Mechanism of dexamethasone inhibition of chemotactic factor induced granulocyte aggregation

The reaction of FMLP with granulocytes causes aggregation and degranulation and enhances adherence to endothelium. To evaluate whether prevention of granule extrusion could impair these granulocyte activities, granulocytes were treated with either dexamethasone or hydrocortisone prior to treatment with FMLP. Dexamethasone was added to suspensions of cytochalasin B-treated granulocytes; it markedly impaired the aggregation response of the granulocytes of FMLP. When cytochalasin-B was not used, granulocyte aggregation in response to FMLP or PMA was inhibited by dexamethasone. Although dexamethasone prevented aggregation of cells following stimulation with FMLP or PMA, it failed to prevent the aggregation of granulocytes induced by rabbit lactoferrin. Adherence of granulocytes to human endothelial monolayers was enhanced by FMLP; dexamethasone inhibited the enhancement. However, with the addition of human lactoferrin to the granulocytes exposed to dexamethasone, the cells were able to adhere as well to endothelium as the cells exposed to FMLP but free of dexamethasone. When cytochalasin- B-treated granulocytes were incubated with dexamethasone or hydrocortisone prior to the addition of FMLP, the subsequent release of lactoferrin was substantially blocked, whereas the release of the primary granule products, lysozyme and beta-glucuronidase, was attenuated but not completely blocked. Thus, corticosteroids might block chemotactic-factor-induced granulocyte aggregation by selectively preventing release of specific granule products that contribute to and sustain aggregation.     

Early transmissibility assessment of the N501Y mutant strains of SARS-CoV-2 in the United Kingdom,October to November 2020

Two new SARS-CoV-2 lineages with the N501Y mutation in the receptor-binding domain of the spike protein spread rapidly in the United Kingdom. We estimated that the earlier 501Y lineage without amino acid deletion Δ69/Δ70, circulating mainly between early September and mid-November, was 10% (6–13%) more transmissible than the 501N lineage, and the 501Y lineage with amino acid deletion Δ69/Δ70, circulating since late September, was 75% (70–80%) more transmissible than the 501N lineage.     

Isolation and characterization of an age-related antigen present on senescent human red blood cells

Autologous membrane-bound IgG was isolated from a subpopulation of human red blood cells (RBC) with specific density greater than 1.110, by affinity chromatography of purified RBC membrane glycoprotein preparations using immobilized wheat germ agglutinin and immobilized anti-human immunoglobulin (Ig) as immunoabsorbents. The Ig-containing population thus obtained, when further separated by chromatography on Sephadex G-200 in the presence of chaotropic agents, yielded four peaks (Ia, Ib, II, and III). Double immunodiffusion revealed the presence of Ig in the first three peaks (IgM in peak Ia, IgA in Ib, and IgG in II) but not in peak III. Peak III was precipitated by the Ig-containing peaks (Ia, Ib, and II) in immunodiffusion assays, suggesting that the antigenic membrane determinants responsible for the binding of autologous Ig to senescent human RBC were contained in this peak (III). Peaks Ia, Ib and II precipitate purified asialoglycophorin; peak III was reactive with purified autoantibodies directed against asialoglycophorin. These results suggest that an age-related antigenic determinant(s) present on senescent human RBC is exposed by desialylation of the major sialoglycoprotein component of the RBC membrane.     

Concordance of a point mutation 5' to the G gamma globin gene with G gamma beta +. Hereditary persistence of fetal hemoglobin in the black population

Hereditary persistence of fetal hemoglobin (HPFH) is a genetically heterogeneous and clinically benign condition characterized by persistent expression of fetal hemoglobin (Hb F) into adulthood. In the G gamma beta + type, no major deletions in the globin gene cluster occur; adult heterozygotes produce approximately 20% Hb F, which results from overproduction of G gamma chains, with no apparent increase in production from the adjacent A gamma gene. We have recently described a point mutation 202 base pairs 5' to the cap site of the G gamma gene in an individual with G gamma beta + HPFH. This mutation abolishes a normal ApaI restriction endonuclease site, and thus can be detected by blotting of genomic DNA. We present here further data on the ApaI mutation: (1) It occurs in six of seven families with G gamma beta + HPFH. (2) In three families, detailed haplotype analysis using 11 polymorphic restriction sites in the beta globin cluster has been done. The two that carry the missing ApaI site are identical but the third, which has a normal ApaI pattern, differs from the other two in at least two sites, one of which is a new polymorphic Nco I site between the delta and beta globin genes. This suggests the possibility of a different HPFH mutation in the third family. (3) The haplotype of the G gamma beta + HPFH chromosome carrying the ApaI mutation is different from that of 108 beta A chromosomes of black individuals that have been tested. (4) The G gamma ApaI site is normal in 61 beta A and 109 beta S alleles from non-HPFH black individuals, including 22 who share the same haplotype for the intragenic G gamma, A gamma HindIII polymorphisms. These data add support to the possibility that the -202 mutation is actually causative of the G gamma beta + HPFH phenotype.     

