Haemoglobin Inkster (α285 aspartic acid → valineβ2) Coexisting with β-Thalassaemia in a Caucasian Family

S ummary . Haemoglobin Inkster, a new α-chain variant, was discovered in a family which also had the gene for β-thalassaemia. The amino acid abnormality was in αTp-9 which contains 29 amino-acid residues. Structural studies were facilitated by cleavage of the abnormal α-chains with cyanogen bromide followed by tryptic digestion. The substitution was shown to be valine for aspartic acid at position 85 in the α-chain. Affected individuals had no haematological abnormalities. Individuals with both β-thalassaemia and Hb Inkster had slightly lower percentages of Hb Inkster than those found in persons heterozygous for the Hb Inkster gene alone. 'Interaction' between thalassaemia and variant haemoglobin genes involving different haemoglobin loci has been reported in another family with β-thalassaemia and an α-chain haemoglobin mutant, as well as in the converse situation of coexisting β-thalassemia and a β-chain haemoglobin mutant. This decrease in the mutant haemoglobin percentage differs from the more common 'interaction' of thalassaemia and mutant haemoglobin genes involving the same haemoglobin locus, in which the mutant haemoglobin percentage is increased. The mechanism for the 'interaction' is unknown, but the presence of an unusually low percentage of a haemoglobin variant should warrant investigation for coexisting thalassaemia involving a different haemoglobin locus.     

Activation of neutrophil function via CD66: differential effects upon β2 integrin mediated adhesion

We investigated the potential role of transforming growth factor-beta (TGF-β) on spontaneous and cytokine-induced proliferation of B-cell chronic lymphocytic leukaemia (B-CLL) cells in vitro . Purified B lymphocytes from 21 B-CLL patients were cultured for 5 d in the presence of medium alone, IL-2 and/or IL-10, in the presence or absence of TGF-β, and proliferation was measured by ³H-thymidine incorporation. TGF-β inhibited B-cell proliferation in the majority of patients (15/21) but no inhibition was detected in 6/21 patients whatever the type of stimulant used. Addition of neutralizing antibodies to TGF-β increased spontaneous and cytokine-induced proliferation; this effect was dose dependent and specific because addition of an irrelevant chicken IgG had no effect on B-CLL proliferation. In resistant patients, neutralizing antibodies to TGF-β did not increase the proliferation. The expression of TGF-β receptors on B-CLL cells was significantly lower than the one observed on normal CD5⁺ B lymphocytes for which the sensitivity to TGF-β inhibition was more marked than in CLL. In addition, we found a strong correlation between the response of leukaemic B cells to TGF-β inhibitory action and the expression of TGF-β receptors on these cells. In summary, TGF-β appears to function in CLL as a negative regulator of B lymphocytes but loss of responsiveness to this factor accompanied by a decrease of TGF-β receptor expression, might provide a selective advantage to B-CLL lymphocytes.     

Haemoglobin D Iran: β222 Glutamic Acid→Glutamine (B4)

A new haemoglobin D (Iran) has been described. The substitution was in the beta chain at residue 22 (B₄), which is the most frequently mutated area in human haemoglobin. No clinical symptoms were associated with this haemoglobin.     

β2 glycoprotein-I antigen is increased in primary hyperlipidaemia

Summary. In order to determine whether elevated levels of β₂ glycoprotein-I (β₂GPI) are associated with increased plasma lipids, we measured plasma β₂ GPI antigen levels in 47 patients with primary hyperlipidaemia (20 severe hypercholesterolaemia, nine severe hypertriglyceridaemia, and 18 mixed hyperlipidaemia) and 34 normal healthy subjects. Mean β₂GPI levels were significantly increased in each patient group (302.3, 272.9 and 299.1 mg/1. respectively) compared to controls (199.6 mg/1) (p<0.01). Significant correlations were demonstrated between β₂GPI levels and triglyceride and total cholesterol levels in the control group (r = 0.387, r =0.559: P<0.5), but were not observed in all patient groups. These results indicate that β₂GPI is increased in hyperlipidaemia and that its distribution between plasma lipid fractions is perturbed. Plasma lipid levels should therfore be considered when interpreting results of β₂GPI antigen assays.     

