The influence of uridine diphosphate glucuronosyl transferase 1A promoter polymorphisms, beta-globin gene haplotype, co-inherited alpha-thalassemia trait and Hb F on steady-state serum bilirubin levels in sickle cell anemia.

PURPOSE: Homozygosity for the (AT)7 allele of uridine diphosphate glucuronosyl transferase 1A (UGT1A1) gene polymorphism is associated with increased bilirubin levels in sickle cell anemia (SCA). In the present study, in addition to UGT1A1 promoter genotype, serum bilirubin level was related to other genetic modifiers -beta(S)-globin gene haplotype, Hb F, co-inherited alpha-thal trait, age and gender. METHODS: The patients were randomly selected from the sickle cell clinic, Medical College of Georgia. UGT1A1 promoter polymorphisms were determined using automated sequencing. Other investigations were with standard techniques. RESULTS: There were 67 SCA patients (41 males and 26 females), aged 2-44 yr (mean of 20.6 +/- 10.7). Ten (14.9%) patients were homozygous for the (AT)6 UGT1A1 allele, 35 (52.2%) were heterozygous for (AT)6 and (AT)7 alleles while 22 (32.8%) were homozygous for (AT)7. Serum bilirubin was significantly higher in the homozygous (AT)7 group (3.7 +/- 1.5, 3.8 +/- 2.3 and 5.6 +/- 2.4 mg/dL, respectively). It was also significantly higher in males than females and in patients aged >10 yr. There was a significant negative linear correlation (r = -0.304, P = 0.016) of serum bilirubin with Hb F. The beta-globin haplotype and co-existing alpha-thal trait did not have any significant influence on serum bilirubin levels. Patients on hydroxyurea were older, had lower Hb F, but higher mean serum bilirubin. The latter also was signifcantly higher among those with UGT1A1 (AT)7 homozygosity. CONCLUSIONS: Apart from UGT1A1 (AT)7 homozygosity, Hb F, age and gender are the other factors that significantly influence serum bilirubin level in SCA.

Regulation of primary Strongyloides ratti infections in mice: a role for interleukin-5.

C57BL/6 mice genetically deficient in interleukin-5 (IL-5-/-) and normal C57BL/6 (IL-5+/+) mice were infected with larvae of a homogonic strain of the nematode Strongyloides ratti. In primary infections both male and female IL-5-/- mice released two to four times more eggs and larvae than IL-5+/+ mice. IL-5-/- mice harboured about 60% more intestinal worms, which were more fecund, than IL-5+/+ mice. The duration of the infection was similar in normal and IL-5-deficient mice. Both IL-5-/- and IL-5+/+ mice resisted a secondary infection. IL5-/- mice lost more weight during the infection than normal mice and took longer to regain their initial weight after expelling the worms. The number of eosinophils increased in the bone marrow, peritoneal cavity and small intestine of IL-5+/+ mice, but not IL-5-/- mice, following infection. No significant differences between infected IL-5+/+ and IL-5-/- mice in mast cells or other leucocytes were observed in the peritoneal cavity. Thus, IL-5 functions to protect the host in a primary infection of S. ratti by limiting the number and fecundity of worms establishing in the small intestine. This protection is correlated with elevated blood and tissue eosinophilia which occurs in normal mice but not in IL-5-/- mice.

Long-term stroke risk in children with sickle cell disease screened with transcranial Doppler.

Stroke is an important complication of sickle cell disease. Stroke prediction is clinically important because it offers the possibility of primary prevention. In 1992, transcranial Doppler (TCD) evidence of elevated intracranial internal carotid or middle cerebral artery velocity was demonstrated to be associated strongly with an increased risk of ischemic stroke. This study extends the original study and includes 125 more children, longer follow-up, and intracranial hemorrhage in the stroke-risk model. Elevated time averaged mean maximum blood flow velocity, especially when velocity is 200 cm/sec or greater by TCD, was associated strongly with stroke risk. The cases not predicted by TCD point to the need for more information on the optimal timing of TCD surveillance for stroke risk.

Sickle cell disease and tonsillectomy. Preoperative management and postoperative complications.

