Hemoglobin Koln in a Black: Preand PostSplenectomy Red Cell Survival (DF32P and 51Cr) and the Pathogenesis of Hemoglobin Instability

A 17-yr-old black male with hemolysisand pigmenturia but no anemia was foundto have hemoglobin Köln (₂₂^{98 valmet}[FG5]). Splenectomy was done because ofcomplicating thrombocytopenia. Thrombokinetic studies with ⁵¹Cr tagged plateletssuggested hypersplenism, and after surgerythe platelet count returned to normal. Thered cell t ⁵¹Cr was more than doubled,but the red cell life span (DF³²P) wasmore modestly improved (30.6 47.2days). The "elution" of ⁵¹Cr from the redcells presplenectomy was 5.6%/day,whereas after surgery it was normal(1.9%/day), accounting for the disparitybetween the survival methods. Study of theisolated cyanferri derivative of hemoglobinKöln by ultracentrifugation at various saltconcentrations and various pH’s indicatedan increased tendency to dimer formationunder conditions where normal hemoglobin is a tetramer. This results from thesite and type of amino acid substitution andaccounts in part for its instability.

Submitted on January 8, 1973 Revised on April 30, 1973 Accepted on May 4, 1973     

Hemoglobin L Ferrara in a Jewish Family Associated with a Hemolytic State in the Propositus

A Jewish family in which Hb L Ferrara (₂^{47 Asp Gly} ₂) occurred is reported. Studies of some of the properties of this hemoglobin demonstratedthat its oxygen equilibria, number of readily reactive-SH groups, and spectro-photometric tyrosine titration were indistinguishable from Hb A. Nevertheless,Hb LF was more unstable than Hb A at 55 C. The propositus had acceleratedblood destruction although six other heterozygotes for Hb LF did not. Asecond defect in red cell enzymes or red cell lipids of the propositus was notdemonstrable with the technics used but the possibility that the simultaneousoccurrence of Hb LF and an otherwise "silent" red cell defect may lead to ahemolytic state remains an attractive explanation. The data provided by thisfamily study did not permit a definite conclusion about the relationship ofclinically evident hemolysis in the propositus to the presence of the abnormalhemoglobin.

Submitted on January 21, 1969 Accepted on April 7, 1969     

Hemoglobin D Iran agr2A beta222-GlurarrGln in Association With Thalassemia

A 25-yr-old Indian (Asiatic) woman investigated for a life-long anemia was foundto have a hitherto undescribed structuralhemoglobin variant ₂^A₂^22-glugln, whichwas found independently and designatedHb D Iran by Rahbar in members of afamily from Iran. In the present case, Hb DIran was found in association with high A₂thalassemia. The replacement of glutamicacid by glutamine at 22 (helical residueB4) was demonstrated by thin-layer chromatography after automated Edman sequencing. This is the fourth substitution tobe described at 22; the previous substitutions were (1) G Coushatta (ala); (2) ESaskatoon (lys); (3) G Taipei (gly). Helicalresidue B4 is an external residue, does notparticipate in - or protein-heme contactsand hemoglobin D Iran resembles Hb A invisible spectra, O₂ equilibria in dilute solutions and heat stability. Since the propositus and her mother who was also heterozygous for thalassemia had similardegrees of anemia, no interaction betweenthis variant and thalassemia was evident.The presence of this Hb variant in Iraniansand in Western Indians may reflect themigrations of populations in these areascenturies ago.

Submitted on November 15, 1972 Revised on February 5, 1973 Accepted on February 6, 1973     

Hemoglobin Rush [beta101 (G3) Glutamine]: A New Unstable Hemoglobin Causing Mild Hemolytic Anemia

A mild hemolytic anemia in a 43-yr-old blackwoman was attributed to the presence of an abnormal hemoglobin (Hb Rush) which migratedcathodically to Hb A at pH 8.0. Its structuralabnormality was found to be in the -chain,101 (G3) glu gln. Another electrophoreticband at pH 8.0 proved to be a hybrid tetramer(^A₂^ARush). Hb Rush is heat unstable. Alikely explanation of the instability is thepresence of an uncovered positive charge in thecentral cavity where normally glutamic acid inposition 101 neutralizes arginine in position104 contributing to the net neutrality in thisregion. This neutrality is disturbed by the substitution of glutamic acid by glutamine in HbRush.