Renal Metabolism of β2-Microglobulin

beta 2-microglobulin (beta 2M) is a protein of 11,800 daltons which occurs in the plasma of normal individuals at concentrations of approximately 2 microgram/ml. It is presumed to be relatively freely filterable. More than 99% of the filtered beta 2M is taken up by an active reabsorptive mechanism and catabolized by the renal tubule. The data presented here demonstrate that renal extraction is only slightly diminished by complete ureteral obstruction. The renal extraction of beta 2M is greater than can be accounted for by filtration alone. These data indicate that some uptake of beta 2M occurs from the peritubular capillary circulation. The loading of animals with beta 2M is associated with a marked tubular proteinuria suggesting that this protein may play a part in inducing tubular injury.     

Long-term prognostic value of serum β2 microglobulin in myelomatosis

The prognostic value of serum beta 2 microglobulin (s beta 2m) measured at entry, at plateau and at 3-monthly follow-up times has been assessed for patients in the Medical Research Council's 4th and 5th Myelomatosis Trials. Analysis of 1014 patients confirmed the value of presentation s beta 2m of predicting survival in the short term but showed that predictive value was lost for subsequent survival in those patients who had survived for at least 2 years. However, measurements of s beta 2m taken during follow-up were predictive of subsequent survival. The predictive power of these follow-up measurements was very similar to that for the presenting measurement, and again they were only of value in predicting survival in the next 2 years.     

β2-glycoprotein 1 (β2GP1) enhances cardiolipin binding activity but is not the antigen for antiphospholipid antibodies

Some investigators have reported that a serum protein, beta 2-glycoprotein 1 (beta 2GP1), either alone or in combination with negatively charged phospholipid, may be the antigen for anticardiolipin (aCL) antibodies. To examine these reports further, ELISA tests, inhibition experiments, Ouchterlony and Western blot techniques were used to examine anticardiolipin binding to beta 2GP1. Sera from patients with the antiphospholipid syndrome (APS) and syphilis were studied, as well as whole IgG immunoglobulin and affinity purified (a.p.) IgG aCL antibodies. Results showed no binding of aCL antibodies to beta 2GP1 in the absence of cardiolipin. beta 2GP1 caused enhanced binding of aCL antibodies to cardiolipin, but this enhancement was not observed in inhibition experiments. Binding to cardiolipin occurred in the absence of beta 2GP1. Enhancement of cardiolipin binding activity by beta 2GP1 was observed for APS, but not for syphilis. We conclude that beta 2GP1 is not the antigen for aCL antibodies, nor is it likely that the antibody recognizes shared beta 2GP1-cardiolipin epitopes. Instead, this protein may make cardiolipin more available for aCL binding on solid surfaces by some yet undefined mechanism. This effect may not extend to aqueous suspensions.     

Significance of β2-Microglobulin in Liver Diseases

Serum levels of beta 2-microglobulin (beta 2-M) were found to be significantly elevated in acute viral hepatitis, chronic persistent or active hepatitis and liver cirrhosis. beta 2-M values were significantly lower in chronic persistent hepatitis than in the three other groups. Serum beta 2-M was normal in 75 asymptomatic carriers of HBsAg. Steroid therapy was followed by reduction of serum beta 2-M levels in 11 cases of chronic active hepatitis. Variations of beta 2-M were independent from that of transaminases, bilirubin and gamma-globulins.     

Pretreatment serum β2-microglobulin in multiple myeloma

Serum beta 2-microglobuline (S-beta 2m) was evaluated in 121 untreated patients with multiple myeloma. Values greater than 3 mg/l were found in 82% of the patients. Mean S-beta 2m values of the total group of patients correlated with clinical stage. However, there was no correlation if values were corrected for S-creatinine. Seventy-nine patients had normal (less than or equal to 106 mumol/l) and 52 patients abnormal S-creatinine. Patients with S-beta 2m values below 7 X 6 mg/l had an estimated median survival of 44 months compared to 12 months for patients with levels above 7 X 6 mg/l. If S-beta 2m values in patients with normal S-creatinine were combined with values corrected for S-creatinine from patients with elevated S-creatinine a beta 2m cut off level of 6 X 6 mg/l gave a median probable survival of 43 months compared to 14 months. We conclude that pretreatment S-beta 2 microglobulin is a useful marker for predicting survival in multiple myeloma. The problem of the relationship between S-beta 2m and S-creatinine is discussed.     