BACKGROUND: Patients with sickle cell disease are recognized as having a relatively higher risk for postoperative complications, including fever, atelectasis, pneumonia, or sickle cell vas-occlusion. OBJECTIVE: To present a protocol for preoperative management of patients with sickle cell disease undergoing tonsillectomy, including the use of transfusions and intravenous hydration. DESIGN: Retrospective chart review. SETTING: Academic, tertiary care referral medical center. PATIENTS: Seventy-five patients with sickle cell disease who underwent tonsillectomy with or without adenoidectomy were included for review. Preoperative management was documented, and risk factors were assessed. Intraoperative management was reviewed, and postoperative complications were identified and compared with preoperative data and management. RESULTS: Preoperative management consisted of transfusions to a hemoglobin S ratio (hemoglobin S-total hemoglobin) less than 40% or a hemoglobin level greater than 100 g/L. Aggressive intravenous hydration of 1.5 times the maintenance fluid was given 24 hours before surgery. Increased complications were associated with a preoperative hemoglobin S ratio greater than 40% (P < .05) and an age younger than 4 years (P < .05). Operative time, technique, and blood loss were not statistically significant risk factors. The average length of hospitalization was 4.8 days. CONCLUSIONS: Children with sickle cell disease presenting for elective tonsillectomy should be given a transfusion to a hemoglobin S ratio less than 40% in an attempt to reduce postoperative complications. Additional factors, such as age and presence of obstructive sleep apnea, only increase the potential risks.

Inhibition of nitric oxide: effects on interleukin-1 beta-enhanced ovulation rate, steroid hormones, and ovarian leukocyte distribution at ovulation in the rat.

The ovulatory process resembles an inflammatory reaction with an infiltration of leukocytes, production of inflammatory mediators such as cytokines, and a general edema and hyperemia. Nitric oxide (NO), a potent vasodilator and the main mediator of macrophage tumoricidal and bacteriocidal activities, is known to participate in inflammatory reactions and has been shown to mediate the interleukin-1 beta (IL-1 beta)-directed tissue-remodeling events within the ovary. The regulation by NO of ovulation rate, leukocyte distribution, and steroid release in the rat ovary was investigated through use of a combination of in vivo and in vitro models of ovulation and a competitive inhibitor, N-omega-nitro-L-arginine methyl ester (L-NAME), of the NO synthase (NOS) enzyme. Subcutaneous L-NAME (1.5 x 10(-4) mol/kg) administration significantly reduced the in vivo ovulation rate of eCG/hCG-primed rats (L-NAME-treated: 10.6 +/- 1.8 [mean +/- SEM] oocytes per ovary [O/O], 11.0 +/- 1.2 rupture sites per ovary [RS/O]; saline-treated: 18.0 +/- 1.8 O/O, 19.4 +/- 1.1 RS/O; p < 0.01) at 20 h post-hCG. These results were reflected in vitro, where addition of L-NAME (3.5 x 10(-5) mol/L) to LH (0.1 microgram/ml)-perfused ovaries decreased ovulation rate from 8.2 +/- 1.6 to 2.7 +/- 1 ovulations per ovary (p < 0.05) and simultaneously decreased nitrite accumulation at the completion of perfusions from 16.5 +/- 1.9 to 4.1 +/- 0.5 nmol/ml (p < 0.001). The addition of L-NAME to LH+IL-1 beta (4 ng/ml)-perfused ovaries decreased ovulation rate from 15.2 +/- 2.4 to 0.8 +/- 0.8 ovulations per ovary (p < 0.001) and simultaneously decreased nitrite accumulation at 22 h from 22.8 +/- 2.2 to 1.9 +/- 0.6 nmol/ml (p < 0.001). Studies analyzing and manipulating perfusion flow rate indicated that the L-NAME effects on ovulation rate are primarily due to a reduction in flow rate resulting from inhibition of NO, which may be a consequence of the known vasoconstrictor effects of NOS inhibitors. The observed reduction of in vivo ovulation rate by NO inhibition at 20 h post-hCG was associated with a significant reduction in thecal MCA149+ neutrophils at 12 h post-hCG, the expected time of ovulation (L-NAME-treated: 98.4 +/- 9.2 cells per thecal area; saline-treated: 211.5 +/- 11.5 cells per thecal area; p < 0.001), while ED1+ monocytes/macrophages underwent similar but nonsignificant changes. Plasma (20 h post-hCG) and perfusate progesterone were not different with L-NAME treatment, while perfusate estradiol levels were markedly reduced upon addition of L-NAME, suggesting a role for NO in ovulation but not in the process of luteinization. In summary, deprivation of NO by use of the competitive inhibitor, L-NAME, led to fewer ovulations, reduced accumulation of nitrite, a decreased neutrophil count in the theca of preovulatory follicles, and reduced estradiol secretion, while progesterone release remained unaffected. The NO pathway may therefore play an important role in the regulation of ovulation and the mediation of IL-1 beta's pro-ovulatory effects. There are likely to be primarily vascular effects, but also a nonvascular component, to the NO regulation of ovulation, with both components indirectly affecting ovulatory leukocyte distribution and steroid secretion.