Submitted on May 10, 1973 Revised on June 25, 1973 Accepted on July 16, 1973     

Further Cases of Hb Q-H Disease (Hb Q-agr Thalassemia)

Case reports of four patients, all Chinese, with Hb Q-H disease—also calledHb Q- thalassemia—are presented. Three were siblings. Symptoms of chronichemolytic anemia with jaundice and hepatosplenomegaly were present in allfour subjects. The red blood cells were microcytic. Slight hypochromia waspresent in three of the cases. Poikilo- and anisocytosis with target cells andsmall intraerythrocytic crystals were found in the blood. Starch-gel electrophoresis revealed the presence of a large amount of Hb Q, a small amount ofHb H, and a minor slow-moving hemoglobin component with a mobility asmuch behind Hb A₂ as Hb Q was behind Hb A. A small amount of Hb "Bart’s"was probably also present. The minor slow-moving component was thought torepresent Hb ₂^Q₂^A₂ or Hb Q₂. Hb A and Hb A₂ were not seen except afterrecent blood transfusion. Study of hemoglobin polypeptide chains showed thepresence of normal ^A-chains and abnormal ^Q-chains, without demonstrable^A-chains in the first three patients. In Patient #4 normal ^A-chains weredemonstrable only after recent blood transfusion. The mother of the threesiblings was heterozygous for Hb A; the father had -thalassemia trait.

Submitted on September 7, 1965 Accepted on April 23, 1966     

Synthesis of Hemoglobin Abraham Lincoln (beta 32 leu rarr pro)

Globin chain synthesis was studied in vitrowith reticulocytes from a patient heterozygous for Hb Abraham Lincoln, an unstable beta chain variant. Synthesis of-chains by the reticulocytes exceededtotal -chain synthesis, and a substantialfraction (about 16%) of radioactivityincorporated into globin was recoveredfrom the cells as uncombined subunits.In a time-course study, the ratio of : -chain specific activity was found to increase progressively in a nearly linearmanner, suggesting that a fraction ofnewly synthesized -chains had undergone rapid destruction. The specific activity of the abnormal -chain was nearlythree times that of ^A. The rate of synthesis of the -chain of Hb AbrahamLincoln appeared to be approximately halfthat of the -chain of Hb A.

Submitted on September 13, 1973 Revised on November 1, 1973 Accepted on November 6, 1973     

Bone Marrow and Peripheral Blood Globin Synthesis in an American Black Family With Beta Thalassemia

Synthesis of globin chains in bonemarrow and peripheral blood samplesfrom a black family with mild betathalassemia was compared with similar studies in white people. Blood andbone marrow were incubated with ¹⁴C-leucine, globin chains were isolated,and / and / ratios were calculated.The results of studies of globin synthesis in homozygotes of differentraces were similar, despite the differences in severity of clinical disease. Inthe heterozygotes, there was a significant defect in beta synthesis in theperipheral blood of white subjects,while in two of three black patients the/ ratio was in the normal range. Although there was no evidence of segregation of an alpha thalassemia genein this black family to explain the unusual / ratios, the presence of sucha gene in the heterozygotes could notbe excluded.

Submitted on September 2, 1971 Revised on November 5, 1971 Accepted on December 15, 1971     

Hemoglobin Andrew-Minneapolis agrA2beta144 Lys rarr Asn2: A New High-Oxygen-Affinity Mutant Human Hemoglobin

The functional properties and primarystructure of a new -chain mutant ofhuman hemoglobin are described. Themutant was transmitted as an autosomaldominant characteristic. Affected members of the kindred exhibited markederythrocytosis due to the high oxygenaffinity of the resultant hemoglobin. Theabnormality is associated with a substitution of an asparaginyl residue for lysinein the 144 position of the -chain,^A₂^144LysAsn₂, presumably due to anAAA/G to AAA/U transversion. The mutant hemoglobin displayed a profound increase in oxygen affinity, with a P₅₀ of thefresh whole blood of 14 mm Hg. The isolated mutant hemoglobin exhibited nearnormal heme—heme interaction, a half-normal Bohr effect, and normal reactivitywith 2,3-diphosphoglycerate.

