeta+, 4 HbS beta(o), 4 HbSS, mean age 38.2 years). Six out of the 21 patients developed one episode of ACS (two patients had positive blood cultures, for Mycoplasma pneumoniae and Haemophilus influenzae respectively). The aim of our study was to evaluate the therapeutic efficacy of red cell-exchange during ACS. This procedure decreases HbS levels. The patients who underwent erythro-exchange showed a dramatic clinical and radiographic improvement with stabilized HbS levels between 20% and 30%. During follow up (14-32 months), none of the 6 patients developed viral complications related to transfusion therapy, alloimmunization or recurrence of ACS. In conclusion, in regard to the pre- and post-red cell-exchange clinical and laboratory data, we can say that red cell-exchange provides a dramatic resolution of the episode of ACS, minimizes the development of iron overload, and rapidly decreases HbS and hematocrit levels. In light of our results, we hypothesize that ACS episodes are secondary to pulmonary damage and to a gradual worsening related to age, and that there is some evidence that individuals affected by SCD in the third to fourth decade of life are more susceptible to ACS and/or other severe disease-related complications, needing repeated and strict clinical follow up.

Sickle cell anemia and related hemoglobinopathies.

The pathophysiology, laboratory investigation, clinical manifestation, and treatment for the most common serious genetic disease in the United States have been discussed. It is unfortunate how uninformed the medical community is about this illness. Treatment for a chronic disease such as sickle cell disease requires that the physician be knowledgeable and compassionate. Although there is no cure, a great deal can be done to help patients cope with their illness. Recent advances in prevention of pneumococcal infection, proper use of transfusion therapy, and prenatal diagnosis are such examples.

[Acute cerebrovascular accident associated with drepanocytosis complicated by pneumococcal meningitis in two children]. / Accidente cerebrovascular agudo asociado a drepanocitosis complicada con meningitis neumocócica en dos niños.

INTRODUCTION: Between 6% and 17% of the patients with drepanocytosis will have an acute cerebrovascular accident (ACVA). Precipitating factors have been described including bacterial meningitis, upper respiratory tract obstruction, dehydration, intense exercise, postoperatively, and hypoxia due to altitude. CLINICAL CASES: We report two Negro children with drepanocytosis who, at the ages of 8 and 20 months, had pneumococcal meningitis complicated by an ACVA. The 20 month old boy had been previously diagnosed as having drepanocytosis; the 8 month old girl was diagnosed when she was admitted to hospital with meningitis. In both cases the clinical features of the ACVA were focal epileptic seizures followed by hemiplegia. On cranial CT and MR regions of focal ischaemia of the hemisphere were observed. The boy of 20 months made an excellent recovery with no long term sequelae. The 8 month old girl had severe permanent sequelae: hemiparesia, blindness, mental deficiency and epileptic encephalopathy. CONCLUSIONS: We present two Negro children with drepanocytosis who had ischemic ACVAs, a common complication of the disease, during the course of pneumococcal meningitis but with very different clinical courses. Recent increases in immigration will mean that previously rare illnesses will be more commonly seen in our environment, and should be considered in the differential diagnosis.

Transgenic sickle mice are markedly sensitive to renal ischemia-reperfusion injury.

Ischemic injury is invoked as a mechanism contributing to end-organ damage and other complications of sickle cell disease (SCD). However, the intrinsic sensitivity of tissues in SCD to ischemic insults has never been addressed. We examined the effect of renal ischemia in a transgenic mouse expressing human sickle hemoglobin. Twenty-four hours after bilateral, total renal artery occlusion for 15 minutes, transgenic sickle mice exhibited worse renal function and more marked histological injury. With bilateral renal ischemia of greater duration (22.5 minutes), and after 6 hours, transgenic sickle mice exhibited massive vascular congestion, sickling of red blood cells, more marked histological injury in the kidney, and more prominent congestion in the capillary beds in the lungs and heart. Additionally, serum amyloid P-component, the murine homologue of C-reactive protein, was markedly increased in transgenic sickle mice as compared to wild-type mice. Twenty-four hours after bilateral renal ischemia for 22.5 minutes, transgenic sickle mice exhibited 28% mortality, with no mortality observed in any other group. With bilateral renal ischemia of short or long duration, renal expression of caspase-3 was most prominent in transgenic sickle mice subjected to ischemia. Thus, renal ischemia in this murine model induces more severe renal injury and extrarenal complications. We conclude that tissues in SCD exhibit heightened vascular congestion and sensitivity to ischemia and that clinically apparent or silent episodes of ischemia may contribute to the complications of SCD.