Alpha thalassemia and stroke risk in sickle cell anemia.

In an effort to identify possible risk factors for stroke in Sickle Cell Anemia (Hb SS), we analyzed the distribution of alpha-globin gene deletions in a group of Hb SS patients with and without stroke. The group with stroke consisted of 44 patients, (27 male, 17 female) with a mean of 7.5 years at time of stroke. The control group (non-stroke) had 256 Hb SS patients (126 male, 130 female) with a mean age of 7.7 years. There were 9 patients with heterozygous alpha-thalassemia in the stroke group (20.5%). In the control group, there were 93 patients with heterozygous alpha-thalassemia and 5 with homozygous alpha-thalassemia. The incidence of alpha-thalassemia in Hb SS patients without stroke (38%) was comparable to that reported for the African-American population in general. The incidence in the stroke population (20.5%) was significantly lower (P = 0.02) These results indicate that alpha-thalassemia is associated with a lower risk of stroke in Hb SS. This observation should be confirmed in studies involving larger numbers of patients. Possible protective effects of alpha-thalassemia are unknown but may be related to decreased hemolysis and more favorable rheologic properties of red blood cells.

Three sickle cell anemia patients each with a different alpha chain variant. Diagnostic complications.

We have studied three sickle cell anemia patients who also carried a heterozygosity for one of the following alpha chain abnormalities: Hb G-Philadelphia [alpha 68(E17)Asn-->Lys], Hb Montgomery [alpha 48 (CE6)Leu-->Arg], and Hb Chicago [alpha 136(H19)Leu-->Met]. Electrophoretic analyses alone may result in incomplete and incorrect information. Confirmation of the diagnosis of Hb SS or Hb SC disease by one of the fast high performance liquid chromatographic procedures is recommended.

Spleen in sickle cell anemia: comparative studies of Nigerian and U.S. patients.

Anecdotal reports have attributed persistent splenomegaly in African sickle cell anemia (SS) patients to the effects of malaria. However, no comparative studies of patients in malarial and nonmalarial regions have been conducted, and few studies of malaria antibody titers have been reported. In the present study, age- and sex-matched Nigerian patients (n = 310), while it was found only in 8% of U.S. patients (n = 100) from Georgia. There was significant linear correlation between spleen size and Hb levels and with serum immunoglobulins in the Nigerian group. However, serum complement levels (C3 and C4) were not affected by spleen size. In both groups, patients with splenomegaly had fewer circulating pitted red cells than their counterparts without splenomegaly. The mean +/- SE of IgG-specific malaria antibody titer among the Nigerian patients without palpable spleens was 9,386 +/- 2,036; 9,334 +/- 2,980 in those with spleens between 1 and 5 cm, 16,201 +/- 4,502 in those with spleens between 6 and 10 cm, and 22,445 +/- 8,456 in those with spleens above 10 cm. Coexistent alpha-thalassemia did not influence the prevalence of splenomegaly among the Nigerian SS patients. This study provides additional evidence that malaria plays a significant role in the persistence of splenomegaly in African patients.

Postnatal changes in the quantities of globin chains and hemoglobin types in two babies with Hb H disease.