Submitted on January 17, 1974 Accepted on April 15, 1974     

Autosomal recessive chronic granulomatous disease caused by defects in NCF-1, the gene encoding the phagocyte p47-phox: mutations not arising in the NCF-1 pseudogenes

Chronic granulomatous disease (CGD) is a primary immunodeficiencycaused by defects in any one of 4 genes encoding phagocyte NADPHoxidase subunits. Unlike other CGD subtypes, in which there isgreat heterogeneity among mutations, 97% of affectedalleles in patients previously reported with A47⁰ CGDcarry a single mutation, a GT deletion (GT) in exon 2 of thep47-phox gene, NCF-1. This unusually highincidence results from recombination events between NCF-1and its highly homologous pseudogenes, in which GT originates. In 50 consecutive patients with A47⁰ CGD, 4 were identified whowere heterozygous for GT in NCF-1, and for the firsttime, 2 were identified whose DNA appeared normal at this position. Toavoid co-amplification of pseudogene sequence and to enable theidentification of mutations in these patients, allele-specificpolymerase chain reaction was used to amplify alleles not containingGT. In each of the 4 patients who were heterozygous for GT, anadditional novel mutation was identified. These were 2 missensemutations, G125  A in exon 2 (predicting Arg42  Gln)and G784  A in exon 8 (Gly262  Ser), and 2 splice junction mutations at the 5' end of intron 1, gt  at and gtg  gtt. The first of 2 patients who appeared normal at the GT position was a compound heterozygote with the G125  A transition on one allele and a deletion of G811 on the other. In the second of these patients, only a single defect was detected, G574  A,which predicts Gly192  Ser but is likely to result indefective splicing because it represents the final nucleotide of exon 6.     

Sickle Hemoglobin in Combination With Hb JBangkok (agrA2beta56 glyrarrasp2)

Several members of a black family fromSouthern Illinois were found to be heterozygous for HB J^Bangkok (^A₂^{56 glyasp}₂), ahemoglobin abnormality previously described only in individuals of Thai, Chinese, or Indonesian ancestry. In two children (ages 3 and 8) Hb J^Bangkok waspresent in combination with sickle hemoglobin. Neither of these children demonstrated evidence of hemolytic disease,enlargement of the liver or spleen, orsymptomatic sickle crises. The Hb J comprised 54%-59% of the total in all familymembers having this variant, in commonwith previous reports of this hemoglobin.Hb A₂ and alkali-resistant hemoglobinwere present in normal concentrations inall of the family members studied. Deoxygenated mixtures of Hbs S and J^Bangkokexhibited minimum gelation concentrations similar to those of equivalent mixtures of Hbs S and A.

Submitted on March 27, 1974 Accepted on May 14, 1974     

CD4+ CD25+ Treg cells inhibit human memory gammadelta T cells to produce IFN-gamma in response to M tuberculosis antigen ESAT-6

T cells play an important role in innate immunity against infections; however, the regulation of these cells remains largely unknown. In the present study, we show that ESAT-6, an antigen of Mycobacterium tuberculosis, induces IFN- secretion by human T cells. In addition, ESAT-6 also induces the activation and proliferation of T cells. Phenotypic analysis indicates that IFN-–producing T cells are mainly effector memory cells with the surface phenotype of CD45RA^–CD62L^–CCR7^–. These results were further confirmed by the fact that naive T cells from cord blood did not produce IFN- in response to ESAT-6. Further studies indicated that stimulation with ESAT-6 directly induced purified T cells to produce IFN-, independent of both antigen-presenting cells and CD4⁺ T cells. Unexpectedly, depletion of CD4⁺ T cells markedly enhanced IFN- production by T cells, indicating that CD4⁺ T cells regulate the response of T cells. Importantly, CD4⁺CD25⁺ T regulatory (Treg) cells but not CD4⁺CD25^– T cells significantly inhibited IFN- production by T cells. Taken together, these data demonstrate for the first time that Treg cells can play an important role in the regulation of immune responses of antigen-specific human memory T cells.     

