Arvind K. Shah

Causes of death in sickle cell disease: an autopsy study

Summary. More precise analysis of causes of death is needed to focus research efforts and improve morbidity and mortality in sickle cell disease. In this study, the morphological evidence of the cause of death was studied in 306 autopsies of sickle cell disease, which were accrued between 1929 and 1996. The most common cause of death for all sickle variants and for all age groups was infection (33–48%). The terminal infection was heralded by upper respiratory tract syndromes in 72·6% and by gastroenteritis in 13·7%. The most frequent portal of entry in children was the respiratory tract but, in adults, a site of severe chronic organ injury. Other causes of death included stroke 9·8%, therapy complications 7·0%, splenic sequestration 6·6%, pulmonary emboli/thrombi 4·9%, renal failure 4·1%, pulmonary hypertension 2·9%, hepatic failure 0·8%, massive haemolysis/red cell aplasia 0·4% and left ventricular failure 0·4%. Death was frequently sudden and unexpected (40·8%) or occurred within 24 h after presentation (28·4%), and was usually associated with acute events (63·3%). This study shows that the first 24 h after presentation for medical care is an especially perilous time for patients with sickle cell disease and an acute event. Close monitoring and prompt aggressive treatment are warranted.

BFU‐E colony growth in response to hydroxyurea: Correlation between in vitro and in vivo fetal hemoglobin induction

Patients with sickle‐cell anemia treated with hydroxyurea may have significant reduction in frequency and severity of pain episodes. However, previous clinical trials show a variable response to hydroxyurea. Criteria which can be used to select patients who are likely to respond to hydroxyurea treatment would be useful. Our laboratory has previously demonstrated an inverse linear relationship between the total number of burst‐forming unit‐erythroid (BFU‐E) colonies and fetal hemoglobin levels in sickle‐cell patients treated with hydroxyurea. In the present report, an in vitro cell culture system was established to evaluate the effects of hydroxyurea on BFU‐E colony growth and induction of fetal hemoglobin production. Five Hb SS patients who were not previously treated with hydroxyurea and three Hb SS patients who failed to respond to hydroxyurea treatment were included in the study. The results show that the number of BFU‐E colonies is decreased from 153.7 to 7.2 per 3 × 105 mononuclear cells, whereas fetal hemoglobin levels were increased from 5.1 to 19.4% in the presence of hydroxyurea in vitro in cultured erythroid progenitors, which were derived from 5 patients before treatment. The number of BFU‐E colonies decreased from 153.7 to 2.0 per 3 × 105 mononuclear cells in the in vitro cultures obtained from serial peripheral blood samples over a 9‐ to 20‐week period of oral hydroxyurea therapy. A simultaneous rise in fetal hemoglobin level from 10.2 to 28.6% in the peripheral blood over the same period of hydroxyurea therapy was also observed. Our results demonstrate that the increase in fetal hemoglobin levels in cells treated with hydroxyurea in vitro is comparable to the rise of fetal hemoglobin production following hydroxyurea therapy in these patients. On the contrary, these findings were not observed in three previously non‐responsive sickle‐cell patients. These results suggest that the changes in number of BFU‐E colonies and fetal hemoglobin levels after in vitro exposure to hydroxyurea may be a useful approach to select sickle‐cell patients who will respond to hydroxyurea therapy. Am. J. Hematol. 56:252–258, 1997.

Mechanism for fetal hemoglobin induction by hydroxyurea in sickle cell erythroid progenitors

