Biomedical subjects
Publications and source records attributed to E Beutler.
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The effect of five different transferrin variants (TFv1, TFv2, TFv3, TFv4, and TFv5) on the hemoglobin level, mean corpuscular volume (MCV), ferritin level, percent transferrin saturation (%TS), and the unsaturated iron binding capacity (UIBC) was investigated in subjects with defined HFE haplotypes, 919 persons undergoing health screening and 113 patients with clinical hemochromatosis. The most common variant is TFv4; the population distribution of this variant was also studied. None of the variants were found to have an effect on any of the parameters of iron metabolism that were investigated. Moreover, the frequency of these variants in patients with clinically significant hemochromatosis was no different from that in the general population. We conclude that these polymorphisms in transferrin do not play a role in the expression of hemochromatosis, nor do they produce any other significant changes in iron metabolism.
Three mutations in the transferrin receptor-2 gene have recently been identified in four Sicilian families with iron overload who had a normal hemochromatosis gene, HFE (C. Camaschella, personal communication). To determine the extent to which mutations in the transferrin receptor-2 gene occur in other populations with iron overload, we have completely sequenced this gene in 17 whites, 10 Asians, and 8 African Americans with iron overload and a C282C/C282C HFE genotype, as well as 4 subjects without iron overload and homozygous for the mutant HFE C282Y genotype, 5 patients with iron overload and homozygous for the mutant HFE C282Y genotype, and 5 normal individuals. None of the individuals exhibited the Sicilian mutations, Y250X in exon 6, M172K in exon 4, and E60X in exon 2. One iron-overloaded individual of Asian descent exhibited a I238M mutation which was subsequently found to be a polymorphism present in the Asian population at a frequency of 0.0192. The presence of the I238M mutation was not associated with an increase in ferritin or transferrin saturation levels. Three silent polymorphisms were also identified, nt 1770 (D590D) and nt 1851 (A617A) and a polymorphism at nt 2255 in the 3' UTR. Thus, mutations in the transferrin receptor-2 gene were not responsible for the iron overload seen in our subjects.
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When rhinoceros species that are browsers in the wild are fed in captivity they become iron overloaded. Presumably, their iron-absorptive mechanisms have evolved to become highly efficient. In humans, mutations of the HFE gene cause increased iron absorption. To determine whether the HFE gene of rhinoceroses has undergone mutation as an adaptive mechanism to improve iron absorption from iron-poor diets, we have sequenced the entire coding region of the HFE genes of four species of rhinoceros. Two of these were browsing species and two were grazing species. Although the HFE gene has been well preserved across species, numerous nucleotide differences were found between rhinoceros and human or mouse, some of which changed deduced amino acids. Of these mutations, only one found in the black rhinoceros appears to be a viable candidate mutation that might adversely affect HFE function. This mutation, S88T, is in a highly conserved region that is involved in the interaction between transferrin receptor and HFE.
We have identified five single nucleotide polymorphisms (SNPs) upstream (5') of the transferrin coding region. One polymorphism is in the 5' UTR at nt +49, and four are in the promoter region at nt -34, -551, -617, and -739, numbering from the start of transcription. The -34 and -617 SNPs are tightly but not completely linked. The -34 polymorphism lies between a conserved Sp1 site and the TATA box. The -617 polymorphism is within the DRII enhancer region. Five haplotypes have been defined from these SNPs by the identification of at least one homozygous individual, and two other haplotypes were deduced from heterozygous individuals. The total iron-binding capacity associated with each transferrin haplotype was haplotype 2 > 1 > 4 > 3. Transferrin promoter haplotype 2 had a significantly higher mean TIBC and haplotype 3 had a significantly lower mean TIBC than the more common haplotype 1. Persons with haplotype 4, which includes the -34T and -617A minor alleles, have a lower mean TIBC but the difference was not statistically significant. In normal individuals, the differences in the haplotypes were not found to be associated with differences in transferrin saturation and ferritin levels. There was no difference in the extent of increase in the mean TIBC levels in individuals with iron deficiency anemia in regard to their haplotype. Furthermore, there was no difference in the relative frequencies of the transferrin haplotypes in the iron-deficient population. In hemochromatosis patients who were homozygous for the C282Y HFE mutation, no particular haplotype was associated with a significant difference in transferrin saturation or ferritin levels. In White patients with Parkinson's disease, a disorder in which there is abnormal iron deposition in the brain, the presence of transferrin haplotype 3 was in slight excess over the normal White population.