The Binding of Cr51 to Hemoglobin I. In Vitro Studies

By a number of different technics, confirmatory evidence has been obtainedthat Cr⁵¹ activity of Hb A₁ tagged with Na₂Cr⁵¹O₄ in a ratio of approximately1 mole chromium to 1000 mole of hemoglobin is largely associated with the polypeptide chains. No significant difference in the site or strength of chromiumbinding could be demonstrated in abnormal hemoglobins resulting from pointamino acid substitutions (Hbs S and C).

The results obtained with Hb F were very suggestive of chain binding,but an apparently weak Hb F-Cr⁵¹ bond or increased elution rate markedlyhampered such studies.

The propensity of Cr⁵¹ for preferential tagging of chains was seen not onlywhen hemoglobin was tagged intact, but also when isolated chains weretagged. This may indicate that there is a specific locus or chemical configuration within the chains where Cr⁵¹ tagging occurs. Knowledge of the exacttotal amino acid sequence of , , and chains should soon be available andmay permit a precise identification of such a Cr⁵¹ binding site.

Whether the results of this type of highly artificial manipulation of hemoglobin has significance in Cr⁵¹ red cell survival studies in clinical situationswhere large amounts of Hb F occur is uncertain. Conflicting results of red cellsurvival studies of the fetal red cells suggests that it may.^27,28 Further investigation of this problem is currently in progress.

Submitted on October 16, 1962 Accepted on March 1, 1963     

A New Abnormal Hemoglobin O Padova, agr 30 (B11) Glu rarr Lys, and a Dyserythropoietic Anemia With Erythroblastic Multinuclearity Coexisting in the Same Patient

A patient with a not previously describedabnormal hemoglobin (^{30Glu Lys}) anddyserythropoietic anemia with erythroblastic multinuclearity is reported. Theheat stability and the functional propertiesof the new abnormal hemoglobin, namedhemoglobin O Padova, are normal, although the replacement lies in the ₁₁ interchain contact. The hemolytic condition,which was much improved by splenectomy, therefore appears to be linked tothe hereditary erythroblastic multinuclearity similar to hereditary erythroblasticmultinuclearity with positive acidifiedserum test (HEMPAS). In addition to theleading features observed in publishedcases of this entity, our case exhibitedsome immunologic peculiarities.

Submitted on November 8, 1973 Accepted on June 11, 1974     

The Interaction of Hemoglobin E With beta Thalassemia: A Study of Hemoglobin Synthesis in a Family of Mixed Burmese and Iranian Origin

A 5-yr-old girl with hemoglobin E- thalassemia was discovered in a family of mixedorigin. The father is Iranian (-thalassemiatrait) and the mother is Burmese (hemoglobin-E trait). Hemoglobin synthesis wasstudied in vitro in the blood of theproposita and family members. In the subjects with hemoglobin E trait the ratio ofthe quantity of hemoglobin A to hemoglobin E was 3:1. However. the ^A/^Esynthesis ratio in reticulocytes was in therange of 1.5-2.18, and the specific activityof ^E was 31%-49% greater than ^A, suggesting instability of hemoglobin E withpreferential destruction of abnormal hemoglobin. The blood of the proposita exhibitedonly hemoglobin F and hemoglobin E andreticulocytes and bone marrow showed no^A synthesis. This Iranian -thalassemiagene is therefore of the ° type. The ^E/synthesis ratio (approximately 0.74) inblood of the proposita was similar to the^A/ ratio in mildly affected relatives withthalassemia trait. These results suggestthat the severity of the hemoglobin E-thalassemia syndrome is attributable toboth instability and defective synthesis ofhemoglobin E in association with absent^A synthesis due to a ° thalassemia gene.