Hydroxyurea (HU) is a widely used cytotoxic agent that is known to induce fetal hemoglobin (HbF) production and is presently used to ameliorate the severity of pain episodes in patients with sickle cell anemia (HbSS). Previously we have shown that HU inhibits growth of burst forming unit–erythroid (BFU‐E) colonies in a dose‐dependent manner, while fetal hemoglobin levels were increased. In the present report, we extended our analysis demonstrating the number of S phase cells is significantly higher for HbSS patients that respond to HU therapy. Studies were completed in vitro using erythroid progenitors derived from umbilical cord samples or peripheral blood from patients with HbS–hereditary persistence of fetal hemoglobin (HbS‐HPFH) or HbSS disease. The effect of HU on (a) S phase erythroid progenitors, (b) BFU‐E colony growth, (c) HbF levels in BFU‐E colonies, and (d) total cellular RNA synthesis was analyzed in vitro for the three groups. The level of S phase erythroid progenitors was similar for all three groups and BFU‐E colony growth was inhibited 92–94% for all samples in a dose‐dependent manner. The HbF levels were increased in BFU‐E colonies from HbSS patients (control, 4.0% ± 1.15% vs. +HU, 22.67% ± 2.03%) whereas HbF levels were decreased in BFU‐E colonies derived from umbilical cord samples (control, 80% ± 9.07% vs. +HU, 35.7% ± 4.81%) or HbS‐HPFH patients (control, 49.67% ± 3.84% vs. +HU, 23.3% ± 0.88%). Total RNA synthesis measured by 3H‐uridine incorporation increased with increasing concentrations of HU; however, actinomycin D inhibited HU‐induced RNA synthesis. These results suggest that HU can inhibit an active globin gene without preference and that newly synthesized RNA is under transcriptional control mechanisms. Am. J. Hematol. 65:227–233, 2000.

Calpromotin, a cytoplasmic protein, is associated with the formation of dense cells in sickle cell anemia

We have tested the hypothesis that dense cell formation in sickle cell disease is associated with increased binding of calpromotin to the membrane, an event that occurs during the activation of calcium‐dependent potassium transport. By SDS polyacrylamide gel electrophoresis, we found that sickle cell membranes contained more calpromotin than did normal membranes when stained with Coomassie brilliant blue or when transferred to nitrocellulose paper and immunostained with horseradish peroxidase. Also, the membranes from dense sickle cells contained significantly (P = 0.00055) higher levels of calpromotin, 2.62 ± 1.59 μg/mg membrane protein, compared to light sickle cells, 1.40 ± 0.70 μg/mg membrane protein, when measured by an enzyme‐linked immunosorbent assay. The ratio of calpromotin associated with dense cell membranes to light cell membranes was significantly greater than 1.0 (P < 0.00005). Transmission electron micrographs of immunogold‐labelled membranes supported the increase in calpromotin binding in dense sickle cell membranes. In addition, the immunogold probe demonstrated clustering, which was not observed in light sickle cell membranes nor in normal membranes. Finally, we incubated HbSS cells in vitro using a repetitive deoxygenation/reoxygenation procedure to produce dense cells and then measured the levels of calpromotin associated with their membranes. As expected, the levels of calpromotin bound to the membrane doubled during the procedure relative to the basal levels at the beginning of the incubation. The correlation coefficient, calculated between the increase in dense cell formation and the increase in calpromotin associated with the membrane, was statistically significant (P = 0.038). The results demonstrate that an increase in calpromotin binding to the membrane is associated with dense cell formation presumably through the activation of the calcium‐dependent potassium channel. Am. J. Hematol. 56:100–106, 1997.

A Simpler Approximation for Areas under the Standard Normal Curve

Abstract A simple approximation for areas under the standard normal curve is presented that is suitable for use when tables and/or calculators are not available or not permitted.

A tungsten-supplemented diet delivered by transplacental and breast-feeding routes lowers intestinal xanthine oxidase activity and affords cytoprotection in ischemia-reperfusion injury to the small intestine

Ischemia-reperfusion injury has been implicated as playing a major role in the development of necrotizing enterocolitis, a major cause of morbidity and mortality in the newborn. A tungsten-supplemented molybdenum-free diet can reduce xanthine oxidase (XO) enzyme activity in the intestine, which in turn reduces the generation of oxygen radicals after an ischemia-reperfusion insult. This study evaluated the ability of this diet to be effective by indirect means, ie, transplacental and breast-feeding routes. XO activity of the intestine was measured in three groups of CD-1 white rats: I, weanlings fed the tungsten diet or standard chow for 1 week; II, 1-day-old rat pups whose mothers were maintained on the tungsten or standard chow for 7 to 10 days prior to term; and III, rat pups at 1 and 3 weeks after birth whose lactating mothers were maintained on the tungsten or standard chow. Some animals from group III also underwent either a 30- or 60-minute episode of occlusion of the superior mesenteric artery (SMA) to evaluate the protective effects of the diet. XO activity was significantly reduced in all groups receiving the tungsten diet (P less than .0001). Blinded histopathologic studies of the entire small bowel showed significantly less villar necrosis (P less than .05) and fibrosis (P less than .0001) in the tungsten-treated group than in the controls. In the 60-minute occlusion study all tungsten-group animals survived, whereas 7 of 12 in the control group died of intestinal infarction within 24 hours (P less than .001).(ABSTRACT TRUNCATED AT 250 WORDS)

