IGHV gene features and MYD88 L265P mutation separate the three marginal zone lymphoma entities and Waldenström macroglobulinemia/lymphoplasmacytic lymphomas.

To clarify the relationships between marginal zone lymphomas (MZLs) and Waldenström macroglobulinemia/lymphoplasmacytic lymphomas (WM/LPLs), immunoglobulin heavy chain variable gene (IGHV) features were analyzed and the occurrence of MYD88 L265P mutations was identified in a series of 123 patients: 53 MZLs from the spleen (SMZLs), 11 from lymph nodes (NMZLs), 28 mucosa-associated lymphatic tissue (MALT) lymphomas and 31 WM/LPLs. SMZLs were characterized by overrepresentation of IGHV1-2 gene rearrangements with a canonical motif, without selection pressure and with long CDR3 segments. NMZLs had increased frequencies of IGHV3 genes. The IGHV gene was unmutated in most cases, often with long CDR3 segments. MALT lymphomas were usually associated with a mutated IGHV gene, but with the absence of selection pressure. WM/LPLs were associated with an IGHV3-23 overrepresentation and high IGHV mutation rate, with features of selection pressure and short CDR3 segments. MYD88 L265P mutations were almost restricted exclusively to WM/LPL patients. Taken all diagnoses together, all patients with MYD88 L265P mutations had an immunoglobulin M peak and almost all patients except one had bone marrow infiltration. These results demonstrate that the history of antigen exposure of the four entities studied was different and MYD88 L265P was specifically associated with WM/LPLs. WM/LPL may thus be functionally associated with constitutive nuclear factor-κB activation.

In vitro evaluation of Haemonetics MCS+ apheresis platelet concentrates treated with photochemical pathogen inactivation following plasma volume reduction using the INTERCEPT Preparation Set.

Pathogen inactivation using the INTERCEPT Blood System requires platelet resuspension in InterSol and reduced plasma. Platelets in plasma collected on the Haemonetics MCS+ were processed on the INTERCEPT Preparation Set for plasma volume reduction and addition of InterSol. The use of the Preparation Set resulted in a mean platelet loss of 5.6 +/- 3.4%. Subsequent photochemical treatment (PCT) with amotosalen and ultraviolet A light, and 7 days of storage, resulted in acceptable changes for platelet swirling, lactate, lactate dehydrogenase (LDH), platelet factor-4 (PF4), p-selectin, glycoprotein V (GpV), pO2, pCO2, tumour necrosis factor-alpha (TNF-alpha) and interleukin-8 (IL-8). All platelet units processed with the Preparation Set and PCT met European requirements for leucoreduction and pH values.

Leukodepletion of platelet concentrates and plasma collected with haemonetics MCS+ apheresis system. Experience of EFS-Alsace.

The prevention of transfusion reactions and transmission of infectious diseases partly relies on the systematic removal of leukocytes from blood products. Apheresis platelets and plasma collected on the Haemonetics MCS+ collection system require filtration to obtain low levels of residual leukocytes. This filtration step is automated for platelet concentrates, whereas plasma filtration requires sterile docking of a leukoreduction filter. Our experience shows residual leukocyte levels of approximately 10(5) for platelets (the French requirements are 10(6) per unit) and 10(2) for apheresis plasma (no existing standard in France). Leukocyte residuals in platelets are highly dependent on the filtration rate, which should be as slow as possible. Whereas the current method of filtration is convenient for platelets, the connection of an in-line filter for plasma causes some organizational problems and is also associated with a loss of plasma. Haemonetics' latest development, the use of a filtering core bowl, should avoid the requirement for the connection of an additional filter for plasma filtration and will ensure continuous filtration of platelets, reducing, even further, the residual leukocyte count in platelet concentrates.

A null HLA-A*68 allele in a bone marrow donor.

The authors describe an A*68 allele present at the molecular level but not expressed at the cell surface. This non expression results from the deletion of one nucleotide in exon 1, which causes a shift of the reading frame leading to an early non-sense codon in the same exon.

An intronic mutation responsible for a low level of expression of an HLA-A*24 allele.

HLA class I typing performed in parallel by molecular biology and serology has revealed cases where an HLA class I allele was identified but the corresponding antigen on the cell surface was not detected. In the present report, we describe three members of a family in whom an HLA-A24 allele identified at the molecular level was typed as A "blank" by lymphocytotoxicity. This serologically blank antigen was nevertheless faintly detectable by isoelectric focusing (IEF) and FACS analyses. Sequencing of the HLA-A*24 allele from the promoter region to the eighth exonic region revealed a point mutation in the acceptor site of the second intron as compared to the normal HLA-A*24 allele. This mutation could lead to incorrect processing of mRNA through a cryptic acceptor site located at the beginning of the third exon and hence to alternative splicing with a frame shift introducing an early stop codon into the fourth exon.

A nucleotide insertion in exon 4 is responsible for the absence of expression of an HLA-A*01 allele.