Hereditary antithrombin deficiency: heterogeneity of the molecular basis and mortality in Dutch families

We studied the molecular basis and genetic heterogeneity of hereditary antithrombin (III) deficiency in nine Dutch families. Polymerase chain reaction (PCR) amplification and direct sequencing of all antithrombin gene exons and flanking intronic regions identified mutations in eight families. Given the opportunity to correlate the molecular basis with survival, we addressed the relevance of molecular defects to mortality in inherited antithrombin deficiency. The defects included single nucleotide deletions (7671 del G, 7768-69 del G) and insertions (5501 ins A, 2463 G-->TC) that lead to frameshifts, a single base substitution [5381 C-->T (129Arg-->stop)] leading to a premature termination codon, and single base substitutions resulting in amino acid substitutions [2652 A-->C (63Tyr-->Ser), 13380 T-->C (421Ile-- >Thr), and 13407 G-->T (430Cys-->Phe)]. All affected individuals were heterozygous for the defects. Previously we found in Dutch families that antithrombin deficiency did not lead to higher mortality compared with the general population. In accordance with these findings, we observed no excess mortality in the nine families [Observed:Expected, 52:52.6; standardised mortality ratio (SMR) 1.0, 95% confidence interval (CI), 0.7-1.3]. Our findings confirmed a considerable genetic heterogeneity underlying antithrombin deficiency. We therefore concluded that the lack of excess mortality in these families is not caused by a Dutch mild defect. We suggest that the longevity is not affected by molecular defects in the antithrombin gene and hypothesize that differences in mortality or natural history between families most likely result from other (genetic) risk factors.     

Beta-thalassemia due to two novel nucleotide substitutions in consensus acceptor splice sequences of the beta-globin gene

We have identified two novel RNA-splicing mutations affecting a critical nucleotide (nt) in the acceptor consensus sequences at both the IVS-1/exon 2 and IVS-2/exon 3 junctions of the human beta-globin gene. Both mutations are single nt substitutions, T to G and C to A, at position -3 adjacent to the invariant AG dinucleotide. For the IVS- 2/exon 3 mutation abnormal splicing into the cryptic splice site at IVS- 2 nt 579 is documented. Identification of these two mutations provides further support for the importance of the location of specific nucleotides within the consensus sequences in splice site selection and RNA processing.     

The regulation of human factor V by a neutrophil protease

Neutrophils activated with serum opsonized zymosan, soluble heat- aggregated IgG, and ionophore A23187 in the presence of calcium release a material capable of initially activating factor V. Subsequent inactivation of factor V was only observed with neutrophil releasate derived from IgG and ionophore. In this study we examine the nature of this neutrophil activity and investigate its role in the regulation of factor V/Va. From early in the fractionation it was apparent that the cells contained different enzymes capable of cleaving factor V. The most active of these was isolated and found to be an isomer of human neutrophil elastase. The purified protease caused a dose-dependent activation of isolated factor V to a maximum of threefold. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, single-chain factor V was cleaved to form intermediates of 100 and 91 kilodaltons (kD). Coagulant activity correlated with the formation of a 97-kD heavy and 77-kD light chain. On prolonged incubation the formed factor Va(e) was inactivated in association with proteolysis of the 97-kD band to smaller peptides and cleavage of the 77-kD light chain to a molecular weight of 75 kD, which is similar to thrombin-activated factor Va light chain. Neutrophil elastase also caused rapid inactivation of thrombin- activated factor V, factor Va(t). These observations suggest that elastase cleaves factor V at sites distinct from that by thrombin and therefore represents a novel factor V activation pattern. It is proposed that upon neutrophil activation elastase is secreted into the plasma milieu to initiate factor V activation. This serves to generate small amounts of thrombin that, in turn, by positive feedback fully activates factor V and thus amplifies the coagulation reaction.     

Thrombomodulin, an endothelial anticoagulant protein, is absent from the human brain

Protein C activation by thrombin is significantly accelerated by the endothelial cell cofactor, thrombomodulin. In this study, we have developed a radioimmunoassay for thrombomodulin and have measured the cofactor content in several human tissues. The assay method detects as little as 2 ng of thrombomodulin. The highest thrombomodulin content was found in lung and placenta, but the antigen was also detected in spleen, pancreas, liver, kidney, skin, heart, and aorta. Unexpectedly, thrombomodulin was absent from brain. Extracts from cerebral cortex, cerebellum, centrum semiovale, midbrain, basal ganglia, pons, and medulla were devoid of thrombomodulin. In contrast, thrombomodulin antigen is present in extracerebral intracranial vessels, including basilar and internal carotid arteries and choroid plexus, as well as in endothelium of the pia-arachnoid.     

Molecular cloning of a cDNA encoding canine factor IX

Factor IX (F.IX) is a vitamin K-dependent plasma protein, a deficiency of which results in hemophilia B. A canine model of hemophilia B exists; attempts to use this model for gene transfer experiments or characterization of the hemophilic defect require elucidation of normal canine F.IX structure. We report the isolation and characterization of the coding region for canine F.IX cDNA. Canine F.IX possesses 86% identity at the amino-acid level with human F.IX. The leader peptide, Gla domain, EGF domains, and the carboxy-terminal portion of the heavy chains show extensive sequence conservation between the canine and human. All Glu residues undergoing gamma-carboxylation in humans are conserved in canines. The complete coding sequence for canine F.IX has been determined, and the derived translation product has been analyzed. A similar approach should allow identification of the causative mutation in canine hemophilia B. Furthermore, this clone may prove a valuable resource in gene transfer experiments for this disease.