We have studied two babies with Hb H disease from birth to about six months of age and analyzed the changes in the relative quantities of the five globin chains (zeta, alpha, beta, G gamma, A gamma) and the four hemoglobins (Hb F, Hb A, Hb Bart's, Hb H) using different high performance liquid chromatography procedures. The types of Hb H disease were -(SEA)/-alpha(3.7 kb) and -(Fil)/-alpha(3.7 kb); the larger -(Fil) deletion includes the functional zeta 2-globin gene, explaining the higher zeta chain level in the baby with the -(SEA)/-alpha(3.7 kb) type. The functional hemoglobin level at birth (Hb A+Hb F) was 11 to 12 g/dl with 3 to 4 g/dl Hb Bart's (gamma 4). Only 5% of the "fast-moving" hemoglobin was Hb H (beta 4). The level of Hb F at birth was low (less than 50% of the total Hb A+Hb F). After birth, the alpha and gamma chain production decreases rapidly resulting in a severe anemia (total functional hemoglobin approximately 7 g/dl) at 30 to 60 days postnatally, improving gradually to 8.5-9.5 g/dl at age of three months. The preferential formation of Hb A over Hb F at birth, and presumably prenatally, has the advantage that the level of the highly unstable Hb H is kept low; it also results in low levels of Hb F impairing the oxygen transfer capability of the fetal blood.

The use of transcranial ultrasonography to predict stroke in sickle cell disease.

BACKGROUND: Stroke, especially cerebral infarction, is a major cause of morbidity and mortality in children with sickle cell disease. Primary prevention of stroke by transfusion therapy may be feasible if there is a way to identify the patients at greatest risk. Transcranial Doppler ultrasonography can measure flow velocity in the large intracranial arteries. The narrowing of these arteries, which leads to cerebral infarction, is characterized by an increased velocity of flow. METHODS: Using transcranial Doppler ultrasonography, we prospectively measured the velocity of cerebral blood flow in children and young adults being followed because of sickle cell disease. The results were classified as either normal or abnormal on the basis of the highest velocity of flow in the middle cerebral artery. Abnormal velocity was defined as a flow greater than or equal to 170 cm per second, a definition determined by post hoc analysis to maximize the predictive success of the test. The end point was a clinically apparent first cerebral infarction. RESULTS: Two hundred eighty-three transcranial ultrasound examinations were performed in 190 patients with sickle cell disease (age at entry, 3 to 18 years). After an average follow-up of 29 months, cerebral infarction was diagnosed in seven patients. In 23 patients the results of the ultrasound examinations were abnormal, and in 167 patients they were normal. The clinical and hematologic characteristics of the two groups were similar, but six of the seven strokes occurred among the 23 patients with abnormal ultrasound results (P less than 0.00001 by Fisher's exact test). In this group, the relative risk of stroke was 44 (95 percent confidence interval, 5.5 to 346). CONCLUSIONS: Transcranial ultrasonography can identify the children with sickle cell disease who are at highest risk for cerebral infarction. Periodic ultrasound examinations and the selective use of transfusion therapy could make the primary prevention of stroke an achievable goal.

A new variant, HB Muscat [alpha 2 beta (2)32(B14)Leu----Val] observed in association with HB S in an Arabian family.

The silent Hb Muscat with a Leu----Val replacement at position beta 32 was discovered by reversed phase high performance liquid chromatography in two members of an Arabian family from Oman; in one person Hb Muscat occurred with Hb S and in the other with Hb A. Hb Muscat is slightly unstable but its presence has no apparent adverse effect on the health of its carriers. Additional hemoglobin abnormalities observed in this family were a common alpha-thalassemia-2 (-3.7 kb) and Hb S. The beta S haplotypes in the heterozygous carriers and the two sickle cell anemia patients were #19 (Benin) and #20 (Bantu); the latter likely originated from an East African population.

Molecular characterization of Hb S(C) beta-thalassemia in American blacks.