Irreversibly sickled cell β‐actin: Defective filament formation

It has been demonstrated that cysteine modification in irreversibly sickled cell β‐actin slows down the remodeling of membrane skeletons [Shartava et al.: J Cell Biol 128:805‐812, 1995]. This slow remodeling can be due to alterations in spectrin‐actin binding and/or actin‐actin interactions in irreversibly sickled cell (ISC) membrane skeletons. In these studies we demonstrate that ISC actin binds spectrin normally. However, ISC β‐actin polymerizes and depolymerizes more slowly than control β‐actin, and forms unusual aggregates when placed under polymerizing conditions. Electron microscopic analysis of actin polymers indicated that ISC actin generates a large amount of aggregates which we conclude are due to the structural modification caused by the disulfide bridge between cysteine264 and cysteine373 in β‐actin. Am. J. Hematol. 55:97‐103, 1997.

The Gardos Channel Is Responsible for CDNB-Induced Dense Sickle Cell Formation

The red blood cells (RBCs) derived from blood taken from homozygous sickle cell (SS) patients demonstrate densities that are inversely proportional to the intracellular reduced glutathione (GSH) content. Addition of 1 mM 1‐chloro‐2,4‐dinitrobenzene (CDNB) to low‐density sickle cells (LDSS), at 4°C, results in a shift of LDSS erythrocytes to high‐density sickle cells (HDSS), with corresponding decreases in GSH. We have previously demonstrated that this CDNB effect was due to increased K+ leakage and that dense cell formation could be inhibited by clotrimazole (specific for the Gardos channel) but not DIOA (specific for the K+–Cl− co‐transport system) at pH 7.4 (Shartava et al. Am. J. Hematol. 1999;62:19–24). Here we demonstrate that clotrimazole (10 μM) inhibits dense cell formation at pH 7.1 and 6.8, while DIOA (1 mM) has no effect. As pH 6.8 is the optimal pH for the K+–Cl− co‐transport system, we can now reasonably conclude that damage to the Gardos channel is responsible for CDNB‐induced dense cell formation. Am. J. Hematol. 64:184–189, 2000.

Overlapping coefficient: the generalized t approach

The two group t test is generalized here to produce a hypothesis testing procedure on the overlapping coefficient of two normally distributed populations with common variance, assuming that the researcher knows the direction of the population means. The confi¬dence intervals are constructed on the overlapping coefficient. An illustrative example is given using the proposed procedures

Kinetics of ibuprofen effect on platelet and endothelial prostanoid release

Reciprocal control of platelet function in the circulation has been proposed for the platelet‐produced platelet proaggregatory prostanoid thromboxane A2 (TxA2) and the vascular endothelium‐produced antiaggregatory prostanoid prostacyclin (PGI2). Forty drug‐free healthy subjects were given a single dose of ibuprofen (0, 8, 10,12, or 14 mg/kg) in a randomized, double‐blind study. Blood samples were drawn 0, 2, 4, and 6 hours and 7 days after dosing for determination in serum (from untreated or in vitro indomethacin‐treated portions of the blood) of TxA2 and PGI2 by radioimmunoassay of their stable metabolites (TxB2 and 6‐keto‐PGFlα). Maximal platelet release of TxA2 (untreated serum) was lower in all drug groups 2, 4, and 6 hours after dosing. There was no significant decrease in PGI2 release. All doses of ibuprofen (except 0 mg/kg) induced essentially identical plasma levels at the times of measurement (postpeak decline), and effects could not be distinguished by dose for 8, 10, 12, or 14 mg/kg at these times. It is concluded that ibuprofen induces antiplatelet effects for at least 6 hours while preserving normal antiplatelet mechanisms.