Submitted on March 19, 1973 Revised on May 4, 1973 Accepted on May 8, 1973     

The Characterization of Hemoglobin Shimonoseki

Hemoglobin Shimonoseki, discovered in western Japan in 1960, has beenfurther characterized as a mutant with abnormal -polypeptide chains on thebasis of:

(1) The presence of a minor hemoglobin component migrating cathodallyat pH 8.6 to Hb A₂, presumably ₂^Sh₂^A₂.

(2) Hybridization studies.

(3) Fingerprinting of isolated -polypeptide chains.

Hemoglobin Sh is characterized by the substitution of arginine for glutamine at residue 54 and can therefore be designated as ₂^{54 Arg}₂^A.

Submitted on December 17, 1963 Accepted on March 7, 1964     

Homozygous State for Hb Constant Spring (Slow-moving Hb X Components)

A 12-yr-old Malay boy who was studied because his youngest brother and both hisparents had the slow-moving Hb Xcomponents (earlier reported to lead to HbH disease when combined with -thalassemia) was found to be homozygous forthe same slow-moving components. He hadsplenomegaly and a just palpable liver, mildanemia with microcytosis, hypochromia,slight morphologic changes of the red bloodcells, and slight reticulocytosis. Of eightchildren in the family, six had the trait forthe abnormality, one was normal, and one,the propositus, was homozygous. Structuralstudies of the isolated abnormal hemoglobinshowed it to be identical to Hb ConstantSpring (Hb CoSp), an -chain variant with172 residues instead of the usual 141, theadditional 31 being attached to the C-terminal end. In addition to the abnormal variant for which the propositus washomozygous, he also had normal Hb A andnormal Hb A₂ with normal -chains. If thetheory that Hb CoSp is due to a structuralmutation affecting the terminator codon iscorrect, this case provides evidence for a duplication of the gene for -chain production.Results of study of several erythrocyteenzymes are also reported.

Submitted on March 15, 1973 Revised on July 30, 1973 Accepted on August 2, 1973     

Globin Chain Synthesis in the Marrow and Reticulocytes of Beta Thalassemia, Hemoglobin H Disease, and Beta Delta Thalassemia

, , and globin chain synthesis inbone marrow and peripheral bloodreticulocytes were studied in two patients with thalassemia major, two withthalassemia intermedia, one with thalassemia minor, one with Hb H disease,and one with homozygous -thalassemia. Nine nonthalassemic patientsserved as controls. In thalassemiamajor, a marked imbalance of - to -chain synthesis was found in the bonemarrow as well as in reticulocytes. Theimbalance, however, was slightly moreevident in the latter. In the patientswith thalassemia intermedia and minorthe - to -globin chain ratios in thereticulocytes were of the same orderof magnitude, despite the marked clinical differences between thalassemiaintermedia and minor. A balanced synthesis was found in the bone marrowof the patient with thalassemia minor.The bone marrow globin synthesis inthalassemia intermedia was not studied. Contrary to that in Hb H diseaseand -thalassemia, the imbalancewas more apparent in the bone marrow. In the latter, no evidence for imbalance was detected in the reticulocytes. These results point out the needfor further studies on globin chain synthesis in the bone marrow and reticulocytes of patients With the variousthalassemia syndromes and the effectof the free globin chain pool on thoseresults.

Submitted on March 9, 1971 Revised on November 29, 1971 Accepted on February 12, 1972     

Adult murine hematopoiesis can proceed without beta1 and beta7 integrins

The function of 41 and 47 integrins in hematopoiesis is controversial. While some experimental evidence suggests a crucial role for these integrins in retention and expansion of progenitor cells and lymphopoiesis, others report a less important role in hematopoiesis. Using mice with a deletion of the 1 and the 7 integrin genes restricted to the hematopoietic system we show here that 41 and 47 integrins are not essential for differentiation of lymphocytes or myelocytes. However, 17 mutant mice displayed a transient increase of colony-forming unit (CFU-C) progenitors in the bone marrow and, after phenylhydrazine-induced anemia, a decreased number of splenic erythroid colony-forming units in culture (CFUe's). Array gene expression analysis of CD4⁺CD8⁺ double-positive (DP) and CD4^–CD8^– double-negative (DN) thymocytes and CD19⁺ and CD4⁺ splenocytes did not provide any evidence for a compensatory mechanism explaining the mild phenotype. These data show that 41 and 47 are not required for blood cell differentiation, although in their absence alterations in numbers and distribution of progenitor cells were observed.      