BACKGROUND: The Toll (Tlr) receptors facilitate innate immunity by detecting products that are unique to invading microorganisms. Stimulation of these receptors can produce severe reactions and death. We propose that synergy between receptors for different microbial products would provide a safety mechanism, preventing inappropriate, potentially fatal reactions by reacting to low concentrations of ligands when more than a single ligand is present. RESULTS: Striking synergy is noted between the ligand for Tlr4, lipopolysaccharide (LPS), and a ligand for Tlr2, muramyl dipeptide (MDP), in the release of tumor necrosis factor from RAW cells. CONCLUSIONS: Synergy between a ligand for Tlr2 and Tlr4 can be demonstrated in a simple in vitro system. The greater sensitivity of MDP-stimulated cells to LPS may explain the data that were once interpreted incorrectly as indicating that Tlr2 is the endotoxin receptor.
We have examined transferrin receptor-1, ferroportin, ceruloplasmin, ferritin light and heavy chains, iron regulatory proteins (IRP)-1 and -2, and hepcidin for mutations that might modulate the iron burden of individuals harboring the common mutant hemochromatosis HFE genotype C282Y/C282Y or cause hemochromatosis independent of mutations in the HFE gene. In a group of white, Asian, and African-American normal and iron-overloaded individuals, the coding and flanking regions of these genes were completely sequenced. Numerous coding region and promoter polymorphisms were detected. These were further examined for association with differences in iron accumulation as measured by plasma transferrin saturation and ferritin levels, but no such association could be documented.
A new spontaneous mutation in the A/J inbred mouse strain, downeast anemia (dea), causes severe hemolytic anemia with extensive tissue iron deposition and marked reticulocytosis. The anemia is present at birth and persists throughout life. The defect is inherited as an autosomal recessive and is transferable through bone marrow stem cells. The red cell morphology is consistent with a nonspherocytic hemolytic anemia, suggestive of a red cell enzymopathy. In linkage analysis, dea is nonrecombinant with the hexokinase-1 gene (Hk1) on mouse Chromosome 10. Expression of Hk1 is markedly decreased in dea erythroid tissues, and the transcript produced is larger than normal. Hexokinase enzyme activity is significantly decreased in dea tissues, including red cells, spleen, and kidney. Southern blot analyses revealed approximately 5.5 kb of additional sequence in the 5' portion of the dea Hk1 gene, which was identified by direct sequencing as an early transposon (ETn) insertion in intron 4. ETn insertions disrupt genes in several mouse models by a variety of mechanisms, including aberrant splicing of ETn sequences into the mRNA. We conclude that the primary gene defect in the dea mutation is in Hk1 and that dea is a model of generalized hexokinase deficiency, the first such model identified to date.
Glucose-6-phosphate dehydrogenase (G6PD) levels are not usually drawn in the evaluation of black neonates with hyperbilirubinemia because of the oft-stated opinion that the levels may be normal at the time of hemolysis and thus will be misleading. In fact, this opinion is not applicable to newborns as many studies have shown that deficiency in the conjugating ability of the liver, not hemolysis, is the main cause of neonatal jaundice associated with G6PD deficiency. We present a case report of a neonate with brisk hemolysis and hyperbilirubinemia in whom the G6PD level was abnormally low at the time of the hemolytic episode. DNA analysis showed him to have the A-(202A,376G) variant and, as well, the UGT1A1 promoter repeat polymorphism associated with Gilbert's disease. This case, as well as a review of the literature, indicates that enzyme levels are not normal in patients with G6PD A- who are undergoing hemolysis.
We conducted an 18-month, placebo-controlled, double-blind study to evaluate cladribine in the treatment of 52 patients with relapsing-remitting multiple sclerosis. Patients received either placebo or cladribine 0.07 mg/kg/day by subcutaneous injection for 5 consecutive days as six monthly courses for a total cumulative dose of 2.1 mg/kg. Analysis of results revealed a statistically significant favorable effect of cladribine on the joint frequency and severity of relapses and magnetic resonance imaging (MRI) findings. MRI-enhancing lesions were completely suppressed in the cladribine patients by the sixth month of treatment. Mild segmental herpes zoster occurred in two cladribine-treated patients and one patient receiving placebo. Otherwise, there were no side effects or adverse events. We conclude that cladribine shows promise as a treatment for relapsing-remitting multiple sclerosis.