An extension of previous reports describing the molecular defects and hematological abnormalities in black patients with Hb S(C) beta-thalassemia living in the Southeastern United States is presented. As many as 58 patients with Hb S-beta(+)-thalassemia, 16 with Hb C-beta(+)-thalassemia and 12 with Hb S-beta(0) -thalassemia have been studied. Patients with Hb S(C) beta(+)-thalassemia type 2 (high Hb A values) were most common; the thalassemia was due to mutations in the promoter of the beta-globin gene [-88 (C----T) and -29 (A----G)] or at the polyadenylation signal (T----C). Two patients with lower Hb A values (type 1) carried a mutation in the first intron of the beta-globin gene (IVS-1-5: G----T). The simultaneous presence of an alpha-thalassemia -2(-alpha/) resulted in some modifications of the hematological parameters, but had a minimal effect on the clinical condition. Patients with Hb S-beta (0) thalassemia had lower hemoglobin values, lower number of red blood cells, and lower MCHC values and suffered more frequently from complications than the patients with Hb S-beta(+)-thalassemia. A total of 17 different beta-thalassemia mutations were observed in 128 chromosomes; two mild beta(+)-thalassemia mutations [-88(C----T) and -29(A----G)] account for more than 80% of the thalassemic chromosomes.

Cerebral vessel stenosis in sickle cell disease: criteria for detection by transcranial Doppler.

Ischemic stroke is a common and disabling complication of sickle cell disease (Hb SS). Most infarctions occur in the presence of intracranial stenotic lesions of the large vessels of the circle of Willis. Transcranial Doppler (TCD), by measuring flow velocity in these arterial segments, can detect focal stenosis on the basis of elevated flow velocity. We report the preliminary results of a prospective study to develop criteria for detection of stenotic lesions based on TCD and identification of patients with Hb SS at risk for stroke. Comparing the TCD findings from six patients with lesions demonstrated by angiography to those from 115 Hb SS children without stroke, we conclude: (a) middle cerebral (MCA), anterior cerebral (ACA), or internal carotid (ICA) artery mean velocities greater than 190 cm/s strongly suggest focal stenosis; (b) MCA or ACA mean velocities of 150 to 190 cm/s suggest abnormality but at present cannot be considered diagnostic of stenosis; (c) mean velocities up to 150 cm/s are possibly due to the effects of low hematocrit and/or young age, and cannot as yet be distinguished from velocity elevations due to vessel stenosis.

Neuropsychologic impairment in children with sickle cell anemia.

In this study, the neuropsychologic functioning of 21 children with sickle cell anemia and 21 sibling controls, age range 7 through 16 years, with no history of neurologic disease, was examined. Outcome measures included tests of intelligence, constructional praxis, memory, and academic learning. On the Wechsler Intelligence Scale for Children--Revised, the sickle cell group had a mean Full Scale IQ of 77.7 (SD 12.4) compared with 94.3 (SD 11.0) for the control group. The profile of test scores was similar for the two groups, with the sickle cell group scoring significantly lower than the control group on almost all cognitive measures. Both groups showed academic achievement to be commensurate with their measured intellectual ability. These results suggest that subtle but significant and widespread neuropsychologic deficits are associated with sickle cell anemia even in the absence of neurologic complications. When and by what process this neuropsychologic impairment is caused needs to be determined.

Severe Hb S-beta zero-thalassaemia with a T----C substitution in the donor splice site of the first intron of the beta-globin gene.

Through direct sequencing and dot-blot analyses with synthetic oligonucleotide probes of amplified DNA, a new nucleotide substitution was discovered in a Black teenager with severe Hb S-beta zero-thalassaemia. The substitution involved a T----C replacement at the second position of the donor splice site of the first intervening sequence of the beta-globin gene. The clinical and haematological observations made in Black subjects with Hb S-beta zero-thalassaemia, with different types of thalassaemia, suggest severe disease resembling sickle cell anaemia. Only an occasional patient had a milder clinical course, perhaps because of a greatly increased production of fetal haemoglobin.

Cerebral infarction in sickle cell anemia: mechanism based on CT and MRI.

We studied 25 patients with sickle cell anemia and cerebral infarction. We classified lesions as to probable mechanism (large versus small vessel disease) based on the CT/MRI appearance of established infarction. Most patients had CT/MRI patterns of major cerebral vessel occlusion (41%) or border-zone (distal insufficiency) infarcts (31%) best explained by large cerebral vessel vasculopathy. Seven of 25 (28%) had either isolated subcortical (12%) or small cortical branch occlusion (16%) consistent with other mechanisms such as small vessel occlusion or embolism. These results suggest that most clinically recognized cerebral infarctions in sickle cell anemia are caused by large vessel disease, but this mechanism may not account for symptoms of cerebral ischemia in all cases.