Compartmentalization regulates the interaction between the platelet integrin αIIbβ3 and ICln

The volume-regulating protein, ICln, interacts with the conserved KxGFFKR α-integrin signature motif. ICln is an abundant protein (4455 ± 650 molecules/platelet) found exclusively in the soluble cytosolic fraction of unactivated platelets. In contrast, its binding partner, the platelet integrin α_IIbβ₃, is present in detergent-insoluble fractions associated with membrane and cytoskeleton subcellular localizations. This study investigated factors that regulate the interaction of ICln with α_IIbβ₃ during platelet activation. His-tagged recombinant ICln bound equally to purified α_IIbβ₃ and to integrin from resting or activated platelets. Binding was not affected by direct integrin activation with Mn⁺⁺ or by inhibitors of integrin occupancy (abciximab, RGD). However, the capacity for interaction between integrin and recombinant ICln was slowly downregulated following prolonged platelet activation for >300 s. In parallel, ICln redistributed to membrane and cytoskeletal platelet subcellular fractions. The time-course of this redistribution preceded the downregulation of integrin binding capacity and suggests that only a short window of opportunity exists for ICln interaction with α_IIbβ₃ to occur. Thus, although ICln has the inherent capacity to bind to α_IIbβ₃ regardless of its activation state, it can only do so following platelet activation. Activation-dependent subcellular redistribution of ICln represents a novel, temporally-regulated mechanism for control of integrin function in platelets.     

Haemoglobin Vanderbilt (α2β289Ser→Arg): a New Haemoglobin with High Oxygen Affinity and Compensatory Erythrocytosis

Haemolysates of family members from three generations, all of whom had polycythaemia, were analysed by polyacrylamide gel electrophoresis at pH 8.8. Two closely spaced major bands were observed, one of which corresponded to Hb A and the other to a new mutant designated Hb Vanderbilt. Whole blood from a heterozygote for Hb Vanderbilt was analysed for oxygen affinity which was found to be much higher than that of normal subjects. Haemoglobin Vanderbilt was separated from Hb A using anion exchange chromatography. Cation exchange chromatography yielded a variant β chain from which a mutant peptide was identified with a structure corresponding to residues β83–89 with a Ser→Arg replacement at position 89. The oxygen affinity of 'stripped' haemolysates from the heterozygote was found to be much less sensitive to added organic phosphates than haemolysates from normal subjects. In whole blood, the decreased sensitivity to 2,3-diphosphoglycerate results in an increased oxygen affinity, thus explaining the clinical observations of tissue hypoxia and compensatory polycythaemia.     

Haemoglobin α2β223Val → Ile produced in Escherichia coli facilitates Hb S polymerization

The doubly substituted variant Hb S-Antilles (beta 6 Glu----Val, beta 23 Val----Ile) produces sickling in heterozygous carriers. The Csat value for pure deoxyHb S-Antilles is nearly half that of deoxyHb S. Dilute solutions of pure Hb S-Antilles have a lower oxygen affinity than those of Hb A or Hb S. The mutant Hb alpha 2 beta 2 23 Val----Ile was synthesized in E. coli. It exhibits a decreased oxygen affinity compared to Hb A and does not polymerize in 1.8 M phosphate buffer. Mixtures of equal amounts of Hb S + Hb beta 23 Val----Ile have a decreased Csat value compared to mixtures of Hb S + Hb A. The beta 23 Val in Hb S contributes to the axial contact joining molecules in each single filament. Substituting Ile for Val at this site increases the strength of this contact through hydrophobic interactions, allowing increased stability of the lateral contact between filaments in pair, which is the specific unit structure of polymers in deoxyHb S.     