HLA class I typing performed in parallel by molecular biology and serology has revealed cases where an HLA class I allele was identified whereas the corresponding antigen was not detected on the cell surface. In the present report, we describe four members of a family in whom an HLA-A1 allele identified at the molecular level was typed as A "blank" by lymphocytotoxicity. This serologically blank antigen was undetectable by isoelectric focusing (IEF). Sequencing of the HLA-A*01 allele from the promoter region to the eighth exonic region revealed insertion of a "C" nucleotide at the beginning of the fourth exon as compared to the common HLA-A*0101 allele. This mutation causes a frame shift, giving rise to an early stop codon in the fourth exon.

Homozygous human TAP peptide transporter mutation in HLA class I deficiency.

Human lymphocyte antigen (HLA) class I proteins of the major histocompatibility complex are largely dependent for expression on small peptides supplied to them by transporter associated with antigen processing (TAP) protein. An inherited human deficiency in the TAP transporter was identified in two siblings suffering from recurrent respiratory bacterial infections. The expression on the cell surface of class I proteins was very low, whereas that of CD1a was normal, and the cytotoxicity of natural killer cells was affected. In addition, CD8+ alpha beta T cells were present in low but significant numbers and were cytotoxic in the most severely affected sibling, who also showed an increase in CD4+CD8+ T cells and gamma delta T cells.

A new DR14 allele (DRB1*1411) containing a short DR11 sequence and its haplotypic association.

In the present work, we describe a new DR14 allele. Sequencing of its second DRB1 exon showed it to be DRB1*1404 from codon numbers 9 to 56 and 61 to 86, and DRB1*11 from codons 57 to 60 inclusive.

Yeast methionyl-tRNA synthetase: analysis of the N-terminal extension and the putative tRNA anticodon binding region by site-directed mutagenesis.

Yeast methionyl-tRNA synthetase has a long N-terminal extension fused to the mononucleotide binding fold that occurs at the N-terminal end of the homologous E coli enzyme. We examined the contribution of this polypeptide region to the activity of the enzyme by creating several internal deletions in MESI which preserve the correct reading frame. The results show that 185 amino acids are dispensable for activity and stability. Removal of the next 5 residues affects the activity of the enzyme. The effect is more pronounced on the tRNA amino-acylation steps than on the adenylate formation step. The Km for ATP and methionine are unaltered, indicating that the global structure of the enzyme is maintained. The Km for tRNA increased slightly by a factor of 3, which indicates that the positioning of the tRNA on the surface of the molecule is not affected. There is, however, a great effect on the Vmax of the enzyme. Examination of the 3-D structure of the homologous E coli methionyl-tRNA synthetase indicates that the amino acid region preceding the mononucleotide binding fold does not participate directly in the catalytic cleft. It could, however, act at a distance by propagating a mutational alteration of the catalytic residues. The tRNA(Met) anticodon binding region of the E coli enzyme has recently been characterized. By mutagenesis of the topologically equivalent region in the yeast enzyme, we could identify residues that alter specifically the aminoacylation of the tRNA. Leu 658 provides a van der Waals contact that is critical for the recognition of the yeast tRNA.

Influence of hemoglobin precipitation on erythrocyte metabolism in alpha and beta thalassemia.

Certain aspects of the metabolism of centrifuged young and old erythrocytes in hemoglobin H disease have been examined and compared with similar studies of beta thalassemia and normal cells. Glycolysis, hexose monophosphate shunt activity (HMPS), potassium flux, and glutathione (GSH) content were measured. The distributions of hemoglobins H and F, as well as the activities of erythrocyte glucose-6-phosphate dehydrogenase (G6PD) and glutamic oxalacetic transaminase (GOT), were utilized for estimations of the relative ages of the cell samples. The young erythrocytes in hemoglobin H disease differed in several respects from older hemoglobin H cells. They contained more soluble hemoglobin H and GSH and, after splenectomy, fewer inclusions. HMPS activity was subnormal in hemoglobin H young cells and rose to normal activity in old cells. Potassium flux tended to increase in old cells when inclusions were present.Beta thalassemia young cells contained less hemoglobin F and, after splenectomy, more inclusions than old cells. In addition, they had markedly increased glycolysis and HMPS activity. GSH was randomly distributed. Potassium flux was increased in younger cells and particularly increased when inclusions appeared in younger cells after splenectomy. The results are interpreted to indicate that inclusion formation is associated with increased erythrocyte cation permeability in the thalassemia syndromes. This is not related to the level of intracellular GSH. The decreased HMPS activity in young hemoglobin H cells may be due to the presence of the extra thiols of soluble hemoglobin H which can act as a reducing agent. The substitution of hemoglobin H for glutathione in this capacity would then spare the NADPH-requiring glutathione reductase system. As a consequence, HMPS activity would decline. However, in older cells the oxidized hemoglobin H precipitates; these must rely upon GSH and glutathione reductase activity for thiol reduction capacity. Accordingly, HMPS activity increases to normal in the old cell population.