Variable protection of beta 3-integrin--deficient mice from thrombosis initiated by different mechanisms

Platelet integrin IIb3 (GPIIb/IIIa) plays a central role inthe initiation of arterial thrombosis, but its contribution todisseminated microvascular thrombosis is less well defined. Therefore,wild-type mice (3^+/+), 3-integrin-deficient mice(3^/), and wild-type mice treated with a hamstermonoclonal antibody (1B5) that blocks murine IIb3 function weretested in models of large-vessel and microvascular thrombosis. In thelarge-vessel model, ferric chloride was used to injure the carotidartery, and the time to thrombosis was measured. In 3^+/+mice, the median time to occlusion was 6.7 minutes, whereas occlusion did not occur in any of the 3^/ mice tested(P < .001). Fab and F(ab')₂ fragments of1B5 increased the median time to occlusion. To initiate systemicintravascular thrombosis, prothrombotic agents were administeredintravenously, and platelet thrombus formation was monitored by thedecrease in circulating platelet count. Three minutes after theinjection of adenosine diphosphate (ADP), collagen + epinephrine,or tissue factor, the platelet counts in 3^+/+ micedecreased by 289, 424, and 429 × 10³/µL, respectively.3^/ mice and wild-type mice pretreated with 1B5 Fab(1 mg/kg, IP) were nearly completely protected from the effects of ADP.In contrast, 3^/ mice were only partially protectedfrom the effects of collagen + epinephrine and minimally protectedfrom the effects of tissue factor. In all cases, less fibrin becamedeposited in the lungs of 3^/ mice than in wild-typemice. These results suggest that though IIb3 plays adominant role in large-vessel thrombosis, it plays a variable role insystemic intravascular thrombosis.     

Coinduction of Embryonic and Adult-Type Globin mRNAs by Sodium Butyrate and Trichostatin A in Two Murine Interleukin-3-Dependent Bone Marrow-Derived Cell Lines

Using an RNase protection assay, globin mRNA species expressed inclones derived from Ba/F3 and B6SUtA cells transfected with theerythropoietin receptor (EpoR) and selected with erythropoietin (Epo)were compared with globin mRNA species induced in corresponding parental cells by sodium butyrate (SB) and trichostatin A (TSA). ^Major/^minor- and -1/-2-globinmRNAs were the major species, with trace amounts of -globin mRNA,formed in Epo-stimulated EpoR⁺ Ba/F3 clones, whereas SBand TSA allowed expression of all species of globin mRNAs, ie, ,h1, ^major/^minor, , and -1/-2,in parental Ba/F3 cells. In contrast, - and -1/-2-globinmRNAs were the major species present in Epo-stimulated EpoR⁺ B6SUtA clones, whereas SB and TSA activated -,h1-, ^S/^T-, and -1/-2-globingenes in parental B6SUtA cells; -globin mRNA was not detected in SB-and TSA-treated B6SUtA cells. Because TSA is a specific inhibitor ofhistone deacetylase, the mimicry of action exhibited by SB and TSAsuggests that the effects of SB are mediated through its ability toinhibit histone deacetylase and that histone deacetylase is an integralpart of the repression of globin genes in theseinterleukin-3-dependent cells. Efficient coinduction of embryonic andadult types of globin mRNA in bone marrow cell lines derived from adultmice indicates that adult hematopoietic precursors possess an embryonicnature. These cell lines are useful models to study the mechanism(s) ofdevelopmental globin gene switching.