Acute Ethanol Intoxication Inhibits Neutrophil β2-Integrin Expression in Rats During Endotoxemia

The effects of acute ethanol intoxication on neutrophil [polymorphonuclear leukocyte (PMN)] adhesion molecule expression and certain other functional properties during endotoxemia were studied in rats to elucidate the mechanisms underlying the immunosuppressive effects of ethanol. Acute ethanol intoxication was induced by an intraperitoneal injection of 20% ethanol at a dose of 5.5 g of ethanol/kg. Control animals received an intraperitoneal injection of saline. Thirty minutes after intraperitoneal injection, animals were given a 90-min intravenous infusion of Escherichia coli endotoxin (total dose of 112.5 μg/rat in 2.5 ml of saline) or saline. Certain rats received granulocyte colony-stimulating factor (G-CSF; 50 μg/kg in 5% dextrose, subcutaneous injection twice daily) or vehicle pretreatment for 2 days before intravenous endotoxin infusion. Endotoxemia significantly upregulated CD11b/c and CD18 expression on PMNs when compared with those of saline-infused rats. Acute ethanol intoxication inhibited this endotoxin-induced upregulation of CD11b/c and CD18 expression on PMNs. Ethanol intoxication also suppressed the phagocytic activities of PMNs in saline-infused rats, but this suppression failed to reach statistical significance in endotoxin-infused rats. Hydrogen peroxide generation by PMNs in saline- or endotoxin-infused rats was not affected by ethanol intoxication. Histological examination showed extensive PMN sequestration in the liver after endotoxin infusion, and ethanol intoxication significantly attenuated this hepatic sequestration of PMNs. G-CSF pretreatment enhanced neutrophil phagocytosis, CD11b/c and CD18 expression in endotoxin-infused rats, and prevented the ethanol-induced inhibition of neutrophil CD18 expression and phagocytosis. The impairment of β₂-integrin expression on PMNs may be one mechanism underlying ethanol-induced defects of neutrophil delivery into tissue sites of infection. G-CSF may be of benefit to the infected alcoholic host by enhancing leukocyte defense functions.     

β2-Microglobulin: Structure, Function and Significance

beta 2-Microglobulin is a low molecular weight protein with sequence homology to immunoglobulins. As a portion of the HLA complex this protein is an important cell-surface structure. Under normal conditions beta 2-microglobulin is synthesized and shed by many cells, particularly lymphocytes, and is detectable in the circulation of normal individuals. Because of its small size it is normally filtered readily at the glomerulus and is catabolized by proximal tubular cells of the kidney. Impaired renal function and hyperproduction of beta 2-microglobulin are both associated with increased serum levels. A function for beta 2-microglobulin as a modulator of lymphocyte surface and as a potential regulator of the immune system is proposed.     

Homozygous deletional α+ thalassaemia associated with unequal expression of the two remaining α1 genes (α1A and α1Q)

S ummary . A Cambodian family presenting several haemoglobinopathies, Hb E, Hb Q and α⁺ thalassaemia, has been investigated. DNA analysis showed that the thalassaemia syndrome corresponds to a leftward type (4.2 kb) deletional from of α⁺ thalassaemia. Genotypes found in the family are: propositus -α^A/-α^Q, β^A/β^E, mother and older sister α^Aα^A/ -α^Q, β^A/β^E; father α^Aα^A/-α^A, β^A/β^A. The propositus consistently presents an α^Q/α^A chain ratio of 60/40 although both chains are products of α₁ loci. The relatively higher expression of the α^Q chain is not observed in the mother and therefore makes it unlikely to reflect anything other than differential expression of the maternal -α^Q/ and paternal -α^A/ haplotypes. This observation raises the possibility that both haplotypes are not strictly identical and that the region of the cross-over event is important for α gene expression.     

Haemoglobin Radcliffe (α2β299(G1)Ala): A High Oxygen-Affinity Variant Causing Familial Polycythaemia

Three members of an Oxfordshire family have polycythacmia. In each case their whole-blood oxygen affinity is increased. This is due to a previously undescribed haemoglobin variant which has been named haemoglobin Radcliffe (α₂β₂^99(G1)Ala). In addition to having a high oxygen affinity haemoglobin Radcliffe shows virtually no haem-haem interaction and a diminished Bohr effect. It is synthesized at the same rate and is as stable as haemoglobin A. X-ray analysis indicates that crystals of deoxyhaemoglobin Radcliffe are isomorphous with those of deoxyhaemoglobin A. Solutions of haemoglobin Radcliffe were also studied by high-resolution proton nuclear magnetic resonance spectroscopy. The structure/function relationships of haemoglobin Radcliffe are discussed in the light of these studies.