Effects of alpha-thalassemia-2 on the developmental changes of hematological values in children with sickle cell disease from Georgia.

The hematology and pathophysiology of sickle cell disease during the postnatal development of younger hemoglobin (Hb) S homozygotes (SS) could be considerably affected by a variability of alpha globin gene numbers. We have documented longitudinal developmental changes of hematological values and hemoglobin composition on 147 patients with SS (alpha alpha/alpha alpha), 64 with SS (-alpha/alpha alpha), and 9 with SS (-alpha/-alpha) between the ages of 1 and 15 years. Non-steady-state data were excluded from these analyses. The number and organization of alpha globin genes was established by gene mapping. As anticipated, mean corpuscular volume and erythrocyte counts correlated with alpha globin gene numbers throughout the 15-year age interval. On the other hand, SS children with alpha alpha/alpha alpha, -alpha/alpha alpha, -alpha/-alpha had similar hemoglobin concentrations up to the ages of 5-10 years. Around the age of 7, the SS patients with -alpha/-alpha developed a higher Hb concentration than that of the SS (-alpha/alpha alpha), which in turn was higher than that of the SS (alpha alpha/alpha alpha). The emergence of this difference coincided with a developmental increase of the mean corpuscular hemoglobin concentration (MCHC) in patients with SS (alpha alpha/alpha alpha) and the decline of Hb F % under 15%. This newly observed developmental change of the MCHC could lead to increased hemolysis and anemia after the age of 5-10 years. It occurs to a smaller extent among SS (-alpha/alpha alpha) or not at all among SS (-alpha/-alpha) such that these two categories of patients have less severe hemolysis and higher hemoglobin levels at older ages. Although the proportion of Hb F was independent of alpha globin gene numbers, the absence of Hb Bart's suggested that alpha-thalassemia promotes the intracellular assembly of Hb F over Hb S tetramers. Thus, the interaction of alpha-thalassemia and Hb F in young SS patients may be more complex than revealed by Hb F levels in cell lysates. Among older SS children (greater than 7 years) alpha-thalassemia and Hb F levels exceeding 15% appear to have additive effects in diminishing the rate of hemolysis.

MR and CT investigation of cerebrovascular disease in sickle cell patients.

Stroke is a common complication of sickle cell disease. Using MR and CT, we studied 10 patients with sickle cell disease and a history of stroke and compared these findings with those of 10 sickle cell patients without stroke. The purpose was to determine if MR could visualize the large vessel vasculopathy previously seen on angiography and to estimate the incidence of asymptomatic abnormalities in the nonstroke group. MR consistently demonstrated the major intracranial arteries and showed three cases with occlusion and three with stenosis of either the internal carotid or middle cerebral arteries. Infarctions were better delineated on MR but were also seen on CT. Seven cases with and two without stroke had high-signal white matter lesions on MR. Further research using cranial MR to develop noninvasive means of identifying sickle cell patients at risk for stroke is warranted.

Different zeta globin gene deletions among black Americans.

Four types of chromosomes with a deletion between the human embryonic zeta and psi zeta globin genes were identified among 2.8% of 321 Black Americans from Georgia. Two deletions of approximately 11 kb which differed by about 300 bp occurred on chromosomes with or without a polymorphic Xba I site 5' to the zeta globin gene [(X+) or (X-)]. The deletions are identifiable in Xba I digests of genomic DNA using an alpha or a zeta globin gene probe which yield fragments of 23 kb from (X+)-zeta alpha alpha chromosomes or 27 kb from (X-)-zeta alpha alpha chromosomes. Digestion with other enzymes and probing with both alpha and zeta probes gave fragments typical of the two zeta globin gene deletions previously identified in Polynesians. Among Black Americans, these zeta globin gene deletions have been found in combination with alpha globin gene deletions in trans but not in cis. Homozygotes have not been found. Hematologic data on carriers of the zeta globin gene deletions in association with Hb AS, SS, and SC suggest that these deletions have no effect on the function of the adult alpha globin genes.