In vitro metabolism of erythrocytes packed at 70 and 90 per cent hematocrits and stored in various CPD-adenine preservatives was studied. It was found that maintenance of acceptable levels of adenosine triphosphate (ATP) for the full 42 days of storage could be accomplished only if glucose levels were doubled from the standard 138.7 mM concentration to 277.5 mM level. If glucose levels were doubled, the amount of adenine could be decreased from 4.07 mM (0.50 mM final concentration) to 2.04 mM (0.25 mM final concentration) with maintenance of ATP at greater than 2.0 mumoles/g Hb. 2,3-diphosphoglycerate concentrations were essentially absent by 21 days in the various media studies. Thus, in vitro levels of ATP appear to be maintained at acceptable levels in a CPD media modified to contain 2 times glucose and 2.04 mM adenine.
Agitation of blood stored in plastic containers has been reported to lead to improved posttransfusion survival and it has been found that, in some media, agitation has improved erythrocyte 2,3-diphosphoglycerate (2,3-DPG) levels. Using CPD II media (CPD with 277.5 mM glucose and 2.04 mM adenine), we were not able to identify any improvement in levels of adenosine triphosphate, 2,3-DPG or glucose in whole blood under various agitation conditions when compared with nonagitated control. The 2,3-DPG level was moderately improved through 28 days in the 90 per cent hematocrit packed erythrocytes but the results were not considered to be significantly beneficial to warrant agitation. Thus, the application of agitation to the CPD II blood storage system was of no great benefit in improving metabolic intermediate levels.
In order to provide data in support of licensure applications for citrate-phosphate-dextrose (CPD) supplemented with adenine, a multi-institutional cooperative effort was organized to determine survivability of red blood cells subjected to prolonged liquid storage. Two manufacturers supplied plastic multiple bag blood storage containers prefilled with modified CPD (glucose 25% greater than the normal concentration) supplemented with adenine (17.0 to 17.3 mg per 63 ml of anticoagulant; 0.25 millimolar approximate final concentration when diluted with 450 ml of whole blood for 35 days showed a mean survival of 80.53 +/- 6.44 per cent (1 SD). Both red blood cell and supernatant plasma biochemical characteristics were comparable to those reported for whole blood stored for 21 days in either acid-citrate-dextrose (ACD) or CPD. Red blood cells from 19 units stored as concentrates for 35 days (Hct 75.03 +/- 3.74%) had a mean survival of 71.38 +/- 10.3 per cent with considerable interdonor variation in survival and interlaboratory variation in some biochemical characteristics. Red blood cells from eight units stored as concentrates (Hct 75.38 +/- 4.30%) for 28 days showed a mean survival of 83.97 +/- 6.10 per cent and biochemical characteristics comparable to those reported for red blood cell concentrates stored in CPD or ACD for 21 days. Modified CPD with adenine as formulated offers an improved anticoagulant for blood banking by extending the permissible red blood cell storage period.
Sixteen glycolytic enzymes were measured in essentially leukocyte-free platelets before storage and daily after room temperature storage or platelets as concentrates. Most of the glycolytic enzymes proved to be very stable to storage. Even the most severely affected enzymes, enolase, triosephosphate isomerase, and lactate dehydrogenase lost less than one-quarter of their enzyme activity. Depletion of glycolytic enzymes does not seem to be a major factor in the storage lesion of platelets.
The metabolism of freshly collected platelets and platelets stored for three or four days at room temperature has been investigated. The levels of glycolytic intermediates were measured in platelets before and after incubation for 15 minutes in a buffered aerobic medium. Platelet intermediates were relatively well maintained, and levels of intermediates that were partially depleted, such as ATP, were restored toward normal after short term incubation even after storage for four days. Incorporation of 32PO4 into fresh and stored platelets during incubation in aerobic buffered medium was also studied. The incorporation pattern was only very modestly influenced by storage. These studies suggest that changes in glycolytic capacity do not play a major role in the loss of viability of platelets during storage at room temperature.
The metabolism of exogenous citrate by freshly collected human platelets has been investigated by measuring the disappearance of citrate from platelet suspensions and by measuring the formation of CO2 and glycogen from purified radioactive citrate. These studies indicate that platelets do not have the capacity to metabolize exogenous citrate.
Platelet concentrates were prepared from blood collected in CPD, CPD-A1, and CPD-A3. CPD-A1 contains 1.25 times as much glucose as does CPD, and sufficient adenine to provide a final concentration of 0.25 mM; CPD-A3 contains twice as much glucose as does CPD, and sufficient adenine to provide a final concentration of 0.5 mM. The consumption of glucose and the production of lactate and pyruvate in the platelet concentrates during room temperature storage was not influenced by glucose or adenine concentration. There was no difference in the rates of fall of the pH of platelets collected in the three preservatives. However, the number of contaminating white blood cells had a significant effect on these parameters. It is suggested that minimizing the number of contaminating white blood cells in platelet preparations may prove to be an important factor in platelet preservation.